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Drug resistance in chronic myeloid leukaemia (CML) may occur via mutations in the causative BCR::ABL1 fusion or BCR::ABL1-independent mechanisms. We analysed 48 patients with BCR::ABL1-independent resistance for the presence of secondary fusion genes by RNA sequencing. We identified 10 of the most frequently detected secondary fusions in 21 patients. Validation studies, cell line models, gene expression analysis and drug screening revealed differences with respect to proliferation rate, differentiation and drug sensitivity. Notably, expression of RUNX1::MECOM led to resistance to ABL1 tyrosine kinase inhibitors in vitro. These results suggest secondary fusions contribute to BCR::ABL1-independent resistance and may be amenable to combined therapies.

Using ex vivo drug screening of primary patient specimens, we identified the combination of the p38 MAPK inhibitor doramapimod (DORA) with the BCL2 inhibitor venetoclax (VEN) as demonstrating broad, enhanced efficacy compared with each single agent across 335 acute myeloid leukemia (AML) patient samples while sparing primary stromal cells. Single-agent DORA and VEN sensitivity was associated with distinct, nonoverlapping tumor cell differentiation states. In particular, increased monocytes, M4/M5 French-American-British classification, and CD14+ immunophenotype tracked with sensitivity to DORA and resistance to VEN but were mitigated with the combination. Increased expression of MAPK14 and BCL2, the respective primary targets of DORA and VEN, were observed in monocytic and undifferentiated leukemias, respectively. Enrichment for DORA and VEN sensitivities was observed in AML with monocyte-like and progenitor-like transcriptomic signatures, respectively, and these associations diminished with the combination. The mechanism underlying the combination’s enhanced efficacy may result from inhibition of p38 MAPK-mediated phosphorylation of BCL2, which in turn enhances sensitivity to VEN. These findings suggest exploiting complementary drug sensitivity profiles with respect to leukemic differentiation state, such as dual targeting of p38 MAPK and BCL2, offers opportunity for broad, enhanced efficacy across the clinically challenging heterogeneous landscape of AML.

Purpose: Targeted therapeutics are a goal of medicine. Methods for targeting T-cell lymphoma lack specificity for the malignant cell, leading to elimination of healthy cells. The T-cell receptor (TCR) is designed for antigen recognition. T-cell malignancies expand from a single clone which expresses one of 48 TCR variable beta (Vβ) genes, providing a distinct therapeutic target. We hypothesized that a monoclonal antibody that is exclusive to a specific Vβ would eliminate the malignant clone while having minimal effects on healthy T-cells. Experimental Design: We identified a patient with large granular T-cell leukemia, and sequenced his circulating T-cell population, 95% of which expressed Vβ13.3. We developed a panel of anti-Vβ13.3 antibodies to test for binding and elimination of the malignant T-cell clone. Results: Therapeutic antibody candidates bound the malignant clone with high affinity. Antibodies killed engineered cell lines expressing the patient TCR Vβ13.3 by antibody-dependent cellular cytotoxicity, TCR-mediated activation-induced cell death, and exhibited specific killing of patient malignant T-cells in combination with exogenous NK cells. EL4 cells expressing the patient’s TCR Vβ13.3 were also killed by antibody administration in an in vivo murine model. Conclusions: This approach serves as an outline for development of therapeutics that can treat clonal T-cell based malignancies and potentially other T-cell-mediated diseases.

PDF file - 73KB, CML (Table 1) and AML (Table 2) Patient sample information.

Sensitivity of CD74-ROS1D2033N to a spectrum of ROS1 kinase inhibitors. Cell growth and viability of Ba/F3 cells expressing native CD74-ROS1 or CD74-ROS1D2033N after 72 h exposure to (A) foretinib, (B) PF-06463922, (C) ceritinib, and (D) brigatinib. Results are shown as mean viability normalized to vehicle-treated control {plus minus} SEM (n = 3 to 6). (E) Table listing inhibitor IC50 (nM) for native versus ROS1D2033N mutant. (F) Graph depicts fold increase in IC50 (nM) for Ba/F3 cells expressing CD74-ROS1D2033N compared to native CD74-ROS1.

Supplementary Figure Legends and Supplementary Tables. Supplementary Table S1. Detailed clinical and diagnostic summary for a patient with ROS1-rearranged lung adenocarcinoma who developed acquired resistance to crizotinib and responded to cabozantinib. Supplementary Table S2. Next-generation sequencing results reveal a novel acquired ROS1 D2033N mutation in the CD74-ROS1 kinase domain.

PDF file - 58KB, Combination index (CI) values.

