44 results on '"Obeng, Esther A."'
Search Results
2. Engineering naturally occurring CD7− T cells for the immunotherapy of hematological malignancies
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Freiwan, Abdullah, Zoine, Jaquelyn T., Crawford, Jeremy Chase, Vaidya, Abishek, Schattgen, Stefan A., Myers, Jacquelyn A., Patil, Sagar L., Khanlari, Mahsa, Inaba, Hiroto, Klco, Jeffery M., Mullighan, Charles G., Krenciute, Giedre, Chockley, Peter J., Naik, Swati, Langfitt, Deanna M., Mamonkin, Maksim, Obeng, Esther A., Thomas, Paul G., Gottschalk, Stephen, and Velasquez, M. Paulina
- Abstract
Chimeric antigen receptor (CAR) T-cell therapy targeting T-cell acute lymphoblastic leukemia (T-ALL) faces limitations such as antigen selection and limited T-cell persistence. CD7 is an attractive antigen for targeting T-ALL, but overlapping expression on healthy T cells leads to fratricide of CD7-CAR T cells, requiring additional genetic modification. We took advantage of naturally occurring CD7− T cells to generate CD7-CAR (CD7-CARCD7−) T cells. CD7-CARCD7− T cells exhibited a predominantly CD4+ memory phenotype and had significant antitumor activity upon chronic antigen exposure in vitro and in xenograft mouse models. Based on these encouraging results, we next explored the utility of CD7− T cells for the immunotherapy of CD19+ hematological malignancies. Direct comparison of nonselected (bulk) CD19-CAR and CD19-CARCD7− T cells revealed that CD19-CARCD7− T cells had enhanced antitumor activity compared with their bulk counterparts in vitro and in vivo. Lastly, to gain insight into the behavior of CD19-CAR T cells with low levels of CD7 gene expression (CD7lo) in humans, we mined single-cell gene and T-cell receptor (TCR) expression data sets from our institutional CD19-CAR T-cell clinical study. CD19-CARCD7lo T cells were present in the initial CD19-CAR T-cell product and could be detected postinfusion. Intriguingly, the only functional CD4+ CD19-CAR T-cell cluster observed postinfusion exhibited CD7lo expression. Additionally, samples from patients responsive to therapy had a higher proportion of CD7lo T cells than nonresponders (NCT03573700). Thus, CARCD7− T cells have favorable biological characteristics and may present a promising T-cell subset for adoptive cell therapy of T-ALL and other hematological malignancies.
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- 2022
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3. Engineering naturally occurring CD7−T cells for the immunotherapy of hematological malignancies
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Freiwan, Abdullah, Zoine, Jaquelyn T., Crawford, Jeremy Chase, Vaidya, Abishek, Schattgen, Stefan A., Myers, Jacquelyn A., Patil, Sagar L., Khanlari, Mahsa, Inaba, Hiroto, Klco, Jeffery M., Mullighan, Charles G., Krenciute, Giedre, Chockley, Peter J., Naik, Swati, Langfitt, Deanna M., Mamonkin, Maksim, Obeng, Esther A., Thomas, Paul G., Gottschalk, Stephen, and Velasquez, M. Paulina
- Abstract
•Naturally occurring CD7 negative T cells are functional effector T cells that can be used for chimeric antigen receptor therapy.•CD7 negative T cells expressing a CD7-CAR have robust antitumor activity and bypass fratricide.
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- 2022
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4. Preferential expansion of CD8+ CD19-CAR T cells postinfusion and the role of disease burden on outcome in pediatric B-ALL
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Talleur, Aimee C., Qudeimat, Amr, Métais, Jean-Yves, Langfitt, Deanna, Mamcarz, Ewelina, Crawford, Jeremy Chase, Huang, Sujuan, Cheng, Cheng, Hurley, Caitlin, Madden, Renee, Sharma, Akshay, Suliman, Ali, Srinivasan, Ashok, Velasquez, M. Paulina, Obeng, Esther A., Willis, Catherine, Akel, Salem, Karol, Seth E., Inaba, Hiroto, Bragg, Allison, Zheng, Wenting, Zhou, Sheng M., Schell, Sarah, Tuggle-Brown, MaCal, Cullins, David, Patil, Sagar L, Li, Ying, Thomas, Paul G., Zebley, Caitlin, Youngblood, Benjamin, Pui, Ching-Hon, Lockey, Timothy, Geiger, Terrence L., Meagher, Michael M., Triplett, Brandon M., and Gottschalk, Stephen
- Abstract
T cells expressing CD19-specific chimeric antigen receptors (CD19-CARs) have potent antileukemia activity in pediatric and adult patients with relapsed and/or refractory B-cell acute lymphoblastic leukemia (B-ALL). However, not all patients achieve a complete response (CR), and a significant percentage relapse after CD19-CAR T-cell therapy due to T-cell intrinsic and/or extrinsic mechanisms. Thus, there is a need to evaluate new CD19-CAR T-cell products in patients to improve efficacy. We developed a phase 1/2 clinical study to evaluate an institutional autologous CD19-CAR T-cell product in pediatric patients with relapsed/refractory B-ALL. Here we report the outcome of the phase 1 study participants (n = 12). Treatment was well tolerated, with a low incidence of both cytokine release syndrome (any grade, n = 6) and neurotoxicity (any grade, n = 3). Nine out of 12 patients (75%) achieved a minimal residual disease-negative CR in the bone marrow (BM). High disease burden (≥40% morphologic blasts) before CAR T-cell infusion correlated with increased side effects and lower response rate, but not with CD19-CAR T-cell expansion. After infusion, CD8+ CAR T cells had a proliferative advantage over CD4+ CAR T cells and at peak expansion, had an effector memory phenotype with evidence of antigen-driven differentiation. Patients that proceeded to allogeneic hematopoietic cell transplantation (AlloHCT) had sustained, durable responses. In summary, the initial evaluation of our institutional CD19-CAR T-cell product demonstrates safety and efficacy while highlighting the impact of pre-infusion disease burden on outcomes. This trial was registered at www.clinicaltrials.gov as #NCT03573700.
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- 2022
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5. Murine foetal liver supports limited detectable expansion of life-long haematopoietic progenitors
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Ganuza, Miguel, Hall, Trent, Myers, Jacquelyn, Nevitt, Chris, Sánchez-Lanzas, Raúl, Chabot, Ashley, Ding, Juan, Kooienga, Emilia, Caprio, Claire, Finkelstein, David, Kang, Guolian, Obeng, Esther, and McKinney-Freeman, Shannon
- Abstract
Current dogma asserts that the foetal liver (FL) is an expansion niche for recently specified haematopoietic stem cells (HSCs) during ontogeny. Indeed, between embryonic day of development (E)12.5 and E14.5, the number of transplantable HSCs in the murine FL expands from 50 to about 1,000. Here we used a non-invasive, multi-colour lineage tracing strategy to interrogate the embryonic expansion of murine haematopoietic progenitors destined to contribute to the adult HSC pool. Our data show that this pool of fated progenitors expands only two-fold during FL ontogeny. Although Histone2B-GFP retention in vivo experiments confirmed substantial proliferation of phenotypic FL-HSC between E12.5 and E14.5, paired-daughter cell assays revealed that many mid-gestation phenotypic FL-HSCs are biased to differentiate, rather than self-renew, relative to phenotypic neonatal and adult bone marrow HSCs. In total, these data support a model in which the FL-HSC pool fated to contribute to adult blood expands only modestly during ontogeny.
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- 2022
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6. Therapeutic approaches targeting splicing factor mutations in myelodysplastic syndromes and acute myeloid leukemia
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Figg, John W., Barajas, Juan M., and Obeng, Esther A.
