233 results on '"Mark D. Minden"'
Search Results
2. The LSC17 Score Correlates with the ELN 2022 Classification of AML and Is an Independent Predictor of Detectable Measurable Residual Disease after Induction Chemotherapy
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Tracy Murphy, Stanley W.K. Ng, Ian King, Tong Zhang, Andrea Arruda, Jaime Claudio, Kristele Pan, Chantal Rockwell, Zhibin Lu, Natalie Stickle, Carl Virtanen, Vikas Gupta, Dawn Maze, Caroline McNamara, Aaron D. Schimmer, Andre C. Schuh, Hassan Sibai, Karen Yee, Dina Khalaf, Brian Leber, Mitchell Sabloff, Anne Tierens, Mark D. Minden, Tracy L. Stockley, Steven Chan, and Jean C.Y. Wang
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Immunology ,Cell Biology ,Hematology ,Biochemistry - Published
- 2022
3. Do Adult Philadelphia-Positive Acute Lymphoblastic Leukemia (Ph+ ALL) Patients Require Allogeneic Transplant in First Remission (CR1)?
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Fatima M Khadadah, Claire Andrews, Eshetu G. Atenafu, Andre C. Schuh, Dawn Maze, Karen Yee, Osamah Jamal S. Jarallah, Mark D. Minden, Aaron D. Schimmer, Steven M. Chan, Vikas Gupta, Dennis Dong Hwan Kim, and Hassan Sibai
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Immunology ,Cell Biology ,Hematology ,Biochemistry - Published
- 2022
4. Clinical and Biological Characteristics and Outcomes of Therapy-Related Acute Lymphoblastic Leukemia (t-ALL) Following Multiple Myeloma Are Distinct in Comparison to t-ALL Following Other Cancers
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Josephine Anne Lucero, Brayan Merchán, Osamah Jamal S. Jarallah, Aniket Bankar, Steven M. Chan, Marta B. Davidson, Vikas Gupta, Dawn Maze, Mark D. Minden, Guillaume Richard-Carpentier, Aaron D. Schimmer, Andre C. Schuh, Karen Yee, and Hassan Sibai
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Immunology ,Cell Biology ,Hematology ,Biochemistry - Published
- 2022
5. Single Centre, Retrospective Analysis of Treatment Outcomes and Prognostic Factors in Blastic Phase Chronic Myeloid Leukemia (CML-BP) Following Systemic Therapy
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Kenny Tang, Dawn Maze, Karen Yee, Hassan Sibai, Marta B. Davidson, Steven M. Chan, Aaron D. Schimmer, Andre C. Schuh, Aniket Bankar, Guillaume Richard-Carpentier, Anne Tierens, Mark D. Minden, Vikas Gupta, Jeffrey H. Lipton, and Dennis Kim
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Immunology ,Cell Biology ,Hematology ,Biochemistry - Published
- 2022
6. Simultaneous Inhibition of SIRT3 and Cholesterol Homeostasis Targets AML Stem Cells By Perturbing Fatty Acid β-Oxidation and Inducing Lipotoxicity
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Cristiana O'Brien, Tianyi Ling, Jacob Berman, Rachel Culp-Hill, Julie A. Reisz, Vincent Rondeau, Soheil Jahangiri, Jonathan St-Germain, Vinitha Macwan, Audrey Astori, Andy G.X. Zeng, Jun Young Hong, Meng Li, Min Yang, Sadhan Jana, John E. Dick, Hening Lin, Ari Melnick, Anastasia N Tikhonova, Andrea Arruda, Mark D. Minden, Brian Raught, Angelo D'Alessandro, and Courtney L Jones
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Immunology ,Cell Biology ,Hematology ,Biochemistry - Published
- 2022
7. Very long chain fatty acid metabolism is required in acute myeloid leukemia
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Jerry Vockley, Kristin J Hope, Kevin A Rea, Tariq A. Akhtar, Aaron D. Schimmer, Al-Walid Mohsen, Nawaz Ahmed, Paul A. Spagnuolo, Angelo D'Alessandro, Richard W. Smith, Matthew Tcheng, David A. Hess, Eric Bohrnsen, Mark D. Minden, Preethi Jayanth, and Alessia Roma
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0301 basic medicine ,Citric Acid Cycle ,Immunology ,Very long chain fatty acid ,Mitochondrion ,Biochemistry ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Cell Line, Tumor ,hemic and lymphatic diseases ,medicine ,Humans ,Glycolysis ,Beta oxidation ,Myeloid Neoplasia ,Chemistry ,Acyl-CoA Dehydrogenase, Long-Chain ,Fatty Acids ,Myeloid leukemia ,Ketone Oxidoreductases ,Cell Biology ,Hematology ,medicine.disease ,Neoplasm Proteins ,Citric acid cycle ,Leukemia, Myeloid, Acute ,Leukemia ,030104 developmental biology ,030220 oncology & carcinogenesis ,Cancer research ,Adenosine triphosphate - Abstract
Acute myeloid leukemia (AML) cells have an atypical metabolic phenotype characterized by increased mitochondrial mass, as well as a greater reliance on oxidative phosphorylation and fatty acid oxidation (FAO) for survival. To exploit this altered metabolism, we assessed publicly available databases to identify FAO enzyme overexpression. Very long chain acyl-CoA dehydrogenase (VLCAD; ACADVL) was found to be overexpressed and critical to leukemia cell mitochondrial metabolism. Genetic attenuation or pharmacological inhibition of VLCAD hindered mitochondrial respiration and FAO contribution to the tricarboxylic acid cycle, resulting in decreased viability, proliferation, clonogenic growth, and AML cell engraftment. Suppression of FAO at VLCAD triggered an increase in pyruvate dehydrogenase activity that was insufficient to increase glycolysis but resulted in adenosine triphosphate depletion and AML cell death, with no effect on normal hematopoietic cells. Together, these results demonstrate the importance of VLCAD in AML cell biology and highlight a novel metabolic vulnerability for this devastating disease.
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- 2021
8. CRISPR screen identifies genes that sensitize AML cells to double-negative T-cell therapy
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Hyeonjeong Kang, Housheng Hansen He, Mark D. Minden, Li Zhang, Jong Bok Lee, Yong Zeng, Dalam Ly, Enoch Tin, Fraser Soares, Nayeema Akhtar, Branson Chen, and Musaddeque Ahmed
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0301 basic medicine ,Adoptive cell transfer ,Myeloid ,T-Lymphocytes ,T cell ,Chronic lymphocytic leukemia ,Immunology ,Biochemistry ,03 medical and health sciences ,0302 clinical medicine ,Mice, Inbred NOD ,hemic and lymphatic diseases ,medicine ,Animals ,Humans ,CRISPR ,Cells, Cultured ,Predictive marker ,Gene Expression Regulation, Leukemic ,business.industry ,Receptors, IgG ,Myeloid leukemia ,Cell Biology ,Hematology ,medicine.disease ,Adoptive Transfer ,Leukemia, Myeloid, Acute ,Leukemia ,030104 developmental biology ,medicine.anatomical_structure ,030220 oncology & carcinogenesis ,Cancer research ,Female ,CRISPR-Cas Systems ,business - Abstract
Acute myeloid leukemia (AML) remains a devastating disease in need of new therapies to improve patient survival. Targeted adoptive T-cell therapies have achieved impressive clinical outcomes in some B-cell leukemias and lymphomas but not in AML. Double-negative T cells (DNTs) effectively kill blast cells from the majority of AML patients and are now being tested in clinical trials. However, AML blasts obtained from ∼30% of patients show resistance to DNT-mediated cytotoxicity; the markers or mechanisms underlying this resistance have not been elucidated. Here, we used a targeted clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) screen to identify genes that cause susceptibility of AML cells to DNT therapy. Inactivation of the Spt-Ada-Gcn5-acetyltransferase (SAGA) deubiquitinating complex components sensitized AML cells to DNT-mediated cytotoxicity. In contrast, CD64 inactivation resulted in resistance to DNT-mediated cytotoxicity. Importantly, the level of CD64 expression correlated strongly with the sensitivity of AML cells to DNT treatment. Furthermore, the ectopic expression of CD64 overcame AML resistance to DNTs in vitro and in vivo. Altogether, our data demonstrate the utility of CRISPR/Cas9 screens to uncover mechanisms underlying the sensitivity to DNT therapy and suggest CD64 as a predictive marker for response in AML patients.
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- 2021
9. Ex-Vivo Testing Using Patient Micro Avatars (PMAs) Predicts Clinical Response in Acute Leukemias
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Alejandra Garcia, Lía Buffa, Magalí Ridano, Paula Scaglia, Maribel Nicola, Sofía Carbajosa, Cintia Garro, Daniela Arroyo, Agustina Conrrero, Juan Pablo Fiorenza, Valentina Alfonso, María Laura Bernaschini, Diego Andino, Natalia Cavallo, Gimena Ferreira, Virginia García, Federico Molineris, Maximiliano Zeballos, Silvina Palmer, Isolda I Fernandez, Maria J Mela Osorio, Carolina Pavlovsky, Juan José García, Martín Alonso, Blanca de los Milagros Rossi, Gustavo Jarchum, Daiana Rodriguez, Andrea Arruda, Mark D. Minden, Nicolás Cazap, Jorge Solimano, Tarek Ali Zaki, Gerardo Gatti, Candelaria Llorens, and Gastón Soria
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Immunology ,Cell Biology ,Hematology ,Biochemistry - Published
- 2022
10. Inhibiting the Mitochondrial RNA Degradosome Complex SUV3 and Pnpase Increases dsRNA in the Cytoplasm, Triggers a Viral Mimicry Response and Kills AML Cells and Progenitors
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Geethu Emily Thomas, Kazem Nouri, Rose Hurren, Yongran Yan, Neil MacLean, Yulia Jitkova, Li Ma, Xiaoming Wang, Chaitra Sarathy, Andrea Arruda, Mark D. Minden, Vito Spadevecchio, and Aaron D. Schimmer
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Immunology ,Cell Biology ,Hematology ,Biochemistry - Published
- 2022
11. Identification of Global miRNA Targetomes within Human Hematopoietic Progenitors and Acute Myeloid Leukemia Uncovers Unique Molecular Networks Centered on Mitochondria Function in Leukemic Stem Cells
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Gabriela Krivdova, Brian Yee, Stefan Aigner, Martino Gabra, Alexander Shishkin, Long Nguyen, Jessica L McLeod, Leah Carley, Leonardo Salmena, Lorien Shakib, Mark D. Minden, Eric L Van Nostrand, Gene W. Yeo, Eric R. Lechman, and John E. Dick
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Immunology ,Cell Biology ,Hematology ,Biochemistry - Published
- 2022
12. Intracellular IL-23 Receptor (IL-23R) Is Necessary for AML Viability and Regulates Mitotic Spindle and Centrosome Formation
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Nathan Duong, Dilshad H. Khan, Geethu Emily Thomas, Rose Hurren, Jong Bok Lee, Jonathan St-Germain, Lily Drimmer, Yongran Yan, Neil MacLean, Marcela Gronda, Brandon D Brown, Brian Raught, Andrea Arruda, Mark D. Minden, Li Zhang, Steven M. Kornblau, Vito Spadevecchio, and Aaron D. Schimmer
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Immunology ,Cell Biology ,Hematology ,Biochemistry - Published
- 2022
13. Multiparameter Flow Cytometry-Based Residual Disease Analysis Identifies Intermediate-Risk AML Patients Who May Benefit from Allogeneic Hematopoietic Stem Cell Transplantation
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Josephine Anne Lucero, Anne Tierens, Muhned Alhumaid, Jose-Mario Capo-Chichi, Tracy L. Stockley, Vikas Gupta, Steven M. Chan, Andre C. Schuh, Mark D. Minden, Jonas Mattsson, Rajat Kumar, Hassan Sibai, and Dennis Dong Hwan Kim
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Immunology ,Cell Biology ,Hematology ,Biochemistry - Published
- 2022
14. Outcome of ALL in Adult Patient with Down Syndrome, Single Center Experience
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Salman Alharbi, Maria Agustina Perusini, Sita Bhella, Mark D. Minden, Dawn Maze, Vikas Gupta, Aaron D. Schimmer, Andre C. Schuh, Karen Yee, Steven M. Chan, Aniket Bankar, Marta B. Davidson, Guillaume Richard-Carpentier, Jad Sibai, and Hassan Sibai
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Immunology ,Cell Biology ,Hematology ,Biochemistry - Published
- 2022
15. Stem Cell-Derived Gene Expression Scores Predict Survival and Leukemic Transformation in Myelofibrosis
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Jessie J.F. Medeiros, Andy G.X. Zeng, Michelle Chan-Seng-Yue, Tristan Woo, Jessica L McLeod, Andrea Arruda, Hubert Tsui, Burhan Goraya, Jaime O. Claudio, Jenny M. Ho, James A. Kennedy, Dawn Maze, Hassan Sibai, Mark D. Minden, Jean C.Y. Wang, John E. Dick, and Vikas Gupta
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Immunology ,Cell Biology ,Hematology ,Biochemistry - Published
- 2022
16. Clinical Outcomes in De Novo Versus Secondary NPM1-Mutated AML
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Elliot C. Smith, Eshetu G. Atenafu, Aniket Bankar, Steven M. Chan, Marta B. Davidson, Vikas Gupta, Mark D. Minden, Guillaume Richard-Carpentier, Aaron D. Schimmer, Andre C. Schuh, Hassan Sibai, Karen Yee, and Dawn Maze
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Immunology ,Cell Biology ,Hematology ,Biochemistry - Published
- 2022
17. CC-90009, a novel cereblon E3 ligase modulator, targets acute myeloid leukemia blasts and leukemia stem cells
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Joshua Hansen, John E. Dick, Philip P Chamberlain, Emily Rychak, Gang Lu, In Sock Jang, Jean C.Y. Wang, Thomas Clayton, Celia Fontanillo, Derek Mendy, Michael Pourdehnad, Jinhong Fan, Stanley W.K. Ng, Eileen Tran, Mark Rolfe, Nathan Mbong, Kai Wang, Chin-Chun Lu, James Carmichael, Christine Surka, Mary E Matyskiela, Daniel W. Pierce, Mark D. Minden, Liqing Jin, Antonia Lopez-Girona, Brian E. Cathers, Elizabeth Anne Tindall, Adrian Contreras, and Christy Hsu
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Models, Molecular ,0301 basic medicine ,Myeloid ,Protein Conformation ,Mice, SCID ,Isoindoles ,01 natural sciences ,Biochemistry ,Mice ,Mice, Inbred NOD ,Acetamides ,Molecular Targeted Therapy ,biology ,Chemistry ,TOR Serine-Threonine Kinases ,Myeloid leukemia ,U937 Cells ,Hematology ,Neoplasm Proteins ,Ubiquitin ligase ,Leukemia, Myeloid, Acute ,Leukemia ,medicine.anatomical_structure ,Neoplastic Stem Cells ,Nuclear Factor 45 Protein ,Stem cell ,Peptide Termination Factors ,Proteasome Endopeptidase Complex ,Ubiquitin-Protein Ligases ,Immunology ,Small Molecule Libraries ,03 medical and health sciences ,Stress, Physiological ,Cell Line, Tumor ,medicine ,Animals ,Humans ,Integrated stress response ,Nuclear Factor 90 Proteins ,Piperidones ,PI3K/AKT/mTOR pathway ,Adaptor Proteins, Signal Transducing ,010405 organic chemistry ,Cereblon ,Ubiquitination ,Cell Biology ,medicine.disease ,Xenograft Model Antitumor Assays ,0104 chemical sciences ,030104 developmental biology ,Proteolysis ,biology.protein ,Cancer research ,CRISPR-Cas Systems ,Protein Processing, Post-Translational - Abstract
A number of clinically validated drugs have been developed by repurposing the CUL4-DDB1-CRBN-RBX1 (CRL4CRBN) E3 ubiquitin ligase complex with molecular glue degraders to eliminate disease-driving proteins. Here, we present the identification of a first-in-class GSPT1-selective cereblon E3 ligase modulator, CC-90009. Biochemical, structural, and molecular characterization demonstrates that CC-90009 coopts the CRL4CRBN to selectively target GSPT1 for ubiquitination and proteasomal degradation. Depletion of GSPT1 by CC-90009 rapidly induces acute myeloid leukemia (AML) apoptosis, reducing leukemia engraftment and leukemia stem cells (LSCs) in large-scale primary patient xenografting of 35 independent AML samples, including those with adverse risk features. Using a genome-wide CRISPR-Cas9 screen for effectors of CC-90009 response, we uncovered the ILF2 and ILF3 heterodimeric complex as a novel regulator of cereblon expression. Knockout of ILF2/ILF3 decreases the production of full-length cereblon protein via modulating CRBN messenger RNA alternative splicing, leading to diminished response to CC-90009. The screen also revealed that the mTOR signaling and the integrated stress response specifically regulate the response to CC-90009 in contrast to other cereblon modulators. Hyperactivation of the mTOR pathway by inactivation of TSC1 and TSC2 protected against the growth inhibitory effect of CC-90009 by reducing CC-90009-induced binding of GSPT1 to cereblon and subsequent GSPT1 degradation. On the other hand, GSPT1 degradation promoted the activation of the GCN1/GCN2/ATF4 pathway and subsequent apoptosis in AML cells. Collectively, CC-90009 activity is mediated by multiple layers of signaling networks and pathways within AML blasts and LSCs, whose elucidation gives insight into further assessment of CC-90009s clinical utility. These trials were registered at www.clinicaltrials.gov as #NCT02848001 and #NCT04336982).
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- 2021
18. CD200 expression marks leukemia stem cells in human AML
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Jenny M. Ho, Kolja Eppert, Mark D. Minden, Liqing Jin, Jessica McLeod, James A. Kennedy, Amanda Mitchell, Stephanie M. Dobson, John E. Dick, Gary D. Bader, Veronique Voisin, and Jean C.Y. Wang
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0301 basic medicine ,Myeloid ,Cellular differentiation ,Population ,CD34 ,Biology ,03 medical and health sciences ,0302 clinical medicine ,hemic and lymphatic diseases ,medicine ,Humans ,education ,education.field_of_study ,Myeloid Neoplasia ,Myeloid leukemia ,Cell Differentiation ,Hematology ,medicine.disease ,Phenotype ,Leukemia, Myeloid, Acute ,Leukemia ,030104 developmental biology ,medicine.anatomical_structure ,030220 oncology & carcinogenesis ,Neoplastic Stem Cells ,Cancer research ,sense organs ,Stem cell ,Nucleophosmin ,Biomarkers - Abstract
The leukemia stem cell (LSC) populations of acute myeloid leukemia (AML) exhibit phenotypic, genetic, and functional heterogeneity that contribute to therapy failure and relapse. Progress toward understanding the mechanistic basis for therapy resistance in LSCs has been hampered by difficulties in isolating cell fractions that enrich for the entire heterogeneous population of LSCs within individual AML samples. We previously reported that CD200 gene expression is upregulated in LSC-containing AML fractions. Here, we show that CD200 is present on a greater proportion of CD45dim blasts compared with more differentiated CD45high cells in AML patient samples. In 75% (49 of 65) of AML cases we examined, CD200 was expressed on ≥10% of CD45dim blasts; of these, CD200 identified LSCs within the blast population in 9 of 10 (90%) samples tested in xenotransplantation assays. CD200+ LSCs could be isolated from CD200+ normal HSCs with the use of additional markers. Notably, CD200 expression captured both CD34– and CD34+ LSCs within individual AML samples. Analysis of highly purified CD200+ LSC-containing fractions from NPM1-mutated AMLs, which are commonly CD34–, exhibited an enrichment of primitive gene expression signatures compared with unfractionated cells. Overall, our findings support CD200 as a novel LSC marker that is able to capture the entire LSC compartment from AML patient samples, including those with NPM1 mutation.