Purpose: Colony-stimulating factor 3 receptor (CSF3R) mutations have been identified in the majority of chronic neutrophilic leukemia (CNL) and a smaller percentage of atypical chronic myeloid leukemia (aCML) cases. Although CSF3R point mutations (e.g., T618I) are emerging as key players in CNL/aCML, the significance of rarer CSF3R mutations is unknown. In this study, we assess the importance of the CSF3R T640N mutation as a marker of CNL/aCML and potential therapeutic target.Experimental Design: Sanger sequencing of leukemia samples was performed to identify CSF3R mutations in CNL and aCML. The oncogenicity of the CSF3R T640N mutation relative to the T618I mutation was assessed by cytokine independent growth assays and by mouse bone marrow transplant. Receptor dimerization and O-glycosylation of the mutants was assessed by Western blot, and JAK inhibitor sensitivity was assessed by colony assay.Results: Here, we identify a CSF3R T640N mutation in two patients with CNL/aCML, one of whom was originally diagnosed with MDS and acquired the T640N mutation upon evolution of disease to aCML. The T640N mutation is oncogenic in cellular transformation assays and an in vivo mouse bone marrow transplantation model. It exhibits many similar phenotypic features to T618I, including ligand independence and altered patterns of O-glycosylation—despite the transmembrane location of T640 preventing access by GalNAc transferase enzymes. Cells transformed by the T640N mutation are sensitive to JAK kinase inhibition to a similar degree as cells transformed by CSF3R T618I.Conclusions: Because of its similarities to CSF3R T618I, the T640N mutation likely has diagnostic and therapeutic relevance in CNL/aCML. Clin Cancer Res; 22(3); 757–64. ©2015 AACR.

Supplemental Figures and Tables. Figure S1 - Immunoblotting characterization of GMB-101 and GMB-475. Figure S2 - Validation of degradation and compound toxicity via comparison of GMB-475 and GMB-651. Figure S3 - Combinations of ATP competitive TKIs with PROTAC. Figure S4 - Schematic of scaffolding roles, VHL ligand and imatinib controls for VHL and pGAB2. Figure S5 - Patient sample sorting workflow and additional patient samples. Table S2 - Patient characteristics for primary samples

Purpose: Rearranged ROS1 is a crizotinib-sensitive oncogenic driver in lung cancer. The development of acquired resistance, however, poses a serious clinical challenge. Consequently, experimental and clinical validation of resistance mechanisms and potential second-line therapies is essential.Experimental Design: We report the discovery of a novel, solvent-front ROS1D2033N mutation in a patient with CD74-ROS1–rearranged lung adenocarcinoma and acquired resistance to crizotinib. Crizotinib resistance of CD74-ROS1D2033N was functionally evaluated using cell-based assays and structural modeling.Results: In biochemical and cell-based assays, the CD74-ROS1D2033N mutant demonstrated significantly decreased sensitivity to crizotinib. Molecular dynamics simulation revealed compromised crizotinib binding due to drastic changes in the electrostatic interaction between the D2033 residue and crizotinib and reorientation of neighboring residues. In contrast, cabozantinib binding was unaffected by the D2033N substitution, and inhibitory potency against the mutant was retained. Notably, cabozantinib treatment resulted in a rapid clinical and near-complete radiographic response in this patient.Conclusions: These results provide the first example of successful therapeutic intervention with targeted therapy to overcome crizotinib resistance in a ROS1-rearranged cancer. Clin Cancer Res; 22(10); 2351–8. ©2015 AACR.

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PDF file - 212KB, SET is overexpressed in primary AML cells and OP449 inhibits growth of primary AML patient cells.

PDF file - 66KB, OP449 in combination with ABL1 tyrosine kinase inhibitors inhibits BCR-ABL1 signaling in CML cells and cellular growth of primary CML blastic phase cells ex vivo.

Purpose: The SET oncoprotein, a potent inhibitor of the protein phosphatase 2A (PP2A), is overexpressed in leukemia. We evaluated the efficacy of SET antagonism in chronic myeloid leukemia (CML) and acute myeloid leukemia (AML) cell lines, a murine leukemia model, and primary patient samples using OP449, a specific, cell-penetrating peptide that antagonizes SET's inhibition of PP2A.Experimental Design:In vitro cytotoxicity and specificity of OP449 in CML and AML cell lines and primary samples were measured using proliferation, apoptosis, and clonogenic assays. Efficacy of target inhibition by OP449 was evaluated by immunoblotting and PP2A assay. In vivo antitumor efficacy of OP449 was measured in human HL-60 xenografted murine model.Results: We observed that OP449 inhibited growth of CML cells including those from patients with blastic phase disease and patients harboring highly drug-resistant BCR-ABL1 mutations. Combined treatment with OP449 and ABL1 tyrosine kinase inhibitors was significantly more cytotoxic to K562 cells and primary CD34+ CML cells. SET protein levels remained unchanged with OP449 treatment, but BCR-ABL1–mediated downstream signaling was significantly inhibited with the degradation of key signaling molecules such as BCR-ABL1, STAT5, and AKT. Similarly, AML cell lines and primary patient samples with various genetic lesions showed inhibition of cell growth after treatment with OP449 alone or in combination with respective kinase inhibitors. Finally, OP449 reduced the tumor burden of mice xenografted with human leukemia cells.Conclusions: We demonstrate a novel therapeutic paradigm of SET antagonism using OP449 in combination with tyrosine kinase inhibitors for the treatment of CML and AML. Clin Cancer Res; 20(8); 2092–103. ©2014 AACR.

