Abstract: Abstract 758 Primary leukemia stem cells (LSCs) reside in an in vivo microenvironment that supports the growth and survival of malignant cells. Despite the increasing understanding of the importance of niche interactions and primary cell biology in leukemia, many studies continue to focus on cell autonomous processes in artificial model systems. The majority of strategies to-date that attempt to define therapeutic targets in leukemia have relied on screening cell lines in culture; new strategies should incorporate the use of primary disease within a physiologic niche. Using a primary murine MLL-AF9 acute myeloid leukemia (AML) model highly enriched for LSCs, we performed an in vivo short hairpin RNA (shRNA) screen to identify novel genes that are essential for leukemia growth and survival. LSCs infected with pools of shRNA lentivirus were transplanted and grown in recipient mice for 2 weeks, after which bone marrow and spleen cells were isolated. Massively parallel sequencing of infected LSCs isolated before and after transplant was used to quantify the changes in shRNA representation over time. Our in vivo screens were highly sensitive, robust, and reproducible and identified a number of positive controls including genes required for MLL-AF9 transformation (Ctnnb1, Mef2c, Ccna1), genes universally required for cell survival (Ube2j2, Utp18), and genes required in other AML models (Myb, Pbx1, Hmgb3). In our primary and validation screens, multiple shRNAs targeting Integrin Beta 3 (Itgb3) were consistently depleted by more than 20-fold over two weeks in vivo. Follow up studies using RNA interference (RNAi) and Itgb3−/− mice identified Itgb3 as essential for murine leukemia cells growth and transformation in vivo, and loss of Itgb3 conferred a statistically significant survival advantage to recipient mice. Importantly, neither Itgb3 knockdown or genetic loss impaired normal hematopoietic stem and progenitor cell (HSPC) function in 16 week multilineage reconstitution assays. We further identified Itgav as the heterodimeric partner of Itgb3 in our model, and found that knockdown of Itgav inhibited leukemia cell growth in vivo. Consistent the therapeutic aims or our study, flow cytometry on primary human AML samples revealed ITGAV/ITGB3 heterodimer expression. To functionally assess the importance of gene expression in a human system, we performed another RNAi screen on M9 leukemia cells, primary human cord blood CD34+ cells transduced with MLL-ENL that are capable of growing in vitro or in a xenotransplant model in vivo. We found that ITGB3 loss inhibited M9 cell growth in vivo, but not in vitro, consistent with the importance of ITGB3 in a physiologic microenvironment. We explored the signaling pathways downstream of Itgb3 using an additional in vivo, unbiased shRNA screen and identified Syk as a critical mediator of Itgb3 activity in leukemia. Syk knockdown by RNAi inhibited leukemia cell growth in vivo; downregulation of Itgb3 expression resulted in decreased levels of Syk phosphorylation; and expression of an activated form of Syk, TEL-SYK, rescued the effects of Itgb3 knockdown on leukemia cell growth in vivo. To understand cellular processes controlled by Itgb3, we performed gene expression studies and found that, in leukemia cells, Itgb3 knockdown induced differentiation and inhibited multiple previously published LSC transcriptional programs. We confirmed these results using primary leukemia cell histology and a model system of leukemia differentiation. Finally, addition of a small molecule Syk inhibitor, R406, to primary cells co-cultured with bone marrow stroma caused a dose-dependent decrease in leukemia cell growth. Our results establish the significance of the Itgb3 signaling pathway, including Syk, as a potential therapeutic target in AML, and demonstrate the utility of in vivo RNA interference screens. Disclosures: Armstrong: Epizyme: Consultancy.

Abstract: Autophagy is a lysosomal pathway involved in degradation of intracellular material. It appears as an adaptation mechanism that is essential for cellular homeostasis in response to various stress conditions. Over the past decade, many studies have linked alteration of autophagy with cancer initiation and progression, autoimmune, inflammatory, metabolic, and degenerative diseases. This review highlights recent findings on the impact of autophagy on leukemic transformation of normal hematopoietic stem cells and summarizes its role on leukemic cell response to chemotherapy.