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- 2021
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7. Hematopoietic Stem Cells from Mice and Individuals with Sickle Cell Disease Display Premature Senescence and Loss of Function That Is Targetable By Senolytic Therapy
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Barve, Aditya, Dabbah, Mahmoud, Kooienga, Emilia, Obeng, Esther A., Myers, Jacquelyn, Sharma, Akshay, and McKinney-Freeman, Shannon
- Abstract
Sickle cell disease (SCD) is an inherited anemia caused by mutations in the β-globin gene, resulting in aberrant hemoglobin polymerization driving hemolysis, vasooclusion, and systemic inflammation. Allogeneic hematopoietic stem cell (HSC) transplantation is potentially curative, and novel autologous therapies involving gene-editing of mobilized HSCs are being developed. However, many SCD patients are unable to undergo autologous gene-editing due to failure to mobilize sufficient numbers of HSCs. Further, the damaging effects of SCD on the HSC pool remain uncharacterized. Therefore, we interrogated young and aged cohorts of humanized SCD mice and non-SCD controls for HSC frequency and function. Immunophenotypic HSC frequency was mildly elevated (1.3-fold, p=0.00316) in 2-month-old SCD mice, relative to controls (n=5-6/group). In contrast, HSC numbers were halved in 6 and 12-month-old SCD mice (n=8-10/group, p=0.00358 and p=0.0148, respectively). Competitive transplantation of whole bone marrow (WBM) from 2 and 6-month-old SCD mice displayed a profound loss of peripheral blood repopulation, relative to age-matched control mice. Quantitative limiting dilution transplantation of WBM from 2 and 6-month-old SCD mice revealed a 4 and 6-fold loss of functional HSCs, relative to control mice. Thus, despite elevated numbers of phenotypic HSCs in young SCD mice, the HSC pool is depleted of functional repopulating cells during SCD. To understand the loss of functional HSCs, we performed bulk RNA-sequencing of purified HSCs from 6-month-old SCD mice. We observed downregulation of 37 genes encoding ribosomal and histone proteins, suggesting impaired histone/ribosome biogenesis, a phenotype associated with senescence. Consistently, pathway enrichment analysis also revealed perturbed p53 activity and a high degree of association with published molecular signatures of senescence.
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- 2023
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8. Regulation of Alternative Splicing in B-Cell ALL By DYRK1A
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Jin, Qi, Harris, Ethan, Myers, Jacquelyn A., Cotton, Anitria, Wen, Jeremy, Melo-Cardenas, Johanna, Yoshimi, Akihide, Abdel-Wahab, Omar, Obeng, Esther A., and Crispino, John D.
- Abstract
DYRK1A, located in the Down syndrome critical region of chromosome 21, is a serine and threonine kinase that controls multiple cellular processes including apoptosis, cell cycle, transcription and signal transduction. We previously demonstrated that DYRK1A is required for maturation of B cells and is a therapeutic target in B-cell ALL, including both DS and non-DS subtypes. Key substrates within the B-cell lineage include D-type cyclins, FOXO1, and STAT3. Given that several studies have reported that DYRK1A phosphorylates various splicing factors and participates in splicing of Tau in neuronal cells, we investigated the contribution of DYRK1A to splicing in malignant B cells.
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- 2023
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9. SF3B1 K700EAlters the Splicing of Metabolism and Stress Response Genes, Attenuating the Effect of DNMT3A Mutations in MDS
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Wallace, Lashanale, Myers, Jacquelyn A., Engelken, Mark, Cullins, David, Sheppard, Heather, and Obeng, Esther A.
- Abstract
Myelodysplastic syndromes (MDS) are clonal stem cell disorders associated with low blood counts and a high risk of leukemic transformation. Genomic sequencing studies have demonstrated that MDS develops due to the acquisition of somatic mutations in hematopoietic stem cells (HSCs). Splicing factor 3b subunit 1 ( SF3B1) mutations, one of the most frequent mutations in MDS, are associated with lower-risk disease characterized by ineffective erythropoiesis. Our group and others have shown that mutations in SF3B1 cause aberrant mRNA splicing in human and murine HSCs, an expansion of long-term HSCs (LT-HSCs) in primary mice, but a competitive disadvantage in transplantation assays. DNMT3A, de novomethyltransferase 3A, is an epigenetic regulator mutated in ~10-15% of MDS. DNMT3A mutations are associated with unfavorable outcomes in MDS including a higher risk of leukemia transformation and shorter overall survival. Murine models of Dnmt3a mutations revealed that Dnmt3a-mutant HSCs have increased self-renewal over lineage-specific differentiation. SF3B1and DNMT3Amutations co-occur more frequently than expected by chance and arise early in MDS pathogenesis, making them attractive therapeutic targets. MDS patients with SF3B1 and DNMT3Amutations have shorter progression-free survival than those with solely SF3B1mutations. However, the mechanisms by which these mutations cooperate to cause HSC dysfunction and their possible combined role in leukemogenesis are not well understood.
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- 2023
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10. Impaired human hematopoiesis due to a cryptic intronic GATA1 splicing mutation
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Abdulhay, Nour J., Fiorini, Claudia, Verboon, Jeffrey M., Ludwig, Leif S., Ulirsch, Jacob C., Zieger, Barbara, Lareau, Caleb A., Mi, Xiaoli, Roy, Anindita, Obeng, Esther A., Erlacher, Miriam, Gupta, Namrata, Gabriel, Stacey B., Ebert, Benjamin L., Niemeyer, Charlotte M., Khoriaty, Rami N., Ancliff, Philip, Gazda, Hanna T., Wlodarski, Marcin W., and Sankaran, Vijay G.
- Abstract
Studies of allelic variation underlying genetic blood disorders have provided important insights into human hematopoiesis. Most often, the identified pathogenic mutations result in loss-of-function or missense changes. However, assessing the pathogenicity of noncoding variants can be challenging. Here, we characterize two unrelated patients with a distinct presentation of dyserythropoietic anemia and other impairments in hematopoiesis associated with an intronic mutation in GATA1 that is 24 nucleotides upstream of the canonical splice acceptor site. Functional studies demonstrate that this single-nucleotide alteration leads to reduced canonical splicing and increased use of an alternative splice acceptor site that causes a partial intron retention event. The resultant altered GATA1 contains a five–amino acid insertion at the C-terminus of the C-terminal zinc finger and has no observable activity. Collectively, our results demonstrate how altered splicing of GATA1, which reduces levels of the normal form of this master transcription factor, can result in distinct changes in human hematopoiesis.
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- 2019
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11. A Murine Model Harboring Cooperating DNMT3A and SF3B1 Mutations Phenocopies SF3B1 Driven Myelodysplastic Syndrome
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Wallace, Lashanale, Leal-Cervantes, Ana, Metidji, Amina, Myers, Jacquelyn, Rolle, Chandra, Tillman, Heather, and Obeng, Esther A.
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- 2022
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12. A Murine Model Harboring Cooperating DNMT3A and SF3B1 Mutations Phenocopies SF3B1 Driven Myelodysplastic Syndrome
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Wallace, Lashanale, Leal-Cervantes, Ana, Metidji, Amina, Myers, Jacquelyn, Rolle, Chandra, Tillman, Heather, and Obeng, Esther A.
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- 2022
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13. The development of therapy related myeloid neoplasms in childhood cancer survivors
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Obeng, Esther A. and Gruber, Tanja A.
- Abstract
Childhood cancer survivors exposed to chemotherapy show signs of accelerated aging and are at risk of developing secondary malignancies; however, the mechanisms responsible for these long-term adverse effects are not clear. In a recent study, Bertrums et al.show that exposure to chemotherapy results in an increase in mutational age of normal hematopoietic stem cells.