Although the use of ATP-competitive tyrosine kinase inhibitors of oncoprotein BCR-ABL1 has enabled durable responses in patients with chronic myeloid leukemia (CML), issues of drug resistance and residual leukemic stem cells remain. To test whether the degradation of BCR-ABL1 kinase could offer improved response, we developed a series of proteolysis-targeting chimera (PROTAC) that allosterically target BCR-ABL1 protein and recruit the E3 ligase Von Hippel-Lindau, resulting in ubiquitination and subsequent degradation of the oncogenic fusion protein. In both human CML K562 cells and murine Ba/F3 cells expressing BCR-ABL1, lead compound GMB-475 induced rapid proteasomal degradation and inhibition of downstream biomarkers, such as STAT5, and showed increased sensitivity compared with diastereomeric controls lacking degradation activity. Notably, GMB-475 inhibited the proliferation of certain clinically relevant BCR-ABL1 kinase domain point mutants and further sensitized Ba/F3 BCR-ABL1 cells to inhibition by imatinib, while demonstrating no toxicity toward Ba/F3 parental cells. Reverse phase protein array analysis suggested additional differences in levels of phosphorylated SHP2, GAB2, and SHC associated with BCR-ABL1 degradation. Importantly, GMB-475 reduced viability and increased apoptosis in primary CML CD34+ cells, with no effect on healthy CD34+ cells at identical concentrations. GMB-475 degraded BCR-ABL1 and reduced cell viability in primary CML stem cells. Together, these findings suggest that combined BCR-ABL1 kinase inhibition and protein degradation may represent a strategy to address BCR-ABL1–dependent drug resistance, and warrant further investigation into the eradication of persistent leukemic stem cells, which rely on neither the presence nor the activity of the BCR-ABL1 protein for survival.Significance:Small-molecule–induced degradation of BCR-ABL1 in CML provides an advantage over inhibition and provides insights into CML stem cell biology.

Identifying resistance mutations in a drug target provides crucial information. Lentiviral transduction creates multiple types of mutations due to the error-prone nature of the HIV-1 reverse transcriptase (RT). Here we optimized and leveraged this property to identify drug resistance mutations, developing a technique we term LentiMutate. This technique was validated by identifying clinically relevant EGFR resistance mutations, then applied to two additional clinical anticancer drugs: imatinib, a BCR-ABL inhibitor, and AMG 510, a KRAS G12C inhibitor. Novel deletions in BCR-ABL1 conferred resistance to imatinib. In KRAS-G12C or wild-type KRAS, point mutations in the AMG 510 binding pocket or oncogenic non-G12C mutations conferred resistance to AMG 510. LentiMutate should prove highly valuable for clinical and preclinical cancer-drug development.Significance:LentiMutate can evaluate a drug's on-target activity and can nominate resistance mutations before they occur in patients, which could accelerate and refine drug development to increase the survival of patients with cancer.

Imatinib and other BCR-ABL1 inhibitors are effective therapies for chronic myelogenous leukemia (CML), but these inhibitors target additional kinases including KIT, raising the question of whether off-target effects contribute to clinical efficacy. On the basis of its involvement in CML pathogenesis, we hypothesized that KIT may govern responses of CML cells to imatinib. To test this, we assessed the growth of primary CML progenitor cells under conditions of sole BCR-ABL1, sole KIT, and dual BCR-ABL1/KIT inhibition. Sole BCR-ABL1 inhibition suppressed mature CML progenitor cells, but these effects were largely abolished by stem cell factor (SCF) and maximal suppression required dual BCR-ABL1/KIT inhibition. In contrast, KIT inhibition did not add to the effects of BCR-ABL1 inhibition in primitive progenitors, represented by CD34+38− cells. Long-term culture-initiating cell assays on murine stroma revealed profound depletion of primitive CML cells by sole BCR-ABL1 inhibition despite the presence of SCF, suggesting that primitive CML cells are unable to use SCF as a survival factor upon BCR-ABL1 inhibition. In CD34+38+ cells, SCF strongly induced pAKTS473 in a phosphoinositide 3-kinase (PI3K)–dependent manner, which was further enhanced by inhibition of BCR-ABL1 and associated with increased colony survival. In contrast, pAKTS473 levels remained low in CD34+38− cells cultured under the same conditions. Consistent with reduced response to SCF, KIT surface expression was significantly lower on CD34+38− compared with CD34+38+ CML cells, suggesting a possible mechanism for the differential effects of SCF on mature and primitive CML progenitor cells. Cancer Res; 73(18); 5775–86. ©2013 AACR.

ALK is expressed in the B-ALL sample with the ALK A348D mutation.

PDF file, 421K, Supplementary Figure 1: Specificity of imatinib, dasatinib, PPY-A, BAW667, and a SCF-blocking antibody on Mo7ep210BCR-ABL1 cells. Supplementary Figure 2: Comparison of CFU-GM colony formation upon sole BCR-ABL1 inhibition (PPY-A), sole KIT inhibition or dual BCR-ABL1/KIT inhibition. Supplementary Figure 3: Inhibition of KIT in human cells through use of a lentiviral vector for simultaneous expression of shKIT and GFP . Supplementary Figure 4: Analysis of CML CFU-GM colony growth following removal of the individual cytokines. Supplementary Figure 5: BCR-ABL1 and KIT signaling influence activation of AKT associated with a reduction of Foxo3A. Supplementary Table 1. Activity profile of BAW667.

