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The 1,287 genes targeted by the shRNA of the leukemia library.

Standard hematologic parameters in SIRT5-/- mice and SIRT5+/+ littermates.

Plasmids.

Patient sample information.

Antibodies.

We discovered that the survival and growth of many primary acute myeloid leukemia (AML) samples and cell lines, but not normal CD34+ cells, are dependent on SIRT5, a lysine deacylase implicated in regulating multiple metabolic pathways. Dependence on SIRT5 is genotype agnostic and extends to RAS- and p53-mutated AML. Results were comparable between SIRT5 knockdown and SIRT5 inhibition using NRD167, a potent and selective SIRT5 inhibitor. Apoptosis induced by SIRT5 disruption is preceded by reductions in oxidative phosphorylation and glutamine utilization, and an increase in mitochondrial superoxide that is attenuated by ectopic superoxide dismutase 2. These data indicate that SIRT5 controls and coordinates several key metabolic pathways in AML and implicate SIRT5 as a vulnerability in AML.Significance:Reducing SIRT5 activity is detrimental to the survival of AML cells regardless of genotype, yet well tolerated by healthy hematopoietic cells. In mouse models, disrupting SIRT5 inhibits AML progression. SIRT5 controls several metabolic pathways that are required for leukemia cell survival. These results identify SIRT5 as a therapeutic target in AML.See related commentary by Li and Melnick, p. 198.

All supplemental figures and legends.

Next generation sequencing for 52 myeloid malignancies-related genes.

Oligonucleotide sequences.

Ranked list of 1,287 genes from shRNA library screen in primary AML cells. The 1,287 genes assessed with an shRNA library screen were sorted by the second highest percentile fold change present in 2 shRNA and across 2 samples, with the 34 genes showing a fold change in the top 2 percent in more than 2 samples listed first.

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.

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.

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).

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).

PDF file - 45K

We discovered that the survival and growth of many primary acute myeloid leukemia (AML) samples and cell lines, but not normal CD34+ cells, are dependent on SIRT5, a lysine deacylase implicated in regulating multiple metabolic pathways. Dependence on SIRT5 is genotype agnostic and extends to RAS- and p53-mutated AML. Results were comparable between SIRT5 knockdown and SIRT5 inhibition using NRD167, a potent and selective SIRT5 inhibitor. Apoptosis induced by SIRT5 disruption is preceded by reductions in oxidative phosphorylation and glutamine utilization, and an increase in mitochondrial superoxide that is attenuated by ectopic superoxide dismutase 2. These data indicate that SIRT5 controls and coordinates several key metabolic pathways in AML and implicate SIRT5 as a vulnerability in AML. Significance: Reducing SIRT5 activity is detrimental to the survival of AML cells regardless of genotype, yet well tolerated by healthy hematopoietic cells. In mouse models, disrupting SIRT5 inhibits AML progression. SIRT5 controls several metabolic pathways that are required for leukemia cell survival. These results identify SIRT5 as a therapeutic target in AML. See related commentary by Li and Melnick, p. 198.

Introduction Vessel wall magnetic resonance imaging can improve the evaluation of intracranial atherosclerotic disease. However, pathological validation is needed to improve vessel wall magnetic resonance imaging techniques. Human pathology samples are not practical for such analysis, so an animal model is therefore needed. Materials and methods Watanabe heritable hyperlipidemic rabbits and apolipoprotein E knockout rabbits were evaluated against New Zealand white wild-type rabbits. Evaluation of intracranial arteries was performed with vessel wall magnetic resonance imaging and pathological analysis, rating the presence and severity of disease in each segment. Two-tailed t-tests were performed to compare disease occurrence and severity prevalence among rabbit subtypes. Sensitivity and specificity were calculated to assess the diagnostic accuracy of vessel wall magnetic resonance imaging. Results Seventeen rabbits (five Watanabe heritable hyperlipidemic, four apolipoprotein E knockout and eight New Zealand white) were analysed for a total of 51 artery segments. Eleven segments (five Watanabe heritable hyperlipidemic and six apolipoprotein E knockout) demonstrated intracranial atherosclerotic disease on pathology. Disease model animals had lesions more frequently than New Zealand white animals ( PConclusion Intracranial atherosclerotic disease can be reliably produced and detected using 3T vessel wall magnetic resonance imaging-compatible Watanabe heritable hyperlipidemic and ApoE rabbit models. Further analysis is needed to characterize better the development and progression of the disease to correlate tissue-validated animal findings with those in human vessel wall magnetic resonance imaging studies.

BackgroundBetter understanding of vessel biology and vascular pathophysiology is needed to improve understanding of cerebrovascular disorders. Tissue from diseased vessels can offer the best data. Rabbit models can be effective for studying intracranial vessels, filling gaps resulting from difficulties acquiring human tissue. Spatially-resolved transcriptomics (SRT) in particular hold promise for studying such models as they build on RNA sequencing methods, augmenting such data with histopathology.MethodsRabbit brains with intact arteries were flash frozen, cryosectioned, and stained with H&E to confirm adequate inclusion of intracranial vessels before proceeding with tissue optimization and gene expression analysis using the Visium SRT platform. SRT results were analyzed with k-means clustering analysis, and differential gene expression was examined, comparing arteries to veins.ResultsCryosections were successfully mounted on Visium proprietary slides. Quality control thresholds were met. Optimum permeabilization was determined to be 24 minutes for the tissue optimization step. In analysis of SRT data, k-means clustering distinguished vascular tissue from parenchyma. When comparing gene expression traits, the most differentially expressed genes were those found in smooth muscle cells. These genes were more commonly expressed in arteries compared to veins.ConclusionsIntracranial vessels from model rabbits can be processed and analyzed with the Visium SRT platform. Face validity is found in the ability of SRT data to distinguish vessels from parenchymal tissue and differential expression analysis accurately distinguishing arteries from veins. SRT should be considered for future animal model investigations into cerebrovascular diseases.