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- 2020
19. Updates from Ongoing, First-in-Human Phase 1 Dose Escalation and Expansion Study of TTI-621, a Novel Biologic Targeting CD47, in Patients with Relapsed or Refractory Hematologic Malignancies
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Ian W. Flinn, Yaping Shou, Bob Uger, James M. Foran, Mary-Elizabeth M. Percival, Diego Villa, Kathleen Large, Catherine Diefenbach, Stephen M. Ansell, Youn H. Kim, Ahmed Sawas, Craig Okada, Kerry J. Savage, Gloria H. Y. Lin, Tatyana Feldman, Oleg E. Akilov, Steven M. Horwitz, Michael B. Maris, Lubomir Sokol, Mark D. Minden, Deepa Jagadeesh, Penka S. Petrova, Tina Catalano, Matthew Mei, and Naomi Molloy
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Oncology ,medicine.medical_specialty ,business.industry ,Immunology ,Cell Biology ,Hematology ,First in human ,Biochemistry ,Refractory ,Internal medicine ,Dose escalation ,Medicine ,In patient ,business - Abstract
Background CD47 is an innate immune checkpoint that binds signal regulatory protein alpha (SIRPα) and delivers a "do not eat" signal to suppress macrophage phagocytosis. Overexpression of CD47 can allow tumor cells to escape immune surveillance. TTI-621 (SIRPαFc) is a fusion protein consisting of the CD47 binding domain of human SIRPα linked to the Fc region of human IgG1, designed to enhance phagocytosis and antitumor activity by blocking the CD47-SIRPα interaction between malignant cells and macrophages, and engaging Fcγ receptors. Here we report updates from the first-in-human study of TTI-621 (NCT02663518) in hematologic malignancies. Methods Study Part 1 tested increasing weekly intravenous doses of TTI-621 based on 3+3 escalation. The maximum tolerated dose was initially determined to be 0.2 mg/kg based on dose limiting toxicity (DLT) of thrombocytopenia [Grade (Gr) 4 of any duration]. Expanded testing followed in patients (pts) with hematologic malignancies, including leukemia, lymphoma, and multiple myeloma. In Part 2, most pts received 0.2 mg/kg. However, based on investigator discretion, a subset of pts received escalating doses up to 0.5 mg/kg. In Part 3, pts with T-cell lymphomas received stepwise dose escalations from 0.2 to 0.5 mg/kg over the first 5−8 weeks. In over 200 pts tested in Parts 1−3, thrombocytopenia did not increase with dose, typically recovered within 2−4 days, and was not associated with clinical sequelae. Part 4 was then undertaken to optimize TTI-621 dosing and is currently escalating doses in a 3+3 manner through pre-planned dose levels (0.5, 0.7, 1, and 1.4 mg/kg) in pts with cutaneous T-cell lymphoma (CTCL). The DLT criteria was modified to require Gr 4 thrombocytopenia lasting >72 hours. Safety monitoring includes weekly clinical laboratories and assessments of adverse events (AEs) per CTCAE v 4.03. Blood samples are obtained for pharmacokinetics (PK) and for pharmacodynamic (PD) assessments of receptor occupancy (RO) on normal peripheral T cells. Disease assessments are performed per Olsen's criteria. Results In Parts 1−3 (n=214), the most common related AEs were infusion-related reaction (IRR, 43%; 3% Gr ≥3), thrombocytopenia (30%; 22% Gr ≥3), chills (21%; 0% Gr ≥3), and fatigue (15%; 1% Gr ≥3). Objective responses to single agent TTI-621 were achieved in 14/71 (20%) NHL pts including CTCL (n=42, 1 CR, 7 PRs), PTCL (n=22, 2 CRs, 2 PRs) and DLBCL (n=7, 1 CR, 1 PR). In Part 4, as of July 10, 2020, 15 pts (9M/6F, median age 67 years) have enrolled into 4 dose cohorts (0.5−1.4 mg/kg). CTCL subtypes include mycosis fungoides (MF, n=10) and Sézary syndrome (n=5) with advanced (≥IIB) disease in 9 (60%) pts who received a median of 3 (range 1−12) prior systemic therapies. Related AEs have occurred in 11 (73%) pts including IRR (n=10) and thrombocytopenia (n=3); Gr ≥3 AEs have occurred in 4 (27%) pts including thrombocytopenia (n=3), IRRs (n=2), and exfoliative dermatitis (n=1). Thrombocytopenia generally occurred on dosing days, recovered in 2-4 days, and has not worsened with increasing doses. IRRs typically occurred during initial infusions. The Gr 3 IRR events occurred in 2 pts in the 1 and 1.4 mg/kg cohorts; low Gr IRRs have occurred across doses in 8 pts. IRRs typically resolved without recurrence and low Gr events often resolved allowing for completion of infusions. For initial infusions, the Gr 3 IRRs prompted increasing infusion times from 1 hour up to 4 hours and discretional use of steroid pre-medication. The exfoliative dermatitis occurred Day 80 and led to treatment discontinuation in 1 pt with MF whose underlying disease confounded the etiology. PK results reveal dose dependent increases in exposure; PD studies indicate ~60% RO at end of infusion up to 1 mg/kg. Antitumor activity to date includes 1 PR and 1 skin CR in 6 evaluable pts in the 1 mg/kg cohort; 2 responding pts bridged to allogeneic transplantation. The mean % change in mSWAT scores were -0.4%, -27%, and -37% for 0.5, 0.7 and 1 mg/kg cohorts, respectively. 1.4 mg/kg cohort results will be presented at the meeting. Conclusions In Parts 1−3, TTI-621 doses of 0.05 to 0.5 mg/kg were well-tolerated and demonstrated single agent activity in multiple hematologic malignancies. Preliminary data from Part 4 dose optimization indicate that weekly infusions of TTI-621 up to 1.4 mg/kg are well-tolerated without dose limiting or cumulative thrombocytopenia. Antitumor activity was seen at 1 mg/kg; dose escalation is continuing at 2 mg/kg. Disclosures Horwitz: Janssen: Consultancy; Verastem: Consultancy, Research Funding; Daiichi Sankyo: Research Funding; GlaxoSmithKline: Consultancy; ASTEX: Consultancy; Portola: Consultancy, Research Funding; C4 Therapeutics: Consultancy; Beigene: Consultancy; Myeloid Therapeutics: Consultancy; Vividion Therapeutics: Consultancy; Infinity/Verastem: Research Funding; Aileron: Consultancy, Research Funding; ADCT Therapeutics: Consultancy, Research Funding; Affirmed: Consultancy; Trillium: Consultancy, Research Funding; Corvus: Consultancy; Millenium/Takeda: Consultancy, Research Funding; Kyowa Hakka Kirin: Consultancy, Research Funding; Innate Pharma: Consultancy; Forty Seven: Consultancy, Research Funding; Seattle Genetics: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Kura Oncology: Consultancy; Miragen: Consultancy; Mundipharma: Consultancy. Foran:Agios: Honoraria, Research Funding; H3Biosciences: Research Funding; Xencor: Research Funding; Trillium: Research Funding; Takeda: Research Funding; Kura Oncology: Research Funding; Aptose: Research Funding; Aprea: Research Funding; Actinium: Research Funding; Boehringer Ingelheim: Research Funding; Abbvie: Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Revolution Medicine: Consultancy. Sawas:Flatiron Health: Current Employment; Gilead: Speakers Bureau; Seattle Genetics: Speakers Bureau; Daiichi Sankyo: Speakers Bureau; Affimed: Research Funding; Roche: Current equity holder in publicly-traded company. Feldman:AstraZeneca: Consultancy; Viracta: Research Funding; Trillium: Research Funding; Pfizer: Research Funding; Janssen: Speakers Bureau; Bayer: Consultancy, Honoraria; Kyowa Kirin: Consultancy, Research Funding; Eisai: Research Funding; Cell Medica: Research Funding; Amgen: Research Funding; Pharmacyclics: Honoraria, Other, Speakers Bureau; Abbvie: Honoraria; Takeda: Honoraria, Other: Travel expenses; Celgene: Honoraria, Research Funding; Kite: Honoraria, Other: Travel expenses, Speakers Bureau; BMS: Consultancy, Honoraria, Research Funding; Corvus: Research Funding; Rhizen: Research Funding; Seattle Genetics, Inc.: Consultancy, Honoraria, Other: Travel expenses, Research Funding, Speakers Bureau; Portola: Research Funding. Sokol:EUSA Pharma: Consultancy, Honoraria, Speakers Bureau; Kyowa/Kirin Inc.: Membership on an entity's Board of Directors or advisory committees; Kymera Therapeutics: Membership on an entity's Board of Directors or advisory committees. Mei:Sanofi: Consultancy; Morphosys: Membership on an entity's Board of Directors or advisory committees. Flinn:Gilead Sciences: Consultancy, Research Funding; F. Hoffmann-La Roche: Research Funding; Roche: Consultancy, Research Funding; Vincera Pharma: Consultancy; Agios: Research Funding; Iksuda Therapeutics: Consultancy; Great Point Partners: Consultancy; Genentech, Inc.: Research Funding; Forty Seven: Research Funding; Forma Therapeutics: Research Funding; ArQule: Research Funding; Kite Pharma: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; TG Therapeutics: Consultancy, Research Funding; Trillium Therapeutics: Research Funding; Triphase Research & Development Corp.: Research Funding; Curio Science: Consultancy; Constellation Pharmaceuticals: Research Funding; MorphoSys: Consultancy, Research Funding; Curis: Research Funding; Nurix Therapeutics: Consultancy; Novartis: Research Funding; Pfizer: Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy, Research Funding; Portola Pharmaceuticals: Research Funding; Seattle Genetics: Consultancy, Research Funding; Teva: Research Funding; Loxo: Research Funding; BeiGene: Consultancy, Research Funding; Calithera Biosciences: Research Funding; Johnson & Johnson: Other; Rhizen Pharmaceuticals: Research Funding; Yingli Pharmaceuticals ≠: Consultancy, Research Funding; Verastem: Consultancy, Research Funding; Karyopharm Therapeutics: Research Funding; AstraZeneca: Consultancy, Research Funding; Incyte: Research Funding; Takeda: Consultancy, Research Funding; IGM Biosciences: Research Funding; Infinity Pharmaceuticals: Research Funding; Unum Therapeutics: Consultancy, Research Funding; Juno Therapeutics: Consultancy, Research Funding; Acerta Pharma: Research Funding; Celgene: Research Funding; Merck: Research Funding. Villa:Celgene: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Roche: Consultancy, Honoraria, Research Funding; AZ: Consultancy, Honoraria, Research Funding; Kite/Gilead: Consultancy, Honoraria; Nano String: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Sandoz Canada: Consultancy, Honoraria; Immunovaccine: Consultancy, Honoraria; Purdue Pharma: Consultancy, Honoraria. Percival:Pfizer: Research Funding; Trillium: Research Funding; Nohla Therapeutics: Research Funding; Biosight: Research Funding; Oscotec: Research Funding; Cardiff Oncology: Research Funding; Genentech: Membership on an entity's Board of Directors or advisory committees; Glycomimetics: Research Funding. Jagadeesh:Seattle Genetics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Regeneron: Research Funding; Verastem: Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Research Funding; Debiopharm Group: Research Funding. Savage:Merck, BMS, Seattle Genetics, Gilead, AstraZeneca, AbbVie, Servier: Consultancy; Roche (institutional): Research Funding; Merck, BMS, Seattle Genetics, Gilead, AstraZeneca, AbbVie: Honoraria; BeiGene: Other: Steering Committee. Akilov:Mallinckrodt: Consultancy; Medivir: Consultancy; Seattle Genetics, Inc.: Consultancy; Kyowa Hakko Kirin: Consultancy; Soligenix: Honoraria; Pfizer: Research Funding; Actelion: Consultancy, Research Funding; Trillium Therapeutics Inc.: Consultancy, Research Funding. Diefenbach:Trillium: Research Funding; Millenium/Takeda: Research Funding; Seattle Genetics: Consultancy, Research Funding; Merck: Consultancy, Research Funding; MEI: Research Funding; LAM Therapeutics: Research Funding; Incyte: Research Funding; Genentech, Inc.: Consultancy, Research Funding; Denovo: Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding. Kim:Kyowa-Kirin Pharma: Research Funding; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees; Medivir: Membership on an entity's Board of Directors or advisory committees; Horizon Pharma: Consultancy, Research Funding; Merck: Research Funding; miRagen: Research Funding; Trillium: Research Funding; Galderma: Membership on an entity's Board of Directors or advisory committees, Research Funding; Eisai: Membership on an entity's Board of Directors or advisory committees, Research Funding; Corvus: Research Funding; Elorac: Research Funding; Forty Seven Inc: Research Funding; Neumedicine: Consultancy, Research Funding; Innate Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Solingenix: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Portola: Research Funding. Lin:Trillium Therapeutics Inc.: Current Employment. Catalano:Trillium Therapeutics Inc.: Current Employment. Petrova:Trillium Therapeutics Inc.: Current Employment. Uger:Trillium Therapeutics Inc.: Current Employment. Molloy:Trillium Therapeutics Inc.: Current Employment. Large:Trillium Therapeutics Inc.: Current Employment. Shou:Trillium Therapeutics Inc.: Current Employment. Ansell:Affimed: Research Funding; Bristol Myers Squibb: Research Funding; Regeneron: Research Funding; Trillium: Research Funding; ADC Therapeutics: Research Funding; Seattle Genetics: Research Funding; Takeda: Research Funding; AI Therapeutics: Research Funding.
- Published
- 2020
20. Impact of preleukemic mutations and their persistence on hematologic recovery after induction chemotherapy for AML
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Andrea Arruda, Vikas Gupta, Tracy Murphy, Jinfeng Zou, Caroline J McNamara, Anne Tierens, Karen Yee, Mark D. Minden, Hassan Sibai, Mariam Korulla, Suzanne Kamel-Reid, Dawn Maze, Andre C. Schuh, Steven M. Chan, Tracy Stockley, Scott V. Bratman, Georgina S. Daher-Reyes, and Aaron D. Schimmer
- Subjects
Oncology ,medicine.medical_specialty ,Myeloid ,Treatment outcome ,medicine.disease_cause ,Persistence (computer science) ,Internal medicine ,Antineoplastic Combined Chemotherapy Protocols ,Biomarkers, Tumor ,medicine ,Humans ,Retrospective Studies ,Mutation ,business.industry ,High-Throughput Nucleotide Sequencing ,Induction chemotherapy ,Induction Chemotherapy ,Hematology ,Variant allele ,Prognosis ,medicine.disease ,Stimulus Report ,Blood Cell Count ,Leukemia, Myeloid, Acute ,Leukemia ,Treatment Outcome ,medicine.anatomical_structure ,Disease remission ,business - Abstract
Key Points DNMT3A R882, TET2, ASXL1, and SRSF2 mutations identified at the time of diagnosis are associated with delayed count recovery. Persistence of preleukemic mutations in remission at high variant allele frequency is associated with delayed count recovery.
- Published
- 2019
21. A stemness screen reveals C3orf54/INKA1 as a promoter of human leukemia stem cell latency
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Sasan Zandi, Kerstin B. Kaufmann, Mike J. Tsay, Laura García-Prat, Stanley W.K. Ng, Chiara Pastrello, Mark D. Minden, John E. Dick, Gabriela Krivdova, Jean C.Y. Wang, Elvin Wagenblast, Eric R. Lechman, Stephanie Z. Xie, Qiang Liu, Peter van Galen, Igor Jurisica, Amanda Mitchell, Erno Wienholds, Shin-ichiro Takayanagi, and Christian A. Cumbaa
- Subjects
0301 basic medicine ,Myeloid ,Immunology ,CD34 ,Myeloid leukemia ,Cell Biology ,Hematology ,Biology ,medicine.disease ,Biochemistry ,Cell biology ,03 medical and health sciences ,Leukemia ,Haematopoiesis ,030104 developmental biology ,0302 clinical medicine ,medicine.anatomical_structure ,030220 oncology & carcinogenesis ,Cord blood ,medicine ,Stem cell ,Progenitor cell - Abstract
There is a growing body of evidence that the molecular properties of leukemia stem cells (LSCs) are associated with clinical outcomes in acute myeloid leukemia (AML), and LSCs have been linked to therapy failure and relapse. Thus, a better understanding of the molecular mechanisms that contribute to the persistence and regenerative potential of LSCs is expected to result in the development of more effective therapies. We therefore interrogated functionally validated data sets of LSC-specific genes together with their known protein interactors and selected 64 candidates for a competitive in vivo gain-of-function screen to identify genes that enhanced stemness in human cord blood hematopoietic stem and progenitor cells. A consistent effect observed for the top hits was the ability to restrain early repopulation kinetics while preserving regenerative potential. Overexpression (OE) of the most promising candidate, the orphan gene C3orf54/INKA1, in a patient-derived AML model (8227) promoted the retention of LSCs in a primitive state manifested by relative expansion of CD34+ cells, accumulation of cells in G0, and reduced output of differentiated progeny. Despite delayed early repopulation, at later times, INKA1-OE resulted in the expansion of self-renewing LSCs. In contrast, INKA1 silencing in primary AML reduced regenerative potential. Mechanistically, our multidimensional confocal analysis found that INKA1 regulates G0 exit by interfering with nuclear localization of its target PAK4, with concomitant reduction of global H4K16ac levels. These data identify INKA1 as a novel regulator of LSC latency and reveal a link between the regulation of stem cell kinetics and pool size during regeneration.
- Published
- 2019
22. Distinct patterns of clonal evolution in patients with concurrent myelo- and lymphoproliferative neoplasms
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John E. Dick, Mark D. Minden, Anne Tierens, Stephanie M. Dobson, Suzanne Kamel-Reid, Mahadeo A. Sukhai, Tracy Stockley, Vikas Gupta, James A. Kennedy, Jessie J. F. Medeiros, and Andrea Arruda
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business.industry ,Immunology ,Cell Biology ,Hematology ,Biology ,Biochemistry ,Somatic evolution in cancer ,03 medical and health sciences ,0302 clinical medicine ,Text mining ,Increased risk ,hemic and lymphatic diseases ,030220 oncology & carcinogenesis ,Myeloid cells ,In patient ,Stem cell ,Overproduction ,business ,030215 immunology - Abstract
TO THE EDITOR: BCR-ABL -negative myeloproliferative neoplasms (MPNs) are a group of clonal stem cell disorders characterized by the overproduction of mature myeloid cells. However, MPN patients also have a 2.8- to 3.4-fold increased risk of developing a lymphoproliferative disorder (LPD) compared
- Published
- 2018
23. Venetoclax enhances T cell-mediated anti-leukemic activity by increasing ROS production
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Michael Andreeff, Marcela Gronda, Yoosu Na, Rose Hurren, Xiaoming Wang, Neil MacLean, Andrea Arruda, Sara Mirali, Hyeonjeong Kang, Dilshad H. Khan, Zoe Alaniz, Marina Konopleva, Li Zhang, Mark D. Minden, Yulia Jitkova, Jongbok Lee, Aaron D. Schimmer, and Mingjing Xu
- Subjects
Adult ,Immunobiology and Immunotherapy ,T-Lymphocytes ,T cell ,Immunology ,Azacitidine ,Respiratory chain ,Antineoplastic Agents ,Biochemistry ,Cell therapy ,chemistry.chemical_compound ,Immune system ,hemic and lymphatic diseases ,Tumor Cells, Cultured ,medicine ,Humans ,Cytotoxicity ,Cells, Cultured ,Immunity, Cellular ,Sulfonamides ,Myeloid Neoplasia ,Chemistry ,Venetoclax ,Cell Biology ,Hematology ,Bridged Bicyclo Compounds, Heterocyclic ,Leukemia, Myeloid, Acute ,medicine.anatomical_structure ,Mechanism of action ,Cancer research ,medicine.symptom ,Reactive Oxygen Species ,medicine.drug - Abstract
The combination of venetoclax and the hypomethylating agent azacytidine induces a high rate of complete responses in treatment-naïve older patients with acute myeloid leukemia (AML). Lee and colleagues elucidate a novel mechanism of action for the synergy of these agents, demonstrating that venetoclax enhances reactive oxygen species (ROS) generation and enhances T-cell effector function while azacytidine renders AML cells more susceptible to T cell–mediated cytotoxicity., Key Points A Bcl-2 inhibitor, venetoclax, increases the effector activity of antileukemic T cells without inducing T-cell apoptosis.Venetoclax increases T-cell effector function in a reactive oxygen species–dependent manner., Visual Abstract, Venetoclax, a Bcl-2 inhibitor, in combination with the hypomethylating agent azacytidine, achieves complete remission with or without count recovery in ∼70% of treatment-naive elderly patients unfit for conventional intensive chemotherapy. However, the mechanism of action of this drug combination is not fully understood. We discovered that venetoclax directly activated T cells to increase their cytotoxicity against acute myeloid leukemia (AML) in vitro and in vivo. Venetoclax enhanced T-cell effector function by increasing reactive oxygen species generation through inhibition of respiratory chain supercomplexes formation. In addition, azacytidine induced a viral mimicry response in AML cells by activating the STING/cGAS pathway, thereby rendering the AML cells more susceptible to T cell–mediated cytotoxicity. Similar findings were seen in patients treated with venetoclax, as this treatment increased reactive oxygen species generation and activated T cells. Collectively, this study presents a new immune-mediated mechanism of action for venetoclax and azacytidine in the treatment of AML and highlights a potential combination of venetoclax and adoptive cell therapy for patients with AML.
- Published
- 2021
24. Myelodysplastic Syndromes: A More Global 5-Hydroxymethylcytosine Deficiency Disorder Than Suggested By the Presence of TET2 Mutations
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Yihong Guan, Torsten Haferlach, Hetty E. Carraway, Courtney E Hershberger, Mark D. Minden, Babal K. Jha, Jaymeson Gordon, Jae-Sook Ahn, Diego Coutinho, Simona Pagliuca, Ying Ni, Ilana Zalcberg, Carmelo Gurnari, Valeria Visconte, Jaroslaw P. Maciejewski, Richard A. Padgett, Hyeoung-Joon Kim, Dennis Dong Hwan Kim, and Joop H. Jansen
- Subjects
5-Hydroxymethylcytosine ,chemistry.chemical_compound ,chemistry ,business.industry ,Myelodysplastic syndromes ,Immunology ,Cancer research ,Medicine ,Cell Biology ,Hematology ,business ,medicine.disease ,Biochemistry - Abstract
The high frequency of TET2 mutations in myelodysplastic syndromes (MDS) and the sole function of TET-dioxygenases as 5-hydroxymethylcytosine (5-hmC) hydroxylases emphasize the key role of this gene in disease pathogenesis. However, the broad down-regulation of 5-hmC argues for a role of DNA demethylation in MDS beyond TET2 lesions, which albeit the high frequency, do not convey any impact on survival outcomes. In fact, decrease in 5-hmC levels is by far more widely spread than TET2 lesions pointing towards other pathways affecting TET2 activity, thereby obscuring a precise determination of its mutational and clinical consequences. Herein, we investigated TETs expression to identify factors explaining the widespread deficiency of 5-hmC in MDS possibly determining clinical phenotypes and prognosis. An integrative data analysis of genomic studies (whole genome and deep targeted NGS), RNA-sequencing and 5-hmC quantification was performed on 1,665 patients with MDS and 91 healthy controls (HC). Meta-analytic studies of 5-hmC levels in myeloid neoplasia (n=598) and data of RNA-sequencing of fractionated CD34 (GSE63569) were also included as confirmatory cohorts. We started by analyzing the clinical impact of TET2 mutations carried by 23% of our study population. No impact on survival was found in carriers of TET2 lesions including those with biallelic, truncating or missense mutations compared to wild-type (WT) (Fig1A). By using 5-hmC levels as a functional readout of TET activity, we found a TET deficiency in about 70% of patients, a proportion higher than one would conclude by considering the mere presence of TET2 mutations (Fig1B). To explain the decrease in 5-hmC levels in WT cases, we next examined transcriptome modifications. Analysis of the expression of TET family of genes showed that MDS patients had lower TET2 mRNA levels in total and in CD34+ cells as compared to HC, irrespective of their TET2 status. Therefore, we reasoned that TET2 deficiency is more ubiquitously involved in MDS pathogenesis than what would be expected by the only estimation of mutant cases. Indeed, "low expressor" status (defined by TET2 expression < 25%ile of HC) was found in 74% of MDS. Along with variable 5-hmC levels, concomitant differences in TET1/TET3 expression were also investigated. While TET1 levels were too low to be evaluated, TET3 expression levels were markedly higher in all and in WT MDS compared to HC, possibly in an attempt to compensate TET2 dysfunction (Fig1C). In addition, TET3 expression did not correlate with TET2 mutational burden, confuting a compensatory feedback mechanism in TET2 mutant cases. Further uni- and multivariate analyses showed that elevated TET3 levels compensated TET2 deficiency in terms of clinical outcomes (Fig1D) and linear regression analyses confirmed that indeed lack of compensation by TET3 (low TET3 expression) was associated with high risk features. To explore whether other factors might be associated with low TET2 levels, we studied TET2 expression in WT cases as to the presence of other mutations. We found that TET2 expression was significantly lower in patients harboring DNMT3A (P< 0.0001), SF3B1 (P< 0.0001) and SRSF2 (P= 0.04) compared to HC. However, lack of correlation between levels of TET2 and mutational burden failed to prove a direct relationship of these mutations (Fig1E). Decreased hydroxylation of 5-mC may also be caused by endogenous L-2-hydroxyglutarate (L2HG) produced via malate shunt. Accordingly, L2HG dehydrogenase (L2HGDH) levels catabolizing L2HG and malate dehydrogenases (MDH1/2) supplying L2HG, would influence TET2 activity in a reciprocal fashion. Consistently we found that MDH1/2 levels were increased in MDS and that L2HGDH showed also a likely compensatory increase to handle elevated L2HG loads. Further, linear regression analyses revealed that L2HGDH levels were correlated inversely with TET2 and positively with TET3 expression in WT cases (Fig1F). In sum, MDS can be considered a wide-ranging 5-hmC deficiency disorder driven by direct or indirect loss of TET2functions by mutations or down-modulation due to a variety of mechanisms. Disease phenotypes and outcomes are both influenced by counteracting factors such as expression of TET3. Application of precision therapeutic approaches should be informed by the analyses of all these factors. Figure 1 Figure 1. Disclosures Carraway: Astex: Other: Independent review committee; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Stemline: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Agios: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Other: Independent review committee; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Other: Independent review committee; Celgene, a Bristol Myers Squibb company: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Kim: Paladin: Consultancy, Honoraria, Research Funding; Bristol-Meier Squibb: Research Funding; Pfizer: Honoraria; Novartis: Consultancy, Honoraria, Research Funding. Minden: Astellas: Consultancy. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Maciejewski: Bristol Myers Squibb/Celgene: Consultancy; Novartis: Consultancy; Regeneron: Consultancy; Alexion: Consultancy.