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- 2022
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14. Congenital macrothrombocytopenia with focal myelofibrosis due to mutations in human G6b-B is rescued in humanized mice
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Hofmann, Inga, Geer, Mitchell J., Vögtle, Timo, Crispin, Andrew, Campagna, Dean R., Barr, Alastair, Calicchio, Monica L., Heising, Silke, van Geffen, Johanna P., Kuijpers, Marijke J.E., Heemskerk, Johan W.M., Eble, Johannes A., Schmitz-Abe, Klaus, Obeng, Esther A., Douglas, Michael, Freson, Kathleen, Pondarré, Corinne, Favier, Rémi, Jarvis, Gavin E., Markianos, Kyriacos, Turro, Ernest, Ouwehand, Willem H., Mazharian, Alexandra, Fleming, Mark D., and Senis, Yotis A.
- Abstract
Unlike primary myelofibrosis (PMF) in adults, myelofibrosis in children is rare. Congenital (inherited) forms of myelofibrosis (cMF) have been described, but the underlying genetic mechanisms remain elusive. Here we describe 4 families with autosomal recessive inherited macrothrombocytopenia with focal myelofibrosis due to germ line loss-of-function mutations in the megakaryocyte-specific immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing receptor G6b-B (G6b, C6orf25, or MPIG6B). Patients presented with a mild-to-moderate bleeding diathesis, macrothrombocytopenia, anemia, leukocytosis and atypical megakaryocytes associated with a distinctive, focal, perimegakaryocytic pattern of bone marrow fibrosis. In addition to identifying the responsible gene, the description of G6b-B as the mutated protein potentially implicates aberrant G6b-B megakaryocytic signaling and activation in the pathogenesis of myelofibrosis. Targeted insertion of human G6bin mice rescued the knockout phenotype and a copy number effect of human G6b-B expression was observed. Homozygous knockin mice expressed 25% of human G6b-B and exhibited a marginal reduction in platelet count and mild alterations in platelet function; these phenotypes were more severe in heterozygous mice that expressed only 12% of human G6b-B. This study establishes G6b-B as a critical regulator of platelet homeostasis in humans and mice. In addition, the humanized G6bmouse will provide an invaluable tool for further investigating the physiological functions of human G6b-B as well as testing the efficacy of drugs targeting this receptor.
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- 2018
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15. Congenital macrothrombocytopenia with focal myelofibrosis due to mutations in human G6b-B is rescued in humanized mice
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Hofmann, Inga, Geer, Mitchell J., Vögtle, Timo, Crispin, Andrew, Campagna, Dean R., Barr, Alastair, Calicchio, Monica L., Heising, Silke, van Geffen, Johanna P., Kuijpers, Marijke J. E., Heemskerk, Johan W. M., Eble, Johannes A., Schmitz-Abe, Klaus, Obeng, Esther A., Douglas, Michael, Freson, Kathleen, Pondarré, Corinne, Favier, Rémi, Jarvis, Gavin E., Markianos, Kyriacos, Turro, Ernest, Ouwehand, Willem H., Mazharian, Alexandra, Fleming, Mark D., and Senis, Yotis A.
- Abstract
Unlike primary myelofibrosis (PMF) in adults, myelofibrosis in children is rare. Congenital (inherited) forms of myelofibrosis (cMF) have been described, but the underlying genetic mechanisms remain elusive. Here we describe 4 families with autosomal recessive inherited macrothrombocytopenia with focal myelofibrosis due to germ line loss-of-function mutations in the megakaryocyte-specific immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing receptor G6b-B (G6b, C6orf25, or MPIG6B). Patients presented with a mild-to-moderate bleeding diathesis, macrothrombocytopenia, anemia, leukocytosis and atypical megakaryocytes associated with a distinctive, focal, perimegakaryocytic pattern of bone marrow fibrosis. In addition to identifying the responsible gene, the description of G6b-B as the mutated protein potentially implicates aberrant G6b-B megakaryocytic signaling and activation in the pathogenesis of myelofibrosis. Targeted insertion of human G6b in mice rescued the knockout phenotype and a copy number effect of human G6b-B expression was observed. Homozygous knockin mice expressed 25% of human G6b-B and exhibited a marginal reduction in platelet count and mild alterations in platelet function; these phenotypes were more severe in heterozygous mice that expressed only 12% of human G6b-B. This study establishes G6b-B as a critical regulator of platelet homeostasis in humans and mice. In addition, the humanized G6b mouse will provide an invaluable tool for further investigating the physiological functions of human G6b-B as well as testing the efficacy of drugs targeting this receptor.
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- 2018
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16. Mutant SF3B1 splices a more leukemogenic EVI1
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Obeng, Esther A.
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- 2022
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17. Venetoclax-Based Combination Therapy As a Bridge to Allogeneic Hematopoietic Stem Cell Transplant in Children with Relapsed/Refractory AML
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Pfeiffer, Thomas, Li, Ying, Karol, Seth E, Rubnitz, Jeffrey E, Epperly, Rebecca, Madden, Renee, Mamcarz, Ewelina, Obeng, Esther A, Qudeimat, Amr, Sharma, Akshay, Srinivasan, Ashok, Suliman, Ali, Talleur, Aimee C, Velasquez, Mireya Paulina, Gottschalk, Stephen, Triplett, Brandon M., and Naik, Swati
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- 2022
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18. Splicing factor deficits render hematopoietic stem and progenitor cells sensitive to STAT3 inhibition
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Potts, Kathryn S., Cameron, Rosannah C., Metidji, Amina, Ghazale, Noura, Wallace, LaShanale, Leal-Cervantes, Ana I., Palumbo, Reid, Barajas, Juan Martin, Gupta, Varun, Aluri, Srinivas, Pradhan, Kith, Myers, Jacquelyn A., McKinstry, Mia, Bai, Xiaoying, Choudhary, Gaurav S., Shastri, Aditi, Verma, Amit, Obeng, Esther A., and Bowman, Teresa V.
- Abstract
Hematopoietic stem and progenitor cells (HSPCs) sustain lifelong hematopoiesis. Mutations of pre-mRNA splicing machinery, especially splicing factor 3b, subunit 1 (SF3B1), are early lesions found in malignancies arising from HSPC dysfunction. However, why splicing factor deficits contribute to HSPC defects remains incompletely understood. Using zebrafish, we show that HSPC formation in sf3b1homozygous mutants is dependent on STAT3 activation. Clinically, mutations in SF3B1are heterozygous; thus, we explored if targeting STAT3 could be a vulnerability in these cells. We show that SF3B1heterozygosity confers heightened sensitivity to STAT3 inhibition in zebrafish, mouse, and human HSPCs. Cells carrying mutations in other splicing factors or treated with splicing modulators are also more sensitive to STAT3 inhibition. Mechanistically, we illustrate that STAT3 inhibition exacerbates aberrant splicing in SF3B1mutant cells. Our findings reveal a conserved vulnerability of splicing factor mutant HSPCs that could allow for their selective targeting in hematologic malignancies.
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- 2022
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19. CD45RA-Depleted Haploidentical Transplantation Combined with NK Cell Addback Results in Promising Long-Term Outcomes in Pediatric Patients with High-Risk Hematologic Malignancies
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Naik, Swati, Talleur, Aimee C, Li, Ying, Madden, Renee, Mamcarz, Ewelina, Qudeimat, Amr, Sharma, Akshay, Srinivasan, Ashok, Suliman, Ali, Epperly, Rebecca, Obeng, Esther A, Velasquez, Mireya Paulina, Hijano, Diego, Marón, Gabriela M., Metais, Jean-Yves, Gottschalk, Stephen, and Triplett, Brandon M.
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- 2022
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20. CD45RA Depleted T-Cell Addback and Prophylactic Blinatumomab Administration Following Tcrαβ/CD19-Depleted Haploidentical Transplantation in Pediatric Patients with High Risk Acute Leukemia
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Naik, Swati, Li, Ying, Madden, Renee, Mamcarz, Ewelina, Srinivasan, Ashok, Sharma, Akshay, Talleur, Aimee C, Qudeimat, Amr, Suliman, Ali, Epperly, Rebecca, Obeng, Esther A, Velasquez, Mireya Paulina, Hijano, Diego, Marón, Gabriela M., Metais, Jean-Yves, Gottschalk, Stephen, and Triplett, Brandon M.