Background Chronic phase chronic myeloid leukemia (CP-CML) is characterized by overproduction of differentiating myeloid cells, while blast phase CML (BP-CML) cells exhibit a differentiation block. Tyrosine kinase inhibitors (TKIs) are effective in CP-CML, but resistance is common in BP-CML and can occur without explanatory BCR-ABL1 kinase mutations (BCR-ABL1-independent resistance). Similarly, CML stem/progenitor cells (LSPCs) are insensitive to TKIs, and residual leukemia persists in the majority of CML patients on TKI therapy. We previously reported overlap between the transcriptomes of CD34 + cells from BP-CML and TKI-naïve CP-CML patients with primary TKI resistance, pointing to commonalities between LSPC quiescence, BCR-ABL1-independent TKI resistance, and BP-CML. Results To identify common mechanisms, we performed a meta-analysis of published CML transcriptomes. We identified a small set of genes with consistently low expression in LSPC quiescence, BCR-ABL1-independent TKI resistance, and BP-CML, including Membrane Spanning 4-Domains A3 (MS4A3), a signaling protein previously reported to inhibit hematopoietic cell cycle progression. Low MS4A3 in CD34 + cells from TKI-naïve CP-CML patients was associated with shorter survival on subsequent TKI therapy, suggesting that MS4A3 governs TKI response. To understand the function of MS4A3, we lentivirally introduced MS4A3 shRNA or an MS4A3 expression vector into CML CD34 + LSPCs. MS4A3 knockdown increased clonogenicity and imatinib resistance, while ectopic MS4A3 expression had opposite effects. MS4A3 KD also increased LSPC persistence ex vivo in LTC-IC assays, and in vivo in NSG mice xenografts, while modulating MS4A3 expression had no effect on normal CD34 + cells. We next generated Ms4a3+/+│-/-; Scl-tTA+; TRE-BCR-ABL1+ compound transgenic mice. Upon BCR-ABL1 induction, Ms4a3-/-; Scl-tTA+; TRE-BCR-ABL1+ mice developed leukocytosis comparable to Ms4a3+/+ controls. However, BM of Ms4a3-/-; Scl-tTA+; TRE-BCR-ABL1+ mice showed increased short-term HSCs and multipotent progenitor cells, and reduced granulocyte-macrophage progenitors. When Lin - BM cells from leukemic mice were transplanted into irradiated recipients, Ms4a3-/-; Scl-tTA+; TRE-BCR-ABL1+ cells showed increased engraftment and myeloid leukocytosis, validating our observations in human cells. To determine how MS4A3 is downregulated in CML, we expressed BCR-ABL1in 32D-cl3 cells. p210 BCR-ABL1 drastically reduced Ms4a3 expression, while kinase-inactive p210 BCR-ABL1-K271R had no effect. Moreover, we found that suppression of C/EBPε by MECOM reduces MS4A3, consistent with previous reports of MECOM as a driver of TKI resistance and progression to BP. Treatment of CML CD34 + cells with a library of epigenetic pathway inhibitors revealed that MS4A3 is suppressed by both DNA methylation and PRC2/EZH2-mediated H3K27 trimethylation, which was confirmed by patch-PCR and ChIPseq. These data indicate that multi-levelled mechanisms cooperate in the suppression of MS4A3 in CML. To determine how MS4A3 regulates clonogenicity and TKI response, we expressed MS4A3-EGFP fusion protein in LAMA-84 CML cells. We found that MS4A3 resides on the plasma membrane and in endosomes. Surface protein biotin labelling and tandem mass spectrometry ± MS4A3 KD showed that MS4A3 controls endocytosis of membrane proteins, including common β chain (βc) cytokine receptors. Specifically, MS4A3 promotes endocytosis of βc cytokine receptors upon GM-CSF/IL-3 stimulation of primary LSPCs and enhances downstream signaling and differentiation, suggesting that restoring MS4A3 expression has therapeutic efficacy. To test this, we manufactured a prototype MS4A3 protein-loaded liposomal nanoparticle (NP) using coating with the CD34 CD62L for targeted delivery to CD34 + cells. Compared to MS4A3-free NPs, MS4A3 NPs increased CD34 +CD38 + and CD34 -CD38 + at the expense of CD34 +CD38 - cells, reduced clonogenicity, and increased sensitivity to TKIs, mimicking ectopic MS4A3 expression. Conclusion MS4A3 governs response to differentiating myeloid cytokines, providing a unifying mechanism for the differentiation block characteristic of primitive LSPCs and BP-CML cells. We posit that LSPCs downregulate MS4A3 to evade βc cytokine-induced differentiation to maintain a primitive, TKI-insensitive state. MS4A3 re-expression or delivery of ectopic MS4A3 may eliminate LSPCs. Figure 1 Figure 1. Disclosures Druker: Aptose Therapeutics: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Cepheid: Consultancy, Membership on an entity's Board of Directors or advisory committees; EnLiven: Consultancy, Research Funding; Blueprint Medicines: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Aileron: 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; Bristol-Myers Squibb: Research Funding; ALLCRON: Consultancy, Membership on an entity's Board of Directors or advisory committees; GRAIL: Current equity holder in publicly-traded company; Iterion Therapeutics: Membership on an entity's Board of Directors or advisory committees; Merck & Co: Patents & Royalties; Nemucore Medical Innovations, Inc.: Consultancy; Novartis Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Pfizer: Research Funding; Recludix Pharma, Inc.: Consultancy; The RUNX1 Research Program: Membership on an entity's Board of Directors or advisory committees; Third Coast Therapeutics: Membership on an entity's Board of Directors or advisory committees; VB 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: Agios: Research Funding; Astrazeneca: Research Funding; Array: Research Funding; Genentech: Research Funding; Janssen: Research Funding; Takeda: Research Funding; Gilead: Research Funding; Incyte: Research Funding; Petra: Research Funding; Seattle Genetics: Research Funding; Constellation: Research Funding; Schrodinger: Research Funding. Oehler: BMS: Consultancy; OncLive: Honoraria; Pfizer: Research Funding; Takeda: Consultancy; Blueprint Medicines: Consultancy. Radich: BMS: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Deininger: Sangamo: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Part of a Study Management Committee, Research Funding; Incyte: Consultancy, Honoraria, Research Funding; Fusion Pharma, Medscape, DisperSol: Consultancy; Novartis: Consultancy, Research Funding; SPARC, DisperSol, Leukemia & Lymphoma Society: Research Funding; Blueprint Medicines Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Part of a Study Management Committee, Research Funding.

Standard of care for AML includes chemotherapy and stem cell transplant, with 5-year survival rates Citation Format: Dongqing Yan, Anca Franzini, Anthony D. Pomicter, Brayden J. Halverson, Orlando Antelope, Clinton C. Mason, Jonathan M. Ahmann, Anna V. Senina, Courtney L. L. Jones, Matthew S. Zabriskie, Hein Than, Michael J. Xiao, Alexandria van Scoyk, Ami B. Patel, William L. L. Heaton, Shawn C. Owen, Joshua L. Andersen, Christina M. Egbert, Julie A. Reisz, Angelo D'Alessandro, James E. Cox, Kevin C. Gantz, Hannah M. Redwine, Siddharth M. Iyer, Jamshid S. Khorashad, Nima Rajabi, Christian A. Olsen, Thomas O'Hare, Michael W. Deininger. A critical role for SIRT5 in acute myeloid leukemia metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB109.

Chronic myelomonocytic leukemia (CMML) is a hematologic malignancy nearly confined to the elderly. Previous studies to determine incidence and prognostic significance of somatic mutations in CMML have relied on candidate gene sequencing, although an unbiased mutational search has not been conducted. As many of the genes commonly mutated in CMML were recently associated with age-related clonal hematopoiesis (ARCH) and aged hematopoiesis is characterized by a myelomonocytic differentiation bias, we hypothesized that CMML and aged hematopoiesis may be closely related. We initially established the somatic mutation landscape of CMML by whole exome sequencing followed by gene-targeted validation. Genes mutated in ⩾ 10% of patients were SRSF2, TET2, ASXL1, RUNX1, SETBP1, KRAS, EZH2, CBL and NRAS, as well as the novel CMML genes FAT4, ARIH1, DNAH2 and CSMD1. Most CMML patients (71%) had mutations in ⩾ 2 ARCH genes and 52% had ⩾ 7 mutations overall. Higher mutation burden was associated with shorter survival. Age-adjusted population incidence and reported ARCH mutation rates are consistent with a model in which clinical CMML ensues when a sufficient number of stochastically acquired age-related mutations has accumulated, suggesting that CMML represents the leukemic conversion of the myelomonocytic-lineage-biased aged hematopoietic system.