- Published
- 2021
25. Phase 3, Open-Label, Randomized Study of Gilteritinib and Azacitidine Vs Azacitidine for Newly Diagnosed FLT3-Mutated Acute Myeloid Leukemia in Patients Ineligible for Intensive Induction Chemotherapy
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Pau Montesinos, Kamel Laribi, Wen-Chien Chou, Jessica K. Altman, Anne-Marie Watson, Tomoki Naoe, Ramon V. Tiu, Elizabeth Shima Rich, Elżbieta Patkowska, Jordi Esteve, Stanley Gill, Violaine Havelange, Je-Hwan Lee, Jason E. Hill, Ruishan Wu, Shufang Liu, Eunice S. Wang, Mark D. Minden, Michael Heuser, and Nisha Philipose
- Subjects
Oncology ,medicine.medical_specialty ,business.industry ,Immunology ,Azacitidine ,Gilteritinib ,Induction chemotherapy ,Myeloid leukemia ,Cell Biology ,Hematology ,Newly diagnosed ,Biochemistry ,law.invention ,Randomized controlled trial ,law ,Internal medicine ,medicine ,In patient ,Open label ,business ,medicine.drug - Abstract
Background: Gilteritinib (GIL), a FLT3 inhibitor, shows efficacy/safety in patients (pts) with FLT3mut+ relapsed/refractory (R/R) AML. For pts with newly diagnosed (ND) AML with FLT3 mutations unable to receive intensive induction chemotherapy (IIC), survival is limited, and therapy options are few. In preclinical studies, GIL plus azacitidine (AZA) impeded tumor growth and induced apoptosis/differentiation of FLT3-ITD AML cell lines. Antitumor effects appeared synergistic in xenografted mouse models (data on file). We investigated GIL+AZA vs AZA in adults with ND FLT3mut+ AML ineligible for IIC (NCT02752035). Aim/Objective: To compare efficacy and safety/tolerability of GIL+AZA and AZA in pts with ND FLT3mut+ AML ineligible for IIC. Methods: Pts were initially randomized (1:1:1) to GIL (120 mg/day orally on Days 1-28) plus AZA (75 mg/m 2/day SC/IV on Days 1-7), AZA (same regimen), or GIL (same regimen) during 28-day cycles. The GIL arm was removed due to preferred therapy changes. Pts were then randomized (2:1) to GIL+AZA or AZA alone. The primary endpoint was overall survival (OS) and key secondary endpoint was event-free survival (EFS). Treatment failure date was randomization date if complete remission (CR) was not achieved after 6 cycles. Subgroup/sensitivity analyses were prespecified. Response rates, safety/tolerability, and pharmacokinetic endpoints were analyzed. Data from long-term follow-up ≤3 yrs were obtained, including subsequent AML therapy. These results are from an interim analysis at 70 deaths (~50% of total deaths). Results: As of August 26, 2020, 123 pts were randomized to GIL+AZA (n=74) and AZA (n=49); 39 (52.7%) and 31 (63.3%) deaths, respectively, occurred. Median age was 78 yrs with GIL+AZA and 76 yrs with AZA; ECOG PS ≥2 was 47.3% and 32.7%, and FLT3-ITD alone was in 78.4% and 81.6% of pts, respectively (TKD alone 18.9% vs 14.3%; ITD with TKD 2.7% vs 4.1%). Median follow-up was 9.76 mo for GIL+AZA and 17.97 mo for AZA. Median exposure duration was 112 days for GIL in the GIL+AZA arm (n=73); AZA exposure was 98 and 99 days in the GIL+AZA and AZA (n=47) arms, respectively. Subsequent AML therapy was received by 20.3% pts on GIL+AZA and 44.9% pts on AZA; median time to first subsequent therapy was 8.2 and 4.5 mo, respectively. In the AZA arm, 22 pts received subsequent AML therapy, including 10 pts on GIL and 4 pts on other FLT3 inhibitors. Median OS was 9.82 mo for GIL+AZA and 8.87 mo for AZA (HR 0.916 [95% CI 0.529, 1.585]; P=.753). Patient subgroups with improved OS with GIL+AZA vs AZA included pts with ECOG PS 0-1 (HR 0.811 [95% CI 0.409, 1.608]) and high FLT3-ITD allelic ratio ≥0.5 (HR 0.580 [95% CI 0.285, 1.182]). Median EFS was 0.03 mo in both arms (HR 1.175 [95% CI 0.764, 1.807]; P=.459). In sensitivity analyses, median EFS with events based on composite CR (CRc; CR+CRi+CRp) was 5.03 mo for GIL+AZA and 3.29 mo for AZA (HR 0.924 [95% CI 0.576, 1.482]; P=.767). Although CR rates for both arms were similar (16.2% vs 14.3%), CRc rates were significantly higher for GIL+AZA vs AZA (58.1 vs 26.5%, difference 31.4% [95% CI 13.1, 49.7]; P No substantial differences in GIL trough concentrations at steady state (C trough) were seen between GIL+AZA and GIL alone (prior to arm removal). However, on Cycle 1 Day 15, median GIL C trough was 579 ng/mL (GIL+AZA and GIL arms) in contrast to C trough of 279 ng/mL observed with GIL monotherapy in the ADMIRAL trial in pts with R/R AML. Reasons for this difference are being evaluated. No apparent relationship was seen between C trough and response rates/grade of thrombocytopenia or neutropenia. Conclusions: In this trial of pts with ND FLT3mut+ AML ineligible for IIC, GIL+AZA led to significantly higher CRc rates but similar OS vs AZA alone. Pts with ECOG PS 0-1 and high FLT3-ITD allelic ratio appeared to have greater benefit with GIL+AZA. No new safety signals were seen. Curiously, C trough values in pts with ND FLT3mut+ AML ineligible for IIC were 2-fold greater than in pts with R/R FLT3mut+ AML. These results support the safety, tolerability, and activity of GIL+AZA vs AZA. Disclosures Wang: GlaxoSmithKline: Consultancy, Honoraria, Other: Advisory Board; Genentech: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Other: Advisory Board; Jazz Pharmaceuticals: Consultancy, Honoraria, Other: Advisory Board; Pfizer: Consultancy, Honoraria, Other: Advisory Board, Speakers Bureau; Takeda: Consultancy, Honoraria, Other: Advisory board; Mana Therapeutics: Consultancy, Honoraria; Kite Pharmaceuticals: Consultancy, Honoraria, Other: Advisory Board; Kura Oncology: Consultancy, Honoraria, Other: Advisory board, steering committee, Speakers Bureau; Stemline Therapeutics: Consultancy, Honoraria, Other: Advisory board, Speakers Bureau; BMS/Celgene: Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees; DAVA Oncology: Consultancy, Speakers Bureau; Rafael Pharmaceuticals: Other: Data safety monitoring committee; Gilead: Consultancy, Honoraria, Other: Advisory board; Daiichi Sankyo: Consultancy, Honoraria, Other: Advisory board; PTC Therapeutics: Consultancy, Honoraria, Other: Advisory board; Genentech: Consultancy; MacroGenics: Consultancy. Montesinos: Stemline/Menarini: Consultancy; Sanofi: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Daiichi Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Forma Therapeutics: Consultancy; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Glycomimetics: Consultancy; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Tolero Pharmaceutical: Consultancy; Karyopharm: Membership on an entity's Board of Directors or advisory committees, Research Funding; Teva: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Agios: Consultancy; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Astellas Pharma, Inc.: Consultancy, Honoraria, Other: Advisory board, Research Funding, Speakers Bureau. Minden: Astellas: Consultancy. Lee: Korean Society of Hematology: Membership on an entity's Board of Directors or advisory committees; Astellas Pharma, Inc.: Consultancy, Honoraria, Other: Advisory board; AbbVie: Honoraria, Other: Advisory board. Heuser: Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees, Research Funding; BergenBio: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Research Funding; Tolremo: Membership on an entity's Board of Directors or advisory committees; BMS/Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer Pharma AG: Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astellas: Research Funding. Naoe: Fuji Film: Other: Study funding for JAGSE; Astellas Pharma, Inc.: Consultancy, Other: Study funding for JAGSE; Bristol-Myers: Honoraria; Nippon Shinyaku: Honoraria; Daichi Sankyo: Other: Study funding for JAGSE; Otsuka Pharma: Honoraria. Chou: Abbvie: Honoraria, Other: Advisory Board, Research Funding; Celgene: Honoraria, Other: Advisory Board, Research Funding; IQVIA: Honoraria, Other: Advisory Board; Pfizer: Honoraria, Other: Advisory Board; Novartis: Honoraria, Other: Advisory Board; Bristol Myers Squibb: Honoraria, Research Funding; Kirin: Honoraria, Research Funding. Laribi: IQONE: Other: Personal Fees; Takeda: Other: Personal Fees, Research Funding; AstraZeneca: Other: Personal Fees; BeiGene: Other: Personal Fees; Astellas Phama, Inc.: Other: Personal Fees; Jansen: Research Funding; AbbVie: Other: Personal Fees, Research Funding; Le Mans Hospital: Research Funding; Novartis: Other: Personal Fees, Research Funding. Esteve: Jazz: Consultancy; Novartis: Research Funding; Bristol Myers Squibb/Celgene: Consultancy; Pfizer: Consultancy; Abbvie: Consultancy; Novartis: Consultancy, Research Funding; Astellas: Consultancy. Altman: ALZ Oncology: Research Funding; Amgen: Research Funding; Biosight: Consultancy, Other: Travel fees, Research Funding; Kartos: Research Funding; Boehringer Ingelheim: Research Funding; Daiichi Sankyo: Consultancy; Syros: Consultancy; BMS: Research Funding; GlycoMimetics: Other: Participation on an advisory board; Aprea: Research Funding; Kura: Research Funding; Kura Oncology: Consultancy; AbbVie: Consultancy, Other: Advisory Board, Research Funding; Theradex: Consultancy, Other: Advisory boards; Fujifilm: Research Funding; Astellas: Consultancy, Other: Advisory Board, Research Funding; Immunogen: Research Funding. Havelange: Astellas Pharma, Inc.: Consultancy; Novartis: Honoraria, Other: Travel fees, Participation on an advisory board; BMS: Other: Travel fees, Participation on an advisory board; Incyte: Other: Advisory board; Abbvie: Other: Advisory board. Watson: Astellas Pharma, Inc.: Consultancy; Roche, Amgen: Other: Travel support. Patkowska: Astellas Pharma, Inc.: Consultancy, Other: Travel fees; Pfizer: Other: Travel fees; Jazz Pharmaceuticals: Other: Travel fees; Angelini Pharma: Honoraria, Other: Travel fees; Novartis: Honoraria, Other: Travel fees; Bristol-Myers Squibb: Other: Travel fees; AMGEN: Honoraria; Servier: Honoraria, Other: Travel fees; KCR US, Inc.: Consultancy. Liu: Astellas Pharma, Inc.: Current Employment. Wu: Astellas: Current Employment. Philipose: Astellas Pharma Global Development: Current Employment. Hill: Astellas Pharma Global Development: Current Employment; Ligacept, LLC: Current holder of individual stocks in a privately-held company, Other: Stockholder. Gill: Astellas Pharma Global Development: Current Employment. Rich: Astellas Pharma Global Development, Inc.: Current Employment. Tiu: Astellas Pharma, Inc.: Current Employment.
- Published
- 2021
26. Impact of FLT3 Mutation Clearance after Front-Line Treatment with Gilteritinib Plus Azacitidine, or Gilteritinib or Azacitidine Alone in Patients with Newly Diagnosed AML: Results from the Phase 2/3 Lacewing Trial
- Author
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Jason E. Hill, Ruishan Wu, Elizabeth Shima Rich, Jessica K. Altman, Mark D. Minden, and Eunice S. Wang
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Oncology ,medicine.medical_specialty ,business.industry ,Immunology ,Azacitidine ,Gilteritinib ,Front line ,Cell Biology ,Hematology ,Newly diagnosed ,Biochemistry ,Internal medicine ,Flt3 mutation ,medicine ,In patient ,business ,medicine.drug - Abstract
Background: The presence of measurable residual disease (MRD) after achievement of remission with induction therapy is a prognostic marker of relapse risk in patients with acute myeloid leukemia (AML). Gilteritinib is an oral FLT3 inhibitor approved as a single agent for the treatment of patients with FLT3-mutated (FLT3mut+) relapsed or refractory AML. Evaluation of gilteritinib in the front-line setting is under way. We evaluated FLT3 internal tandem duplication (FLT3-ITD) mutation clearance using two different thresholds and correlated mutation clearance with survival outcomes in patients with newly diagnosed AML ineligible for intensive chemotherapy who were treated with front-line gilteritinib plus azacitidine (AZA) or either agent alone in the phase 2/3 LACEWING trial. Methods: Adult patients with newly diagnosed FLT3mut+ AML ineligible for intensive induction chemotherapy received 28-day cycles of once-daily gilteritinib plus AZA in the Safety Cohort (80 or 120 mg/day gilteritinib plus 75 mg/m 2 AZA, Days 1-7) and in Arm AC (120 mg/day gilteritinib plus 75 mg/m 2 AZA, Days 1-7), gilteritinib (120 mg/day) alone in Arm A, or AZA (75 mg/m 2, Days 1-7) alone in Arm C. A subset of patients who had a best overall response of composite complete remission (CRc; defined as the sum of patients who achieved complete remission with or without complete hematologic or platelet recovery) and who had bone marrow-derived DNA samples available at baseline and at least one additional post-baseline timepoint were assessed for FLT3-ITD mutation clearance using next-generation sequencing. An Illumina ® sequencing platform was used to quantify FLT3-ITD and total FLT3 alleles. The FLT3-ITD variant allelic frequency (VAF) was defined as the ratio of FLT3-ITD to total FLT3 frequency. Data were analyzed using two different mutation clearance thresholds, FLT3-ITD VAF Results: The median age of patients enrolled in LACEWING was 77 years (range, 59-90), with 73% of patients aged >75 years. Although baseline characteristics of the overall LACEWING population were generally well balanced across treatment arms, higher proportions of patients treated with gilteritinib plus AZA (47%) or gilteritinib alone (59%) had an Eastern Cooperative Oncology Group (ECOG) performance status of ≥2 compared with patients treated with AZA alone (33%). Overall, 40 patients who achieved CRc and had sufficient DNA samples from bone marrow aspirates obtained at baseline and at least one additional post-baseline timepoint were included in the analysis (Safety Cohort, n=8; Arm A, n=7; Arm AC, n=17; and Arm C, n=8). Across both thresholds, the proportions of patients with FLT3 mutation clearance did not markedly differ between patients treated with gilteritinib or AZA (Table). In patients who received gilteritinib, FLT3-ITD mutation clearance using either threshold was associated with a similar increase in median overall survival (OS) compared to patients who did not achieve mutation clearance (Figure). Conclusions: Regardless of MRD threshold, rates of MRD negativity were not substantially different between newly diagnosed FLT3mut+ AML patients ineligible for intensive induction chemotherapy who received gilteritinib alone, gilteritinib plus AZA, or AZA alone. Advanced age coupled with a worse baseline ECOG performance score at baseline may have compromised treatment response and achievement of FLT3 mutation clearance in patients treated with gilteritinib. The mutation clearance thresholds used in this analysis showed similar median OS in patients who received gilteritinib. Figure 1 Figure 1. Disclosures Wang: Pfizer: Consultancy, Honoraria, Other: Advisory Board, Speakers Bureau; Genentech: Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Consultancy, Honoraria, Other: Advisory Board; Novartis: Consultancy, Honoraria, Other: Advisory Board; Kura Oncology: Consultancy, Honoraria, Other: Advisory board, steering committee, Speakers Bureau; Jazz Pharmaceuticals: Consultancy, Honoraria, Other: Advisory Board; Takeda: Consultancy, Honoraria, Other: Advisory board; Kite Pharmaceuticals: Consultancy, Honoraria, Other: Advisory Board; Stemline Therapeutics: Consultancy, Honoraria, Other: Advisory board, Speakers Bureau; Mana Therapeutics: Consultancy, Honoraria; BMS/Celgene: Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees; DAVA Oncology: Consultancy, Speakers Bureau; Rafael Pharmaceuticals: Other: Data safety monitoring committee; Gilead: Consultancy, Honoraria, Other: Advisory board; Daiichi Sankyo: Consultancy, Honoraria, Other: Advisory board; PTC Therapeutics: Consultancy, Honoraria, Other: Advisory board; Genentech: Consultancy; MacroGenics: Consultancy. Altman: Kartos: Research Funding; Theradex: Consultancy, Other: Advisory boards; Biosight: Consultancy, Other: Travel fees, Research Funding; Daiichi Sankyo: Consultancy; AbbVie: Consultancy, Other: Advisory Board, Research Funding; BMS: Research Funding; Amgen: Research Funding; Astellas: Consultancy, Other: Advisory Board, Research Funding; Fujifilm: Research Funding; ALZ Oncology: Research Funding; Immunogen: Research Funding; GlycoMimetics: Other: Participation on an advisory board; Syros: Consultancy; Kura Oncology: Consultancy; Boehringer Ingelheim: Research Funding; Aprea: Research Funding; Kura: Research Funding. Minden: Astellas: Consultancy. Wu: Astellas: Current Employment. Rich: Astellas Pharma Global Development, Inc.: Current Employment. Hill: Ligacept, LLC: Current holder of individual stocks in a privately-held company, Other: Stockholder; Astellas Pharma Global Development: Current Employment.
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- 2021
27. Proteomics and Phospho-Proteomics Reveal Predictive Signatures of Response and Mechanisms of Resistance to Midostaurin Plus Chemotherapy in FLT3 Mutant Positive Acute Myeloid Leukemia
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Jane Theaker, Andrea Arruda, John G. Gribben, Andrew Hugin Thompson, Ryan Smith, Luis Miguel Veiga Veiga Nobre, Pedro R. Cutillas, Arran Dokal, Francesca Patella, Salvatore Federico Pedicona, Mark D. Minden, Bela Wrench, Calum Greenhalgh, Pedro Casado-Izquierdo, and David Britton
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Chemotherapy ,business.industry ,medicine.medical_treatment ,Immunology ,Mutant ,Myeloid leukemia ,Cell Biology ,Hematology ,Proteomics ,Biochemistry ,chemistry.chemical_compound ,chemistry ,Cancer research ,Medicine ,Midostaurin ,business - Abstract
Background: Midostaurin is approved for FLT3 mutant-positive (FLT3+) acute myeloid leukemia (AML), however efficacy has also been observed in a subpopulation of FLT3 mutant-negative AML, suggesting that FLT3 mutation is not the only determinant conferring midostaurin sensitivity. Casado et al previously described phosphoprotein signatures significantly associated with ex vivo responses to midostaurin in primary AML blasts (Casado et al Leukaemia 2018). In the current study, we tested whether our signatures could group FLT3+ patients based on clinical responses to midostaurin plus chemotherapy. Methods: FLT3+ bone marrow (BM) and peripheral blood (PB) specimens were collected at diagnosis, post-treatment and relapse (n=54 cases) from the Leukemia Tissue Bank at Princess Margaret Cancer Centre. All patients in this study were treated with standard chemotherapy plus midostaurin. Protein/phosphoprotein-signatures for BM and PB samples were analysed independently. Case-studies with multiple post-treatment time-points or relapse events following second line treatments were also analysed. Peptides (proteomics) and enriched phosphopeptides (phosphoproteomics) were quantified using liquid chromatography - tandem mass spectrometry. A classification machine learning (ML) algorithm was trained to group patients based on response to treatment as a function of protein/phosphoprotein-signature status. Other features (e.g. genetic mutations, HSC-transplant) were also analysed. Differential survival analysis between patient groups was carried out with Kaplan-Meier and Log Rank test methods. Pathways upregulated in post-treatment or relapse specimens, particularly from those cases that responded poorly to chemo + midostaurin (i.e. early relapse / refractory disease) were investigated using enrichment statistical methods including kinase-substrate enrichment analysis (KSEA) and gene ontology analysis and identified as potential mechanisms of resistance. Statistical significance of enrichment was determined using parametric methods and p-values adjusted for multiple testing using the Benjamini-Hochberg method. Results: ML models were developed based on the ex-vivo phosphoproteomics signatures described in the Casado et al study, from which we trained a predictive model (model 1). Patients positive for model 1 exhibited a survival probability of 243 weeks, compared to 126 weeks in signature negative patients (averages by geometric mean, Log Rank p = 9.88e-05). As the patients in the current study received chemotherapy, in addition to midostaurin, we identified a new phosphoproteomic signature consisting of 26 phospho-sites which partially overlapped with the ex-vivo signature. Patients positive for this new phosphoproteomic signature showed a markedly longer survival time than negative patients (269 vs 76 weeks, Log Rank p = 1.30e-05 for PB and 241 vs 56, Log Rank p = 2.13e-09 for BM specimens, Table). A proteomic signature was also identified in the current study. Positive patients showed a longer survival time than negative patients (330 vs 173 weeks, Log Rank p = 5.0e-04 for PB and 460 vs 156, Log Rank p = 5.2e-06 for BM specimens, Table), however this was less differentiating than the phosphoproteomic signature. Pathways upregulated in post-treatment or relapse specimens from early relapse or refractory cases were associated with molecular functions such as cell proliferation, anti-apoptosis, non-homologous end-joining, transcriptional regulation, spliceosome and cytoskeleton remodelling. Conclusions: We have identified protein and phosphoprotein signatures with the potential to further stratify AML for midostaurin treatment. Phosphoproteomic signatures differentiated according to response better than the proteomic signatures. Pathways upregulated in relapse/refractory cases may have a role in resistance and this will be determined in follow up studies. Analysis will also be performed on FLT3 mutant-negative cases to validate the signatures and elucidate mechanisms of resistance in this group. Disclosures Veiga Nobre: Kinomica Ltd.: Current Employment. Minden: Astellas: Consultancy. Gribben: Janssen: Honoraria, Research Funding; AZ: Honoraria, Research Funding; Abbvie: Honoraria; BMS: Honoraria; Gilead/Kite: Honoraria; Morphosys: Honoraria; Novartis: Honoraria; Takeda: Honoraria; TG Therapeutics: Honoraria. Britton: Kinomica Ltd.: Current Employment, Current equity holder in publicly-traded company.