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- 2022
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21. Preferential expansion of CD8+CD19-CAR T cells postinfusion and the role of disease burden on outcome in pediatric B-ALL
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Talleur, Aimee C., Qudiemat, Amr, Métais, Jean-Yves, Langfitt, Deanna, Mamcarz, Ewelina, Crawford, Jeremy Chase, Huang, Sujuan, Cheng, Cheng, Hurley, Caitlin, Madden, Renee, Sharma, Akshay, Suliman, Ali, Srinivasan, Ashok, Velasquez, Paulina, Obeng, Esther, Willis, Catherine, Akel, Salem, Karol, Seth E., Inaba, Hiroto, Bragg, Allison, Zheng, Wenting, Zhou, Sheng, Schell, Sarah, Tuggle-Brown, MaCal, Cullins, David, Patil, Sagar L, Li, Ying, Thomas, Paul G., Zebley, Caitlin, Youngblood, Benjamin, Pui, Ching-Hong, Lockey, Timothy, Geiger, Terrence L., Meagher, Michael, Triplett, Brandon M., and Gottschalk, Stephen
- Abstract
T cells expressing CD19-specific chimeric antigen receptors (CD19-CARs) have potent antileukemia activity in pediatric and adult patients with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL). However, not all patients achieve a complete response (CR), and a significant percentage relapse after CD19-CAR T-cell therapy due to T-cell intrinsic and/or extrinsic mechanisms. Thus, there is a need to evaluate new CD19-CAR T-cell products in patients to improve efficacy. We developed a Phase 1/2 clinical study to evaluate an institutional autologous CD19-CAR T-cell product in pediatric patients with relapsed/refractory B-ALL. Here we report the outcome of the phase 1 study participants (n=12). Treatment was well tolerated, with low incidence of both cytokine release syndrome (any grade, n=6) and neurotoxicity (any grade, n=3). Nine out of 12 patients (75%) achieved a minimal residual disease-negative CR in the bone marrow. High disease burden (≥40% morphologic blasts) prior to CAR T-cell infusion correlated with increased side effects and lower response rate, but not with CD19-CAR T-cell expansion. Postinfusion, CD8+CAR T cells had a proliferative advantage over CD4+CAR T cells, and at peak expansion had an effector memory phenotype with evidence of antigen-driven differentiation. Patients that proceeded to allogeneic hematopoietic cell transplantation had sustained, durable responses. In summary, initial evaluation of our institutional CD19-CAR T-cell product demonstrates safety and efficacy, while highlighting the impact of pre-infusion disease burden on outcomes. This trial was registered at www.clinicaltrials.govas #NCT03573700.
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- 2022
- Full Text
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22. Selective Targeting of Splicing Factor Mutant Hematopoietic Stem and Progenitor Cells Via STAT3 Inhibition
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Potts, Kathryn S, Cameron, Rosannah C., Ghazale, Noura, Gupta, Varun, Barajas, Juan Martin, Wallace, LaShanale, Leal-Cervantes, Ana, Aluri, Srinivas, Pradhan, Kith, McKinstry, Mia, Bai, Xiaoying, Choudhary, Gaurav S, Shastri, Aditi, Verma, Amit, Obeng, Esther A., and Bowman, Teresa V.
- Abstract
Myelodysplastic syndrome (MDS) is a bone marrow failure disorder driven by dysfunction of hematopoietic stem and progenitor cells (HSPCs). Patient sequencing studies over the last decade have revealed that mutations in splicing machinery predominate in MDS, thus selective targeting of these cells is therapeutically attractive. STAT3 inhibition has been explored previously as a means to eradicate HSPCs in MDS. While efficacy was demonstrated in a subset of samples, the underlying mechanism for this selectivity remains unknown. We examined RNAseq of MDS CD34+ HSPCs with splicing factor mutations versus wildtype, finding alternative splicing and differential expression of STAT3 pathway components. Functionally, we explored if STAT3 signaling represents a novel vulnerability in SF3B1mutant HSPCs using a multi-model approach of in vivozebrafish and mouse systems, and in vitroassays of CRISPR-engineered human leukemia K562 cells and primary MDS samples. Utilizing the small molecule STAT3 inhibitor STATTIC, we found that human cells carrying MDS-associated SF3B1point mutations had heightened sensitivity to STAT3 inhibition compared to wildtype controls. To evaluate the activity of STAT3 inhibition in vivo, we utilized an Mx1-cre conditional knock-in mouse model of mutant SF3B1 (Sf3b1+/K700E). We demonstrated that in vivoSTATTIC treatment selectively depleted Sf3b1mutant cells over wildtype in vivo. RNAseq of sf3b1homozygous mutantzebrafish cells revealed conserved dysregulation of STAT3 pathway splicing and target expression. Diminishing Stat3 (via morpholino knockdown, stable mutants, or STATTIC treatment) decreased HSPCs in sf3b1heterozygotes but not wildtype embryos, demonstrating synthetic lethality between Sf3b1 and Stat3. Our data indicate that SF3B1heterozygosity, regardless of the type of mutation, confers a heightened sensitivity to STAT3 inhibition in zebrafish, mouse, and human HSPCs. Critically, our data indicate that SF3B1-mutant cells can be selectively killed in vivowhile sparing wildtype cells. We sought to rescue HSPCs in sf3b1homozygous mutant zebrafish, however overexpression of ligands Osm and Il6 or wildtype Stat3 was insufficient. Instead, overexpression of constitutively-active Stat3 partially restored HSPCs, indicating that functional Stat3 signaling downstream of Sf3b1 is critical for HSPC formation. To investigate the specificity of the synthetic lethality for SF3B1, we assessed the STAT3 synthetic lethal interaction with other mutated splicing factors in MDS. Similar to SF3B1, we demonstrated STAT3 synthetic lethality with U2AF1and SRSF2heterozygosity in zebrafish and human cells. RNA-sequencing analysis of STATTIC-treated K562 cells revealed an exacerbation of splicing alterations upon STAT3 inhibition that was more pronounced in SF3B1+/K666Ncells compared to wildtype. Even more strikingly, we demonstrated that constitutive activation of STAT3 could partially reverse defective splicing in zebrafish sf3b1homozygous mutant cells. Mechanistically, these data strongly support coordinated splicing dysfunction as the underlying cause for STAT3-SF3B1 synthetic lethality. Together, we demonstrated a conserved and selective synthetic lethal interaction between STAT3 function and splicing factor defects that represents a novel liability for mutant HSPCs with important implications for MDS treatment.
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- 2021
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23. CD45RA-Depleted Haploidentical Transplantation Combined with NK Cell Addback Results in Promising Long-Term Outcomes in Pediatric Patients with High-Risk Hematologic Malignancies
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Naik, Swati, Talleur, Aimee C, Li, Ying, Madden, Renee M., Mamcarz, Ewelina, Qudeimat, Amr, Sharma, Akshay, Srinivasan, Ashok, Suliman, Ali Y, Epperly, Rebecca, Obeng, Esther A., Velasquez, Mireya Paulina, Hijano, Diego, Maron, Gabriela, Gottschalk, Stephen, and Triplett, Brandon M
- Abstract
Novel graft engineering strategies that allow selective T-cell depletion have made haploidentical donor (haplo) hematopoietic cell transplantation (HCT) feasible for patients lacking a matched donor. We hypothesized that selective CD45RA+ cell depletion can limit graft-versus-host disease (GVHD) through removal of naive T cells but allow for rapid immune reconstitution through adoptive transfer of memory T cells, minimizing risk of graft failure, infections and relapse. We performed a prospective clinical trial utilizing CD45RA+ depleted haplo HCT followed by donor NK cell addback to maximize the graft-versus-leukemia effect in children with high-risk hematological malignancies (NCT01807611), and present here an interim analysis.