Extreme mutational selectivity of axitinib limits its potential use as a targeted therapeutic for BCR-ABL1-positive leukemia

BCR-ABL1 point mutation-mediated resistance to tyrosine kinase inhibitor (TKI) therapy in Philadelphia chromosome-positive (Ph(+)) leukemia is effectively managed with several approved drugs, including ponatinib for BCR-ABL1(T315I)-mutant disease. However, therapy options are limited for patients with leukemic clones bearing multiple BCR-ABL1 mutations. Asciminib, an allosteric inhibitor targeting the myristoyl-binding pocket of BCR-ABL1, is active against most single mutants but ineffective against all tested compound mutants. We demonstrate that combining asciminib with ATP-site TKIs enhances target inhibition and suppression of resistant outgrowth in Ph(+) clinical isolates and cell lines. Inclusion of asciminib restores ponatinib’s effectiveness against currently untreatable compound mutants at clinically achievable concentrations. Our findings support combining asciminib with ponatinib as a treatment strategy for this molecularly defined group of patients.

Targeted therapy of chronic myeloid leukemia (CML) is currently based on small-molecule inhibitors that directly bind the tyrosine kinase domain of BCR-ABL1. This strategy has generally been successful, but is subject to drug resistance because of point mutations in the kinase domain. Kinase activity requires transactivation of BCR-ABL1 following an oligomerization event, which is mediated by the coiled-coil (CC) domain at the N terminus of the protein. Here, we describe a rationally engineered mutant version of the CC domain, called CC(mut3), which interferes with BCR-ABL1 oligomerization and promotes apoptosis in BCR-ABL1-expressing cells, regardless of kinase domain mutation status. CC(mut3) exhibits strong proapoptotic and antiproliferative activity in cell lines expressing native BCR-ABL1, single kinase domain mutant BCR-ABL1 (E255V and T315I) or compound-mutant BCR-ABL1 (E255V/T315I). Moreover, CC(mut3) inhibits colony formation by primary CML CD34(+) cells ex vivo, including a sample expressing the T315I mutant. These data suggest that targeting BCR-ABL1 with CC mutants may provide a novel alternative strategy for treating patients with resistance to current targeted therapies.

Mutations in the BCR-ABL1 kinase domain are an established mechanism of tyrosine kinase inhibitor (TKI) resistance in Philadelphia chromosome-positive leukemia, but fail to explain many cases of clinical TKI failure. In contrast, it is largely unknown why some patients fail TKI therapy despite continued suppression of BCR-ABL1 kinase activity, a situation termed BCR-ABL1 kinase-independent TKI resistance. Here, we identified activation of signal transducer and activator of transcription 3 (STAT3) by extrinsic or intrinsic mechanisms as an essential feature of BCR-ABL1 kinase-independent TKI resistance. By combining synthetic chemistry, in vitro reporter assays, and molecular dynamics-guided rational inhibitor design and high-throughput screening, we discovered BP-5-087, a potent and selective STAT3 SH2 domain inhibitor that reduces STAT3 phosphorylation and nuclear transactivation. Computational simulations, fluorescence polarization assays and hydrogen–deuterium exchange assays establish direct engagement of STAT3 by BP-5-087 and provide a high-resolution view of the STAT3 SH2 domain/BP-5-087 interface. In primary cells from chronic myeloid leukemia (CML) patients with BCR-ABL1 kinase-independent TKI resistance, BP-5-087 (1.0 μM) restored TKI sensitivity to therapy-resistant CML progenitor cells, including leukemic stem cells. Our findings implicate STAT3 as a critical signaling node in BCR-ABL1 kinase-independent TKI resistance, and suggest that BP-5-087 has clinical utility for treating malignancies characterized by STAT3 activation.

SummaryWe show that imatinib, nilotinib, and dasatinib possess weak off-target activity against RAF and, therefore, drive paradoxical activation of BRAF and CRAF in a RAS-dependent manner. Critically, because RAS is activated by BCR-ABL, in drug-resistant chronic myeloid leukemia (CML) cells, RAS activity persists in the presence of these drugs, driving paradoxical activation of BRAF, CRAF, MEK, and ERK, and leading to an unexpected dependency on the pathway. Consequently, nilotinib synergizes with MEK inhibitors to kill drug-resistant CML cells and block tumor growth in mice. Thus, we show that imatinib, nilotinib, and dasatinib drive paradoxical RAF/MEK/ERK pathway activation and have uncovered a synthetic lethal interaction that can be used to kill drug-resistant CML cells in vitro and in vivo.

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.

Activation of Janus kinase 2 (JAK2), frequently as a result of the JAK2 V617F mutation, is a characteristic feature of the classical myeloproliferative neoplasms (MPNs) polycythemia vera, essential thrombocythemia, and myelofibrosis, and it is thought to be responsible for the constitutional symptoms associated with these diseases. BMS-911543 is a JAK2-selective inhibitor that induces apoptosis in JAK2-dependent cell lines and inhibits the growth of CD34 + progenitor cells from patients with JAK2 V617F -positive MPN. To explore the clinical potential of this inhibitor, we tested BMS-911543 in a murine retroviral transduction–transplantation model of JAK2 V617F MPN. Treatment was initiated at two dose levels (3 mg/kg and 10 mg/kg) when the hematocrit exceeded 70%. Following the first week, white blood cell counts were reduced to normal in the high-dose group and were maintained well below the levels in vehicle-treated mice throughout the study. However, BMS-911543 had no effect on red blood cell parameters. After 42 days of treatment, the proportion of JAK2 V617F -positive cells in hematopoietic tissues was identical or slightly increased compared with controls. Plasma concentrations of interleukin 6, interleukin 15, and tumor necrosis factor α were elevated in MPN mice and reduced in the high-dose treatment group, whereas other cytokines were unchanged. Inhibitor activity after dosing was confirmed in a cell culture assay using the plasma of dosed mice and phosphorylated signal transducer and activator of transcription 5 flow cytometry. Collectively, these results show that BMS-911543 has limited activity in this murine model of JAK2 V617F -driven MPN and suggest that targeting JAK2 alone may be insufficient to achieve effective disease control.

The mechanisms underlying tyrosine kinase inhibitor (TKI) resistance in chronic myeloid leukemia (CML) patients lacking explanatory BCR-ABL1 kinase domain mutations are incompletely understood. To identify mechanisms of TKI resistance that are independent of BCR-ABL1 kinase activity, we introduced a lentiviral short hairpin RNA (shRNA) library targeting ∼5000 cell signaling genes into K562(R), a CML cell line with BCR-ABL1 kinase-independent TKI resistance expressing exclusively native BCR-ABL1. A customized algorithm identified genes whose shRNA-mediated knockdown markedly impaired growth of K562(R) cells compared with TKI-sensitive controls. Among the top candidates were 2 components of the nucleocytoplasmic transport complex, RAN and XPO1 (CRM1). shRNA-mediated RAN inhibition or treatment of cells with the XPO1 inhibitor, KPT-330 (Selinexor), increased the imatinib sensitivity of CML cell lines with kinase-independent TKI resistance. Inhibition of either RAN or XPO1 impaired colony formation of CD34(+) cells from newly diagnosed and TKI-resistant CML patients in the presence of imatinib, without effects on CD34(+) cells from normal cord blood or from a patient harboring the BCR-ABL1(T315I) mutant. These data implicate RAN in BCR-ABL1 kinase-independent imatinib resistance and show that shRNA library screens are useful to identify alternative pathways critical to drug resistance in CML.