Abstract: Abstract 1817 Azacitidine (AZA) is the first line treatment for IPSS (index prognostic scoring system) high-risk myelodysplastic syndrome (HR-MDS). To date, only Khan et al. (Exp Hematol. 2008) and Hollenbach et al. (PLoS One. 2009) have reported apoptosis as a mechanism of AZA effect on MDS cell lines. Nevertheless, approximately 40% of patients treated with AZA are refractory to this molecule. To investigate the possible mechanisms of AZA resistance in MDS cells, we developped AZA-resistant cell clones (AZA-R) from the well-characterized MDS cell line SKM1. The bulk resistant SKM1 cell line (AZA-R) was obtained following long time exposure of cells to iterative and increasing doses of AZA ranging from 0.1 to 8mM. We first showed that AZA triggered loss of cell metabolism in SKM1 parental cells but not in their AZA-resistant counterpart at a maximally effective dose of 1mM. AZA-mediated loss of cell metabolism accounted mainly for induction of apoptosis as judged by both an increase in caspase 9 and 3 activities triggered by this compound and by a significant protection in the presence of the pan-caspase inhibitor Z-VAD-fmk in SKM1 parental cells. Conversely, no or very few activation of caspases and apoptosis were detected in AZA-R cells strongly suggesting that apoptosis is impaired in AZA-R SKM1 cells. Finally, unlike in SKM1 cells, AZA failed to induce mitochondrial membrane permeabilization in AZA-R SKM1 cells. Importantly, basal autophagy was increased in AZA-R versus AZA-S cells as shown by LC3-I cleavage into LC3-II, p62/SQSTM1 protein expression, cathepsin B activation, mTOR and S6 ribosomal protein dephosphorylation and finally electronic microscopy experiments. In addition, Acadesine, an adenosine derivative and AMPK agonist which targets autophagy was capable to circumvent AZA resistance in both AZA-R SKM1 cells and in medullary cells from five MDS patients resistant to AZA after 6 cycles of Azacitidine. In conclusion, targeting autophagy appears as an attractive therapeutical strategy to circumvent AZA resistance in both established MDS cell lines and cells from MDS patients. Therefore, drugs capable of inducing autophagy or autophagic cell death, such as Acadesine (Robert et al., PLoS One. 2009) which is currently in phase II clinical trials for the treatment of Chronic Lymphoblastic Leukemia could be also beneficial for HR-MDS patients resistant to AZA. Disclosures: Cluzeau: Celgene: Consultancy. Raynaud:Celgene: Consultancy.

Abstract: Abnormal expression of the transcription factor EVI1 through chromosome 3q26 rearrangements has been implicated in the development of one of the most therapeutically challenging high-risk subtypes of acute myeloid leukemia (AML). Here we integrated genomic and metabolic screening of hematopoietic stem cells to reveal that EVI1 overexpression altered cellular metabolism. A pooled shRNA screen targeting metabolic enzymes identified the ATP-buffering, mitochondrial creatine kinase CKMT1 as a druggable dependency in EVI1-positive AML. Of 18 screened AML cell lines harboring various genetic alterations, only the four EVI1-expressing lines exhibited markedly elevated CKMT1 protein expression and activity. Treatment of this cell line panel with either CKMT1-targeting shRNAs or cyclocreatine, an analog of the CKMT1 substrate creatine and inhibitor of the creatine biosynthesis pathway, showed that elevated CKMT1 protein expression correlated with sensitivity to CKMT1 pathway inhibition. Consistent with these data, flow cytometry analysis of a panel of 68 unselected primary AML patient specimens revealed that the four leukemias with the highest levels of EVI1 expression also had elevated CKMT1 protein levels and enhanced sensitivity to cyclocreatine treatment. We next established that enforced EVI1 expression increased CKMT1 protein and mRNA levels and that three independent shRNA molecules targeting EVI1 drastically reduced CKMT1 expression in two EVI1-positive AML cell lines. A luciferase-based reporter system established that RUNX1 represses CKMT1 expression through direct binding to its promoter. ChIP-qPCR approaches were then applied to dissect the sequential events involved in EVI1-induced CKMT1 upregulation and the possible role of RUNX1 as an intermediate. In both primary AML samples and cell lines, we determined that EVI1 represses RUNX1 expression by directly binding to its promoter. This, in turn, eliminates repressive RUNX1 binding at the CKMT1 promoter and thereby promotes CKMT1 expression. Based on these data, we explored the relationship between EVI1 and RUNX1 expression with CKMT1 mRNA levels in two AML transcriptional datasets (GSE14468 and GSE10358). We divided these cohorts into four subgroups with high versus low expression of EVI1 and RUNX1. Consistent with our mechanistic analysis, primary AML samples within the EVI1high/RUNX1low subgroup were significantly more likely to express high levels of CKMT1 than AML samples in the other three subgroups. CKMT1 promotes the metabolism of arginine to creatinine. To determine the effect of CKMT1 suppression on this pathway, we measured the metabolic flux of stable-isotope labeled L-arginine 13C6 through creatine synthesis using mass spectrometry. CKMT1-directed shRNAs or cyclocreatine selectively decreased intracellular phospho-creatine and blocked production of ATP by mitochondria. Salvage of the creatine pathway by exogenous phospho-creatine restored normal mitochondrial function, prevented the loss of viability of human EVI1-positive AML cells induced by cyclocreatine or CKMT1-directed shRNAs, and maintained the serial replating activity of Evi1-transformed bone marrow cells. Primary human EVI1-positive AML is frequently associated with somatic NRAS mutations. Thus, to investigate whether EVI1 over-expression sensitizes primary AMLs to CKMT1 inhibition in vivo, we transplanted primary NrasG12D mutant AMLs with and without elevated Evi1 expression into congenic recipient mice. In this system, Ckmt1 knockdown did not significantly alter the outgrowth of control Nras mutant AML cells compared to a shControl (63% versus 71%). By contrast, NrasG12D AML cells characterized by elevated Evi1 expression were profoundly depleted by Ckmt1 suppression to 2% versus 58% in shControl recipients. Consistent with these results, pharmacologic or genetic inhibition of the CKMT1-dependent pathway blocked disease progression and prolonged the survival of mice injected with human EVI1-positive cells but not with EVI1-negative cells, without noticeable cytotoxic effect on normal murine cells. In conclusion, we have integrated "omic" approaches to identify CKMT1 as a druggable liability in EVI-positive AML. This study supports a potential therapeutic avenue for targeting the creatine kinase pathway in EVI1-positive AML, which remains one of the worst outcome subtypes of AML. Disclosures DeAngelo: Incyte: Consultancy; Novartis: Consultancy; Celgene: Consultancy; Amgen: Consultancy; Baxter: Consultancy; Pfizer: Consultancy; Ariad: Consultancy. Stone:Pfizer: Consultancy; Agios: Consultancy; Jansen: Consultancy; Celator: Consultancy; Merck: Consultancy; Amgen: Consultancy; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Consultancy; Novartis: Consultancy; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Xenetic Biosciences: Consultancy; Sunesis Pharmaceuticals: Consultancy; Seattle Genetics: Consultancy; Roche: Consultancy; Juno Therapeutics: Consultancy; ONO: Consultancy.