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- 2021
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24. CD45RO+ T-Cell Add Back and Prophylactic Blinatumomab Administration Post Tcrαβ/CD19-Depleted Haploidentical Transplantation in Pediatric Patients with High Risk Acute Leukemia
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Naik, Swati, Madden, Renee M., Mamcarz, Ewelina, Srinivasan, Ashok, Sharma, Akshay, Talleur, Aimee C, Epperly, Rebecca, Qudeimat, Amr, Suliman, Ali Y, Obeng, Esther A., Velasquez, Mireya Paulina, Hijano, Diego, Maron, Gabriela, Li, Ying, Gottschalk, Stephen, and Triplett, Brandon M
- Abstract
For patients receiving haploidentical donor (haplo) hematopoietic cell transplant (HCT), depletion of TCRαβ T cells from the haplo-graft allows for excellent outcomes and low rates of graft-versus-host disease (GVHD), albeit with a significant delay in immune reconstitution (IR). The adoptive transfer of donor T-cells has been used to optimize IR but increases the risk of GVHD. CD45RA-depleted (memory) T-cells are associated with low rates of alloreactivity and thereby GVHD but retain specificity for leukemic and viral antigens. We implemented a prospective trial utilizing escalating doses of CD45RA-depeleted T-cell as addback following TCRαβ/CD19-depleted haploHCT to improve IR. Patients with acute lymphoblastic leukemia (ALL) also received prophylactic Blinatumomab (Blina) following infusion of CD45RA-depeleted T-cell to overcome the risk immune escape secondary to HLA-loss and relapse (NCT03849651).
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- 2021
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25. CD45RO+ T-Cell Add Back and Prophylactic Blinatumomab Administration Post Tcrαβ/CD19-Depleted Haploidentical Transplantation in Pediatric Patients with High Risk Acute Leukemia
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Naik, Swati, Madden, Renee M., Mamcarz, Ewelina, Srinivasan, Ashok, Sharma, Akshay, Talleur, Aimee C, Epperly, Rebecca, Qudeimat, Amr, Suliman, Ali Y, Obeng, Esther A., Velasquez, Mireya Paulina, Hijano, Diego, Maron, Gabriela, Li, Ying, Gottschalk, Stephen, and Triplett, Brandon M
- Abstract
Sharma: CRISPR Therapeutics: Other, Research Funding; Novartis: Other: Salary support paid to institution; Vertex Pharmaceuticals/CRISPR Therapeutics: Other: Salary support paid to institution; Spotlight Therapeutics: Consultancy; Medexus Inc: Consultancy; Vindico Medical Education: Honoraria. Gottschalk: Catamaran Bio: Consultancy; Immatics: Membership on an entity's Board of Directors or advisory committees; Other: Other: patents and patent applications in the field of cancer cell and gene therapy ; Novartis: Consultancy; Tidal: Consultancy; Tessa Therapeutics: Consultancy. Triplett: Miltenyi: Other: Travel, meeting registration.
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- 2021
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26. Selective Targeting of Splicing Factor Mutant Hematopoietic Stem and Progenitor Cells Via STAT3 Inhibition
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Potts, Kathryn S, Cameron, Rosannah C., Ghazale, Noura, Gupta, Varun, Barajas, Juan Martin, Wallace, LaShanale, Leal-Cervantes, Ana, Aluri, Srinivas, Pradhan, Kith, McKinstry, Mia, Bai, Xiaoying, Choudhary, Gaurav S, Shastri, Aditi, Verma, Amit, Obeng, Esther A., and Bowman, Teresa V.
- Abstract
Shastri: Guidepoint: Consultancy; Kymera Therapeutics: Research Funding; Onclive: Honoraria; GLC: Consultancy. Verma: Curis: Research Funding; BMS: Research Funding; Stelexis: Consultancy, Current equity holder in publicly-traded company; Eli Lilly: Research Funding; Medpacto: Research Funding; Incyte: Research Funding; GSK: Research Funding; Novartis: Consultancy; Acceleron: Consultancy; Celgene: Consultancy; Stelexis: Current equity holder in publicly-traded company; Throws Exception: Current equity holder in publicly-traded company.
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- 2021
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27. CD45RA-Depleted Haploidentical Transplantation Combined with NK Cell Addback Results in Promising Long-Term Outcomes in Pediatric Patients with High-Risk Hematologic Malignancies
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Naik, Swati, Talleur, Aimee C, Li, Ying, Madden, Renee M., Mamcarz, Ewelina, Qudeimat, Amr, Sharma, Akshay, Srinivasan, Ashok, Suliman, Ali Y, Epperly, Rebecca, Obeng, Esther A., Velasquez, Mireya Paulina, Hijano, Diego, Maron, Gabriela, Gottschalk, Stephen, and Triplett, Brandon M
- Abstract
Sharma: Vindico Medical Education: Honoraria; CRISPR Therapeutics: Other, Research Funding; Vertex Pharmaceuticals/CRISPR Therapeutics: Other: Salary support paid to institution; Novartis: Other: Salary support paid to institution; Spotlight Therapeutics: Consultancy; Medexus Inc: Consultancy. Gottschalk: Novartis: Consultancy; Catamaran Bio: Consultancy; Other: Other: patents and patent applications in the field of cancer cell and gene therapy ; Immatics: Membership on an entity's Board of Directors or advisory committees; Tidal: Consultancy; Tessa Therapeutics: Consultancy. Triplett: Miltenyi: Other: Travel, meeting registration.
- Published
- 2021
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28. Proteasome inhibitors induce a terminal unfolded protein response in multiple myeloma cells
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Obeng, Esther A., Carlson, Louise M., Gutman, Delia M., Harrington, William J., Lee, Kelvin P., and Boise, Lawrence H.
- Abstract
Multiple myeloma (MM) is an incurable plasma cell malignancy. The 26S proteasome inhibitor, bortezomib, selectively induces apoptosis in MM cells; however, the nature of its selectivity remains unknown. Here we demonstrate that 5 different MM cell lines display similar patterns of sensitivity to 3 proteasome inhibitors (PIs) but respond differently to specific NF-κB inhibition. We further show that PIs initiate the unfolded protein response (UPR), a signaling pathway activated by the accumulation of misfolded proteins within the endoplasmic reticulum (ER). Consistent with reports that prosurvival/physiologic UPR components are required for B-cell differentiation into antibody-secreting cells, we found that MM cells inherently expressed the ER chaperones GRP78/Bip and GRP94/gp96. However, bortezomib rapidly induced components of the proapoptotic/terminal UPR, including PERK, the ER stress–specific eIF-2α kinase; ATF4, an ER stress–induced transcription factor; and its proapoptotic target, CHOP/GADD153. Consistent with our hypothesis that PIs induce the accumulation of misfolded ER-processed proteins, we found that the amount of immunoglobulin subunits retained within MM cells correlated with their sensitivity to PIs. These findings suggest that MM cells have a lower threshold for PI-induced UPR induction and ER stress–induced apoptosis because they constitutively express ER stress survival factors to function as secretory cells.
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- 2006
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29. Proteasome inhibitors induce a terminal unfolded protein response in multiple myeloma cells
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Obeng, Esther A., Carlson, Louise M., Gutman, Delia M., Harrington, William J., Lee, Kelvin P., and Boise, Lawrence H.