Summary Ponatinib is the only currently approved tyrosine kinase inhibitor (TKI) that suppresses all BCR-ABL1 single mutants in Philadelphia chromosome-positive (Ph + ) leukemia, including the recalcitrant BCR-ABL1 T315I mutant. However, emergence of compound mutations in a BCR-ABL1 allele may confer ponatinib resistance. We found that clinically reported BCR-ABL1 compound mutants center on 12 key positions and confer varying resistance to imatinib, nilotinib, dasatinib, ponatinib, rebastinib, and bosutinib. T315I-inclusive compound mutants confer high-level resistance to TKIs, including ponatinib. In vitro resistance profiling was predictive of treatment outcomes in Ph + leukemia patients. Structural explanations for compound mutation-based resistance were obtained through molecular dynamics simulations. Our findings demonstrate that BCR-ABL1 compound mutants confer different levels of TKI resistance, necessitating rational treatment selection to optimize clinical outcome.

Despite the well-established success of ABL1 tyrosine kinase inhibitors (TKIs) in the treatment of patients with chronic myeloid leukemia (CML), approximately 20% of patients treated with frontline imatinib develop resistance by 5 years on therapy. The majority (~60%) of such resistant cases are explained by acquired mutations within the BCR-ABL1 kinase domain that compromise inhibitor binding, and nearly all of these mutations are effectively targeted by one or more of the 2nd and 3rd generation ABL1 kinase inhibitors. In contrast, the remaining ~40% of imatinib-resistant cases harbor no explanatory BCR-ABL1 kinase domain mutation, presumably attributable to BCR-ABL1 kinase-independent mechanisms. We hypothesized that resistance in these patients results from acquired auxiliary molecular aberrations which persistently activate signaling pathways downstream despite inhibition of BCR-ABL1 kinase activity. To identify such mechanisms, we performed whole exome sequencing and RNA sequencing on a cohort of 135 CML patients comprising the following subgroups: newly diagnosed/TKI naïve (n=28), BCR-ABL1 kinase-dependent resistance (n=31), and BCR-ABL1 kinase-independent resistance (n=65), and TKI-induced remission (n=7). Resistant patients were required to have demonstrated clinical resistance to one or more ABL1 kinase inhibitors in the form of suboptimal response or loss of cytogenetic response; the subtype of resistance was defined based on the presence or not of an explanatory BCR-ABL1 kinase domain mutation at the time of resistance. The majority of samples collected were from patients with chronic phase CML (n=97), although smaller cohorts of accelerated phase CML, blast crisis CML, and Ph+ ALL were also profiled (n=20, 19, and 9, respectively). Among the 44,413 protein-altering and 902 splice site variants detected across the 120 WES samples, there were on average 908 missense, 146 truncation and 69 splice variants per sample. Genes with truncation and missense variants were compared between BCR-ABL1 kinase-independent and -dependent resistant chronic phase samples. A total of 44 genes were seen with a frequency difference of at least 10%, including PLEKHG5 and NKD2 (30% and 28% difference, respectively), which are involved in regulation of NF-kB and Wnt signaling. Consistent with previous reports, we also detected EZH2 and TET2 as exclusively mutated in the BCR-ABL1 kinase-independent resistance patients (6% and 3%, respectively). Further analyses stratifying variants among resistant patients according to specific ABL1 kinase inhibitor therapy failed and comparing, where available, serial samples from pre- and post-treatment for clonal expansion are underway. Additionally, sufficient material was available to perform ex vivo small-molecule inhibitor screening for 48 patient specimens, the resultant data of which was used to generate putative effective drug target profiles and integrated with exome sequencing variants to prioritize variants of functional relevance (HitWalker; Bottomly et al., Bioinformatics 2013). Among 23 patient samples exhibiting BCR-ABL1 kinase-independent resistance, the mutated genes most frequently ranked in the top 10 functional-prioritized variants were: ABL1 (which included non-kinase domain variants; 34.7%), MAP3K1, MUC4, FGF20 (each 17.4%), ARHGEF15, MEF2A, EPHA8, TYRO3, BMP2K, and IRS1 (each 13.0%). Notably, the top six candidates are members of the neutrophin (ABL1, MAP3K1, and IRS1), EPHA forward (EPHA8, ARHGEF15), and p38 MAPK signaling pathways (MAP3K1 and MEF2A). Taken together, these findings suggest that several of the same pathogenic molecular abnormalities seen in other myeloid malignancies are also present in CML patients with BCR-ABL1 kinase-independent resistance, including a subset which align to persistent re-activation of signaling pathways involved in CML disease pathogenesis and progression. As such, genetic and/or functional profiling of these patients in the clinic may translate to actionable candidates for combination therapy to maximize disease control and improve patient outcomes. Disclosures Agarwal: CTI BioPharma Corp: Research Funding. Radich:Novartis: Consultancy, Research Funding; BMS: Consultancy; Ariad: Consultancy. Deininger:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Consultancy, Research Funding; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Research Funding; CTI BioPharma Corp.: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees. Druker:Pfizer: Patents & Royalties; Dana-Farber Cancer Institute: Patents & Royalties: Millipore royalties via Dana-Farber Cancer Institute; Curis: Patents & Royalties; Array: Patents & Royalties; CTI: Consultancy, Equity Ownership; Pfizer: Patents & Royalties; Curis: Patents & Royalties; Array: Patents & Royalties; Dana-Farber Cancer Institute: Patents & Royalties: Millipore royalties via Dana-Farber Cancer Institute; Oncotide Pharmaceuticals: Research Funding; Novartis: Research Funding; BMS: Research Funding; ARIAD: Patents & Royalties: inventor royalties paid by Oregon Health & Science University for licenses, Research Funding; Roche: Consultancy; Gilead Sciences: Consultancy, Other: travel, accommodations, expenses; D3 Oncology Solutions: Consultancy; AstraZeneca: Consultancy; Ambit BioSciences: Consultancy; Agios: Honoraria; MolecularMD: Consultancy, Equity Ownership, Patents & Royalties; Lorus: Consultancy, Equity Ownership; Cylene: Consultancy, Equity Ownership.