Abstract: Citharoxazole (1), a new batzelline derivative featuring a benzoxazole moiety, was isolated from the Mediterranean deep‐sea sponge Latrunculia (Biannulata) citharistae Vacelet, 1969, together with the known batzelline C ( 2 ). This is the first chemical study of a Mediterranean Latrunculia species and the benzoxazole moiety is unprecedented for this family of marine natural products. The structure was mainly elucidated by the interpretation of NMR spectra and especially HMBC correlations. Copyright © 2011 John Wiley & Sons, Ltd.

Abstract: Imatinib is used to treat chronic myelogenous leukemia (CML), but resistance develops in all phases of this disease. The purpose of the present study was to identify the mode of resistance of newly derived imatinib-resistant (IM-R) and PD166326-resistant (PD-R) CML cells. IM-R and PD-R clones exhibited an increase in viability and a decrease in caspase activation in response to various doses of imatinib and PD166326, respectively, as compared with parental K562 cells. Resistance involved neither mutations in BCR-ABL nor increased BCR-ABL, MDR1 or Lyn expression, all known modes of resistance. To gain insight into the resistance mechanisms, we used pangenomic microarrays and identified 281 genes modulated in parental versus IM-R and PD-R cells. The gene signature was similar for IM-R and PD-R cells, accordingly with the cross-sensitivity observed for both inhibitors. These genes were functionally associated with pathways linked to development, cell adhesion, cell growth, and the JAK-STAT cascade. Especially relevant were the increased expression of the tyrosine kinases AXL and Fyn as well as CD44 and HMGA2. Small interfering RNA experiments and pharmacologic approaches identified FYN as a candidate for resistance to imatinib. Our findings provide a comprehensive picture of the transcriptional events associated with imatinib and PD166326 resistance and identify Fyn as a new potential target for therapeutic intervention in CML. [Mol Cancer Ther 2009;8(7):1924–33]