- Abstract
Multiple myeloma (MM) is an incurable plasma cell malignancy. The 26S proteasome inhibitor, bortezomib, selectively induces apoptosis in MM cells; however, the nature of its selectivity remains unknown. Here we demonstrate that 5 different MM cell lines display similar patterns of sensitivity to 3 proteasome inhibitors (PIs) but respond differently to specific NF-κB inhibition. We further show that PIs initiate the unfolded protein response (UPR), a signaling pathway activated by the accumulation of misfolded proteins within the endoplasmic reticulum (ER). Consistent with reports that prosurvival/physiologic UPR components are required for B-cell differentiation into antibody-secreting cells, we found that MM cells inherently expressed the ER chaperones GRP78/Bip and GRP94/gp96. However, bortezomib rapidly induced components of the proapoptotic/terminal UPR, including PERK, the ER stress–specific eIF-2α kinase; ATF4, an ER stress–induced transcription factor; and its proapoptotic target, CHOP/GADD153. Consistent with our hypothesis that PIs induce the accumulation of misfolded ER-processed proteins, we found that the amount of immunoglobulin subunits retained within MM cells correlated with their sensitivity to PIs. These findings suggest that MM cells have a lower threshold for PI-induced UPR induction and ER stress–induced apoptosis because they constitutively express ER stress survival factors to function as secretory cells.
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- 2006
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30. Splicing Control in Normal and Aberrant Erythropoiesis
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Obeng, Esther
- Abstract
Alternative splicing is employed by all eukaryotic cells to increase proteome diversity and to regulate gene expression. RNA sequencing analysis of purified populations of erythroblasts at different stages of maturation has led to the identification of a dynamic alternative splicing program that directly modulates the protein isoform expression of cytoskeletal proteins and genes involved in RNA processing, heme biosynthesis, and iron transport. Regulated interactions of multiple RNA-binding proteins and cis-regulatory sequences located within exons or their flanking introns promote or inhibit functional spliceosome assembly at splice junctions, leading to altered exon inclusion or intron retention. Exon skipping regulates tissue and stage specific isoform expression of red cell membrane cytoskeletal proteins including EPB41, ankyrin, and band 3. Intron retention can lead to a frame shift during translation and introduction of a premature termination codon (PTC), that marks the transcript for degradation via the nonsense mediated decay pathway (NMD) upon export from the nucleus into the cytoplasm. Intron retention leading to posttranscriptional regulation of gene expression during terminal erythroid maturation has been identified in genes involved in RNA processing and iron transport including SF3B1, SNRNP70, SLC25A37and SLC25A28. Mutations that alter mRNA splice sites or introduce PTCs lead to a variety of congenital anemias including beta thalassemia, hereditary pyropoikilocytosis, hereditary elliptocytosis, and hereditary spherocytosis. Aberrant mRNA splicing has subsequently been shown to lead to acquired anemias in subsets of patients with myelodysplastic syndromes (MDS). Somatic missense mutations in components of the spliceosome are the most common category of mutations in MDS. These point mutations lead to changes in the RNA binding specificity of the involved proteins and aberrant splicing of a subset of transcripts. Mutant SF3B1, the most commonly mutated splicing factor in MDS, has been shown to cause aberrant pre-mRNA splicing and an increase in transcripts predicted to undergo NMD due to use of upstream, cryptic 3‘ splice sites. Our group and others evaluating the strong genotype-phenotype association between SF3B1point mutations and subtypes of MDS with ring sideroblasts have shown that the expression of the mitochondrial iron transporter, ABCB7, is decreased in samples from SF3B1-mutant MDS patients due to cryptic 3‘ splice site selection and introduction of a PTC between exons 8 and 9. The identification and functional validation of additional aberrantly spliced mutant-SF3B1 target genes is ongoing, with the goal of understanding how point mutations in a core component of the mRNA splicing machinery can lead to such specific effects on erythroid maturation.
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- 2018
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31. Splicing Control in Normal and Aberrant Erythropoiesis
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Obeng, Esther
- Abstract
No relevant conflicts of interest to declare.
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- 2018
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32. Integrated Genomic and Proteomic Analysis of Murine CLL-like Cells Reveals SF3B1 Mutation to Impact DNA Damage Response and BCR Signaling
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Wang, Lili, Yin, Shanye, Martinez, Aina zurita, Regis, Fara Faye, Brooks, Angela, Herman, Sarah E. M., Ten Hacken, Elisa, Obeng, Esther, Campagna, Dean, Fleming, Mark D., Ebert, Benjamin L., Wiestner, Adrian, Leshchiner, Ignaty, Getz, Gad, Carrasco, Ruben D., and Wu, Catherine J.
- Abstract
Wiestner: Pharmacyclics LLC, an AbbVie Company: Research Funding. Wu:Neon Therapeutics: Equity Ownership.
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- 2018
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33. Integrated Genomic and Proteomic Analysis of Murine CLL-like Cells Reveals SF3B1Mutation to Impact DNA Damage Response and BCR Signaling
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Wang, Lili, Yin, Shanye, Martinez, Aina zurita, Regis, Fara Faye, Brooks, Angela, Herman, Sarah E.M., Ten Hacken, Elisa, Obeng, Esther, Campagna, Dean, Fleming, Mark D., Ebert, Benjamin L., Wiestner, Adrian, Leshchiner, Ignaty, Getz, Gad, Carrasco, Ruben D., and Wu, Catherine J.
- Abstract
Collective large-scale sequencing efforts have unexpectedly revealed the high frequency of mutations in the splicing factor genes (SF3B1, U2AF1, SRSF2, ZRSR2) in various solid and hematological cancers, suggesting the association of splicing dysregulation with tumorigenesis. Mutations in SF3B1occur in 5-20% of patients with chronic lymphocytic leukemia (CLL) and are associated with poorer overall survival and chemotherapy resistance. These mutations are restricted to hotspots (>50% at K700E site) and strongly co-occur with ATMmutations (loss-of-function) and deletion of 11q (ATMminimal deleted region). Numerous studies including ours have demonstrated that somatic alterations in this gene cause RNA splicing dysregulation, however, how this splicing factor mutation alone and in combination with ATMdeletion impacts cellular processes and contributes to CLL remains to be fully defined.
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- 2018
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34. Congenital Thrombocytopenia and Myelofibrosis Due to Germline Mutations in G6b-B— a Megakaryocyte-Specific Immunoreceptor Tyrosine-Based Inhibitory Motif (ITIM) Receptor
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Hofmann, Inga, Crispin, Andrew, Campagna, Dean, Calicchio, Monica, Schmitz-Abe, Klaus, Obeng, Esther A., Markianos, Kyriacos, Senis, Yotis A., and Fleming, Mark D.
- Abstract
Unlike primary myelofibrosis (PMF) in adults, which is one of the three classical BCR-ABL-negative myeloproliferative neoplasms (MPN), myelofibrosis in children is rare, occurring most commonly in patients with acute leukemia, particularly acute megakaryoblastic leukemia. Congenital or inherited forms of myelofibrosis (cMF) are exceedingly rare, and may occur in the context of patients with inherited platelet disorders. Here we describe 3 families with inherited thrombocytopenia and cMF due to germline null mutations in the megakaryocyte-specific immunoreceptor tyrosine-based inhibitory motif (ITIM) receptor G6b-B.
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- 2017
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35. Synthetic Lethal Interactions of MDS-Associated Spliceosomal Gene Mutations Identifies the Basis for Their Mutual Exclusivity
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Lee, Stanley Chun-Wei, Dilai, Khrystyna, Obeng, Esther A., Kim, Eunhee, Micol, Jean-Baptiste, Yoshimi, Akihide, Willekens, Christophe, Inoue, Daichi, Saada, Véronique, Cho, Hana, Chung, Young Rock, Palacino, James, Seiler, Michael, Buonamici, Silvia, Smith, Peter, Ebert, Benjamin L., Bradley, Robert, and Abdel-Wahab, Omar
- Abstract
Palacino: H3 Biomedicine Inc.: Employment. Seiler:H3 Biomedicine: Employment. Buonamici:H3 Biomedicine: Employment. Smith:H3 Biomedicine Inc.: Employment.