The imatinib paradigm in chronic myelogenous leukemia (CML) established continuous BCR-ABL inhibition as a design principle for ABL tyrosine kinase inhibitors (TKI). However, clinical responses seen in patients treated with the ABL TKI dasatinib despite its much shorter plasma half-life and the apparent rapid restoration of BCR-ABL signaling activity following once-daily dosing suggested acute, potent inhibition of kinase activity may be sufficient to irrevocably commit CML cells to apoptosis. To determine the specific requirements for ABL TKI-induced CML cell death for a panel of clinically important ABL TKIs (imatinib, nilotinib, dasatinib, ponatinib, and DCC-2036), we interrogated response of CML cell lines and primary CML cells following acute drug exposure using intracellular fluorescence-activated cell sorting and immunoblot analyses of BCR-ABL signaling, apoptosis measurements, liquid chromatography/tandem mass spectrometry of intracellular drug levels, and biochemical TKI dissociation studies. Importantly, significant intracellular TKI stores were detected following drug washout, levels of which tracked with onset of apoptosis and incomplete return of BCR-ABL signaling, particularly pSTAT5, to baseline. Among TKIs tested, ponatinib showed the most robust capacity for apoptotic commitment showing sustained suppression of BCR-ABL signaling even at low intracellular levels following extensive washout, consistent with high-affinity binding and slow dissociation from ABL kinase. Together, our findings suggest commitment of CML cells to apoptosis requires protracted incomplete restoration of BCR-ABL signaling mediated by intracellular retention of TKIs above a quantifiable threshold. These studies refine our understanding of apoptotic commitment in CML cells and highlight parameters important to design of therapeutic kinase inhibitors for CML and other malignancies. Cancer Res; 73(11); 3356–70. ©2013 AACR.

BCR-ABL1 compound mutations can confer high-level resistance to imatinib and other ABL1 tyrosine kinase inhibitors (TKIs). The third-generation ABL1 TKI ponatinib is effective against BCR-ABL1 point mutants individually, but remains vulnerable to certain BCR-ABL1 compound mutants. To determine the frequency of compound mutations among chronic myeloid leukemia patients on ABL1 TKI therapy, in the present study, we examined a collection of patient samples (N = 47) with clear evidence of 2 BCR-ABL1 kinase domain mutations by direct sequencing. Using a cloning and sequencing method, we found that 70% (33/47) of double mutations detected by direct sequencing were compound mutations. Sequential, branching, and parallel routes to compound mutations were common. In addition, our approach revealed individual and compound mutations not detectable by direct sequencing. The frequency of clones harboring compound mutations with more than 2 missense mutations was low (10%), whereas the likelihood of silent mutations increased disproportionately with the total number of mutations per clone, suggesting a limited tolerance for BCR-ABL1 kinase domain missense mutations. We conclude that compound mutations are common in patients with sequencing evidence for 2 BCR-ABL1 mutations and frequently reflect a highly complex clonal network, the evolution of which may be limited by the negative impact of missense mutations on kinase function.

Tyrosine kinase inhibitor (TKI) therapy targeting the BCR-ABL1 kinase is effective against chronic myeloid leukaemia (CML), but is not curative for most patients. Minimal residual disease (MRD) is thought to reside in TKI-insensitive leukaemia stem cells (LSCs) that are not fully addicted to BCR-ABL1. Recent conceptual advances in both CML biology and therapeutic intervention have increased the potential for the elimination of CML cells, including LSCs, through simultaneous inhibition of BCR-ABL1 and other newly identified, crucial targets.

ABL001 (Fig. 1) is a first-in-class allosteric inhibitor of BCR-ABL1 that occupies the vestigial myristoyl pocket, inducing an autoinhibited kinase conformation (Sellers, et al. Proceedings of the 106th Annual Meeting of the AACR; 2015 Apr 18-22; Philadelphia, PA). Phase 1 clinical trials with single-agent ABL001 in relapsed patients with chronic or accelerated phase chronic myeloid leukemia (CML) previously treated with at least two different tyrosine kinase inhibitors (TKIs) are underway. Patients who exhibit relapsed disease associated with the presence of the T315I mutation after at least one TKI are also eligible if no other effective therapy exists. The unexplored possibility of combining an allosteric inhibitor (e.g. ABL001) with a clinically proven BCR-ABL1 TKI such as nilotinib could have important implications for maximum disease control. Given the remote location of the myristoyl allosteric pocket relative to the catalytic site in the kinase domain of BCR-ABL1, a reasonable expectation is that ABL001 should be insulated from point mutations that impart resistance to TKIs such as nilotinib, dasatinib or ponatinib. However, pre-clinical studies demonstrate that the high level of potency exerted on native BCR-ABL1 is not uniformly maintained against a panel of clinically observed point mutations that impart resistance to non-allosteric BCR-ABL1 TKIs (Wylie, et al. Blood (2014) 124, 21, Abstract 398). For example, the T315I and F359V mutations confer substantial in vitro resistance to ABL001 in cell line experiments. The mechanistic reasons for these differences are under investigation. We will report on: (1) Ba/F3 cell line resistance assays predictive of clinical mutation-based escape mechanisms starting from cells expressing native BCR-ABL1 or BCR-ABL1T315I, for ABL001 as a single agent and in combination with a BCR-ABL1 TKI (nilotinib, dasatinib or ponatinib) (2) profiling of ABL001 against T315I-inclusive and non-T315I compound mutations, as a single agent and in combination with a BCR-ABL1 TKI (nilotinib, dasatinib or ponatinib) and (3) Molecular Dynamics studies probing the structural reasons for the selectivity profile of ABL001. In summary, our results to date on ABL001 suggest that single-agent ABL001 retains activity against a subset of non-T315I compound mutants. Preliminary findings indicate that the in vitro resistance profile of ABL001 is non-overlapping with that of TKIs that bind in the catalytic site. In contrast to ABL001, it is known that some myristoyl pocket binders activate rather than induce autoinhibition of BCR-ABL1. Current computational modeling efforts are focused on exploring a similar bifurcation from the perspective of native BCR-ABL1 as compared to ABL001-resistant BCR-ABL1 mutants such as T315I and F359V. Results of ongoing experiments with T315I-inclusive compound mutants will be reported. Our ultimate interest is to identify ABL001-sensitive compound mutants and make this information available to clinicians. Figure 1. ABL001 Chemical Structure Figure 1. ABL001 Chemical Structure Disclosures Druker: Millipore: Patents & Royalties; ARIAD: Research Funding; Fred Hutchinson Cancer Research Center: Research Funding; Aptose Therapeutics, Inc (formerly Lorus): Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oregon Health & Science University: Patents & Royalties; Henry Stewart Talks: Patents & Royalties; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; McGraw Hill: Patents & Royalties; Cylene Pharmaceuticals: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; CTI Biosciences: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Oncotide Pharmaceuticals: Research Funding; MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals: Research Funding; Roche TCRC, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sage Bionetworks: Research Funding; AstraZeneca: Consultancy. Deininger:Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Purpose: Chronic myelomonocytic leukemia (CMML) is a clinically heterogeneous myelodysplastic/myeloproliferative neoplasm with short overall survival. Emerging data based on sequencing of candidate genes with a known role in myeloid leukemia have identified recurrent CMML mutations, some of which have been associated with poor prognosis. A comprehensive evaluation of the mutational landscape of CMML and its prognostic significance is lacking. Patients and Methods. We comprehensively characterized the mutational landscape of CMML by a 2-step design. We initially performed whole exome sequencing (WES) of paired leukemia and germline DNA from 21 patients with a confirmed CMML diagnosis (discovery cohort) to identify genes with somatic mutations. From this discovery cohort, 215 genes showing potential mutations as well as 61 genes selected from the literature were examined by targeted resequencing in a second cohort of 69 clinically annotated CMML patients, using two independent platforms for orthogonal confirmation Blood or marrow samples from 22 young and 17 old controls were included as controls. Results. We identified 22 genes with mutations in >3% of CMML patients, and 67/69 patients (97%) had one or more mutations in at least one of these genes. SRSF2, ASXL1 and TET2 were the most frequently mutated genes. Several novel CMML genes were identified, including FAT4 with a mutation prevalence of 10% and BCR, CBFA2T3, and TRPM1 with a prevalence of 3 – 9% (see Table). Total deleterious mutations per patient ranged from 0 – 11, with significant exclusion or association of various combinations of mutations in SETBP1, ASXL1, KRAS, TET2, and EZH2 observed. In univariate analysis hemoglobin < 9g/dL, white blood cell count > 15x109/L, no treatment with hypomethylating agents, mutations in ASXL1, EZH2, or NRAS and mutations in growth factor signaling genes were associated with shorter overall survival. In multivariate analysis, mutations in NRAS (P Conclusion. (i) The mutational landscape of CMML is complex and involves mutations in multiple genes, many affected with relatively low prevalence. (ii) Mutations in FAT4, a putative tumor suppressor and key regulator of the Hippo pathway, occur in approximately 10% of patients. (iii) Absence of therapy with hypomethylating agents and mutations in NRAS or the functional group of growth factor signaling genes are predictive of poor survival. Table. Count and prevalence of N=69 CMML patients having a deleterious mutation in one of the genes observed mutated at a prevalence ≥ 10%. Gene # CMML Patients with Mutation (%) SRSF2 34 (49%) TET2 28 (41%) ASXL1 25 (36%) RUNX1 14 (20%) SETBP1 11 (16%) KRAS 10 (14%) EZH2 8 (12%) FAT4 7 (10%) CBL 7 (10%) NRAS 7 (10%) Figure. Overall survival of N=48 CMML patients by HMA use and presence of deleterious mutation in NRAS. Figure. Overall survival of N=48 CMML patients by HMA use and presence of deleterious mutation in NRAS. Disclosures Mason: Agilent, Inc.: Research Funding. Deininger:Celgene: Research Funding; Agilent, Inc.: Research Funding.