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- 2021
28. Enasidenib in Combination with Venetoclax in IDH2-Mutated Myeloid Malignancies: Preliminary Results of the Phase Ib/II Enaven-AML Trial
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Caroline J McNamara, Tracy Murphy, Courtney D. DiNardo, Charina Cameron, Severine Cathelin, Mark D. Minden, Hassan Sibai, Aaron D. Schimmer, Andre C. Schuh, Steven M. Chan, Dawn Maze, Karen W.L. Yee, and Vikas Gupta
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Myeloid ,business.industry ,Venetoclax ,education ,Immunology ,Cell Biology ,Hematology ,Enasidenib ,Biochemistry ,IDH2 ,chemistry.chemical_compound ,medicine.anatomical_structure ,chemistry ,Phase (matter) ,Cancer research ,Medicine ,business ,health care economics and organizations - Abstract
BACKGROUND: Isocitrate dehydrogenase 2 (IDH2) mutations are found in about 10-15% of acute myeloid leukemia (AML) cases. Enasidenib (ENA) is a first-in-class mutant IDH2 inhibitor that induces differentiation of IDH2-mutated leukemic cells. However, the clinical efficacy of single agent ENA therapy in relapsed or refractory (R/R) AML is limited, underscoring the need for combination therapy. Preclinical studies have shown that IDH-mutated leukemic cells are particularly sensitive to BCL2 inhibition with venetoclax (VEN), a finding supported by clinical studies of VEN combination therapies. We previously demonstrated that the combination of ENA and VEN can be more effective than each drug alone, in reducing leukemic burden in patient-derived xenograft models of IDH2-mutated AML. Here, we report preliminary safety and efficacy results of an ongoing open-label, single-arm, phase Ib/II trial (NCT04092179) of ENA in combination with VEN in patients with IDH2-mutated myeloid malignancies. METHODS: Patients 18 years of age or above with IDH2-mutated R/R AML or high-risk MDS/MPN and an ECOG performance status of 0-2 were eligible. Patients previously treated with an IDH2 or BCL2 inhibitor were excluded. Participants received VEN continuously starting on cycle 1 day 1 with a 3-day ramp-up to a target dose of 400 mg daily (dose level 0) in cohorts 1 and 2. ENA was administered at 100 mg daily continuously starting on cycle 1 day 15. Each cycle was 28 days. Concurrent use of a moderate or strong CYP3A4 inhibitor was allowed with 50% dose reduction of VEN after completion of the first 2 cycles at 100% target dose. Interruptions of VEN and/or ENA were permitted for management of adverse events (AEs). Primary endpoints were 1) safety and tolerability and 2) overall response rate (ORR) defined as complete remission (CR) + CR with incomplete blood count recovery (CRi) + morphologic leukemia-free state (MLFS) + partial remission (PR) by revised IWG criteria. Secondary endpoints include pharmacokinetic (PK) profiles of VEN, duration of response, overall survival (OS), and IDH2 mutant allele burden in bone marrow by ddPCR. RESULTS: The study opened to recruitment in November of 2020. As of July 28, 2021 (data cutoff), 11 patients were enrolled on study; 10 with R/R AML and 1 with very-high risk MDS by IPSS-R. Six patients had a R140Q mutation, and 5 had a R172K mutation. Median age was 72 years (range: 32 - 80); 6 patients were male. Participants had received a median of 2 prior lines of therapies (range: 1 - 4). Six of 10 AML patients had primary refractory disease. The MDS patient experienced secondary azacitidine failure. Key treatment emergent grade ≥ 3 AEs regardless of attribution were: febrile neutropenia (n=3), intracranial hemorrhage (n=3), lung infection (n=2), other infection (n=2), elevated AST/ALT (n=2), sepsis (n=1), leukocytosis (n=1), TRALI (n=1), and small bowel obstruction (n=1). No cases of differentiation or tumor lysis syndrome were observed. No patients discontinued the study due to AEs. The addition of ENA, a known inhibitor of CYP3A4, did not significantly affect the PK profiles of VEN. Nine of the AML patients completed at least 1 cycle of treatment and are considered evaluable for efficacy. One AML patient died from intracranial hemorrhage prior to completion of cycle 1. Median duration of observation is 3.5 months. Of the evaluable patients, CR was achieved in 2 patients (22%) and CRi in 3 patients (33%) for an ORR of 55% (Fig. 1). Median number of cycles to response was 3. All responders remain in remission and on study with a median of 6 cycles received to date (range: 3 - 8). Of the remaining 4 patients, 2 patients (22%) remain on study with stable disease, and 2 patients (22%) experienced progressive disease and died (one after 7 cycles and the other after 1 cycle). Median OS for the entire cohort has not been reached. A sustained reduction in mutant IDH2 allele frequency in bone marrow correlated with response (Fig. 2). For the MDS patient, no response was observed after 1 cycle of treatment. CONCLUSIONS: VEN in combination with ENA is a well-tolerated regimen with no dose-limiting toxicities observed at the current dose level. The preliminary efficacy of this combination is encouraging with an ORR of 55% in evaluable R/R AML patients, with some responders achieving deep molecular remissions. Patient enrollment in dose-escalation cohorts is ongoing. Figure 1 Figure 1. Disclosures Chan: AbbVie: Research Funding; BMS: Research Funding. Gupta: Sierra Oncology: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Constellation Pharma: Consultancy, Honoraria; Incyte: Honoraria, Research Funding; AbbVie: Consultancy, Honoraria; Roche: Consultancy; BMS-Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy. Maze: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene-BMS: Honoraria; Takeda: Research Funding; PharmaEssentia: Research Funding; Kronos Bio: Research Funding. Minden: Astellas: Consultancy. Schimmer: Takeda Pharmaceuticals: Consultancy, Research Funding; Medivir AB: Research Funding; Novartis: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Otsuka Pharmaceuticals: Consultancy, Honoraria; UHN: Patents & Royalties. Schuh: AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astellas: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Agios: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Servier: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; GlycoMimetics: Research Funding; Kite/Gilead: Research Funding; Jazz: Membership on an entity's Board of Directors or advisory committees; Teva: Honoraria, Membership on an entity's Board of Directors or advisory committees. Yee: Pfizer: Membership on an entity's Board of Directors or advisory committees; Forma Therapeutics: Research Funding; F. Hoffmann La Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding; Tolero: Research Funding; Genentech: Research Funding; Janssen: Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Shattuck Labs: Membership on an entity's Board of Directors or advisory committees; Onconova: Research Funding; Bristol-Myers Squibb/Celgene: Membership on an entity's Board of Directors or advisory committees; Otsuka: Membership on an entity's Board of Directors or advisory committees; Paladin: Membership on an entity's Board of Directors or advisory committees; Geron: Research Funding; MedImmune: Research Funding; Jazz: Research Funding; TaiHo: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria; Astex: Membership on an entity's Board of Directors or advisory committees, Research Funding. DiNardo: Agios/Servier: Consultancy, Honoraria, Research Funding; Notable Labs: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria; AbbVie: Consultancy, Research Funding; Novartis: Honoraria; Foghorn: Honoraria, Research Funding; Forma: Honoraria, Research Funding; GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Research Funding; ImmuneOnc: Honoraria, Research Funding; Celgene, a Bristol Myers Squibb company: Honoraria, Research Funding. OffLabel Disclosure: Enasidenib is approved for the treatment of relapsed or refractory AML as single agent. Venetoclax, in combination with azacitidine or low-dose cytarabine, is approved for the treatment of newly diagnosed AML patients who are 75 years or older, or who have comorbidities that preclude use of intensive induction chemotherapy.
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- 2021
29. Inhibiting Mitochondrial Complex II Exposes a Novel Metabolic Vulnerability in Acute Myeloid Leukemia
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Alessia Roma, Julie A. Reisz, Matthew Tcheng, Angelo D'Alessandro, Praveen P.N. Rao, Nawaz Ahmed, Kristin J Hope, Preethi Jayanth, Jessica Luc, Paul A. Spagnuolo, Mark D. Minden, Andrew C. Doxey, and Sarah Walker
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Immunology ,Vulnerability ,Cancer research ,Myeloid leukemia ,Cell Biology ,Hematology ,Mitochondrial Complex II ,Biology ,Biochemistry - Abstract
Acute myeloid leukemia (AML) is a hematological malignancy, characterized by an increased reliance on mitochondria-related energetic pathways including oxidative phosphorylation (OXPHOS). Consistent with this, the electron transport chain (ETC), a component of OXPHOS has been demonstrated to be a suitable anti-leukemia target, with ETC complex I inhibitors currently in clinical development. Relative to its counterparts, complex II (CII) is unique in that it directly links the ETC to the tricarboxylic acid (TCA) cycle through succinate dehydrogenase (SDH) activity. Moreover, it is the only ETC complex with elevated activity in AML, relative to normal hematopoietic samples, with indirect inhibition selectively targeting AML cells. However, direct CII inhibition in AML has not been previously investigated, nor have the mechanisms underlying the divergent fates of AML and normal cells upon CII inhibition. A genetic approach was first used to assess the effects of CII impairment on AML growth in vitro and in vivo. Using lentiviral mediated shRNA we generated AML cell lines lacking succinate dehydrogenase assembly factor 1 (Sdhaf1). Sdhaf1 knockdown suppressed CII activity, cell proliferation and clonogenic growth across all three cell lines and delayed leukemia growth in vivo. To recapitulate these effects through a pharmacological approach, we aimed to identify a novel CII inhibitor, since currently available inhibitors are only effective at high doses and are neurotoxic. Through an in silico structural screen and molecular docking study, shikonin was identified as a small molecule that selectively binds to CII. Shikonin inhibited CII activity in the AML cells lines and patient-derived samples, and selectively killed AML cells (EC 50: 1.0μM ± 0.04) while sparing normal progenitors. In murine engraftment models, shikonin (2.0-3.0 mg/kg, 3x/week for 5 weeks) significantly reduced engraftment of patient-derived AML cells but had no effect on normal hematopoiesis. To further characterize the mechanisms governing the divergent cell fates of CII inhibition, we performed stable isotope metabolic tracing using 13C 6- glucose and 13C 5, 15N 2-glutamine in patient-derived AML cells and normal mobilized peripheral blood mononuclear cells (MNCs). Both pharmacological and genetic loss of CII resulted in TCA cycle truncation by impairing oxidative metabolism of both glucose and glutamine. In Sdhaf1 knockdown and primary AML cells, this led to a depletion in steady state levels of TCA metabolites proceeding SDH. Inhibition of CII most notably suppressed levels of aspartate, a nucleotide precursor whose levels dictate the proliferative capacity of a cell under ETC dysfunction. Remarkably, MNCs maintained aspartate levels despite inhibition of CII, which was attributed to reductive carboxylation of glutamine, an alternate metabolic pathway that can regenerate TCA intermediates when OXPHOS is impaired. In contrast, while reductive carboxylation was also active in AML cells after CII inhibition, this activity was insufficient to maintain aspartate levels and resulted in metabolite depletion and cell death. Thus, loss of CII activity results in diverse cell fates whereby normal haematopoietic, but not AML cells sufficiently use reductive carboxylation of glutamine to overcome TCA cycle truncation, sustain aspartate levels and avert cell death. This is further evident through modulation of glutamine entry into the TCA cycle, where supplementation of cell-permeable α-ketoglutarate abrogated shikonin-mediated cell death while concomitant treatment with the glutaminase inhibitor CB-839, sensitized cells. Together, these results expose reductive carboxylation to support aspartate biosynthesis, as a novel metabolic vulnerability in AML that can be pharmacologically targeted through CII inhibition for clinical benefit. Disclosures Minden: Astellas: Consultancy. D'Alessandro: Omix Thecnologies: Other: Co-founder; Rubius Therapeutics: Consultancy; Forma Therapeutics: Membership on an entity's Board of Directors or advisory committees.
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- 2021
30. A Phase 3, Randomized, Double-Blind, Placebo-Controlled Study to Assess the Efficacy and Safety of Entospletinib in Combination with Intensive Induction and Consolidation Chemotherapy in Adults with Newly Diagnosed N ucleophosmin 1-mutated Acute Myeloid Leukemia
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Pavan Kumar, Gordon L. Bray, Wendy Stock, Eytan M. Stein, John C. Byrd, Thomas Oellerich, Jorge E. Cortes, Mark D. Minden, Jenna Elder, and Jorge F. DiMartino
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Oncology ,medicine.medical_specialty ,Entospletinib ,business.industry ,Immunology ,Placebo-controlled study ,Myeloid leukemia ,Consolidation Chemotherapy ,Cell Biology ,Hematology ,Newly diagnosed ,Biochemistry ,Double blind ,Internal medicine ,Medicine ,business - Abstract
Background: Spleen tyrosine kinase (SYK) is a component of both lymphoid and myeloid cell signaling pathways and has been implicated in the pathogenesis of a subset of acute myeloid leukemia (AML) defined by dysregulated expression of the HOXA9 and MEIS1 transcription factors. Entospletinib (ENTO) is an oral, selective SYK inhibitor that is acceptably tolerated when administered with intensive induction and consolidation in newly diagnosed AML patients. In a phase 2 study, following induction with cytarabine and daunorubicin (7+3) plus ENTO, higher rates of complete response (CR) or CR with incomplete hematologic recovery (CRi) were observed in patients with rearrangements of the KMT2A (MLL) gene (MLL-r) and mutations of the nucleophosmin 1 (NPM1) gene, both of which are associated with aberrant expression of HOXA9 and MEIS1, as compared to patients without these mutations. In an exploratory analysis, patients with HOXA9/MEIS1 expression levels above the median experienced superior overall survival (OS) as compared to patients with expression levels below the median. In the AGILITY trial, we hypothesize that the addition of ENTO to intensive induction/consolidation in newly diagnosed patients with NPM1-mutated AML will improve the rate of CR without evidence of measurable residual disease (MRD-negative CR) post-induction and duration of event-free survival (EFS). Methods: AGILITY will be a global, multi-center, double-blind, placebo-controlled trial of ENTO in combination with cytarabine plus daunorubicin or idarubicin induction (7+3) and age-adjusted high-dose cytarabine (HiDAC) consolidation in newly diagnosed AML patients aged 18-75 years who are candidates for intensive induction and harbor a documented NPM1 mutation based on local or central mutation testing. Patients with co-mutated FLT3 (internal tandem duplication or tyrosine kinase domain) and for whom midostaurin with 7+3 is indicated are excluded. Patients will be stratified based on age ( An independent data-monitoring committee will monitor emerging safety and efficacy data from this trial on an ongoing basis. Disclosures Byrd: Vincerx Pharmaceuticals: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Novartis, Trillium, Astellas, AstraZeneca, Pharmacyclics, Syndax: Consultancy, Honoraria; Newave: Membership on an entity's Board of Directors or advisory committees. Cortes: Bristol Myers Squibb, Daiichi Sankyo, Jazz Pharmaceuticals, Astellas, Novartis, Pfizer, Takeda, BioPath Holdings, Incyte: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Bio-Path Holdings, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Sun Pharma: Consultancy, Research Funding. Minden: Astellas: Consultancy. Oellerich: Roche: Consultancy; Gilead: Research Funding; Kronos Bio, Inc.: Consultancy; Merck KGaA: Consultancy, Research Funding. Stein: Syros Pharmaceuticals, Inc.: Consultancy; Daiichi Sankyo: Consultancy; PinotBio: Consultancy; Celgene: Consultancy; Bristol Myers Squibb: Consultancy; Jazz Pharmaceuticals: Consultancy; Foghorn Therapeutics: Consultancy; Blueprint Medicines: Consultancy; Gilead Sciences, Inc.: Consultancy; Abbvie: Consultancy; Janssen Pharmaceuticals: Consultancy; Genentech: Consultancy; Syndax Pharmaceuticals: Consultancy; Agios Pharmaceuticals, Inc: Consultancy; Novartis: Consultancy; Astellas: Consultancy. Elder: PharPoint Research, Inc.: Current Employment. Kumar: Kronos Bio, Inc.: Current Employment, Current equity holder in publicly-traded company. Bray: Kronos Bio, Inc.: Consultancy. DiMartino: Kronos Bio, Inc.: Current Employment, Current equity holder in publicly-traded company. Stock: Pfizer: Consultancy, Honoraria, Research Funding; amgen: Honoraria; agios: Honoraria; jazz: Honoraria; kura: Honoraria; kite: Honoraria; morphosys: Honoraria; servier: Honoraria; syndax: Consultancy, Honoraria; Pluristeem: Consultancy, Honoraria. OffLabel Disclosure: Entospletinib is an investigational therapy
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- 2021
31. Clinical Significance of Clonal Hematopoiesis in the Setting of Autologous Stem Cell Transplantation for Lymphoma
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Sharon Ben Barouch, Andrea Arruda, Kit I. Tong, John E. Dick, Mileidys Alvarez, Vishal Kukreti, Christine Chen, John Kuruvilla, Armand Keating, Jesse Joynt, Robert Kridel, Anca Prica, Jessie J. F. Medeiros, Mark D. Minden, Sagi Abelson, Mehran Bakhtiari, Sita Bhella, Tracy Lackraj, and Michael Crump
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Autologous stem-cell transplantation ,business.industry ,Immunology ,Clonal hematopoiesis ,Cancer research ,Medicine ,Clinical significance ,Cell Biology ,Hematology ,business ,medicine.disease ,Biochemistry ,Lymphoma - Abstract
Introduction : Peripheral blood samples of healthy individuals may harbour detectable mutations in genes recurrently mutated in myeloid malignancies, a situation referred to as clonal hematopoiesis (CH). Risk factors for CH include increasing age as well as previous exposure to cytotoxic therapy. CH has been associated with an increased risk of overall mortality, including in the setting of autologous stem cell transplantation (ASCT) for non-Hodgkin lymphoma (Gibson et al, JCO, 2017). The excess mortality is largely driven by cardio-vascular disease, but may also be additionally attributable to an increased risk of myeloid malignancies that arise through the selection of CH subclones. Herein, we aimed to investigate the prognostic implications of CH after ASCT in an independent and diversified, large cohort of lymphoma patients using ultra-deep, highly sensitive error-correction sequencing. Methods : DNA was obtained from 420 residual apheresis products obtained from patients who had undergone autologous stem cell transplantation for lymphoma at the Princess Margaret Cancer Center between 2002 and 2018. Target DNA sequences corresponding to regions recurrently mutated in myeloid neoplasms (affecting n = 36 genes) were captured using single molecule molecular inversion probes (smMIPs) that incorporate molecular tagging. Single nucleotide variants and short insertions and deletions were identified using SmMIP-tools (Medeiros et al, bioRxiv, 2021), which implements a series of steps including probabilistic modeling of allele-specific error rates and generation of consensus sequences to suppress next-generation sequencing-associated errors. Given the high sensitivity and precision of our method, we did not prespecify a variant allele fraction cut-off. Results : All patients had relapsed/refractory lymphoma, except for 98 (23.3%) mantle cell lymphoma patients and one patient with extranodal NK/T-cell lymphoma where ASCT was part of frontline management. The most common conditioning regimens were high-dose melphalan and etoposide (77.5%) and high-dose melphalan and Ara-C (16.4%). We identified 275 high-confidence mutations in 181 out of 420 patients (43.1%), with 64 of these 181 patient samples (35.4%) having more than one mutation. The median age was higher in patients with CH than in patients without (55 years vs. 51, P = 0.002). The most frequently mutated gene were PPM1D (11.9%), followed by TET2 (11.4%), DNMT3A (8.8%), ASXL1 (5.2%) and TP53 (4.5%). The lymphoma subtype with the highest prevalence of CH was T-cell lymphoma (CH found in 72.2% of cases), followed by transformed indolent lymphoma (51.4%), mantle cell lymphoma (47.5%), diffuse large B-cell lymphoma (40.4%) and Hodgkin lymphoma (33.3%). While there was no difference in the number of CD34+ cells infused for patients with and without CH, the median time to neutrophil engraftment and the median time to platelet engraftment were significantly longer in patients with CH (11 days vs. 10 days, P = 0.025; and 14 days vs. 13 days, P < 0.001, respectively). The median follow-up of living patients was 4.2 years. Patients with CH had inferior 5-year OS from the time of first relapse (38.9% vs. 45.5%, P = 0.037) and from the time of ASCT (51.2% vs. 59.1%, P = 0.017, see figure). Five-year OS from ASCT was 47.5% vs. 53.7% in patients with 1 mutation and > 1 mutation, respectively, compared to 59.1% in patients without CH (P = 0.005). The presence of CH did not have an impact on the risk of post-ASCT relapse. In multivariate Cox regression analysis in which CH and age (as a continuous variable) were included, CH remained significantly associated with adverse OS post-ASCT (HR 1.39, 95% 1.02-1.91, P = 0.038). Only seven patients out of 420 (1.7%) developed a therapy-related myeloid neoplasm (TMN). The cumulative incidence of TMN was not significantly increased in patients with CH (10-year cumulative incidence 3.3% vs. 3.0% in those without CH, P = 0.433). Conclusions : Our results show that CH was associated with delayed neutrophil and platelet engraftment. Moreover, CH conferred an increased risk of death after ASCT that was not explained by lymphoma relapse. The risk of TMN was low in our cohort and CH was not a risk factor for TMN, an observation that is distinct from prior observations (e.g. Gibson et al, JCO, 2017 and Husby et al, Leukemia, 2020). Our results raise the possibility that the risk of TMN may be modulated by factors other than CH. Figure 1 Figure 1. Disclosures Minden: Astellas: Consultancy. Kuruvilla: Janssen: Honoraria, Research Funding; Antengene: Honoraria; AstraZeneca: Honoraria, Research Funding; Amgen: Honoraria; Incyte: Honoraria; Novartis: Honoraria; Karyopharm: Honoraria, Other: Data and Safety Monitoring Board; Pfizer: Honoraria; AbbVie: Honoraria; TG Therapeutics: Honoraria; Medison Ventures: Honoraria; Merck: Honoraria; Gilead: Honoraria; BMS: Honoraria; Roche: Honoraria, Research Funding; Seattle Genetics: Honoraria. Crump: Roche: Research Funding; Epizyme: Research Funding; Kyte/Gilead: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Prica: Kite Gilead: Honoraria; Astra-Zeneca: Honoraria. Chen: Beigene: Membership on an entity's Board of Directors or advisory committees; Astrazeneca: Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy. Kridel: Gilead Sciences: Research Funding.