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- 2016
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36. Synthetic Lethal Interactions of MDS-Associated Spliceosomal Gene Mutations Identifies the Basis for Their Mutual Exclusivity
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Lee, Stanley Chun-Wei, Dilai, Khrystyna, Obeng, Esther A., Kim, Eunhee, Micol, Jean-Baptiste, Yoshimi, Akihide, Willekens, Christophe, Inoue, Daichi, Saada, Véronique, Cho, Hana, Chung, Young Rock, Palacino, James, Seiler, Michael, Buonamici, Silvia, Smith, Peter, Ebert, Benjamin L., Bradley, Robert, and Abdel-Wahab, Omar
- Abstract
Mutations in genes encoding RNA splicing factors constitute the most common class of alterations in patients with myelodysplasticsyndromes (MDS). These occur predominantly as heterozygous point mutations at restricted residues in SF3B1, SRSF2, and U2AF1in a mutually exclusive manner, suggesting that spliceosomal gene mutations confer gain-of-function with converging biological effects. However, recent studies suggest that mutations in each splicing factor result in distinct alterations in pre-mRNA splicing. It is therefore unclear if such mutual exclusivity is due to overlapping biological effects and/or synthetic lethal interactions. Furthermore, although cells bearing mutant splicing factors have been shown to require the wildtype allele for survival, whether these mutations can exist in a homozygous state is unknown. Here we addressed these questions by analyzing the effects of expressing SF3B1and SRSF2mutations simultaneously or in a homozygous state in vivo.
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- 2016
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37. Heterozygous Hotspot SF3B1 Mutations Found in Myelodysplastic Syndromes Downregulate Genes Involved in Differentiation of Erythroid Cells
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Buonamici, Silvia, Darman, Rachel, Perino, Samantha, Agrawal, Anant, Peng, Shouyong, Seiler, Michael, Lim, Kian Huat, Bhavsar, Erica B., Feala, Jacob, Obeng, Esther A., Bailey, Suzanna, Chan, Betty, Fekkes, Peter, Keaney, Gregg F., Kumar, Pavan, Kunii, Kaiko, Lee, Linda, Mackenzie, Crystal, Matijevic, Mark, Mizui, Yoshiharu, Myint, Khin, Park, Eunice, Pazolli, Ermira, Thomas, Michael, Wang, John, Warmuth, Markus, Yu, Lihua, Zhu, Ping, Furman, Richard R., Ebert, Benjamin L, and Smith, Peter G
- Abstract
Buonamici: H3 Biomedicine: Employment. Darman:H3 Biomedicine: Employment. Perino:H3 Biomedicine: Employment. Agrawal:H3 Biomedicine: Employment. Peng:H3 Biomedicine: Employment. Seiler:H3 Biomedicine: Employment. Feala:H3 Biomedicine: Employment. Bailey:H3 Biomedicine: Employment. Chan:H3 Biomedicine: Employment. Fekkes:H3 Biomedicine: Employment. Keaney:H3 Biomedicine: Employment. Kumar:H3 Biomedicine: Employment. Kunii:H3 Biomedicine: Employment. Lee:H3 Biomedicine: Employment. Mackenzie:Eisai: Employment. Matijevic:Eisai: Employment. Mizui:H3 Biomedicine: Employment. Myint:Eisai: Employment. Park:H3 Biomedicine: Employment. Pazolli:H3 Biomedicine: Employment. Thomas:H3 Biomedicine: Employment. Wang:H3 Biomedicine: Employment. Warmuth:H3 Biomedicine: Employment. Yu:H3 Biomedicine: Employment. Zhu:H3 Biomedicine: Employment. Furman:Acerta Pharma BV: Research Funding; Gilead: Consultancy; Pharmacyclics LLC, an AbbVie Company: Consultancy, Honoraria, Speakers Bureau. Ebert:Celgene: Consultancy; H3 Biomedicine: Consultancy; Genoptix: Consultancy, Patents & Royalties. Smith:H3 Biomedicine: Employment.
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- 2015
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38. Expressionof Sf3b1- K700Ein Murine B Cells Causes Pre-mRNA Splicing and Altered B Cell Differentiation and Function
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Wang, Lili, Gambe, Rutendo, Fan, Jean, Wan, Youzhong, Brooks, Angela N, Sun, Jing, Obeng, Esther A., Neuberg, Donna S, Meyerson, Matthew, Fleming, Mark D., Ebert, Benjamin L., Carrasco, Ruben D., and Wu, Catherine J
- Abstract
No relevant conflicts of interest to declare.
- Published
- 2015
- Full Text
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39. Expressionof Sf3b1-K700Ein Murine B Cells Causes Pre-mRNA Splicing and Altered B Cell Differentiation and Function
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Wang, Lili, Gambe, Rutendo, Fan, Jean, Wan, Youzhong, Brooks, Angela N, Sun, Jing, Obeng, Esther A., Neuberg, Donna S, Meyerson, Matthew, Fleming, Mark D., Ebert, Benjamin L., Carrasco, Ruben D., and Wu, Catherine J
- Abstract
Mutations in the RNA splicing factor SF3B1have been identified by large-scale sequencing as putative drivers in chronic lymphocytic leukemia (CLL), but their precise roles in the pathogenesis of CLL remains unknown. Although prior transcriptomic studies using primary CLL samples have led to the appreciation of altered RNA splicing in association with these mutations, understanding of their impact on cellular function has been complicated by their variable mutant allele frequency across samples as well as their common co-occurrence with other heterogeneous gene mutations. We therefore generated a mouse line that conditionally expresses the commonly occurring Sf3b1-K700E mutation at its endogenous murine locus. We obtained B-cell lineage specific expression of the mutant allele by crossing heterozygous floxed Sf3b1-K700E mice with homozygous CD19-Cre knockin mice. We confirmed that expression of the mutant allele was uniquely present as a heterozygous mutation in B cells, but not in other cell lineages.