Mutations in the BCR-ABL1 kinase domain are a well-established mechanism of tyrosine kinase inhibitor (TKI) resistance, but fail to explain many cases of clinical TKI failure. In the remaining patients, resistance occurs via activation of alternative signaling pathways that maintain survival despite BCR-ABL1 inhibition (BCR-ABL1-independent resistance). STAT3 mediates TKI resistance in chronic myeloid leukemia (CML) cells cultured in the presence of bone marrow-derived factors (Bewry et al., 2008; Traer et al., 2012; Nair et al., 2012), and also plays a critical role in survival of CML cells with BCR-ABL1-independent resistance (Eiring et al. #31, ASH 2012). While targeting transcription factors is notoriously difficult, our combination of synthetic chemistry, in vitro reporter assays, and computational modeling has led to a low micromolar mechanism-based STAT3 inhibitor, which, in combination with TKIs, shows promise as a treatment for CML patients with BCR-ABL1-independent resistance. The original compound of the series, SF1-066 (10 µM; Fletcher et al., 2009), combines with TKIs to reduce survival of CML CD34+ cells exhibiting BCR-ABL1-independent resistance (Eiring et al. #31, ASH 2012). To improve the potency and selectivity of SF1-066, we synthesized successive STAT3 inhibitor libraries and ranked candidates by structure-activity relationship using a luciferase-based reporter screen (Kraft et al. #2445, ASH 2012). This reporter assay quantifies STAT3 transcriptional activity in TKI-resistant AR230R cells, which grow in the continuous presence of imatinib (1.0 µM), lack BCR-ABL1 kinase domain mutations, and exhibit high levels of pSTAT3Y705, thereby enabling convenient, high-throughput screening for potency and selectivity in the context of endogenous STAT3 activation. Among three sequential STAT3 inhibitor libraries, BP5-087 emerged as the new lead compound. Fluorescence polarization assays verified that BP5-087 was 5-fold more effective than SF1-066 in outcompeting an SH2 peptide probe, and computational simulations predicted better overall binding of BP5-087 (-9.6 kcal/mol) versus SF1-066 (-7.6 kcal/mol) to the STAT3 SH2 interface. In AR230R cell growth assays, BP5-087 was effective at a 5-fold lower dose compared to SF1-066, with minimal effects on TKI-sensitive parental controls. Therefore, we tested BP5-087 in the context of primary TKI resistance. BP5-087 (1 µM) in combination with imatinib (2.5 µM) reduced colony formation and increased apoptosis of CD34+ cells from CML patients with BCR-ABL1-independent resistance. These cells have no BCR-ABL1 kinase domain mutations and undergo BCR-ABL1 kinase inhibition as detected by immunoblot analyses. In contrast, BP5-087 had no effect on CD34+ cells from newly diagnosed CML patients or normal individuals. Immunofluorescence demonstrated that dual treatment of TKI-resistant CML CD34+ cells resulted in reduced levels of nuclear pSTAT3Y705, consistent with an inhibitor of STAT3 dimerization. In more primitive CML stem cells, long term culture-initiating cell (LTC-IC) assays revealed that neither inhibitor alone had any effect on colony formation of primitive LTC-IC progenitors, whereas imatinib (2.5 µM) in combination with BP5-087 (1.0 µM) reduced LTC-IC colony formation by 66%. Consistent with this observation, immunofluorescence showed high levels of pSTAT3Y705 in primitive TKI-resistant CD34+CD38- cells when cultured in the presence but not absence of TKIs. To test the feasibility of BP5-087 for in vivo use, we treated mice orally with BP5-087 (25 mg/kg/day) for 4 weeks and observed no changes in body weight, peripheral blood cellularity, or bone marrow colony forming ability. Mass spectrometry confirmed that BP5-087 is orally bioavailable. In summary, BP5-087 is a systematically-derived, direct inhibitor of STAT3 that, in combination with TKIs, reduces survival of CML cells with BCR-ABL1-independent resistance. Further rounds of structure-activity optimization may reveal an inhibitor with a clinically-relevant effective concentration. Disclosures: Deininger: Bristol Myers Squibb: Advisory Boards Other, Consultancy, Research Funding; Ariad Pharmaceuticals: Advisory Boards, Advisory Boards Other, Consultancy; Novartis: Advisory Boards, Advisory Boards Other, Consultancy, Research Funding; Celgene: Research Funding; Gilead Sciences: Research Funding.