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- 2021
32. Sirtuin 3 Inhibition Targets AML Stem Cells through Perturbation of Fatty Acid Oxidation
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Courtney L. Jones, Angelo D'Alessandro, Min Yang, Jacob Berman, Vincent Rondeau, Cristiana O'Brien, Hening Lin, Ari Melnick, Jun Young Hong, Rachel Culp-Hill, Tianyi Ling, Meng Li, Mark D. Minden, Julie A. Reisz, and Andrea Arruda
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biology ,Biochemistry ,Chemistry ,Immunology ,Sirtuin ,biology.protein ,Cell Biology ,Hematology ,Stem cell ,Perturbation (geology) ,Beta oxidation - Abstract
Acute myeloid leukemia (AML) in adults has a 5-year survival of approximately 30% and a high rate of disease recurrence in part due to our inability to eliminate the disease-initiating leukemic stem cells (LSCs) (Shlush et al. Nature, 2017). Previous studies have shown that LSCs uniquely rely on oxidative phosphorylation (OXPHOS) for survival (Lagadinou et al. Cell Stem Cell, 2013). Thus, novel therapies that are designed to target LSC metabolism have the potential to improve patient outcomes. Work from our group and others has demonstrated that a critical metabolite for OXPHOS regulation in LSCs is the coenzyme NAD + (Jones et al. Cell Stem Cell, 2020; Mitchell et al. Blood Advances 2019). One family of NAD + dependent proteins important in cancer biology, and AML specifically (Yan et al. Blood Cancer Discovery, 2021), are sirtuins. To determine if any sirtuins are important in LSC function we knocked down each sirtuin family member (sirtuin 1-7) with siRNA in four primary AML specimens and measured viability and colony forming ability. Knockdown of sirtuin 3 (SIRT3) decreased viability and colony forming potential of all AML specimens tested. SIRT3 is a mitochondrial de-acetylase with a multi-faceted role in metabolic regulation and oncogenesis (Finley, et al. Trends in Molecular Medicine, 2016). SIRT3 interacts with pathways upstream of OXPHOS including the tricarboxylic acid (TCA) and fatty acid oxidation (FAO). Importantly, a SIRT3 inhibitor (YC8-02) has been developed and has been shown to be effective pre-clinically for the treatment of B-cell lymphoma (Li et al. Cancer Cell, 2019). To further understand the significance of SIRT3 in LSCs, we assessed viability and colony forming potential upon YC8-02 treatment. LSCs were enriched from primary specimens based upon relative reactive oxygen species (ROS) level as previously described (Lagadinou et al. Cell Stem Cell, 2013). LSCs and blasts enriched from ten primary AML, and four AML cell lines (MOLM13, TEX, OCI-AML2, OCI-AML3) were cultured for 48 hours with or without YC8-02 before assessing viability and colony forming ability. YC8-02 treatment resulted in a significant decrease in colony forming potential of AML cells compared to control (data not shown). Similarly, LSCs, blasts, and cell lines showed a significant decrease in viability upon YC8-02 treatment (Fig 1A and data not shown). Cord blood and mobilized peripheral blood samples conversely did not show a change in colony forming potential following SIRT3 knockdown or YC8-02 treatment, respectively (data not shown). To assess YC8-02's effect on LSC function, three AML samples were treated with 10µM of drug for 24 hours and transplanted into NSG-S mice. YC8-02 treatment resulted in a significant decrease in AML engraftment, indicating a decrease in LSC function (Fig 1B). To determine the mechanism by which SIRT3 inhibition causes cell death, LSCs enriched from three primary specimens were treated with YC8-02; metabolite and lipid levels were determined by mass spectrometry. This analysis revealed a significant accumulation of fatty acids post YC8-02 treatment. To further characterize these changes, MOLM13 cells were treated with 13C 16-palmitic acid following 4 hours of incubation with 10µM YC8-02. Cells were collected 4 and 16 hours after introduction of palmitic acid and metabolic tracing was assessed by mass spectrometry. We found an accumulation of long and very long chain fatty acids and a decrease in TCA cycle intermediates (Fig 1C). FAO normally supplies TCA with intermediate acetyl-CoA; thus, these data indicate a decrease in FAO upon YC8-02 treatment. Accordingly, we measured changes in OXPHOS in response to treatment with YC8-02, in primary LSCs (Fig 1D) and AML cell lines (data not shown) and found a significant decrease in basal oxygen consumption. Further, ATP levels were significantly decreased upon YC8-02 treatment in LSCs (Fig 1E). In conclusion, we show that SIRT3 plays a pivotal role in FAO and LSC function. When SIRT3 is inhibited, FAO activity decreases resulting in the accumulation of long and very long chain fatty acids. This change in FAO activity reduces the availability of products for the TCA cycle, limiting necessary intermediates for OXPHOS, decreasing ATP production, and ultimately causing cell death. Therefore, our data suggests that SIRT3 is a potential therapeutic target for LSCs and should be considered in future pre-clinical and clinical investigations. Figure 1 Figure 1. Disclosures Melnick: Constellation: Consultancy; Epizyme: Consultancy; Daiichi Sankyo: Research Funding; Sanofi: Research Funding; Janssen Pharmaceuticals: Research Funding; KDAC Pharma: Membership on an entity's Board of Directors or advisory committees. Minden: Astellas: Consultancy. D'Alessandro: Omix Thecnologies: Other: Co-founder; Rubius Therapeutics: Consultancy; Forma Therapeutics: Membership on an entity's Board of Directors or advisory committees.
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- 2021
33. KDM6 Demethylases Integrate DNA Repair Gene Regulation: Loss of KDM6A Sensitizes AML to PARP Inhibition and Potentiates with BCL2 Blockade
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Liberalis Debraj Boila, Liqing Jin, Alex Murison, Subham K. Bandyopadhyay, Subhadeep Ghosh, Siva Sai Naga Anurag Muddineni, Andy G.X. Zeng, Sayantani Sinha, Shankha Subhra Chatterjee, Mayukh Biswas, Nathan Mbong, Olga I. Gan, Andrea Arruda, Wasim Shaikh, Anwesha Bose, Satyaki Bhowmik, Sayan Chakraborty, James A. Kennedy, Amanda Mitchell, Eric R. Lechman, Debasis Banerjee, Michael Milyavsky, Mark D. Minden, John E. Dick, and Amitava Sengupta
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hemic and lymphatic diseases ,Immunology ,Cell Biology ,Hematology ,Biochemistry - Abstract
Acute myeloid leukemia (AML) is a heterogeneous, aggressive hematological malignancy with dismal prognosis where limited targeted therapies are currently available. Poly-(ADP-ribose)-polymerase (PARP) inhibition has emerged as an important therapeutic arsenal to target homologous recombination-deficient tumors. However, molecular understanding of PARP blockade in the context of epigenetic derangements and transcriptional plasticity in human elderly AML pathogenesis remains unexplored. KDM6 proteins are H3K27 demethylases that critically regulate chromatin architecture in multi-cellularity and tumorigenesis (Tran, Mol Cell Biol 2020). KDM6A escapes X-chr inactivation, and Utx-/- female mice spontaneously develop aging associated myeloid leukemia (Gozdecka, Nat Genet 2018; Sera, Blood 2021). In addition, KDM6A loss of function mutation is implicated in AML relapse (Stief, Leukemia 2020). In contrast, KDM6B primarily exerts an oncogenic function in heme-malignancies. Together, KDM6A and KDM6B play cell type-specific function in leukemia, and KDM6 proteins and associated signaling emerge as important focal point for developing molecular targeted therapy. We identify that KDM6 demethylase activity critically regulates DNA damage repair (DDR) gene expression program in AML. Transcriptome analysis indicated a significant downregulation of expression of DDR genesets in both KDM6A deficient human AML and Utx -/- pre-leukemic cells. Lentiviral shRNA screening performed in response to low-dose γ-irradiation in AML stem cells, revealed a radioprotective function of KDM6A. Expression of KDM6s is regulated by genotoxic stress in a time-dependent manner, and deficiency of JmjC catalytic function impaired DDR transcriptional activation and compromised repair potential. Mechanistically, quantitative ChIP experiments also revealed co-operation between KDM6A and SWI/SNF facilitating dynamic chromatin remodeling at TSS/promoter to induce DDR gene expression. To interrogate changes in chromatin accessibility we performed ATAC-seq analysis in KDM6 deficient AML. Motif enrichment highlighted that while KDM6A depletion led to reduced chromatin access to 140 transcription factors (TFs), only 56 TF binding sites showed increased accessibility. Overall, changes in chromatin accessibility, associated with a reduced binding of DDR regulatory TFs in KDM6 deficient AML, account for a compromised DDR function. In agreement with these findings an array of KDM6 deficient AML cells were more sensitive to PARP inhibition, and pre-clinical mice models xenotransplanted with KDM6A loss of function AML line showed an increased susceptibility to PARP blockade in vivo. FLT3-ITD positive AML with a lower KDM6A expression was more sensitive to olaparib. In addition, olaparib administration significantly reduced bone marrow engraftment of patient-derived xenografts of KDM6A-mutant primary AML. Interestingly, KDM6A expression is upregulated in venetoclax-resistant monocytic-AML compared to venet-sensitive primitive-AML. Using venet responsive isogenic lines we demonstrated that attenuation of KDM6 function increased mitochondrial activity, intracellular ROS levels, de-repressed BCL2 expression, and sensitized AML cells to venetoclax. Additionally, KDM6 loss resulted in transcriptional repression of BCL2A1, commonly associated with venet resistance (Zhang, Nat Cancer 2020). Corroborating these results, dual targeting of PARP with BCL2 was superior to PARP or BCL2 inhibitor monotherapy in inducing primary AML apoptosis, and KDM6A loss further enhanced this synergism. In sum, our study illustrates a molecular mechanistic rationale in support for a novel combination targeted therapy for AML, and posit KDM6A as a molecular determinant for therapeutic efficacy. Intriguingly, KDM6A functions as a gatekeeper of BCL2 and BCL2A1 expression. Similar to TET2 although bi-allelic Utx loss causes evolution to myeloid neoplasms, minimal KDM6 activity is important for survival of human AML cells. KDM6s have been implicated in solid tumors, and both PARP and BCL2 inhibitors are being tested in cancer patients, underscoring a wider scope of application. To conclude, KDM6A unfolds to be a central regulator for susceptibility of AML to both PARP and BCL2 inhibition, expanding the possibility to characterize effective combination targeted therapy for AML in clinical settings. Disclosures Minden: Astellas: Consultancy. Dick: Celgene, Trillium Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding.
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- 2021
34. Transcriptomic Features of Immune Exhaustion and Senescence Predict Outcomes and Define Checkpoint Blockade-Unresponsive Microenvironments in Acute Myeloid Leukemia
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Ivana Gojo, Joshua F. Zeidner, Sarah K. Tasian, Francesco Mazziotta, Sergio Rutella, Vedran Radojcic, Michael Kramer, Jayakumar Vadakekolathu, Martin Bornhäuser, Heidi Altmann, Mark D. Minden, Andrea Arruda, Rupkatha Mukhopadhyay, Leo Luznik, Stephen Reeder, Hanna A. Knaus, and Tung On Yau
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Senescence ,Transcriptome ,Immune system ,business.industry ,Immunology ,Cancer research ,Medicine ,Myeloid leukemia ,Cell Biology ,Hematology ,business ,Biochemistry ,Blockade - Abstract
Acute myeloid leukemia (AML) is a molecularly and clinically heterogeneous disease. Reinstating immunological control of AML is highly desirable to eradicate chemotherapy-resistant clones and provide long-term disease control. We recently identified bone marrow (BM) microenvironmental transcriptomic profiles that stratify patients with newly diagnosed AML into an immune-infiltrated and an immune-depleted subtype and that refine the accuracy of survival prediction beyond that afforded by current prognosticators (Vadakekolathu J et al., 2020). We have also shown that CD8 + T cells from patients with AML exhibit features of immune exhaustion and senescence (IES), including heightened expression of killer cell lectin-like receptor subfamily G member 1 (KLRG1) and B3GAT1 (encoding CD57) (Knaus H et al., 2018). Whether deranged T-cell functions affect the likelihood of responding to antitumor therapy, including immune checkpoint blockade (ICB), is an outstanding question in AML. In the current study, we analyzed 183 BM samples collected longitudinally at time of AML onset, response assessment and disease relapse from multiple cohorts of patients with AML treated with standard-of-care induction chemotherapy, and from 33 elderly AML patients with newly diagnosed or chemotherapy-refractory/relapsed AML treated with azacitidine, and the PD-1 checkpoint inhibitor pembrolizumab (NCT02845297). Primary patient specimens and associated clinical data were obtained via informed consent in accordance with the Declaration of Helsinki on research protocols approved by the Institutional Review Boards of the participating Institutions. RNA (150-200 ng) was extracted from BM aspirates and was processed on the nCounter FLEX analysis system (NanoString Technologies, Seattle, WA) using the PanCancer Immune profiling panel, as previously published (Vadakekolathu J et al., 2020). The correlation between transcriptomic features of IES, clinical characteristics, therapeutic response and patient outcome was validated using publicly available RNA-sequencing and NanoString data from 1,698 patients with AML, including samples from the TCGA-AML (n=147 cases), Beat-AML Master Trial (n=264 cases, of which 240 with survival data and 195 with chemotherapy response data) and Children's Oncology Group (COG)-TARGET AML series (n=145 cases). We initially showed that, compared with their non-senescent CD8 +CD57 -KLRG1 - counterpart, senescent CD8 +CD57 +KLRG1 + T cells are functionally impaired in terms of their ability to effect AML-blast killing mediated by an anti-CD33/CD3 bi-specific T-cell engager antibody construct (kindly provided by Amgen, USA; effector/target [E/T] ratio = 1:5). We then used gene set enrichment analysis (GSEA) to derive a transcriptomic signature of IES encompassing natural killer (NK)-cell and stem-like CD8 + T-cell markers, and showed that IES states correlate with lymphoid infiltration, adverse-risk molecular lesions (TP53 and RUNX1 mutations), experimental gene signatures of leukemia stemness (LSC17 score; Ng et al., 2016) and poor outcome in response to standard induction chemotherapy (Fig. 1A). In independent validation cohorts of children and adults with AML, the IES score was higher at baseline in patients with primary induction failure (following a standard 2 cycles of chemotherapy) compared with complete remission, increased in post-chemotherapy BM specimens, and predicted survival with greater accuracy than the ELN cytogenetic risk classifier (Fig. 1B). In the immunotherapy setting, high IES scores at baseline defined a checkpoint blockade-unresponsive AML tumor microenvironment and correlated with significantly shorter overall survival (9.1 versus 15.56 months in patients with high and low IES scores, respectively; HR = 3.32 (95% CI = 1.19-9.25); log-rank P = 0.021; Fig. 1C). Finally, the IES-related gene set also predicted for long-term outcomes and objective responses, based on RECIST criteria, to single-agent nivolumab or pembrolizumab, or combination anti-PD-1 + anti-CTLA-4, in 106 patients with melanoma (PRJEB23709 and GSE93157 series), a tumor type known to derive durable clinical benefit from ICB (Fig. 1D). Our findings encourage the pursuit of immune senescence reversal as a strategy to functionally reinvigorate T cells and could inform the delivery of ICB and other T cell-targeting immunotherapies to patients who are likely to benefit. Figure 1 Figure 1. Disclosures Radojcic: Syndax Pharmaceuticals: Research Funding; Regeneron Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Allakos: Membership on an entity's Board of Directors or advisory committees. Minden: Astellas: Consultancy. Tasian: Aleta Biotherapeutics: Consultancy; Gilead Sciences: Research Funding; Kura Oncology: Consultancy; Incyte Corporation: Research Funding.
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- 2021
35. Single-Cell Proteogenomic Sequencing Allows Early Detection of Relapse Clone with CN-LOH at FLT3-ITD Locus from Initial Diagnosis in AML
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Hyewon Lee, Troy Ketela, TaeHyung Kim, Myung Hee Chang, Jose-Mario Capo-Chichi, Mark D. Minden, Zhaolei Zhang, Young Seok Yoo, Gurbaksh Basi, Adam C. Smith, Anne Tierens, and Dennis Dong Hwan Kim
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Genetics ,medicine.anatomical_structure ,Immunology ,Cell ,medicine ,Clone (cell biology) ,Early detection ,Locus (genetics) ,Cell Biology ,Hematology ,Biology ,Biochemistry ,Flt3 itd - Abstract
Introduction FLT3-ITD is one of the most common and clinically relevant mutations in acute myeloid leukemia (AML). A high FLT3-ITD allelic ratio (AR) (>0.5) is strongly associated with poor prognosis. FLT3-ITDs are heterogeneous mutations, varying in sizes and locations with some patients having multiple FLT3-ITDs. Unfortunately, conventional clinical techniques are often not adequate in measuring these characteristics at sufficient resolution. The current study aimed to characterize an AML case with 2 FLT3-ITDs at diagnosis, map the multi-omic evolution of AML clones, and understand genetic patterns underlying AML relapse using single-cell proteogenomic sequencing (ScPGseq). Methods Leukemic samples from a 46-year old female patient diagnosed with de novo AML obtained at initial diagnosis and relapse were archived and used in this study. Based on the 2017 ELN risk stratification, the patient was classified into a low-risk group due to normal karyotype/low FLT3-ITD AR/NPM1+. The patient achieved complete remission (CR) after induction chemotherapy in combination with midostaurin. The patient relapsed 2 months after 4 cycles of consolidation, with a CR duration of 6 months. After relapse, the disease was refractory despite reinduction and salvage therapies, even with gilteritinib (Fig A). ScPGseq was performed using the Mission Bio's AML panel and antibody oligoconjugates for 16 cell surface proteins. Raw sequencing reads were processed using Mission Bio's Tapestri pipeline and results were exported from Tapestri Insights (v3.0.2). All downstream computational and statistical analyses were performed using R and Python. Results Consistent with clinical sequencing (R 2 = 0.997) and PCR, ScPGseq identified 5 mutations in FLT3 (21bp and 39bp ITDs and D835Y), NPM1, and WT1 at diagnosis (2367 cells). At relapse, 1 FLT3-ITD (21bp) and mutations in NPM1 and WT1 were detected while the other FLT3-ITD (39bp) and FLT3 D835Y were absent (2611 cells). Clonal analyses of mutation patterns identified 4 AML clones at diagnosis (C1-4) and 1 at relapse (C3R; Fig B). Two closely related clones, C3 and C3R (WT1+/NPM1+/21bp FLT3-ITD+) differed in zygosity of 21bp FLT3-ITD, where nearly all cells in C3R carries homozygous FLT3-ITD. SNP array confirmed the presence of copy neutral loss of heterozygosity (CN-LOH) in chr13q. Analysis of 16 cell surface proteins along with clonal information identified 3 clusters including one devoted to non-leukemic fractions (i.e., no mutations) (top) and another nearly exclusively consisting of mutant cells from diagnosis, which we termed "monocyte-like signature" (right bottom). The other cluster was composed of nearly all mutant cells from the relapse sample and about 1/3 of mutant cells from diagnosis, which we termed "immature myeloid cell signature" (left) (Fig C). When investigating further, C3 cells with immature myeloid cell signature (98/287 cells, 34.1%) exhibited much higher allelic burden of (mean 73% vs. 56%, adj. p < 1.1e-6) and were significantly enriched with homozygous 21bp FLT3-ITDs compared to C3 cells with monocyte-like signature (189/287 cells, 65.9%) (41/98 cells, 42% vs. 11/189 cells, 5.8%, adj. p = 2.7e-13). No other mutations showed similar patterns. It indicates that C3R existed from the initial diagnosis at low frequency, instead of losing heterozygosity during relapse. Without the single cell proteogenomic analyses, C3R cells, present in The multi-omic information elucidates the complete clonal history of this AML (Fig D). Starting with WT1 and NPM1 mutations (C1), 3 FLT3 mutations (2 ITDs and D835Y) were subsequently acquired in 3 subclones (C2-4). A subset of C3 cells (WT1+/NPM1+/21bp FLT3-ITD+) further gained CN-LOH in chr13q (C3R at diagnosis). When treated, AML cells without homozygous 21bp FLT3-ITD were cleared. Cells with homozygous 21bp FLT3-ITD survived/escaped from the treatment and became the dominant clone at relapse (C3R at relapse). Conclusion The current study demonstrates that ScPGseq allows 1) simultaneous and comprehensive analyses of multiple FLT3-ITDs at the single-cell level 2) early detection of relapse clone with subclonal homozygous 21bp FLT3-ITD from the initial diagnosis, which explains one of the mechanisms of relapse in AML cases with low FLT3-ITD AR 3) multi-omic clonal analyses, which further refine clonal models relying only on mutation profiles. Figure 1 Figure 1. Disclosures Minden: Astellas: Consultancy. Kim: Pfizer: Honoraria, Research Funding; Bristol-Meier Squibb: Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Paladin: Honoraria, Research Funding.