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- 2015
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40. Heterozygous Hotspot SF3B1 Mutations Found in Myelodysplastic Syndromes Downregulate Genes Involved in Differentiation of Erythroid Cells
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Buonamici, Silvia, Darman, Rachel, Perino, Samantha, Agrawal, Anant, Peng, Shouyong, Seiler, Michael, Lim, Kian Huat, Bhavsar, Erica B., Feala, Jacob, Obeng, Esther A., Bailey, Suzanna, Chan, Betty, Fekkes, Peter, Keaney, Gregg F., Kumar, Pavan, Kunii, Kaiko, Lee, Linda, Mackenzie, Crystal, Matijevic, Mark, Mizui, Yoshiharu, Myint, Khin, Park, Eunice, Pazolli, Ermira, Thomas, Michael, Wang, John, Warmuth, Markus, Yu, Lihua, Zhu, Ping, Furman, Richard R., Ebert, Benjamin L, and Smith, Peter G
- Abstract
Heterozygous mutations in several core members of the spliceosome complex have been reported in Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML). In particular high frequency SF3B1 hotspot mutations, a component of the U2 complex involved in the interaction with the branch point (BP) and recognition of the 3' splice sites (ss) during splicing, have been identified in Refractory Anemia with Ringed Sideroblasts (RARS) a subtype of MDS. Using computational analyses of RNAseq from several cancer types including RARS, we identified that SF3B1 hotspot mutations induce aberrant 3'ss selection by recognizing a cryptic AG located between 15 to 24 nucleotides upstream of the canonical AG. Experimental confirmation of these motif features was performed using minigenes in SF3B1 mutant cells. Furthermore, we discovered that SF3B1 mutant utilized a different BP from that used by SF3B1 wild-type providing novel mechanistic insights into changes in function induced by the hotspot mutations. The induction of aberrant splicing can introduce premature termination codons thus targeting mRNA for degradation by Nonsense Mediated Decay (NMD). We predicted that close to 50% of the aberrantly spliced genes would be subject to NMD and showed (using isogenic Nalm-6 cells engineered by AAV homology to express SF3B1K700Eor SF3B1K700K) that several of these genes were downregulated at the transcript and protein levels. These downregulated genes/proteins might be involved in the pathogenesis of SF3B1 mutant cancers. Interestingly, pathway analysis of genes differentially expressed or aberrantly spliced in SF3B1 mutant compared to wild-type in RARS samples identified cell differentiation and epigenetics as the primary misregulated pathways. To study the impact of SF3B1 mutations on differentiation, we used the TF-1 differentiation cell model where erythroid differentiation is induced by treatment with erythropoietin (EPO). EPO treatment, as expected, induced erythroid differentiation in TF-1 cells transduced with SF3B1WT, but a block in erythroid differentiation was observed in TF-1 cells transduced with SF3B1K700E(the most common mutation in MDS and chronic lymphocytic leukemia (CLL)). Intriguingly, SF3B1G742D, which is found mutated in CLL but not MDS, did not block differentiation in this myeloid differentiation model, implying that specific SF3B1 mutations and splicing aberrations have important context dependent effects. Pathway analysis comparing SF3B1K700Evs. SF3B1WTor SF3B1G742Didentified several genes involved in heme biosynthesis or downstream of GATA1 to be downregulated (such as, AHSP, ALAS2, CCL5, CD36, EPOR, GP1BB, HBB, HBE1, HBG1, PRG2) in SF3B1K700Ecells only. This is consistent with the role of SF3B1K700Ein RARS. In our analyses, we also identified that ABCB7 is aberrantly spliced and that the aberrant transcript is subject to NMD, causing downregulation of the canonical transcript and protein. ABCB7 is a mitochondrial transporter important in cellular iron metabolism and in heme production; moreover, partial loss of function mutation in ABCB7 has been identified in X-linked sideroblastic anemia and ataxia, demonstrating an iron overload phenotype in cells with defective ABCB7. Interestingly, when ABCB7 was knocked down in TF-1 cells we observed block in differentiation similar to that observed in SF3B1K700Ecells suggesting a link between SF3B1 mutation and ABCB7 levels and impaired differentiation. Taken together, these data suggest that SF3B1 mutations induce aberrant splicing and as a consequence downregulation of several genes that contribute to the block in erythroid differentiation, one of the key biological defects observed in MDS.
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- 2015
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41. Mutant Splicing Factor 3b Subunit 1 (SF3B1) Causes Dysregulated Erythropoiesis and a Stem Cell Disadvantage
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Obeng, Esther A., McConkey, Marie E., Campagna, Dean, Schneider, Rebekka K., Chen, Michelle C., Schmidt, Paul J, Mullally, Ann, Fleming, Mark D., and Ebert, Benjamin L.
- Abstract
No relevant conflicts of interest to declare.
- Published
- 2014
- Full Text
- View/download PDF
42. Cancer-Associated Mutations in SF3B1 Exhibit Neomorphic Splicing Activity and Block Erythroid Differentiation
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Buonamici, Silvia, Perino, Samantha, Lim, Kian Huat, Feala, Jacob, Obeng, Esther A., Aicher, Michelle, Aird, Daniel, Bailey, Suzanna, Berkenblit, Anna, Chan, Betty, Erik, Corcoran, Corson, Laura, Darman, Rachel, Fekkes, Peter, Furman, Richard R., Keaney, Gregg F, Kumar, Pavan, Kunii, Kaiko, Lee, Linda, Mackenzie, Crystal, Park, Eunice, Puyang, Xiaoling, Selvaraj, Anand, Thomas, Michael, Wang, John, Warmuth, Markus, Yu, Lihua, Zhu, Ping, Mizui, Yoshiharu, Ebert, Benjamin L., and Smith, Peter G
- Abstract
Buonamici: H3 Biomedicine: Employment. Perino:H3 Biomedicine: Employment. Lim:H3 Biomedicine: Employment. Feala:H3 Biomedicine: Employment. Aicher:H3 Biomedicine: Employment. Aird:H3 Biomedicine: Employment. Bailey:H3 Biomedicine: Employment. Berkenblit:H3 Biomedicine: Employment. Chan:H3 Biomedicine: Employment. Erik:H3 Biomedicine: Employment. Corson:H3 Biomedicine: Employment. Darman:H3 Biomedicine: Employment. Fekkes:H3 Biomedicine: Employment. Furman:Pharmacyclics: Consultancy, Speakers Bureau. Keaney:H3 Biomedicine: Employment. Kumar:Eisai: Employment. Kunii:H3 Biomedicine: Employment. Lee:H3 Biomedicine: Employment. Mackenzie:Eisai: Employment. Park:H3 Biomedicine: Employment. Puyang:H3 Biomedicine: Employment. Selvaraj:H3 Biomedicine: Employment. Thomas:H3 Biomedicine: Employment. Wang:H3 Biomedicine: Employment. Warmuth:H3 Biomedicine: Employment. Yu:H3 Biomedicine: Employment. Zhu:H3 Biomedicine: Employment. Mizui:H3 Biomedicine: Employment. Smith:H3 Biomedicine: Employment.
- Published
- 2014
- Full Text
- View/download PDF
43. Mutant Splicing Factor 3b Subunit 1 (SF3B1) Causes Dysregulated Erythropoiesis and a Stem Cell Disadvantage
- Author
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Obeng, Esther A., McConkey, Marie E., Campagna, Dean, Schneider, Rebekka K., Chen, Michelle C., Schmidt, Paul J, Mullally, Ann, Fleming, Mark D., and Ebert, Benjamin L.
- Abstract
Recurrent, heterozygous, somatic mutations in components of the mRNA spliceosome complex were recently identified in over 60% of myelodysplastic syndrome (MDS) patients. Splicing factor mutations are thought to be founding mutations in MDS based on their allele fraction at diagnosis. Splicing factor 3b Subunit 1 (SF3B1) is the most frequently mutated splicing factor in MDS. SF3B1 mutations are highly associated (70 – 85% of cases) with refractory anemia with ring sideroblasts (RARS), a morphologic subtype of MDS characterized by the presence of erythroid precursors with perinuclear iron-laden mitochondria in the bone marrow. The pathophysiological role of SF3B1 mutations in MDS has yet to be elucidated. To explore the biology of SF3B1 mutations, we generated a heterozygous conditional knock-in mouse model of the most common SF3B1 mutation, K700E.
- Published
- 2014
- Full Text
- View/download PDF
44. Cancer-Associated Mutations in SF3B1 Exhibit Neomorphic Splicing Activity and Block Erythroid Differentiation
- Author
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Buonamici, Silvia, Perino, Samantha, Lim, Kian Huat, Feala, Jacob, Obeng, Esther A., Aicher, Michelle, Aird, Daniel, Bailey, Suzanna, Berkenblit, Anna, Chan, Betty, Erik, Corcoran, Corson, Laura, Darman, Rachel, Fekkes, Peter, Furman, Richard R., Keaney, Gregg F, Kumar, Pavan, Kunii, Kaiko, Lee, Linda, Mackenzie, Crystal, Park, Eunice, Puyang, Xiaoling, Selvaraj, Anand, Thomas, Michael, Wang, John, Warmuth, Markus, Yu, Lihua, Zhu, Ping, Mizui, Yoshiharu, Ebert, Benjamin L., and Smith, Peter G
- Abstract
Recently, heterozygous mutations in several spliceosome genes have been observed in hematological and solid cancers, but their functional role in these diseases is not well understood. Among these, SF3B1 is the most commonly mutated spliceosome gene in myelodysplastic syndromes (MDS) and chronic lymphocytic leukemia (CLL). SF3B1 is part of the U2 complex involved in the recognition of the 3’ splice sites (3’ss) during early spliceosome assembly.
- Published
- 2014
- Full Text
- View/download PDF
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