Introduction BCR-ABL1 kinase domain mutations are detected in 30-60% of patients who develop resistance to tyrosine kinase inhibitors (TKIs) such as imatinib. However, the underlying mechanism(s) of resistance in the remaining patients are not known. To identify BCR-ABL1-independent mechanisms of resistance to TKIs, we used K562 cells that were adapted for long-term growth in 1 µM imatinib (K562-R). These cells lack BCR-ABL1 kinase domain mutations and survive despite continued suppression of BCR-ABL1 kinase activity. To screen for novel genes associated with BCR-ABL1-independent resistance, parental K562 and K562-R cells were lentivirally infected with a pooled shRNA library containing 27,000 shRNAs targeting ∼5,000 genes with known roles in cell signaling (Cellecta, Human Module 1). Using an unbiased, customized algorithm, we identified 18 genes with multiple shRNA hits that were depleted more than three-fold in K562-R cells compared to parental controls. Among the top five genes were RAN and XPO1 (CRM1), both of which encode components of a nucleocytoplasmic transport complex. Parental K562 cells also showed depletion for RAN and XPO1 shRNAs, suggesting a potential role in pathogenesis of CML and magnified activity in TKI resistance. Recently, enhanced expression of XPO1 was shown in Philadelphia chromosome positive leukemia and investigated as a potential therapeutic target in blast crisis CML (Walker et al. ASH 2012). Methods To validate the data from our shRNA library screen, we cloned a RAN shRNA into a tet-inducible lentiviral vector (Cellecta). Transduced parental and K562-R cells were treated with/out imatinib (2.5 mM) in the presence or absence of doxycycline. Parental and K562-R cells were treated with various concentrations of the oral XPO1 inhibitor Selinexor (KPT-330), which is now being evaluated in Phase 1 clinical trials in patients with solid and hematological malignancies. Following 72 hours of treatment, proliferation was measured by MTS assay (Promega). Additionally, CD34+ cells from newly diagnosed CML patients were lentivirally transduced with RAN shRNA and treated with/out imatinib (2.5 mM) in the presence or absence of doxycycline (n=3) or KPT-330 (n=1). Survival was assessed by colony formation assays. Normal cord blood CD34+cells were used as controls. Results Transduction of parental or K562-R cells with the inducible RAN shRNA and treatment with doxycycline reduced RAN mRNA levels by 57 and 76% respectively compared to the untreated control. This level of RAN suppression was sufficient to reduce the proliferation of parental K562 by 54%. It also reduced the IC50 of imatinib by > 3-fold in K562-R cells. In CD34+ cells from newly diagnosed CML patients, RAN shRNA alone had no significant effect on colony formation, whereas RAN shRNA in combination with imatinib (2.5 µM) reduced colony formation by 46% compared to imatinib alone. The IC50 of KPT-330 was 30-35% less in K562-R cells compared to parental controls suggesting a higher sensitivity of K562-R cells to XPO1 inhibition. We observed that 25 nM KPT-330 reduced colony formation by 52% in primary CML cells without reducing cord blood CD34+colonies. Combination of 2.5 µM imatinib and 25 nM KPT-330 reduced the colony formation significantly compared to imatinib alone in primary CML cells with no change in the control. Conclusion An unbiased, shRNA library screen and subsequent validations with primary CML cells suggested that the activity of nucleocytoplasmic transport is crucial for the survival of CML cells. Genetic and pharmacologic disruption revealed that nucleocytoplasmic transport is a target in both TKI sensitive and resistant CML cells. A deeper understanding of this pathway in CML will be important for understanding BCR-ABL1 independent resistance to TKI treatment Disclosures: Kauffman: Karyopharm Therapeutics Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties. Schacham:Karyopharm: Membership on an entity’s Board of Directors or advisory committees. Chenchik:Cellecta: Membership on an entity’s Board of Directors or advisory committees. Bonneau:Cellecta: Membership on an entity’s Board of Directors or advisory committees. Deininger:Bristol-Myers Squibb: Advisory Boards Other, Consultancy, Research Funding; Ariad Pharmaceuticals: Advisory Boards, Advisory Boards Other, Consultancy; Novartis: Advisory Boards, Advisory Boards Other, Consultancy, Research Funding; Celgene: Research Funding; Gilead Sciences: Research Funding.

Abstract 31 In CML, TKI resistance in the absence of BCR-ABL1 kinase mutations is mechanistically unclear. Since extrinsic signals from the bone marrow (BM) microenvironment protect CML cells from TKIs in a STAT3-dependent manner (Bewry et al. 2008; Traer et al. 2011), we hypothesized that overt resistance may occur when CML cells maintain intrinsic STAT3 activation in the absence of extrinsic signals. We also asked whether combined targeting of STAT3 and BCR-ABL1 could produce synthetic lethality to overcome TKI resistance. To model extrinsic BCR-ABL1 kinase-independent resistance, we grew CML cell lines and CMLCD34+ progenitor cells from newly diagnosed patients in the presence of conditioned medium (CM) derived from HS-5 BM stromal cells. To model intrinsic resistance, we used the imatinib-resistant K562R and AR230R cell lines. Our models of TKI resistance proliferate in 1.0–2.5 μM imatinib and exhibit pSTAT3Y705 activation despite suppression of BCR-ABL1 kinase activity. To investigate the role of pSTAT3Y705 in TKI resistance, we used shRNA-mediated knockdown (shSTAT3), a dominant-negative mutant (dnSTAT3), and pharmacologic STAT3 inhibitors developed in our laboratories to block STAT3 activity. Compared to scrambled controls in the presence of HS-5 CM, shSTAT3 reduced colony formation (33.8% reduction, p Preliminary immunofluorescence data indicates that pSTAT3Y705 levels are higher in drug-resistant CML CD34+CD38− stem cells than in CD34+CD38+ progenitor cells, suggesting a potential role for pSTAT3Y705 in TKI resistance of primitive CML stem cells. To investigate whether pharmacologic STAT3 inhibition in combination with TKIs could overcome resistance, we tested a library of compounds that interfere with STAT3 dimerization. The most selective of these inhibitors, S3I-201.1066 (SF1-066), was biochemically shown to disrupt STAT3 dimerization and transcriptional activation. Compared to cells treated with 1.0 μM imatinib alone, addition of 10 μM SF1-066 impaired the clonogenicity of K562R cells by 68.6% (p We next developed a series of 14 second-generation STAT3 inhibitors and tested them using a luciferase reporter harboring sequential STAT3 cis-inducible elements to measure endogenous STAT3 activity in AR230R cells. Two lead compounds, BP2-047 and BP3-163, reduced luciferase activity by 69.2% (p Altogether, our data identifies STAT3 as a universal target in TKI-resistant CML cells, and suggests that simultaneous pharmacologic inhibition of STAT3 and BCR-ABL1 may be a suitable therapeutic strategy for treatment of patients with TKI resistance despite inhibition of BCR-ABL1 kinase activity. Disclosures: No relevant conflicts of interest to declare.