Supplementary Tables 1-4, Figure 1 from The ABL Switch Control Inhibitor DCC-2036 Is Active against the Chronic Myeloid Leukemia Mutant BCR-ABLT315I and Exhibits a Narrow Resistance Profile

Results of LentiMutate on KRAS-G12C cDNA and AMG 510 in the H358 cell line. Values shown are the averages of two biological replicates. The fold enrichment of "All nucleotides" is the sum of substitution rates for all nucleotides in the with doxycycline replicates divided by the sum of substitution rates for all nucleotides without doxycycline (parental) at one nucleotide position. Nucleotide w/ max error refers to the highest substitution rate for a single nucleotide at one nucleotide position.

PDF file - 1469K, Different ABL TKIs demonstrate potency differences in relative cellular and biochemical target inhibition (S1); Incomplete restoration of BCR-ABL signaling activity following washout of dasatinib or ponatinib tracks with intracellular drug retention and commitment to apoptosis in LAMA cells (S2); Incomplete restoration of BCR-ABL signaling activity following washout of nilotinib, imatinib, or DCC-2036 tracks with intracellular drug retention and commitment to apoptosis in LAMA cells (S3); BCR-ABL signaling is partially attenuated in conditions of continuous low concentrations of ABL TKIs that induce apoptosis (S4); Commitment to apoptosis in primary CML cells tracks with intracellular retention of ABL TKIs (S5); Intracellular retention of dasatinib, nilotinib, and imatinib is reduced following washout in primary CML cells harboring the resistant BCR-ABLT315I mutant (S6).

Expression of RUVBL1 variants identified by LentiMutate confer resistance to compound B Individual cDNAs were expressed in the H1993 (left) or H2009 (right) cell lines under a doxycycline-inducible promoter. Values shown are averages of two biological replicates {plus minus} the standard deviation.

PDF file - 337K, Summary of mean fluorescence intensity values for levels of CrkL and STAT5 phosphorylation in K562 cells following acute ABL TKI exposure followed by standard and expanded washout (S1); Kinase:inhibitor dissociation off-rates for ABL TKIs (S2).

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Tyrosine kinase inhibitors (TKIs) targeting BCR-ABL1 have revolutionized therapy for chronic myeloid leukemia (CML), paving the way for clinical development in other diseases. Despite success, targeting leukemic stem cells and overcoming drug resistance remain challenges for curative cancer therapy. To identify drivers of kinase-independent TKI resistance in CML, we performed genome-wide expression analyses on TKI-resistant versus sensitive CML cell lines, revealing a nuclear factor-kappa B (NF-κB) expression signature. Nucleocytoplasmic fractionation and luciferase reporter assays confirmed increased NF-κB activity in the nucleus of TKI-resistant versus sensitive CML cell lines and CD34+ patient samples. Two genes that were upregulated in TKI-resistant CML cells were proteasome 26S subunit, non-ATPases 1 (PSMD1) and 3 (PSMD3), both members of the 19S regulatory complex in the 26S proteasome. PSMD1 and PSMD3 were also identified as survival-critical genes in a published small hairpin RNA library screen of TKI resistance. We observed markedly higher levels of PSMD1 and PSMD3 mRNA in CML patients who had progressed to the blast phase compared with the chronic phase of the disease. Knockdown of PSMD1 or PSMD3 protein correlated with reduced survival and increased apoptosis in CML cells, but not in normal cord blood CD34+ progenitors. Luciferase reporter assays and immunoblot analyses demonstrated that PSMD1 and PSMD3 promote NF-κB protein expression in CML, and that signal transducer and activator of transcription 3 (STAT3) further activates NF-κB in scenarios of TKI resistance. Our data identify NF-κB as a transcriptional driver in TKI resistance, and implicate PSMD1 and PSMD3 as plausible therapeutic targets worthy of future investigation in CML and possibly other malignancies.

Chronic myeloid leukemia (CML) is successfully treated with BCR-ABL1 tyrosine kinase inhibitors, but a significant percentage of patients develop resistance. Insulin receptor substrate 1 (IRS1) has been shown to constitutively associate with BCR-ABL1, and IRS1-specific silencing leads to antineoplastic effects in CML cell lines. Here, we characterized the efficacy of NT157, a pharmacological inhibitor of IGF1R-IRS1/2, in CML cells and observed significantly reduced cell viability and proliferation, accompanied by induction of apoptosis. In human K562 cells and in murine Ba/F3 cells, engineered to express either wild-type BCR-ABL1 or the imatinib-resistant BCR-ABL1T315I mutant, NT157 inhibited BCR-ABL1, IGF1R, IRS1/2, PI3K/AKT/mTOR, and STAT3/5 signaling, increased CDKN1A, FOS and JUN tumor suppressor gene expression, and reduced MYC and BCL2 oncogenes. NT157 significantly reduced colony formation of human primary CML cells with minimal effect on normal hematopoietic cells. Exposure of primary CML cells harboring BCR-ABL1T315I to NT157 resulted in increased apoptosis, reduced cell proliferation and decreased phospho-CRKL levels. In conclusion, NT157 has antineoplastic effects on BCR-ABL1 leukemogenesis, independent of T315I mutational status.

The Philadelphia chromosome (Ph) resulting from the t(9;22) translocation generates the oncogenic BCR::ABL1 fusion protein that is most commonly associated with chronic myeloid leukemia (CML) and Ph-positive (Ph+) acute lymphoblastic leukemia (ALL). There are also rare instances of patients (≤1%) with newly diagnosed acute myeloid leukemia (AML) that harbor this translocation (Paietta et al.