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- 2021
36. Nicotinamide Phosphoribosyltransferase Inhibitors Induce Apoptosis of AML Stem Cells through Dysregulation of Lipid Metabolism
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Jean C.Y. Wang, Mark D. Minden, Severine Cathelin, Veronique Voisin, Qiang Liu, Amit Subedi, Changjiang Xu, Gary D. Bader, John E. Dick, David Sharon, Eric R. Lechman, Angelo D'Alessandro, and Steven M. Chan
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Chemistry ,Immunology ,Nicotinamide phosphoribosyltransferase ,Cell Biology ,Hematology ,CD38 ,Biochemistry ,Sterol regulatory element-binding protein ,chemistry.chemical_compound ,Downregulation and upregulation ,Apoptosis ,Cancer research ,Cytotoxic T cell ,NAD+ kinase ,Stem cell - Abstract
Current chemotherapeutic regimens for acute myeloid leukemia (AML) often fail to eliminate leukemic stem cells (LSCs) which contribute to disease relapse. A key step towards the development of more effective therapies is the identification of vulnerabilities that are unique to LSCs. Here, we sought to identify LSC-specific metabolic dependencies by performing a flow cytometry-based screen of 110 metabolically-focused drugs against a primary human AML sample. This sample harbored distinct subsets defined by CD34 and CD38 expression, and LSC activity assayed by xenotransplantation was restricted to the CD34+CD38- fraction. Through this screen, we found that inhibitors of nicotinamide phosphoribosyltransferase (NAMPT), which catalyzes the rate-limiting step in the NAD+ salvage pathway, preferentially depleted CD34+CD38- cells, implicating NAMPT inhibitors as potential anti-LSC agents. To evaluate the therapeutic potential of NAMPT inhibitors, we focused on KPT-9274, a small-molecule NAMPT inhibitor currently under clinical development for other cancer types. Treatment with KPT-9274 depleted the CD34+CD38- fraction across multiple primary human AML samples through induction of apoptosis. The preferential sensitivity of CD34+CD38- cells to NAMPT inhibition correlated with a lower basal level of intracellular NAD+ and greater dependency on NAMPT activity for NAD+ generation relative to the other fractions. In contrast, normal CD34+ HSPCs were largely resistant to the cytotoxic effects of KPT-9274 due to their capacity to utilize the Preiss-Handler pathway for NAD+ generation. Consistent with the in vitro findings, KPT-9274 treatment significantly reduced LSC activity as determined by secondary engraftment potential in 2 of 3 patient-derived xenograft (PDX) models of human AML and had minimal impact on normal HSC activity in mice engrafted cord blood cells. To gain mechanistic insights into how NAMPT inhibition induces cell death, we performed transcriptomic analysis of sorted CD34+CD38- cells treated with KPT-9274. This analysis revealed a striking upregulation of genes involved in cholesterol and lipid synthesis including the stearoyl-CoA desaturase (SCD) gene. The upregulated genes were highly enriched for known targets of the sterol regulatory element binding protein (SREBP) transcription factors. Functional studies demonstrated that this transcriptional response was protective against the cytotoxic effect of NAMPT inhibition in AML cells. To uncover the metabolic basis of this protective effect, we performed global metabolomic profiling of AML cells treated with KPT-9274 and observed a decrease in the ratio of monounsaturated fatty acids (MUFAs) to saturated fatty acids (SFAs) upon drug treatment. This drop in MUFA:SFA ratio reflected a reduction in SCD activity which catalyzes the desaturation of SFAs to MUFAs in a NADPH-dependent reaction. Since depletion of intracellular MUFAs could trigger apoptosis, we hypothesized that the SREBP response might protect against cell death through upregulation of SCD activity and consequent increase in MUFA synthesis. In line with this hypothesis, we found that exogenous oleic acid, a MUFA, completely rescued cell death induced by KPT-9274, while treatment with SCD inhibitors sensitized AML cells to the cytotoxic effects of NAMPT inhibition. To explore the translational application of our findings, we tested whether dipyridamole (DP), a clinically approved anti-platelet agent with inhibitory activity against SREBP signaling, can be repurposed to enhance the anti-leukemic effects of KPT-9274. We showed that treatment with DP, at non-toxic concentrations, potentiated the cytotoxicity of KPT-9274 against AML cells in vitro. Importantly, in vivo combination treatment with KPT-9274 and DP effectively targeted LSC activity in a PDX model that was refractory to KPT-9274 as single agent. In summary, our findings demonstrate that LSCs are preferentially dependent on NAMPT activity for survival over non-LSCs and normal HSCs. We further uncovered that NAMPT inhibition results in dysregulation of lipid homeostasis and induces a lipogenic response coordinated by SREBPs that protects AML cells against NAD+ depletion. These findings offer insights into drug combination strategies to enhance the efficacy of NAMPT inhibitors and provide the rationale for testing NAMPT inhibitors in the treatment of AML in clinical trials. Disclosures Dick: Bristol-Myers Squibb/Celgene: Research Funding. Wang:Trilium therapeutics: Patents & Royalties: There is an existing license agreement between TTI and University Health Network and J.C.Y.W. may be entitled to receive financial benefits further to this license and in accordance with UHN's intellectual property policies. .
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- 2020
37. Preliminary Results from a Phase 1 Study of Cfi-400495, a PLK4 Inhibitor, in Patients with Acute Myeloid Leukemia and High Risk MDS
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Caroline J McNamara, Mark R. Bray, Dina Khalaf, Graham C. Fletcher, Tracy Murphy, Dawn Maze, Mark D. Minden, Andre C. Schuh, Steven M. Chan, Debbie Valiquette, Aaron D. Schimmer, Hassan Sibai, Vikas Gupta, Karen W.L. Yee, Kylie Martin, and Brian Leber
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education.field_of_study ,medicine.medical_specialty ,medicine.diagnostic_test ,business.industry ,education ,Immunology ,Population ,Phases of clinical research ,Myeloid leukemia ,Decitabine ,Induction chemotherapy ,Cell Biology ,Hematology ,Biochemistry ,Bone marrow examination ,Tolerability ,Internal medicine ,Medicine ,In patient ,business ,health care economics and organizations ,medicine.drug - Abstract
Introduction: CFI-400945 is a first-in-class, potent, selective, orally active inhibitor of Polo-like kinase 4 (PLK4) (Ki=0.26nM), a master regulator of centriole duplication, necessary for genomic integrity (Mason et al. Cancer Cell 2014; 26:163-76). CFI-400945 has activity in leukemia cell lines and primary leukemia samples including those with complex karyotype, inversion 3 and monosomy 7 (Minden. personal communications). This suggests that CFI-400945 may provide an effective treatment of patients with AML. The objectives of this phase 1 trial was to establish the safety, tolerability, and recommend phase II dose (RP2D) of CFI-400945 in patients with acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Methods: Patients with relapsed/refractory AML or MDS and patients with untreated AML who refused induction chemotherapy or who are not candidates for intensive chemotherapy were eligible. Dose escalation followed a standard 3+3 design with a starting dose of 64 mg orally once daily. Plasma levels of CFI-400945 free base were measured on Days 1, 2, & 29 of Cycle 1 and Day 15 on all subsequent cycles. Peripheral blood and/or bone marrow were obtained at baseline, Day 8 of Cycle 1 and Day 1 of each subsequent cycle prior to dosing for pharmacodynamic monitoring. Safety assessments using the NCI CTCAE version 4.03 were performed. Results: From May 2018 to June 2019, nine patients have been enrolled on study across three pre-defined dose levels (64 mg [n=3], 96 mg [n=4], and 128 mg [n=2]). Three patients had untreated AML, five patients had relapsed/refractory AML and one patient had myelodysplastic syndrome/myeloproliferative disorder (MDS/MPN). Patient characteristics at diagnosis are outlined in Table 1. Six (67%) patients had baseline high throughput sequencing; the most frequent mutations were TP53 (33%), TET2 (33%), KRAS (33%) and DNMT3A (33%). A total of 20 cycles were administered with a median of 1 cycle (range, 0 to 7 cycles). The most common non-hematological drug related toxicities of any grade, which occurred in over 20%, were diarrhea (44%), headache (44%), colitis (33%), vomiting (33%), bilirubin increase (22%), dizziness (22%), fatigue (22%), and nausea (22%). One patient on the 96 mg dose level was not evaluable for DLT and hence, replaced. Both patients treated at the 128 mg/day dose level developed DLTs, consisting of grade 3 colitis and grade 5 sepsis and colitis. Pharmacokinetic profile indicated low interpatient variability between patients. Maximum exposure did not correlate with toxicity Six patients were evaluable for disease response. Two (33%) achieved complete remission (CR), 3 pts (50%) had stable disease (with one patient having a 78% reduction in marrow blast count). The patient with MDS/MPN who did not complete 1 cycle of therapy progressed to AML (Figure 1). Both patients who obtained a CR had an early response within 2 cycles. One CR has been durable for 218 days with no measurable residual disease (MRD) by flow cytometry. The additional patient, who obtained a CR with incomplete platelets recovery, with subsequent best response of CR, had a sustained response for 91 days before relapse was confirmed by bone marrow examination (Figure 1). Conclusion: Single agent CFI-400945 has activity in patients with poor risk AML. The RP2D in this population is 96 mg once daily. Dose expansion is occurring at the RP2D level. A phase 2 study with CFI-400945 single agent or in combination study with azacitidine or decitabine is planned. Disclosures Leber: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Alexion: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Lundbeck: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda/Palladin: Honoraria, Membership on an entity's Board of Directors or advisory committees; Treadwell: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS/Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Otsuka Pharmaceutical: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Bray:Treadwell Therapeutics: Current Employment; TIO Discovery: Current Employment. Gupta:Pfizer: Consultancy; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sierra Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol MyersSquibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Research Funding. Maze:Novartis: Honoraria; Pfizer: Consultancy; Takeda: Research Funding. McNamara:Novartis: Honoraria. Schimmer:Jazz: Honoraria; Otsuka: Honoraria; Medivir AB: Research Funding; AbbVie Pharmaceuticals: Other: owns stock ; Takeda: Honoraria, Research Funding; Novartis: Honoraria.
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- 2020
38. Prognostic Role of Multiparameter Flow Cytometry-Based Measurable Residual Disease Assessment in Patients with Acute Myeloid Leukemia Harboring DNMT3A/TET2/ASXL1 Mutation
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Vikas Gupta, Georgina S. Daher-Reyes, Hassan Sibai, Aaron D. Schimmer, Caroline J McNamara, Anne Tierens, Dawn Maze, Mark D. Minden, Muhned Alhumaid, Steven M. Chan, Andre C. Schuh, Karen W.L. Yee, Dennis Dong Hwan Kim, Tracy Murphy, and Igor Novitzky-Basso
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Oncology ,medicine.medical_specialty ,education.field_of_study ,Multivariate analysis ,Proportional hazards model ,business.industry ,Immunology ,Population ,Induction chemotherapy ,Myeloid leukemia ,Cell Biology ,Hematology ,medicine.disease ,Lower risk ,Biochemistry ,Hematologic disease ,Internal medicine ,medicine ,Cumulative incidence ,education ,business - Abstract
BACKGROUND: Multiparameter flow cytometry (MFC) has increasingly been used for measurable residual disease (MRD) assessment in patients with acute myeloid leukemia (AML), while next-generation sequencing (NGS)-based MRD monitoring tool is in clinical development for its application. Clonal hematopoiesis (CH), in which leukemia-associated somatic mutations gene are present in individuals with no apparent hematologic disease, adds a challenge in the detection of MRD. In patients with AML, CH could be potentially pre-leukemic, while persistent mutations in DNMT3A, TET2 orASXL1 (DTA) in remission marrow are usually removed from the analysis of residual leukemic cells. However, reports suggest that persistent DTA mutations in remission may be correlated with an increased relapse risk. In the patients with DTA mutations, the use of NGS for MRD monitoring is limited or modified due to the presence of CH clone in the remission marrow. We evaluated whether MFC-MRD can be adjunctive to predict the risk of AML relapse in this population of 221 patients with DTA mutation (DNMT3A (n=123), ASXL1 (n=56) or TET2 (n=100). METHODS: The present study evaluated long-term outcomes in AML patients who achieved first complete remission (CR1) and compared outcomes according to MFC-based MRD status (was defined as negative if patients achieved 0.1 or less) assessed at the time of CR1. A total of 435 patients diagnosed with AML and treated with induction chemotherapy between 2015 and 2018 were included. MFC-MRD was assessed in 336 patients in CR1 (77%). NGS was performed using samples obtained at the time of initial diagnosis and used for mutational subgroup classification. Overall survival (OS) was calculated as the date of CR1 to the date of death and censored on the date of the last follow-up. Relapse-free survival (RFS) was defined as the time from the date of CR1 to the date of relapse or death from any cause. Cumulative incidence of relapse (CIR) and non-relapse mortality (NRM) were calculated considering competing risk. The Kaplan-Meier method using a log-rank test and a multivariate Cox proportional hazard model was used for analyses of time-to-event endpoints. For CIR and NRM, Gray test was performed for the risk factors and the Fine-Gray model was adopted for the multivariate model. RESULTS: According to the MFC-MRD status, i.e., the group with positive MRD (MRDpos; n=118, 35%) vs. those with negative MRD (MRDneg; n=218, 65%), we evaluated OS, RFS, and CIR. The MFC-MRDneg group showed better OS at 2 years 67.0% than the MFC-MRDpos group 40.7% (p We divided the groups according to the number of induction treatment courses, AML type, cytogenetics risk, and age ( Also, we evaluated MFC-MRD status at CR by mutational profile subgroup. Long-term outcomes such as OS, RFS, CIR or NRM were compared by the mutational subgroup. It consistently showed a trend of superior OS, RFS and lower risk of CIR in patients with MFC-MRDneg compared to MFC-MRDposTab1. Of interest, in the subgroup of patients carrying any DTA mutations (n=221), those with MFC-MRDneg (n=103) showed better OS (HR 1.61 [1.01-2.55%]; p=0.042), RFS (HR 1.66 [1.06-2.61%]; p=0.026) and CIR (HR 1.99[1.03-3.83%]; p=0.04) compared to those MFC-MRDpos (n=64; Fig 1). Multivariate analysis confirmed that the MFC-MRDneg is an independent prognostic factor in patients with DTAmutwith respect to OS: MFC-MRDpos (HR 1.63, p=0.04) and age (≥60; HR 2.04, p=0.008) for OS; for RFS, MFC-MRDpos (HR 1.71, p=0.02) and age (≥60; HR 2.32, p= 0.001); for CIR, MFC-MRDpos (HR 2.31, p=0.01) and HCT (HR 0.14, p= Conclusion: These findings suggest that in AML patients with DTAmut, MFC-MRD status at the time of remission assessment can be a tool for MRD assessment when NGS-based MRD assessment is limited. Further study is strongly warranted to reach a clearer conclusion with multiple cohorts. Disclosures Schimmer: Takeda: Honoraria, Research Funding; Novartis: Honoraria; Jazz: Honoraria; Otsuka: Honoraria; Medivir AB: Research Funding; AbbVie Pharmaceuticals: Other: owns stock . Tierens:Amgen: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Astellas Pharma: Membership on an entity's Board of Directors or advisory committees. McNamara:Novartis: Honoraria. Maze:Pfizer: Consultancy; Novartis: Honoraria; Takeda: Research Funding. Gupta:Pfizer: Consultancy; Bristol MyersSquibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sierra Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Research Funding.
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- 2020
39. Elevated Expression of Mir-130a in t(8,21) AML Reinforces the Aberrant Molecular Program of AML1-ETO
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Eric R. Lechman, Leonardo Salmena, Veronique Voisin, Schoof E Erwin, Jessica McLeod, Mark D. Minden, Sajid A. Marhon, Gabriela Krivdova, John E. Dick, and Martino Gabra
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Mir 130a ,Immunology ,Cancer research ,Cell Biology ,Hematology ,Biology ,Biochemistry ,Aml1 eto - Abstract
Background: Deregulation of self-renewal and differentiation programs are central to the pathogenesis of hematologic malignancies. MicroRNAs (miRNAs) represent a large class of post-transcriptional regulators that mediate repression of multiple target mRNAs and are frequently deregulated in acute myeloid leukemia (AML). From our previous in vivo miRNA enforced expression screen in human hematopoietic stem and progenitor cells (HSPC), we identified miR-130a as a regulator of self-renewal and lineage specification. Enforced expression of miR-130a in human cord blood (CB) derived HSPC caused an expansion of HSC, block in erythroid differentiation and abnormal myelopoiesis in xenografts. Thus, we examined miR-130a expression in AML and found miR-130a to be specifically upregulated in t(8,21) AML. The translocation t(8,21) is one of the most common karyotypic abnormalities in AML, accounting up to 10% of all AML cases. The consequence of this translocation is a fusion of AML1 and ETO genes, resulting in a formation of the AML1-ETO (AE) oncofusion protein, which acts as a dominant repressor of the wild type AML1/RUNX1. The ETO moiety mediates the recruitment of the nuclear corepressor (NCoR) and histone deacetylases (HDAC1-3) to block RUNX1 target gene expression. This prevents myeloid maturation, apoptosis and promotes leukemogenesis. Here we investigated the molecular mechanism of miR-130a in t(8,21) AML and how it contributes to the leukemogenesis of this AML subtype. Results: Using the TCGA dataset and our PMCC patient cohort, we identified miR-130a to be upregulated in t(8,21) AML and high miR-130a expression was associated with worse patient overall survival. To interrogate the functional significance of elevated miR-130a in t(8,21) AML, we performed knock-down (KD) experiments in the Kasumi-1 cell line, which represents a well characterized model system for t(8,21) AML. Notably, KD of miR-130a induced a significant reduction in the CD34+ cell population and an increase in differentiated CD11b+CD15+ and pro-apoptotic annexin V+ cells. We next examined the impact of miR-130a KD in CD34+ blasts from primary t(8,21) AML patient samples. In line with our findings in the Kasumi-1 cells, miR-130a KD decreased the proportion of CD34+ cells and increased the proportion of differentiated CD11b+CD15+ blasts. To investigate the effect of miR-130a KD on leukemic engraftment in vivo, we transduced CD34+ blasts from 2 patient samples and transplanted them into NSG-GF mice. miR-130a KD decreased leukemic engraftment and the proportion of transduced cells, corroborating the functional significance of high miR-130a expression in t(8,21) AML. To investigate the mechanistic action of miR-130a, we performed label-free semi-quantitative proteomics in human CB derived HSPC to uncover miR-130a targets. Surprisingly, we found the beta subunit of RUNX1, CBFb, and Transducin Beta Like 1 X-Linked Receptor 1, TBL1XR1, to be among the most repressed targets. TBL1XR1 is a component of the nuclear receptor corepressor (NCoR) complex and is involved in NCoR degradation. Thus, we performed western and immunoprecipitations (IP) assays in Flag-AE Kasumi-1 cells following miR-130a KD to examine changes in the expression of proteins associated with the AE complex. We observed increased expression of CBFβ, TBL1XR1 and CEBPA with miR-130a KD. Notably, miR-130a KD resulted in a dramatic decrease of NCoR protein levels. IP of Flag-AE showed decreased association of CBFβ and NCoR with AE, despite unaltered protein levels of AE. To investigate changes in binding occupancy of Flag-AE after miR-130a KD, we performed Cleavage Under the Targets and Release Using Nuclease (CUT&RUN) assay. Surprisingly, we observed 2-fold gain of AE sites in miR-130a KD sample compared to control. De novo motif enrichment analysis showed loss of motives for ETS and HOX transcription factors known to associate with AE following miR-130a KD. Genomic distribution of the peaks revealed a dramatic shift of AE peaks away from the promoter region to introns in miR-130a KD. Pathway enrichment analysis of the unique peaks gained in miR-130a KD showed stress responses and organelle disassembly, in line with the differentiation phenotype observed with miR-130a KD. Collectively, we uncovered a novel mechanism by which miR-130a reinforces the aberrant AE molecular program by controlling the composition and binding of the AE complex. Disclosures Dick: Bristol-Myers Squibb/Celgene: Research Funding.
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- 2020
40. The Metabolic Enzyme Hexokinase 2 Localizes to the Nucleus in AML and Normal Hematopoietic Stem/Progenitor Cells to Maintain Stemness
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Geethu Emily Thomas, Grace Egan, Laura Garcia Prat, Botham Aaron, Veronique Voisin, Elias Orouji, Jordan M Chin, Boaz Nachmias, Kerstin B Kaufmann, Fieke W Hoff, Neil Maclean, Rose Hurren, Xiaoming Wang, Marcela Gronda, Andrea Arruda, Mark D. Minden, Gary D Bader, Steven M. Kornblau, John E. Dick, and Aaron D Schimmer
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Immunology ,Cell Biology ,Hematology ,Biochemistry - Abstract
Mitochondrial metabolites affect epigenetic marks, but it is largely unknown whether mitochondrial metabolic enzymes can directly localize to the nucleus to regulate stem cell function in AML. Here, we discovered that the mitochondrial enzyme, Hexokinase 2 (HK2), localizes to the nucleus in AML and normal hematopoietic stem cells to maintain stem cell function. We searched for mitochondrial enzymes moonlighting in the nucleus using 8227 AML cells, a low passage primary AML culture model arranged in a hierarchy with functionally defined stem cells in the CD34+CD38-fraction. By immunoblotting and confocal microscopy, we detected HK2 in the nucleus of 8227 cells with higher expression in the nucleus of stem cells vs bulk cells. HK2 is the first and rate-limiting enzyme in glycolysis and phosphorylates glucose. In contrast, other metabolic enzymes including phosphofructokinase, fumarase, pyruvate kinase 2, glucose phosphate isomerase, enolase1, citrate synthase, aconitase 2, and succinate dehydrogenase were not detected in the nucleus of these cells. We also detected HK2, but not these other metabolic enzymes, in the nucleus of OCI-AML2, U937, NB4 and TEX leukemia as well as 8 of 9 primary AML samples. Next, we tested whether nuclear HK2 was functionally important to maintain stem cell function in AML. We over-expressed HK2 tagged with nuclear localizing signals (PKKKRKV and PAAKRVKLD) in 8227 and NB4 leukemia cells. We confirmed selective over-expression of HK2 in the nucleus of these cells without increasing levels in the cytoplasm or mitochondria. Over-expression of nuclear HK2 increased clonogenic growth and inhibited retinoic acid-mediated cell differentiation without changing basal proliferation. Over expression of HK2 also increased engraftment of 8227 cells into mouse marrow. We evaluated the selective inhibition of nuclear HK2 by over-expressing HK2 with an outer mitochondrial localization signal while knocking down total endogenous HK2 with shRNA targeting the 3'UTR of HK2. Selective depletion of nuclear HK2 reduced clonogenic growth, increased AML differentiation after treatment with retinoic, and decreased the percentage of CD34+CD38- 8227 stem cells without changing basal proliferation. To determine whether nuclear HK2 maintains stemness through its kinase activity, we over-expressed a kinase dead double mutant of nuclear HK2(D209A D657A). Nuclear kinase dead HK2 increased clonogenic growth and inhibited differentiation after retinoic acid treatment, demonstrating that HK2 maintains stemness independent of its kinase function. To understand nuclear functions of HK2, we used proximity-dependent biotin labeling (BioID) and mass spectrometry to identify proteins that interact with nuclear HK2 and identified proteins related to chromatin organization and regulation. Therefore, we examined the impact of nuclear HK2 on chromatin accessibility using ATAC-seq. Over expression of nuclear HK2 enhanced chromatin accessibility, whereas the selective knockdown of nuclear HK2 compacted chromatin. In summary, we discovered that HK2 localizes to nucleus of AML cells and functions independent of its kinase activity to maintain the stem/progenitor state of AML. Thus, we define a new role for mitochondrial enzymes in the regulation of leukemic stemness and differentiation. Disclosures Dick: Bristol-Myers Squibb/Celgene: Research Funding. Schimmer:Takeda: Honoraria, Research Funding; Novartis: Honoraria; Jazz: Honoraria; Otsuka: Honoraria; Medivir AB: Research Funding; AbbVie Pharmaceuticals: Other: owns stock .