Abstract 2445 Constitutive activation of signal transducer and activator of transcription 3 (STAT3) correlates with drug resistance and a poor prognosis in many cancers. STAT3 signaling is mediated by phosphorylation at tyrosine-705 (p STAT3Y705), dimerization, and nuclear transactivation. In chronic myeloid leukemia (CML), pSTAT3Y705 is demonstrable under two distinct resistance scenarios: (1) extrinsic resistance, in which BCR-ABL1 kinase-independent survival signals originating from the bone marrow (BM) microenvironment activate pSTAT3Y705 in a JAK2- or TYK2-dependent manner, and (2) intrinsic resistance, in which BCR-ABL1 kinase-independent signals activate pSTAT3Y705 in response to kinase inhibition. Based on these observations, we identified TKI-resistant CML as an excellent model for developing and optimizing pharmacologic STAT3 inhibitors. Using K562 and AR230 CML cells that are resistant to 1 μM imatinib (K562R and AR230R; intrinsic resistance) and primary CML CD34+ progenitor cells exposed to BM stromal-derived conditioned medium (CM; extrinsic resistance), we examined the effects of direct pharmacologic inhibition of STAT3 in TKI-resistant CML. Here, we report the design and validation of next-generation STAT3 inhibitors identified through computational modeling and screening in AR230R CML cells expressing high levels of pSTAT3Y705. We initially examined the effects of an established STAT3 inhibitor, S3I-201.1066 (SF1–066). K562R or AR230R cells were treated with 1 μM imatinib and/or 10 μM SF1–066, followed by culture in methylcellulose medium and scoring after 14–16 days. Combination treatment reduced the clonogenicity of K562R and AR230R cells to 31.4% (p0.05). Next, CD34+ cells from newly diagnosed CML patients (n=4) were cultured for 96 hours in the presence of CM and treated with 2.5 μM imatinib, 10 μM SF1–066 or both. Equal numbers of cells were then plated in colony forming assays. Imatinib combined with SF1–066 reduced colony formation to 54.8% (p Computational modeling informed the design and synthesis of a second-generation, SF1–066-based library. We evaluated compounds with shared molecular functionalities using AR230R cells expressing a luciferase reporter containing sequential STAT3 sis-inducible elements (AR230R-SIE). At 10 μM, we found two inhibitors, BP2–047 and BP3–163, that reduced luminescence by 69.2% (p To account for the reduced luminescence in AR230R-NEG cells and analyze for more potent inhibitors, we performed the luciferase assay with candidate inhibitors at 5 μM. We identified BP5–087 and BP5–088, each with increased potency and STAT3 selectivity compared to SF1–066. These compounds decreased luminescence in AR230R-SIE cells by 36.1% (p0.05). Confirmation of STAT3 binding was obtained using fluorescence polarization assays, in which the EC50 values of BP5–087 and BP5–088 measured 8.5 μM and 4.6 μM, respectively. Studies with mouse and human liver microsomes also revealed that BP5–087 and BP5–088 exhibit enhanced metabolic stability compared to SF1–066. Interestingly, treatment with BP5–087 or BP5–088 (both at 1 and 5 μM) in CML CD34+ progenitors grown in CM showed increased cytoplasmic accumulation of pSTAT3Y705 compared to controls. Thus, we identified BP5–087 and BP5–088 as two of the most potent small-molecule binders of STAT3 reported. These compounds are promising frontrunners toward new therapies for TKI-resistant CML and other diseases in which STAT3 activation drives malignant phenotypes. Disclosures: No relevant conflicts of interest to declare.

Nature Reviews Cancer 12, 513–526 (2012) On page 521 of this article, the passage discussing β-catenin as a regulator of gene expression in chronic myeloid leukaemia should have cited the following article alongside reference 118: Radich, J. P. et al. Gene expression changes associated with progression and response in chronic myeloid leukemia.

Abstract 2481 Ponatinib (AP24534) is a pan-BCR-ABL inhibitor developed for treatment-refractory chronic myeloid leukemia (CML) and has significant activity in patients who fail second-line dasatinib and/or nilotinib tyrosine kinase inhibitor (TKI) therapy. A pivotal phase II trial (clinicaltrials.gov NCT01207440) is underway. BCR-ABL kinase domain mutation-mediated ponatinib resistance has been investigated in vitro (Cancer Cell 16, 2009, 401). Here, we developed ponatinib-resistant, BCR-ABL+ cell lines lacking a kinase domain mutation and investigated mechanisms of resistance to ponatinib and other TKIs. Methods: Four BCR-ABL+ CML cell lines (K562, AR230, BV173, and 32D(BCR-ABL)) were maintained in liquid culture containing ponatinib (0.1 nM) for 10 days. The ponatinib concentration was increased in small increments for a minimum of 90 days, yielding corresponding ponatinib-resistant cell lines. BCR-ABL kinase domain sequencing of sensitive and resistant cells confirmed BCR-ABL to be unmutated. Real-time qPCR was used to compare the expression of BCR-ABL in ponatinib-sensitive and -resistant cell lines. Immunoblot analysis (total and tyrosine-phosphorylated BCR-ABL) was used to the compare levels of BCR-ABL protein and to determine whether resistance to ponatinib corresponded with reduced (partially BCR-ABL-independent) or complete inhibition of BCR-ABL tyrosine phosphorylation (fully BCR-ABL-independent). Cell proliferation assays were performed on resistant and sensitive cell lines in the presence of ponatinib, nilotinib, and dasatinib. A small-molecule inhibitor screen composed of >90 cell-permeable inhibitors that collectively target the majority of the tyrosine kinome as well as other kinases (Blood 116, 2010, abstract 2754) is currently being applied to the 32D(BCR-ABL)R cell line in the presence of 24 nM ponatinib to assess synthetic lethality, with results analyzed using a companion drug sensitivity algorithm. As a second strategy to generate resistant lines, N-ethyl-N-nitrosourea (ENU) mutagenesis was done to investigate BCR-ABL kinase domain-mediated resistance in myeloid K562, AR230, BV173, and 32D(BCR-ABL) cells. After ENU exposure, cells were washed and cultured in 96-well plates with escalating ponatinib. Results: The four BCR-ABL+ cell lines initially grew in the presence of 0.1 nM but not 0.5 nM ponatinib. Upon gradual exposure to escalating ponatinib, each of the cell lines exhibited a degree of adaptation to growth in the presence of the inhibitor (range: 10 to 240-fold). Real-time qPCR showed a modest two-fold increase in BCR-ABL expression level in K562R, AR230R and BV173R cell lines relative to the respective parental lines. Based on immunoblot analysis, cell lines segregated into two categories of ponatinib resistance: partially (K562R and AR230R) or fully BCR-ABL-independent (BV173R and 32D(BCR-ABL)R). Cell proliferation assays showed that ponatinib resistant cell lines also exhibited resistance to nilotinib and dasatinib. The 32D(BCR-ABL)R cell line exhibited a level of ponatinib resistance comparable to that of the Ba/F3 BCR-ABLE255V cell line, which carries the most ponatinib-resistant BCR-ABL mutation. BCR-ABL tyrosine phosphorylation was efficiently blocked by low concentrations of ponatinib ( Conclusions: The ponatinib resistant, BCR-ABL+ cell lines described here exhibit either a partially or fully BCR-ABL independent mechanism of resistance. The molecular details of both processes will be reported, with an emphasis on the striking level of resistance (240-fold over starting conditions) exhibited by the 32D(BCR-ABL)R cell line. Our in vitro results indicate that BCR-ABL independent mechanisms may contribute to ponatinib resistance in myeloid CML cells. Disclosures: No relevant conflicts of interest to declare.