Chronic neutrophilic leukemia (CNL), atypical chronic myeloid leukemia (aCML), and myelodysplastic/myeloproliferative neoplasms, unclassifiable (MDS/MPN-U) are a group of rare and heterogeneous myeloid disorders. There is strong morphologic resemblance among these distinct diagnostic entities as well as a lack of specific molecular markers and limited understanding of disease pathogenesis, which has made diagnosis challenging in certain cases. The treatment has remained empirical, resulting in dismal outcomes. We, therefore, performed whole-exome and RNA sequencing of these rare hematologic malignancies and present the most complete survey of the genomic landscape of these diseases to date. We observed a diversity of combinatorial mutational patterns that generally do not cluster within any one diagnosis. Gene expression analysis reveals enrichment, but not cosegregation, of clinical and genetic disease features with transcriptional clusters. In conclusion, these groups of diseases represent a continuum of related diseases rather than discrete diagnostic entities.

Identifying resistance mutations in a drug target provides crucial information. Lentiviral transduction creates multiple types of mutations due to the error-prone nature of the HIV-1 reverse transcriptase (RT). Here we optimized and leveraged this property to identify drug resistance mutations, developing a technique we term LentiMutate. This technique was validated by identifying clinically relevant EGFR resistance mutations, then applied to two additional clinical anticancer drugs: imatinib, a BCR-ABL inhibitor, and AMG 510, a KRAS G12C inhibitor. Novel deletions in BCR-ABL1 conferred resistance to imatinib. In KRAS-G12C or wild-type KRAS, point mutations in the AMG 510 binding pocket or oncogenic non-G12C mutations conferred resistance to AMG 510. LentiMutate should prove highly valuable for clinical and preclinical cancer-drug development. Significance: LentiMutate can evaluate a drug's on-target activity and can nominate resistance mutations before they occur in patients, which could accelerate and refine drug development to increase the survival of patients with cancer.

Drug resistance impedes the long-term effect of targeted therapies in acute myeloid leukemia (AML), necessitating the identification of mechanisms underlying resistance. Approximately 25% of AML patients carry FLT3 mutations and develop post-treatment insensitivity to FLT3 inhibitors, including sorafenib. Using a genomewide CRISPR screen, we identified LZTR1, NF1, TSC1 and TSC2, negative regulators of the MAPK and MTOR pathways, as mediators of resistance to sorafenib. Analyses of ex vivo drug sensitivity assays in samples from patients with FLT3-ITD AML revealed that lower expression of LZTR1, NF1, and TSC2 correlated with sensitivity to sorafenib. Importantly, MAPK and/or MTOR complex 1 (MTORC1) activity was upregulated in AML cells made resistant to several FLT3 inhibitors, including crenolanib, quizartinib, and sorafenib. These cells were sensitive to MEK inhibitors, and the combination of FLT3 and MEK inhibitors showed enhanced efficacy, suggesting the effectiveness of such treatment in AML patients with FLT3 mutations and those with resistance to FLT3 inhibitors.

Chronic myeloid leukemia (CML) is successfully treated with BCR-ABL1 tyrosine kinase inhibitors, but a significant percentage of patients develop resistance. Insulin receptor substrate 1 (IRS1) has been shown to constitutively associate with BCR-ABL1, and IRS1-specific silencing leads to antineoplastic effects in CML cell lines. Here, we characterized the efficacy of NT157, a pharmacological inhibitor of IGF1R-IRS1/2, in CML cells and observed significantly reduced cell viability and proliferation, accompanied by induction of apoptosis. In human K562 cells and in murine Ba/F3 cells, engineered to express either wild-type BCR-ABL1 or the imatinib-resistant BCR-ABL1

FLT3 mutations are prevalent in AML patients and confer poor prognosis. Crenolanib, a potent type I pan-FLT3 inhibitor, is effective against both internal tandem duplications and resistance-conferring tyrosine kinase domain mutations. While crenolanib monotherapy has demonstrated clinical benefit in heavily pretreated relapsed/refractory AML patients, responses are transient and relapse eventually occurs. Here, to investigate the mechanisms of crenolanib resistance, we perform whole exome sequencing of AML patient samples before and after crenolanib treatment. Unlike other FLT3 inhibitors, crenolanib does not induce FLT3 secondary mutations, and mutations of the FLT3 gatekeeper residue are infrequent. Instead, mutations of NRAS and IDH2 arise, mostly as FLT3-independent subclones, while TET2 and IDH1 predominantly co-occur with FLT3-mutant clones and are enriched in crenolanib poor-responders. The remaining patients exhibit post-crenolanib expansion of mutations associated with epigenetic regulators, transcription factors, and cohesion factors, suggesting diverse genetic/epigenetic mechanisms of crenolanib resistance. Drug combinations in experimental models restore crenolanib sensitivity. FLT3 is commonly mutated in acute myeloid leukaemia and treatment with FLT3 inhibitors often ends with relapse. Here, the authors perform exome sequencing of samples from patients treated with the FLT3 inhibitor, crenolanib, to show that resistance occurs due to diverse molecular mechanisms, not primarily due to secondary FLT3 mutations.