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- 2020
41. Inferior Outcomes with a High LSC17 Score Can be Improved with Flag-IDA
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Narmin Ibrahimova, Steven M. Chan, Fiona Ferrera, Caroline J McNamara, Zhibin Lu, Jean C.Y. Wang, Vikas Gupta, Mitchell Sabloff, Dina Khalaf, Ian King, Andrea Arruda, Karen W.L. Yee, Tracy Murphy, Mark D. Minden, Jaime O. Claudio, Brian Leber, Tracy Stockley, Natalie Stickle, Stanley W.K. Ng, Hassan Sibai, Chantal Rockwell, Aaron D. Schimmer, Dawn Maze, Tong Zhang, Kristele Pan, Carl Virtanen, Andre C. Schuh, and Anne Tierens
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medicine.medical_specialty ,business.industry ,Immunology ,Induction chemotherapy ,Cell Biology ,Hematology ,Biochemistry ,New diagnosis ,Test (assessment) ,Molecular analysis ,Family medicine ,Cancer centre ,Medicine ,FLAG (chemotherapy) ,In patient ,Treatment resistance ,business ,health care economics and organizations - Abstract
Introduction: Acute myeloid leukemia (AML) is driven by a subpopulation of leukemia stem cells (LSCs), which possess properties such as quiescence and self-renewal that are linked to therapy resistance and relapse. The LSC17 score was derived from genes differentially expressed between functionally validated LSC+ and LSC- fractions from 78 AML patients and is strongly associated with survival and response to standard therapy. A critical advantage of the LSC17 test over cytogenetic and molecular analysis is its rapid turnaround time (24-48h on a NanoString platform), providing clinicians with a rapid and powerful tool for upfront risk stratification. We have developed a clinical assay for the LSC17 score validated in a CAP/CLIA-lab setting. Methods: We conducted a prospective, multicenter validation and feasibility study to test the prognostic value of the LSC17 assay under real-world conditions in AML patients treated with curative intent. Patients with a possible new diagnosis of AML were eligible. Patients with a confirmed diagnosis of acute promyelocytic leukemia were excluded from analysis. Standard prognostic markers including cytogenetics, molecular studies and targeted sequencing using a standard AML panel were performed in parallel to the LSC17 score. Treatment was administered according to physician preference, based on patient history and results of standard prognostic assays, when available. Survival data was censored on June 14th, 2020. Results: 381 patients were recruited to the study between June 2016 and March 2020. 4 patients were excluded for quality control reasons (one sample had insufficient RNA and three samples failed quality control checks). 103 were excluded as they had alternative diagnoses. 84 patients were excluded because they did not receive intensive chemotherapy. LSC17 scores ranged from 0 to 1.25, and were classified as high or low according to the median score of 0.51 from a previously validated reference cohort (Ng et al, Nature 2016). Of the 190 patients included in this analysis, 84 had a low LSC17 score and 106 had a high LSC17 score. The median age was 61 years (range 18-79); 86 (45%) were female. When stratified according to ELN 2017 criteria, 48 (27%), 51 (29%), and 77 (44%) patients had favorable, intermediate, and adverse risk disease, respectively. Low LSC17 score was associated with normal cytogenetics (high vs low, 33% vs 58%; P We first considered response to induction chemotherapy (Table 1). 141 patients had standard induction chemotherapy with 3+7, 40 had Flag-IDA and 9 had CPX-351. High score patients had inferior responses to 3+7 with only 59% achieving complete remission (CR) after 1 cycle of chemotherapy compared to 96% of low score patients; responses for LSC17 high score patients were better in the Flag-IDA group with 80% achieving CR after 1 cycle. When considering overall CR rates after 2 cycles of induction, patients with a high LSC17 score were less likely to achieve CR (high vs low, 87% vs 98%; P=0.02). However, this difference was predominantly observed in patients treated with 3+7 (87% vs 99% CR rate in high vs low score patients, respectively); response rates to Flag-IDA were not significantly different between the 2 groups. Measurable residual disease (MRD) monitoring by flow cytometry was performed at the time of CR in 135 (71%) patients enrolled at Princess Margaret Cancer Centre. Patients with a high LSC17 score were significantly more likely to have MRD compared to low score patients (46% vs 10% respectively, P Conclusion: AML patients with a high LSC17 score have inferior outcomes following 3+7 induction chemotherapy. The LSC17 score should be considered as a tool to identify and stratify high-risk patients to alternative upfront therapies such as Flag-IDA. A risk adapted study is planned to validate these results. Disclosures Gupta: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sierra Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy; Bristol MyersSquibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Research Funding. Maze:Novartis: Honoraria; Takeda: Research Funding; Pfizer: Consultancy. McNamara:Novartis: Honoraria. Schimmer:Medivir AB: Research Funding; AbbVie Pharmaceuticals: Other: owns stock ; Takeda: Honoraria, Research Funding; Novartis: Honoraria; Jazz: Honoraria; Otsuka: Honoraria. Leber:Takeda/Palladin: Honoraria, Membership on an entity's Board of Directors or advisory committees; Treadwell: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BMS/Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Otsuka Pharmaceutical: Honoraria, Membership on an entity's Board of Directors or advisory committees; Lundbeck: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Alexion: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Tierens:Amgen: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Astellas Pharma: Membership on an entity's Board of Directors or advisory committees. Wang:Trilium therapeutics: Patents & Royalties: There is an existing license agreement between TTI and University Health Network and J.C.Y.W. may be entitled to receive financial benefits further to this license and in accordance with UHN's intellectual property policies. .
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- 2020
42. CPX351 Has Short Remission Duration but Is an Effective Bridge to Allogeneic Transplant in High Risk AML: Results from Canadian Real-World Multi-Centre Study
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David Sanford, Aaron D. Schimmer, Caroline J McNamara, Tracy Murphy, Hassan Sibai, Dina Khalaf, Steven M. Chan, Mark D. Minden, Vikas Gupta, Signy Chow, Claire Andrews, Taylor Young, Dennis Dong Hwan Kim, Dawn Maze, Sarit Assouline, Eshetu G. Atenafu, Andre C. Schuh, Karen W.L. Yee, and Joseph Brandwein
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medicine.medical_specialty ,education.field_of_study ,Poor prognosis ,business.industry ,Immunology ,Population ,Cell Biology ,Hematology ,Biochemistry ,Transplantation ,chemistry.chemical_compound ,chemistry ,Median follow-up ,Internal medicine ,Remission duration ,medicine ,Progression-free survival ,Midostaurin ,Multi centre ,business ,education - Abstract
Background and objectives: CPX-351 is a liposomal formulation of daunorubicin and cytarabine in a fixed synergistic ratio of 5:1. It has been approved by both the FDA and EMA for use in high risk AML including therapy related AML (t-AML) and AML with myelodysplastic related change (AML-MRC). When compared with '3+7', CPX-351 resulted in superior OS (9.56 vs 5.96 months) and overall response rate (47.7% v 33.3%; P = .016; Lancet JCO 2018). However, this is little data of CPX-351 in a real world setting. The primary objective of our multi-centre study was to assess outcomes and associated toxicities of CPX-351 in these high risk AML patients. Methods: Patients aged 18 and over who were treated with CPX-351 and who met the WHO criteria for t-AML and AML-MRC were included in the study. Retrospective data was collected from 6 centres throughout Canada. Targeted sequencing was performed on DNA samples using the TruSight Myeloid Sequencing Panel isolated from peripheral blood or bone marrow samples at diagnosis. Overall survival (OS) and progression free survival (PFS) rates were calculated using the Kaplan-Meier method. Results: Fifty patients treated with CPX-351 were identified from six centres. Baseline characteristics are seen in Table 1. Cytogenetics was available in 45 (90%) patients and an abnormal karyotype was seen in 30 (60%) patients. Monosomal (33%) and complex (28%) karyotype were the most common chromosomal abnormalities seen. Targeted sequencing was performed on 64% patients (32/50) and the average number of mutations was 2(0-7). RUNX1 was the most commonly mutated gene found in 22% (7/32), followed by SRSF2 in 19% (6/32) with ASXL1 and NRAS both at 16% (5/32). Other commonly mutated genes were NPM1, IDH2, DNMT3a and TP53 all occurred at a frequency of 12% (4/32). Of the 10 patients (20%) that were FLT3 mutated (80% ITD and 20% TKD), 5 patients received the FLT3 inhibitor midostaurin in combination with CPX-351. Assessing treatment responses, 50% (25/50) of patients achieved a complete remission (CR) or a complete remission with incomplete recovery (CRi). Median neutrophil recovery (≥500/μl) was 32 days (range 18-68) and median platelet recovery (≥50 000/μl) was 34 days (range 19-70). Proven or probable fungal infection was seen in 10 (20%) patients. 30 day mortality was 4% (2/50) and 60-day mortality was 10% (5/50). The median CR duration was short at 7.17 months. Of the 25 patients who did not achieve a CR, 6 (24%) had prior azacitidine use for MDS suggesting CPX-351 may not have activity in this group of patients. Median follow up was 7.43 months (range 0.2 to 18 months). OS was 44% at 12 months (0.28-0.58 95% CI) and 29% at 18 months (Fig 1; 0.13-0.46 95% CI). PFS was 28% at 12 months (0.15-0.43 95% CI) and 18% at 18 months (0.06-0.39 95% CI). There were no differences in OS when stratified by ELN risk (p=0.25), adverse risk cytogenetics (p=0.1485), or poor risk mutations such as FLT3-ITD (p=0.29), RUNX1 (p=0.123) or ASXL1 (p=0.06). This is consistent with previous studies, which suggest that CPX-351 overcomes the poor prognosis associated with these mutations. 18 (36%) patients received an allogeneic stem cell transplant (ASCT) in CR1. Patients who received a allogeneic transplant had significant improvement of OS of 62% at 18 months compared to those who did not receive a transplant of 14.5% at 18 months (Fig 2; p=0.0008). Conclusion Our study reveals that CPX-351 produces high remission rates in patients with AML with a similar efficacy and toxicity profile seen in the Phase 3 trial data (Lancet JCO 2018). However, response rates are short and therefore CPX-351 is most effective when used as a bridge for allogeneic stem cell transplantation in this high risk population. Disclosures Assouline: AbbVie: Consultancy, Honoraria, Speakers Bureau; Pfizer: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Speakers Bureau; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria, Speakers Bureau; Takeda: Research Funding; BeiGene: Consultancy, Honoraria, Research Funding. Brandwein:Celgene: Honoraria; Astellas: Honoraria; Taiho: Honoraria; Roche: Honoraria; Jazz Pharmaceuticals: Honoraria; Pfizer: Honoraria; Amgen: Honoraria. Gupta:Sierra Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Honoraria, Research Funding; Bristol MyersSquibb: Honoraria, Membership on an entity's Board of Directors or advisory committees. Maze:Pfizer: Consultancy; Takeda: Research Funding; Novartis: Honoraria. McNamara:Novartis: Honoraria. Sanford:Astellas: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees. Schimmer:Medivir AB: Research Funding; AbbVie Pharmaceuticals: Other: owns stock ; Takeda: Honoraria, Research Funding; Novartis: Honoraria; Jazz: Honoraria; Otsuka: Honoraria.
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- 2020
43. A Phase 2 Open-Label, Multicenter, Dose Optimization Clinical Study of the Safety, Tolerability, and Pharmacokinetic (PK) and Pharmacodynamic (PD) Profiles of Cfi-400945 As a Single Agent or in Combination with Azacitidine or Decitabine in Patients with Acute Myeloid Leukemia
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Emily Elizabeth Roberts-Thomson, Brian A. Jonas, Mark D. Minden, Tracy Murphy, Mark R. Bray, Gautam Borthakur, Dale L. Bixby, Glenn C. Michelson, Karen W.L. Yee, and Joseph Brandwein
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Oncology ,medicine.medical_specialty ,business.industry ,Immunology ,Azacitidine ,Myeloid leukemia ,Decitabine ,Cell Biology ,Hematology ,Biochemistry ,Clinical study ,Pharmacokinetics ,Internal medicine ,Pharmacodynamics ,Medicine ,In patient ,Open label ,business ,medicine.drug - Abstract
Background: CFI-400945 is a potent, selective, orally administered, first-in-class inhibitor of the serine/threonine kinase, Polo-like kinase 4 (PLK4). PLK4 is a highly conserved master regulator of centriole duplication, and is critical for maintenance of genomic integrity. Aberrant expression of PLK4 results in a number of effects including the centrosome amplification often seen in aneuploid cancers, pointing to a potentially causative role for PLK4 in genome instability and cancer progression. A Phase 1 study has been completed evaluating CFI-400945 as a monotherapy in solid tumors, showing a tolerable safety profile and promising signs of activity. As acute myeloid leukemia (AML) is a disease characterized by genomic instability, it is of significant interest as a potential indication for the clinical evaluation of CFI-400945. pre-clinical studies, CFI-400945 showed potent activity towards leukemia cell lines and primary human samples in vitro, as well as marked efficacy in two subcutaneous models of leukemia, specifically the MV4-11 FLT3-ITD AML. A Phase 1 trial in AML was initiated at the Princess Margaret Cancer Center in 2018, and of six patients evaluable for response, two (33%) achieved complete remission (CR), and 3 patients (50%) had stable disease (with one patient having a 78% reduction in marrow blast count) [re: Murphy et al, ASH 2020]. These promising results have led to a plan for an expanded trial examining CFI-400945 in AML, particularly focused on complex karyotype (CK). Study Design and Methods: The study will have 4 parts, Part 1A (1A): a single agent dose escalation portion, Part 1B (1B): a food effect portion once the MTD of 1A is determined, and combinations with azacitidine (2A), and decitabine (2B). For parts 1A and 1B, patients with relapsed and/or refractory AML, MDS, or CMML after >1 prior therapy will be included. Patients with MDS or CMML must have progressed or had a lack of response after at least 4 cycles of hypomethylating agents. For parts 2A and 2B, patients should have relapsed and/or refractory AML or untreated MDS or CMML. Untreated patients who decline or are ineligible for intensive therapy may be included. The study will use a standard 3 + 3 design. The maximum tolerated dose (MTD) will be defined as the dose level where the number of dose limiting toxicities (DLTs) is Biomarker Selection and companion Diagnostics: No biomarker based pre-selection of patients. PD evaluations will include blast reduction and markers of mitosis. Study Treatment and Endpoints: Part 1: Each cycle will be 28 days (21 days on/7 days off). Starting does of CFI-400945 will be 32mg po. Once the MTD of 1A is determined, 1B will explore the food effect of a high fat meal on the PK of CFI-400945 at the MTD. 2A and 2B will explore the combination of CFI-400945 with standard dose of either azacitidine (2A) or decitabine (2B). The starting dose of CFI-400945 will be 32mg po for 21 days on/7 off. The DLTs will be defined as NCI CTCAE V5.0 Grade >3 related non-hematologic event(s) occurring during cycle 1 or prolonged pancytopenia in the presence of a hypocellular bone marrow > day 42 without evidence of disease. The efficacy endpoints for AML, MDS, and CMML include the overall response rate, and the CR rate per standard criteria. The aim of the food effect part of the study is the asses the effect of high fat food on the PK of CFI-400945. The safety endpoint is the incidence of treatment emergent adverse events. PK endpoints include evaluations of parameters such as half-life, AUC, etc. Exploratory endpoints include eval of minimal residual disease, genomic alterations and other molecular features associated with response and biological effects of PLK4 inhibition. Disclosures Jonas: Amgen: Consultancy, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company), Research Funding; AbbVie: Consultancy, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company), Research Funding; GlycoMimetics: Consultancy, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company), Research Funding; Sigma Tau: Research Funding; Jazz: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Takeda: Consultancy; Tolero: Consultancy; Treadwell: Consultancy; Forty Seven: Research Funding; Accelerated Medical Diagnostics: Research Funding; AROG: Research Funding; Daiichi Sankyo: Research Funding; F. Hoffmann-La Roche: Research Funding; Forma: Research Funding; Genentech/Roche: Research Funding; Hanmi: Research Funding; Incyte: Research Funding; LP Therapeutics: Research Funding; Pfizer: Research Funding; Pharmacyclics: Research Funding. Bixby:GlycoMimetics: Research Funding. Brandwein:Pfizer: Honoraria; Amgen: Honoraria; Celgene: Honoraria; Astellas: Honoraria; Taiho: Honoraria; Roche: Honoraria; Jazz Pharmaceuticals: Honoraria. Michelson:Treadwell Therapeutics: Consultancy. Bray:TIO Discovery: Current Employment; Treadwell Therapeutics: Current Employment. Roberts-Thomson:Treadwell Therapeutics: Current Employment. Borthakur:BioTherix: Consultancy; FTC Therapeutics: Consultancy; Nkarta Therapeutics: Consultancy; Treadwell Therapeutics: Consultancy; PTC Therapeutics: Consultancy; Argenx: Consultancy; Oncoceutics: Research Funding; Xbiotech USA: Research Funding; Polaris: Research Funding; AstraZeneca: Research Funding; BMS: Research Funding; BioLine Rx: Research Funding; Jannsen: Research Funding; Abbvie: Research Funding; Novartis: Research Funding; Incyte: Research Funding; BioLine Rx: Consultancy; PTC Therapeutics: Research Funding; Curio Science LLC: Consultancy; Cyclacel: Research Funding; GSK: Research Funding.
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- 2020
44. Mir-125b Regulates the Self-Renewal of Acute Myeloid Leukemia Stem Cells through PTPN18 and GSK3
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Gabriela Krivdova, Qiang Liu, Eric R. Lechman, Olga I. Gan, Jean C.Y. Wang, Shai Izraeli, Mark D. Minden, John E. Dick, Karin G. Hermans, Aaron Trotman-Grant, and Stephanie M. Dobson
- Subjects
ABL ,Myeloid ,Immunology ,Myeloid leukemia ,Tyrosine phosphorylation ,Cell Biology ,Hematology ,Protein tyrosine phosphatase ,Biology ,Biochemistry ,Haematopoiesis ,chemistry.chemical_compound ,medicine.anatomical_structure ,chemistry ,Cancer research ,medicine ,Kinase activity ,Stem cell - Abstract
Acute myeloid leukemia (AML) is an aggressive hematologic malignancy with poor survival, especially in older patients. Despite high remission rates after chemotherapy, relapse and death are frequent due to persistence of leukemia stem cells (LSCs), which possess properties linked to therapy resistance. Thus, there is an urgent need for a deeper understanding of the unique properties of LSCs. MicroRNAs (miRNAs) are non-coding RNAs that decrease expression of their target mRNAs by post-translational silencing. miRNA profiling of human AML samples fractionated based on LSC activity revealed that miR-125b is expressed at significantly higher levels on cell fractions enriched in LSCs. To evaluate the role of miR-125b in LSCs, expression of miR-125b was enforced in a hierarchical AML model cell line (OCI-AML-8227). miR-125b overexpression (OE) resulted in a significantly lower percentage of CD14+CD15+ differentiated myeloblasts (Figure 1A) and enhanced clonogenic potential in vitro (Figure 1B). Xenotransplantation of four AML patient samples with miR-125b OE revealed a significant increase in the proportion of CD117+ cells, a marker of hematopoietic and leukemic progenitors (Figure 1C). Secondary transplantation of cells harvested from primary engrafted mice at limiting dilution demonstrated a marked increase in LSC frequency with miR-125b OE compared to controls for the two AML samples tested (Figure 1D). Together, these data strongly suggest that miR-125b enhances the self-renewal of LSCs. To investigate the mechanisms by which miR-125b enhances self-renewal, proteomic analysis of miR-125b-OE Ba/F3 cells as well as in silico target prediction were performed and identified PTPN18 as a top putative target for miR-125b. PTPN18 is a tyrosine phosphatase that has been reported to dephosphorylate auto-phosphorylated kinases such as Her2 and Abl to prevent their activation. To evaluate whether PTPN18 OE can rescue the effects miR-125b on LSCs, we carried out transduction of an AML patient sample with control, miR-125b OE, PTPN18 OE, or both miR-125b and PTPN18 OE vectors followed by xenotransplantation. Similar to previous findings, miR-125b OE alone significantly reduced the frequency of CD11b+CD15+ differentiated myeloblasts. Co-transduction of miR-125b/PTPN18 OE vectors resulted in generation of significantly more CD11b+CD15+ cells compared to miR-125b OE alone (Figure 1E), suggesting that suppression of PTPN18 contributes to miR-125b-mediated enhancement of LSC self-renewal. To identify putative phosphotyrosines that might be altered through the miR-125b-PTPN18 signalling axis, we performed immunoprecipitation of phosphotyrosines followed by mass spectrometry in miR-125b-OE Ba/F3 cells and identified increased GSK3 tyrosine phosphorylation as a top target. Additionally, miR-125b OE was confirmed to enhance GSK3 tyrosine phosphorylation, whereas PTPN18 OE reduced it (Figure 1F), together strongly suggesting that miR-125b could enhance tyrosine phosphorylation of GSK3 by silencing PTPN18. GSK3A and GSK3B (GSK3A/B) are paralogous genes that share a high degree of sequence homology and belong to the glycogen synthase kinase 3 (GSK3) family. Tyrosine phosphorylation activates the kinase activity of GSK3, whereas serine phosphorylation inactivates it. We recently identified GSK inhibitors as top candidates targeting LSCs in a stemness-based drug screen using OCI-AML-8227 cells (data not shown). Treatment of OCI-AML-8227 cells with two selective inhibitors of GSK3 selectively reduced the proportion of CD34+ cells while concomitantly increasing expression of myeloid markers CD14 and CD15 (Figure 1G). Overall, our results support an important functional role for PTPN18 and GSK3 in LSC function, and present a potential novel therapeutic target against LSCs. This study highlights the importance of understanding the role of miRNAs and may identify a new druggable vulnerability in LSCs that could lead to the development of new treatment options for AML patients. Figure 1 Disclosures Dick: Bristol-Myers Squibb/Celgene: Research Funding. Wang:Trilium Therapeutics: Patents & Royalties.