The implementation of targeted therapies for acute myeloid leukaemia (AML) has been challenging because of the complex mutational patterns within and across patients as well as a dearth of pharmacologic agents for most mutational events. Here we report initial findings from the Beat AML programme on a cohort of 672 tumour specimens collected from 562 patients. We assessed these specimens using whole-exome sequencing, RNA sequencing and analyses of ex vivo drug sensitivity. Our data reveal mutational events that have not previously been detected in AML. We show that the response to drugs is associated with mutational status, including instances of drug sensitivity that are specific to combinatorial mutational events. Integration with RNA sequencing also revealed gene expression signatures, which predict a role for specific gene networks in the drug response. Collectively, we have generated a dataset—accessible through the Beat AML data viewer (Vizome)—that can be leveraged to address clinical, genomic, transcriptomic and functional analyses of the biology of AML. Analyses of samples from patients with acute myeloid leukaemia reveal that drug response is associated with mutational status and gene expression; the generated dataset provides a basis for future clinical and functional studies of this disease.

// David K. Edwards V 1,* , David Tyler Sweeney 2,* , Hibery Ho 2 , Christopher A. Eide 2 , Angela Rofelty 2 , Anupriya Agarwal 2 , Selina Qiuying Liu 2 , Alexey V. Danilov 2 , Patrice Lee 3 , David Chantry 3 , Shannon K. McWeeney 4 , Brian J. Druker 2,5 , Jeffrey W. Tyner 1 , Stephen E. Spurgeon 2,** and Marc M. Loriaux 2,** 1 Department of Cell, Developmental and Cancer Biology, Oregon Health & Science University, Portland, OR, USA 2 Division of Hematology & Medical Oncology, Oregon Health & Science University Knight Cancer Institute, Portland, OR, USA 3 Array BioPharma, Boulder, CO, USA 4 Department of Bioinformatics and Computational Biology, Oregon Health & Science University, Portland, OR, USA 5 Howard Hughes Medical Institute, Oregon Health & Science University, Knight Cancer Institute, Portland, OR, USA * These authors have contributed equally to this work ** These authors have contributed equally to this work Correspondence to: Marc M. Loriaux, email: // Keywords : colony-stimulating factor 1 receptor; chronic lymphocytic leukemia; tumor-associated macrophages; tumor microenvironment; small-molecule inhibitors Received: September 26, 2017 Accepted: March 01, 2018 Published: May 15, 2018 Abstract In many malignancies, the tumor microenvironment includes CSF1R-expressing supportive monocyte/macrophages that promote tumor cell survival. For chronic lymphocytic leukemia (CLL), these supportive monocyte/macrophages are known as nurse-like cells (NLCs), although the potential effectiveness of selective small-molecule inhibitors of CSF1R against CLL is understudied. Here, we demonstrate the preclinical activity of two inhibitors of CSF1R, GW-2580 and ARRY-382, in primary CLL patient samples. We observed at least 25% of CLL samples showed sub-micromolar sensitivity to CSF1R inhibitors. This sensitivity was observed in samples with varying genetic and clinical backgrounds, although higher white cell count and monocyte cell percentage was associated with increased sensitivity. Depleting CD14-expressing monocytes preferentially decreased viability in samples sensitive to CSF1R inhibitors, and treating samples with CSF1R inhibitors eliminated the presence of NLCs in long-term culture conditions. These results indicate that CSF1R small-molecule inhibitors target CD14-expressing monocytes in the CLL microenvironment, thereby depriving leukemia cells of extrinsic support signals. In addition, significant synergy was observed combining CSF1R inhibitors with idelalisib or ibrutinib, two current CLL therapies that disrupt tumor cell intrinsic B-cell receptor signaling. These findings support the concept of simultaneously targeting supportive NLCs and CLL cells and demonstrate the potential clinical utility of this combination.

Tyrosine kinase inhibitors (TKIs) targeting BCR-ABL1 are remarkably effective therapies in chronic myeloid leukemia (CML). Despite success, TKIs do not target the CML leukemic stem cell (LSC), meaning patients must be treated for life at a high economic burden and often with significant side effects. Our previous work demonstrated a tumor suppressor role for G0/G1 switch gene 2 (G0S2) in CML, which is profoundly downregulated in TKI resistance (>3-fold, p To address this hypothesis, we first assessed ATGL protein expression in CML cell lines (K562 and KU812) in the presence and absence of the BCR-ABL TKI, imatinib. Importantly, ATGL protein expression remained unchanged in graded doses of imatinib, suggesting that ATGL expression occurs in a BCR-ABL1-independent manner. To assess the functional role of ATGL in CML and TKI resistance, we performed shRNA-mediated ATGL knockdown (shATGL) in both cell lines. Although ectopic G0S2 impaired survival of CML cell lines and patient samples, we observed a different phenotype upon ATGL knockdown. Rather, shATGL alone increased colony formation by ~30% in both cell lines in the absence but not presence of imatinib (K562, p=0.04; KU812, p=0.0024). Knockdown of ATGL had no effect on colony formation of normal cord blood CD34 + progenitors (p=0.742). Interestingly, when we ectopically expressed G0S2 into K562 cells with simultaneous ATGL knockdown, the phenotype in colony formation assays mimicked ectopic G0S2 expression, reducing survival by ~50% (p=0.05). These data suggest that the tumor suppressor role of G0S2 in CML is independent of ATGL. However, while ectopic G0S2 expression alone had no effect on apoptosis of CML cell lines, when combined with ATGL knockdown, imatinib-mediated apoptosis was markedly increased. This was similar to data observed in blast phase CML patient samples upon ectopic G0S2 expression. Consistently, RNA sequencing (RNAseq) data for CML patients revealed that ATGL mRNA is highly downregulated in blast phase (n=14) compared with patients in the chronic (n=52) or accelerated (n=11) phases of the disease (p The effect of G0S2 on lipid metabolism was further confirmed with RNAseq and metabolomics/lipidomics analyses. RNAseq revealed no overlapping pathways between K562 cells expressing ectopic G0S2 versus shATGL. Lipidomics analyses revealed that G0S2 knockdown reduced expression of tri- and di-glycerides, whereas ectopic G0S2 promoted triglyceride accumulation in K562 cells. G0S2 knockdown also resulted in substantial changes of phosphatidylethanolamine and phosphatidylcholine expression, implicating G0S2 in the production of lipid bilayer components. Finally, metabolomics data implicated a role for G0S2 as a negative regulator of the mitochondrial electron transport chain. Altogether, these findings suggest that G0S2 and lipid metabolism play a role in regulating leukemic stem and progenitor cell survival as well as TKI response in vitro. Therefore, restoring G0S2 expression and inhibiting FAO, combined with BCR-ABL1 inhibition, may be a novel clinical strategy to induce treatment-free remission in CML patients and eradicate the CML LSC. Disclosures No relevant conflicts of interest to declare.

The development of molecularly-targeted therapies to improve outcomes relative to chemotherapy for acute myeloid leukemia (AML) is impeded by the heterogeneity of genetic aberrations that contribute to disease. Among the multitude of biological mechanisms that lead to AML disease initiation and progression is dysregulation of cytokine signaling pathways, a hallmark of chronic inflammation, which contribute to the growth, survival, and differentiation state of AML cells. We have previously shown that IL-1β, a pro-inflammatory cytokine expressed by many cell types including macrophages and monocytes, stimulates proliferation of leukemic blasts independent of mutational status in primary AML samples via enhanced phosphorylation of p38α MAPK, an effect that can be blocked by IL-1 receptor knockdown or by pharmacologic inhibition (Carey 2017). Additionally, recent studies have shown sensitivity to the approved BCL2 inhibitor venetoclax in AML associates with undifferentiated leukemic cells (Pei 2020; Zhang 2018; Majumder 2020). Based on these associations, we evaluated the combination of doramapimod (DORA), a p38 MAPK inhibitor, with venetoclax (VEN) for potential enhanced sensitivity on primary AML cells. Ex vivo drug screening of primary AML patient samples (n=335) revealed significantly enhanced efficacy of VEN+DORA compared to either single agent (Nemenyi test; p Disclosures Druker: Novartis Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Recludix Pharma, Inc.: Consultancy; EnLiven: Consultancy, Research Funding; Pfizer: Research Funding; The RUNX1 Research Program: Membership on an entity's Board of Directors or advisory committees; Merck & Co: Patents & Royalties; Aileron: Membership on an entity's Board of Directors or advisory committees; ALLCRON: Consultancy, Membership on an entity's Board of Directors or advisory committees; Amgen: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Aptose Therapeutics: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Research Funding; Cepheid: Consultancy, Membership on an entity's Board of Directors or advisory committees; GRAIL: Current equity holder in publicly-traded company; VB Therapeutics: Membership on an entity's Board of Directors or advisory committees; Iterion Therapeutics: Membership on an entity's Board of Directors or advisory committees; Nemucore Medical Innovations, Inc.: Consultancy; Third Coast Therapeutics: Membership on an entity's Board of Directors or advisory committees; Vincerx Pharma: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees. Tyner: Genentech: Research Funding; Takeda: Research Funding; Astrazeneca: Research Funding; Constellation: Research Funding; Agios: Research Funding; Petra: Research Funding; Incyte: Research Funding; Array: Research Funding; Gilead: Research Funding; Janssen: Research Funding; Seattle Genetics: Research Funding; Schrodinger: Research Funding.