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- 2020
45. Geographical Distance from Quaternary Treatment Center Does Not Impact Choice of Upfront Therapy, Clinical Trial Participation and Outcomes in Patients with Newly Diagnosed AML
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Mark D. Minden, Aaron D. Schimmer, Andre C. Schuh, Samantha Aliza Hershenfeld, Hassan Sibai, Caroline McNamara, Vikas Gupta, Steven M. Chan, Karen W.L. Yee, Tracy Murphy, and Dawn Maze
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medicine.medical_specialty ,Performance status ,business.industry ,Immunology ,Induction chemotherapy ,Cell Biology ,Hematology ,Newly diagnosed ,Biochemistry ,Clinical trial ,Treatment center ,Multiple factors ,Family medicine ,Health care ,medicine ,In patient ,business - Abstract
Upfront therapy for newly diagnosed patients with acute myeloid leukemia (AML) includes intensive induction chemotherapy with curative intent, low dose chemotherapy, best supportive care, and clinical trials. The choice between these therapies is influenced by multiple factors including age, cytogenetic and molecular mutations, and performance status. In our single payer provincial health care system, induction chemotherapy and clinical trials are only offered at a small number of specialized quaternary care centers with geographically large catchment areas. As a result, some patients are required to travel long distances for their appointments, which may constitute a barrier to care, especially among elderly patients. We therefore asked whether distance from the quaternary center influences the choice of care for AML. We reviewed the records of patients ≥18 years of age diagnosed with AML from 2015-2017 and assessed at our quaternary care center in Toronto, Canada. We compared upfront therapy choice and survival between patients living close versus distant from the cancer center (empirically defined as 50km) and stratified by age. A total of 675 patients were assessed by our quaternary center for a new diagnosis of AML during the timeframe studied. Of those patients, 477 (71%) patients lived ≤50km, and 198 (29%) patients lived >50km from the quaternary center. The overall median distance from patient residence to the quaternary center was 33.2km (range: 1-1791km), and the median distance of patients in the >50km group was 93km (range: 50.2-1791km). Age, sex, baseline Eastern Cooperative Oncology Group Performance Status (ECOG), and cytogenetic risk were not significantly different between the two groups. There were no differences in the proportion of patients receiving induction chemotherapy or clinical trial as upfront therapy between patients living close versus distant from the quaternary center, even when stratified for age ≥70 years. There was no difference in overall survival between patients living ≤50km versus >50km from the quaternary center either overall, or when stratified by age. In conclusion, geographic distance from treatment center does not appear to impact choice of upfront therapy, access to clinical trials, or clinical outcomes in this study of newly diagnosed patients with AML treated in a single payer environment. Disclosures Gupta: Bristol MyersSquibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Honoraria, Research Funding; Pfizer: Consultancy; Sierra Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees. Maze:Novartis: Honoraria; Pfizer: Consultancy; Takeda: Research Funding. McNamara:Novartis: Honoraria. Schimmer:Takeda: Honoraria, Research Funding; Novartis: Honoraria; Jazz: Honoraria; Otsuka: Honoraria; Medivir AB: Research Funding; AbbVie Pharmaceuticals: Other: owns stock .
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- 2020
46. IPO11 Regulates the Nuclear Import of BZW1/2 and Is Necessary for AML Cells and Stem Cells
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Dilshad H. Khan, Veronique Voisin, Jonathan St-Germain, Andrea Arruda, Geethu E. Thomas, Changjiang Xu, Rose Hurren, Elias Orouji, Mark D. Minden, Boaz Nachmias, Neil MacLean, Brian Raught, Ayesh K. Seneviratne, Gary D. Bader, Xiaoming Wang, Liran I. Shlush, Aaron D. Schimmer, and Aaron Botham
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education.field_of_study ,Gene knockdown ,Myeloid ,Immunology ,Population ,CD34 ,Myeloid leukemia ,Cell Biology ,Hematology ,Biology ,medicine.disease ,Biochemistry ,Haematopoiesis ,Leukemia ,medicine.anatomical_structure ,medicine ,Cancer research ,Stem cell ,education - Abstract
While most patients with acute myeloid leukemia (AML) achieve remission with initial therapy, the majority relapse leading to poor overall survival. Relapse is frequently driven by a rare subset of leukemic stem cells (LSC). Understanding biological mechanisms that maintain LSCs will help identify new therapeutic strategies for this disease. To identify such vulnerabilities, we overlaid the results of a genome-wide CRISPR screen with the expression of genes enriched in functionally defined LSCs. Through our CRISPR screen, we identified 570 genes whose knockout reduced the growth and viability of OCI-AML2 cells. Essential genes for growth and viability by our CRIPSR screen were enriched in the LSC+ population. By overlaying the hits from our CRISPR screen with genes upregulated in LSCs, we identified IPO11, as a top hit, with 7.5-fold increase in the LSC+ fraction compared to the LSC- fraction. IPO11 is a member of the importin-β family of proteins and facilitates the import of protein cargo into the nucleus. Further analysis showed that IPO11 was upregulated in LSC+ (engrafting) vs. LSC- (non-engrafting) primary AML samples, CD34+ vs CD34- AML samples, undifferentiated progenitor vs. myeloid cluster AML samples, and relapse vs de novo AML. IPO11 was increased in AML cells compared to normal hematopoietic cells and increased IPO11 expression was associated with decreased overall survival in AML. By immunoblotting, IPO11 protein was increased in primary AML (n=4) compared to normal hematopoietic cells (n=4). To determine whether IPO11 is necessary for AML growth and viability, we knocked down IPO11 in OCI-AML2, TEX and NB4 leukemia cells with shRNA in lentiviral vectors. Knockdown of IPO11 reduced AML growth and viability by 80-90%. In contrast, knockdown of another importin-β family member, IPO5, that was not a hit in our CRIPSR screen, did not reduce AML growth and viability. Knockdown of IPO11 increased differentiation of AML cells as evidenced by the changes in gene expression, decreased chromatin accessibility, increased CD11b expression and increased non-specific esterase staining. Finally, knockdown of IPO11 reduced the engraftment of TEX cells and the low passage primary AML 8227 cells into immune deficient mice by over 90%. Importantly, IPO11 knockdown reduced engraftment of primary AML cells into mouse marrow. To identify novel cargos of IPO11, we performed proximity-dependent biotin labeling (BioID) coupled with mass spectrometry and identified proteins that interacted with IPO11. Among the top hits were BZW1 and BZW 2 (Basic leucine zipper and W2 domains 1 and 2). BZW1 and BZW2 are members of the bZIP super family of transcription factors. Knockdown of IPO11 reduced levels of BZW1 in the nucleus detected by immunoblotting and confocal microscopy. Commercial antibodies could not detect BZW2. To determine if the nuclear import of BZW1 and 2 were functionally important for the effects of IPO11 on AML stem cell function and differentiation, we knocked down BZW1 and BZW2. Dual knockdown of BZW1 and BZW2 (but not individual) mimicked the effects of IPO11 inhibition and decreased the growth and viability of AML cells. Changes in gene expression after BZW1/2 knockdown were similar to IPO11 knockdown with enrichment in myeloid-differentiated genes. By pathway analysis, we identified that IPO11 knockdown, as well as BZW1/2 knockdown decreased expression of MYC target genes, suggesting a mechanism by which these proteins regulate AML stem cell function. Thus, in summary, we identified IPO11 as an essential gene for the viability of AML cells and stem cells. This work highlights a previously unappreciated role of the protein import pathway in regulating AML stem cell function and highlights a potential new therapeutic target for AML. Disclosures Schimmer: Takeda: Honoraria, Research Funding; Novartis: Honoraria; Jazz: Honoraria; Otsuka: Honoraria; Medivir AB: Research Funding; AbbVie Pharmaceuticals: Other: owns stock .
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- 2020
47. Impact of genomic alterations on outcomes in myelofibrosis patients undergoing JAK1/2 inhibitor therapy
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Jenny M.-Y. Ho, Rebecca Devlin, Tracy Stockley, Jamie Claudio, Hassan Sibai, Suzanne Kamel-Reid, Dawn Maze, Mark D. Minden, Nancy Siddiq, Tony Panzarella, James A. Kennedy, Aaron D. Schimmer, Mahadeo A. Sukhai, Jay Y. Spiegel, Andre C. Schuh, Vikas Gupta, Caroline J McNamara, Mariam Thomas, Justyna Bartoszko, Karen W.L. Yee, and Andrea Arruda
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Oncology ,Genetics ,medicine.medical_specialty ,Mutation ,Ruxolitinib ,Univariate analysis ,Myeloid Neoplasia ,Myeloid ,business.industry ,Anemia ,Hazard ratio ,Hematology ,medicine.disease_cause ,medicine.disease ,03 medical and health sciences ,0302 clinical medicine ,medicine.anatomical_structure ,International Prognostic Scoring System ,030220 oncology & carcinogenesis ,Internal medicine ,medicine ,business ,Myelofibrosis ,030215 immunology ,medicine.drug - Abstract
In myelofibrosis (MF), driver mutations in JAK2, MPL, or CALR impact survival and progression to blast phase, with the greatest risk conferred by triple-negative status. Subclonal mutations, including mutations in high–molecular risk (HMR) genes, such as ASXL1, EZH2, IDH1/2, and SRSF2 have also been associated with inferior prognosis. However, data evaluating the impact of next-generation sequencing in MF patients treated with JAK1/2 inhibitors are lacking. Using a 54-gene myeloid panel, we performed targeted sequencing on 100 MF patients treated with ruxolitinib (n = 77) or momelotinib (n = 23) and correlated mutational profiles with treatment outcomes. Ninety-nine patients had at least 1 mutation identified, 46 (46%) had 2 mutations, and 34 (34%) patients had ≥3 mutations. Seventy-nine patients carried a mutation in JAK2V617F, 14 patients had mutations in CALR, 6 patients had an MPL mutation, and 2 patients were triple negative. No mutation was significantly associated with spleen or anemia response. A high Dynamic International Prognostic Scoring System score and pretreatment transfusion dependence were associated with a shorter time to treatment failure (TTF), and this association retained significance on multivariable analysis. Patients with ASXL1 (hazard ratio [HR], 1.86; P = .03) and EZH2 mutations (HR, 2.94; P = .009) and an HMR profile (HR, 2.06; P = .01) had shorter TTF. On multivariate analysis, ASXL1 or EZH2 mutations were independently associated with shorter TTF and overall survival. These findings help identify patients unlikely to have a durable response with current JAK1/2 inhibitors and provide a framework for future studies.
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- 2017
48. Leveraging increased cytoplasmic nucleoside kinase activity to target mtDNA and oxidative phosphorylation in AML
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Rebecca R. Laposa, Dana Yehudai, Rose Hurren, Xiaoming Wang, Daina Avizonis, Gaëlle Bridon, Shrivani Sriskanthadevan, Aisha Shamas-Din, Mark D. Minden, Marcela Gronda, Veronique Voisin, Thirushi P. Siriwardena, Sanduni U. Liyanage, Changjiang Xu, Aaron D. Schimmer, Gary D. Bader, and Neil MacLean
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DNA Replication ,0301 basic medicine ,Immunology ,Mice, SCID ,Mitochondrion ,Biology ,DNA, Mitochondrial ,Biochemistry ,Oxidative Phosphorylation ,03 medical and health sciences ,chemistry.chemical_compound ,Cell Line, Tumor ,hemic and lymphatic diseases ,Tumor Cells, Cultured ,Animals ,Humans ,Cells, Cultured ,Nucleoside-phosphate kinase ,integumentary system ,Zalcitabine ,Kinase ,Phosphotransferases ,Cytidine ,Cell Biology ,Hematology ,NM23 Nucleoside Diphosphate Kinases ,Molecular biology ,Leukemia, Myeloid, Acute ,030104 developmental biology ,chemistry ,Cell culture ,Signal transduction ,Stem cell ,Nucleoside-Phosphate Kinase ,Nucleoside ,Signal Transduction - Abstract
Mitochondrial DNA (mtDNA) biosynthesis requires replication factors and adequate nucleotide pools from the mitochondria and cytoplasm. We performed gene expression profiling analysis of 542 human acute myeloid leukemia (AML) samples and identified 55% with upregulated mtDNA biosynthesis pathway expression compared with normal hematopoietic cells. Genes that support mitochondrial nucleotide pools, including mitochondrial nucleotide transporters and a subset of cytoplasmic nucleoside kinases, were also increased in AML compared with normal hematopoietic samples. Knockdown of cytoplasmic nucleoside kinases reduced mtDNA levels in AML cells, demonstrating their contribution in maintaining mtDNA. To assess cytoplasmic nucleoside kinase pathway activity, we used a nucleoside analog 2'3'-dideoxycytidine (ddC), which is phosphorylated to the activated antimetabolite, 2'3'-dideoxycytidine triphosphate by cytoplasmic nucleoside kinases. ddC is a selective inhibitor of the mitochondrial DNA polymerase γ. ddC was preferentially activated in AML cells compared with normal hematopoietic progenitor cells. ddC treatment inhibited mtDNA replication, oxidative phosphorylation, and induced cytotoxicity in a panel of AML cell lines. Furthermore, ddC preferentially inhibited mtDNA replication in a subset of primary human leukemia cells and selectively targeted leukemia cells while sparing normal progenitor cells. In animal models of human AML, treatment with ddC decreased mtDNA, electron transport chain proteins, and induced tumor regression without toxicity. ddC also targeted leukemic stem cells in secondary AML xenotransplantation assays. Thus, AML cells have increased cytidine nucleoside kinase activity that regulates mtDNA biogenesis and can be leveraged to selectively target oxidative phosphorylation in AML.
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- 2017
49. Multicenter, Open-Label, 3-Arm Study of Gilteritinib, Gilteritinib Plus Azacitidine, or Azacitidine Alone in Newly Diagnosed FLT3 Mutated (FLT3(mut+)) Acute Myeloid Leukemia (AML) Patients Ineligible for Intensive Induction Chemotherapy: Findings from the Safety Cohort
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Jorge Sierra, Shira Dinner, Erkut Bahceci, Teresa Bernal del Castillo, Olga Salamero, Shufang Liu, Jordi Esteve, Je-Hwan Lee, Pau Montesinos, Goichi Yoshimoto, Kamel Laribi, Elizabeth Shima Rich, Janusz Hałka, Mark D. Minden, Eunice S. Wang, Rik Schots, Clinical sciences, Laboratory of Molecullar and Cellular Therapy, and Hematology
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medicine.medical_specialty ,Combination therapy ,business.industry ,Anemia ,Immunology ,Induction chemotherapy ,Cell Biology ,Hematology ,Neutropenia ,medicine.disease ,Biochemistry ,Tumor lysis syndrome ,03 medical and health sciences ,0302 clinical medicine ,Tolerability ,030220 oncology & carcinogenesis ,Internal medicine ,Cohort ,medicine ,business ,Febrile neutropenia ,030215 immunology - Abstract
Background: Gilteritinib, a highly selective, potent FLT3/AXL inhibitor, showed antileukemic activity with favorable tolerability in a phase 1/2 trial of patients (pts) with FLT3mut+ relapsed/refractory AML (Perl AE, et al. Lancet Oncol. 2017). Gilteritinib plus azacitidine (AZA) synergistically induced apoptosis and inhibited growth in the MV4-11 cell line and in MV4-11 tumors (Ueno Y, et al. Blood. 2016). The safety, tolerability, and efficacy of gilteritinib alone, gilteritinib plus AZA, or AZA alone is being evaluated in pts with newly diagnosed FLT3mut+ AML ineligible for intensive induction chemotherapy; this abstract presents data from the Safety Cohort which evaluated only gilteritinib plus AZA. Methods: Adults with newly diagnosed FLT3mut+ (FLT3-ITD or FLT3-TKD) AML are being enrolled in this ongoing clinical trial (NCT02752035). Prior to initiation of the randomized portion of the study, the appropriate gilteritinib dose for combination therapy was assessed in a Safety Cohort. Patients enrolled in this cohort received escalating doses of oral gilteritinib (80 or 120 mg/day) on Days 1-28 in combination with subcutaneous or intravenous AZA (75 mg/m2/day) on Days 1-7. Observations of dose-limiting toxicities (DLTs) were collected through Cycle 1; treatment was continued in 28-day cycles until lack of clinical benefit or unacceptable toxicity. Safety and tolerability were the primary endpoints of the Safety Cohort; antileukemic response rates (ie, complete remission [CR], CR with incomplete platelet recovery [CRp], CR with incomplete hematological recovery [CRi], partial remission [PR], and overall response rate [ORR]) were also assessed. Results: A total of 15 adult pts (median age, 76 [range: 65-86]) were enrolled into the Safety Cohort (n=9, 80 mg gilteritinib; n=6, 120 mg gilteritinib); 14 pts were FLT3mut+ (ITD alone, n=10; TKD alone, n=3; ITD and TKD, n=1) and 1 pt had no FLT3 mutation. As of 25 June 2018, more than half (n=8/15; 56%, Figure) of the pts had a treatment duration of >6 mo, while 9 pts discontinued treatment (death, n=4; relapse; adverse event [AE]; physician decision; sponsor decision; subject withdrawal, n=1 each) and 6 pts remained on treatment. During the DLT observation period, 1 DLT (tumor lysis syndrome) was observed in a pt who received 80 mg gilteritinib plus AZA; no DLTs were reported in pts who received 120 mg gilteritinib plus AZA. One or more AEs were seen in all 15 pts; 12 (80%) experienced AEs considered at least possibly related to treatment. Adverse events occurring in ≥25% of pts were anemia (n=7), febrile neutropenia and nausea (n=6 each), increased ALT and AST, constipation, diarrhea, neutropenia, thrombocytopenia, and pyrexia (n=5 each), and decreased appetite, fatigue, increased blood creatinine, and hypocalcemia (n=4 each). Grade ≥3 AEs occurring in ≥25% of pts were febrile neutropenia (n=6), anemia and neutropenia (n=5 each), and thrombocytopenia (n=4). Serious AEs occurring in >2 pts were febrile neutropenia (n=5), and anemia and pyrexia (n=3 each). Of the 8 pts with fatal AEs, none of which were related to treatment, 3 occurred early in treatment: septic shock (Day 2), respiratory failure (Day 6), and cerebral hemorrhage in the setting of acute kidney injury and uremia (Day 17). None of the 13 pts with post-baseline lab data had any potentially clinically significant AST/ALT (>3 X ULN) and total bilirubin (>2 X ULN) values; none of the pts had a maximum post-baseline QTcF interval >500 msec. Across the Safety Cohort, a composite complete remission rate of 67% (n=10/15) was observed; 4 pts achieved a best overall response of CR and 6 achieved CRi (Figure). Two additional pts (13%) achieved a best overall response of PR, giving an ORR of 80%. One DLT from the 11 DLT evaluable pts (defined as pts who experienced a DLT, or in the absence of DLT, received at least 23 of 28 doses of gilteritinib and at least 5 of 7 doses of AZA during the DLT observation period) informed the decision to proceed to the randomized portion at a dose of 120 mg gilteritinib for the combination treatment arm. Conclusions: Gilteritinib and AZA were generally well tolerated with no unexpected AEs. This combination therapy induced antileukemic responses in newly diagnosed FLT3mut+ AML pts unfit to receive standard induction chemotherapy. Based on these results, pts are being enrolled into the randomized portion of the study with a dose of 120 mg gilteritinib for the combination treatment arm. Disclosures Wang: Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz: Speakers Bureau; Jazz: Speakers Bureau; Amgen: Consultancy; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Speakers Bureau; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Speakers Bureau; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy. Laribi:Amgen: Other: Personal fees; Takeda: Other: Grant and personal fees; Sandoz: Other: Grant; Novartis: Other: Grant and personal fees; Teva: Other: Grant; Gilead: Other: Personal fees; Hospira: Other: Grant; Roche: Other: Grant. Montesinos:Daiichi Sankyo: Consultancy, Speakers Bureau; Novartis: Research Funding, Speakers Bureau. Liu:Astellas Pharma: Employment. Rich:Astellas Pharma: Employment. Bahceci:Astellas Pharma: Employment.
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- 2018
50. Preclinical Characterization of Cell-Based IL12 Immunotherapy Against Murine Acute Lymphoblastic Leukemia Using Intravital Imaging
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Mark D. Minden, Ruijuan He, Ralph S. DaCosta, Sara Rapic, and Helen Jingshu Jin
- Subjects
education.field_of_study ,medicine.diagnostic_test ,business.industry ,medicine.medical_treatment ,Immunology ,Population ,Cell Biology ,Hematology ,Immunotherapy ,medicine.disease ,Biochemistry ,Flow cytometry ,Leukemia ,medicine.anatomical_structure ,In vivo ,medicine ,Cancer research ,Interleukin 12 ,Bone marrow ,education ,business ,Ex vivo - Abstract
Acute lymphoblastic leukemia (ALL) is a disease of the bone marrow (BM) microenvironment, in which ALL-stroma interactions, signaling molecules, and low oxygen tension all contribute to leukemia development and maintenance. The cytokine interleukin 12 (IL12) is a key primer of the initial specific T-cell immune response for resisting cancer progression, however its potential as an anti-leukemia immunotherapeutic agent has been largely under-examined. One potential reason for this oversight is the inability of current experimental systems to recapitulate the complex BM microenvironmental system ex vivo or obtain the resolution necessary in vivo to study the precise mechanism of effect for such proposed immunotherapies. Recently, our lab has developed a novel femur window chamber (FWC) intravital imaging mouse model capable of long-term spatiotemporal assessment of leukemias within the in vivo femoral BM at cellular resolution. We used this technique to characterize a new cell-based IL12 immunotherapy, in which patients are injected with their own blast cells engineered to express IL12. This approach has shown significant potential in preliminary preclinical trials, although its mechanism of effect is still unknown. Our study sought to establish the precise interaction between the modified IL12-expressing blast cells with the BM ALL population and immune system to determine a) how long post-ALL engraftment IL12 therapy continue to be effective, b) the optimal treatment administration route, and c) whether repeated infusions will yield added benefit. The murine lymphoblastic cell line, 70Z/3, was transfected with either a constitutively active GFP marker (70Z/3-GFP) or a lentivirus expressing mCherry and IL12 (70Z/3-IL12-mCherry). The FWC was installed by shaving the femoral corticalis to reveal the BM cavity and securing a cover glass on top. Mice were then intravenously (IV) injected with 3x106 70Z/3-GFP cells 2 weeks post-surgery. Fluorescent cellular activity within the BM was monitored two times a week for four weeks via confocal microscopy (Zeiss LSM710). 3x106 70Z/3-IL12-mCherry cells were then injected into mice IV, intraperitoneally (IP), or intrafemorally (IF) at each experimental time point following initial inoculation. Leukemic burden was quantified as the percent total GFP signal within the BM space while immune cell activity was monitored using injected fluorescently-conjugated antibodies and ex vivo flow cytometry. Nonfluorescent leukemic and healthy mice were also imaged as controls. Intravital imaging revealed that IV injected 70Z/3-GFP cells first enter the BM, on average, 10 days post-inoculation and achieve 90% infiltration within 3 weeks. Based on this timeline, we evaluated the effect of IL12 treatment administered 1, 2, or 3 weeks post-inoculation, as well as at the time of inoculation itself (co-injection). Mice treated with a single administration of 70Z/3-IL12-mCherry cells at 1 week showed a 26% reduction (p Our study not only demonstrates the ability of cell-based IL12 therapy to elicit a therapeutic response in ALL-bearing mice, it also reveals important insights into the biology of the immunotherapy, which can be applied to further augment the treatment's effect. Disclosures DaCosta: SBI ALApharma Canada: Current Employment, Current equity holder in private company; MolecuLight Inc.: Current Employment, Current equity holder in private company, Patents & Royalties: Device and Method for Fluorescence-Based Imaging and Monitoring.
- Published
- 2020
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