Introduction Despite impressive response rates of >70% in frontline therapy with Azacitidine and Venetoclax, relapsed/refractory (R/R) acute myeloid leukemia (AML) remains a clinical challenge with up-front resistance and relapse developing through mutations of TP53, RAS pathway genes and expression/utilization of alternative BCL2 family members. Through integrative functional genomic analyses performed on a large cohort of primary AML patient samples, we found that the combination of Ruxolitinib (Rux) and Venetoclax (Ven) exhibited broad ex vivo efficacy and synergy. Based on the significant activity seen in both newly diagnosed and R/R AML, we conducted a Phase I study to evaluate the safety and efficacy of Rux+Ven in R/R AML. Methods This Phase I multi-center study will identify a recommended Phase II dose and evaluate the overall safety and preliminary efficacy of Rux+Ven. Eligible patients were >18 years old with R/R AML. There was no restriction on the number of previous therapies or prior exposure to Ven or Rux. We included patients with prior allogeneic transplant if GVHD was controlled and prior MDS patients >75 years old who progressed on a hypomethylating agent (HMA) but had no AML therapy. In cohorts of 3 to 4, patients were assigned to receive Rux+Ven at 1 of 6 dose levels according to the "keyboard" Bayesian toxicity probability interval design applied to a pre-specified target dose-limiting toxicity (DLT) rate of 30%. DLTs were defined as non-disease-related Grade ≥3 non-hematologic toxicities, with exceptions for nausea, vomiting, febrile neutropenia due to disease-related cytopenias, and electrolyte abnormalities that resolve within 48 hours. Hematologic DLT was specified as Grade 4 neutropenia by 42 days post-therapy in the absence of disease. The DLT evaluation period covered the first cycle. Clinical responses were determined using 2017 ELN criteria. Patients could continue study therapy after two cycles if they had a response or were deriving clinical benefit. Results Twenty patients have been enrolled out of the planned accrual of 30. Median age was 73 (range 29-87), 60% of patients had refractory disease, 50% had ≥3 prior therapies, and 35% had prior Ven exposure. At screening, 40% of patients had a complex karyotype and 65% had adverse ELN risk, including 20% with mutated TP53. The numbers of patients treated by dose level (DL) are: 3 on DL0 (200 mg qd Ven and 10 mg bid Rux), 4 on DL1 (400 mg qd Ven and 10 mg bid Rux), 3 on DL2 (400 mg qd Ven and 20 mg bid Rux), and 10 on DL3 (400 mg qd Ven and 30 mg bid Rux), the highest level of the study. Per prescribing guidelines, the cycle 1 Rux doses of 10 patients and Ven doses of 14 patients were reduced because of concomitant CYP3A inhibitor therapy. All 20 patients were DLT evaluable (defined as receiving both study drugs on ≥50% of cycle 1 days) and no DLTs were observed. The median duration of therapy was 55 days (95% CI: 51 - 113 days). The most common hematological AEs, regardless of attribution, were grade 3/4 thrombocytopenia (40%), febrile neutropenia (40%), neutropenia (35%), anemia (35%), and leukopenia (30%). Of the serious AEs recorded in 7 patients (35%), 4 were deemed related to Rux+Ven (3 for febrile neutropenia, 1 for sepsis). There were 2 treatment-emergent deaths, with one ( lung infection) potentially related to study drug. The Clinical Benefit Rate (CBR) was 40% (95% CI: 19.1% - 63.9%) over the first 2 cycles of therapy with five patients (25%) establishing MLFS or better and three achieving a CR or CRi during this time period (Composite Complete Remission rate 15% (95% CI: 3.2% - 37.9%)). For the 5 responders, median number of prior lines was 2, 1 patient had received Ven previously, and none had a TP53 mutation. Ongoing analyses involving ex vivo drug sensitivity, genomic sequencing, CyTOF, and single cell imaging are looking at associations with clinical response. The protocol is being amended to include PK/PD analysis for the final 10 patients. Conclusion The novel, all oral combination of Ven and Rux in heavily pretreated R/R AML patients was well tolerated with no DLTs, promising initial responses, and encouraging duration of therapy. Additional analyses including PK/PD and genomic and immune profiling will give further insight into which patients benefit the most in this poor prognostic group. The well tolerated safety profile makes it a promising combination to advance in the front line setting in combination with HMAs. Figure 1 Figure 1. Disclosures Borate: Astellas: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: 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; Genentech: Membership on an entity's Board of Directors or advisory committees, Other: Advisory Board; Jazz Pharma: Research Funding; Blueprint Medicine: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; incyte: Membership on an entity's Board of Directors or advisory committees, Research Funding; Rampal: Membership on an entity's Board of Directors or advisory committees; Galecto, Inc.: Consultancy; Promedior: Consultancy. Saultz: IKENA: Research Funding. Madanat: Onc Live: Honoraria; Blue Print Pharmaceutical: Honoraria; Stem line pharmaceutical: Honoraria; Geron Pharmaceutical: Consultancy. Druker: VB Therapeutics: Membership on an entity's Board of Directors or advisory committees; Aptose Therapeutics: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Research Funding; Recludix Pharma, Inc.: Consultancy; The RUNX1 Research Program: Membership on an entity's Board of Directors or advisory committees; Merck & Co: Patents & Royalties; Third Coast Therapeutics: Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Amgen: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Pfizer: Research Funding; Iterion Therapeutics: Membership on an entity's Board of Directors or advisory committees; ALLCRON: Consultancy, Membership on an entity's Board of Directors or advisory committees; EnLiven: Consultancy, Research Funding; GRAIL: Current equity holder in publicly-traded company; Novartis Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Nemucore Medical Innovations, Inc.: Consultancy; Cepheid: Consultancy, Membership on an entity's Board of Directors or advisory committees; Aileron: Membership on an entity's Board of Directors or advisory committees; Vincerx Pharma: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees. Tyner: Array: Research Funding; Takeda: Research Funding; Genentech: Research Funding; Constellation: Research Funding; Schrodinger: Research Funding; Petra: Research Funding; Agios: Research Funding; Seattle Genetics: Research Funding; Incyte: Research Funding; Gilead: Research Funding; Janssen: Research Funding; Astrazeneca: Research Funding.