Abstract: Activating mutations in FLT3 occur in up to 35% of patients with AML and correlate with poor prognosis. Therapy directed against FLT3 has been shown to induce response in patients with AML, but these responses are almost always transient. Dual PI3K/mTOR inhibitors have displayed promising results in the treatment of solid tumors, and of hematological cancers. In this report we describe that a dual PI3K/mTOR inhibitor is effective against sorafenib-responsive, and -resistant, AML cell lines both in vitro and in vivo. We generated two cell lines by sustained treatment with sorafenib. Parental cell lines carry the FLT3-ITD mutation and are highly responsive to FLT3 inhibitors, while sorafenib-resistant cell lines display resistance to multiple FLT3 inhibitors. Next generation sequencing did not show any significant difference in the mutational burden in between responsive and resistant cell lines. While next generation sequencing identified FLT3-D835Y with an allele-depth of 67:37 in a resistant cell line, Sanger sequencing and protein mass-spectroscopy did not identify any acquired mutations in the kinase domain of FLT3 in the resistant cells. Moreover, sorafenib treatment effectively blocked FLT3 activation in resistant cells, while it was unable to block colony formation or cell survival, suggesting that the resistant cells are no longer dependent on FLT3. Gene expression analysis of sorafenib-sensitive and -resistant cell lines, as well as of blasts from patients with sorafenib-resistant AML, suggested an enrichment of the PI3K/mTOR pathway that correlated with the resistant phenotype, which was further supported by phospho-specific-antibody array analysis. The selective PI3K/mTOR inhibitor, gedatolisib, efficiently blocked proliferation, colony and tumor formation of resistant cell lines as well as induces apoptosis. Taken together, our data suggest that aberrant activation of the PI3K/mTOR pathway results in FLT3-inhibitors-resistance and a dual specific PI3K/mTOR inhibitor is an effective treatment in both tyrosine kinase inhibitor sensitive and resistant AML. Citation Format: Oscar Lindblad, Eugenia Cordero, Alexandre Puissant, Lucy Macaulay, Nuzhat N. Kabir, Jianmin Sun, Karin Haraldsson, Åke Borg, Fredrik Levander, Kimberly Stegmaier, Kristian Pietras, Lars Rönnstrand, Julhash U. Kazi. Mechanism of sorafenib resistance in acute myeloid leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 310.

Abstract: Background: Acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) are clonal hematopoietic disorders that predominantly occur in older adults. For a limited number of fit patients, induction followed by consolidation chemotherapy and/or allogeneic stem-cell transplant (SCT) leads to cure. However, many AML patients are ineligible for aggressive therapy; up to 30% have primary refractory disease and up to 50% will relapse after front-line therapy, requiring alternative approaches. For these patients, durable long-term remission rate is low, with 5-year overall survival rates lower than 10%. Therefore, there remains an urgent unmet need for novel therapies for such patients. CB-5339 is a second generation, potent and selective, orally bioavailable small molecule inhibitor of valosin containing protein (VCP)/p97. VCP/p97 is a key cellular enzyme involved in cellular stress response pathways critical to cancer cell growth and survival such as protein homeostasis and the response to DNA damage. Inhibition of VCP/p97 in a panel of 131 cancer cell lines representing 16 cancer types revealed AML as the most exquisitely dependent disease (p=0.004) on VCP/p97 function. Further, CB-5339 demonstrated antiproliferative potency in a panel of 16 AML cell lines (IC50:100nM - 500nM). To better characterize the effects of CB-5339 on human leukemia, viability assays were performed on a set of 30 genetically diverse primary AML patient samples. Cellular viability was impacted with a similar potency across samples, irrespective of underlying genetic abnormalities (mean IC50: 423nM). In vivo, CB-5339 treatment resulted in decreased circulating leukemic cells and significantly prolonged survival in an MLL-AF9 syngeneic AML mouse model (p=0.02). In addition, evidence of synergy was exhibited with standard of care AML therapy, a combination of an anthracycline and cytarabine. This triple combination regimen resulted in a 96% mean relative decrease in leukemic burden compared to control mice and significantly prolonged mice survival compared to each regimen alone (p & lt;0.0001). Importantly, CB-5339 was well tolerated as evidenced by stable weight curves and absence of significant myelosuppression. Here, we present a phase 1 study to evaluate CB-5339 in patients with relapsed/refractory (R/R) AML and intermediate or higher-risk MDS. Study design: This is a single arm, open-label, multi-center phase 1 clinical trial. The study includes two parts: 1) a Dose Escalation phase using a single-participant cohort accelerated titration schema in R/R AML or intermediate/higher-risk MDS (IPSS-R) participants and 2) a Dose Expansion phase using a 2 stage design for further assessment of disease specific cohorts and potential combination strategies at the recommended phase 2 dose (RP2D). CB-5339 will be administered orally once-daily (QD) for 4 days, followed by a 3-day treatment-free period weekly, in successive 28-day cycles until progressive disease or intolerable toxicity. Intraparticipant dose escalation will be permitted after Cycle 1. Twice-daily (BID) dosing may also be tested depending on initial PK assessments. The primary objective of this trial is to determine the safety, tolerability, and MTD and/or RP2D of CB-5339. The secondary objectives are to characterize PK properties and antitumor activity of CB-5339. Exploratory objectives will evaluate potential pharmacodynamic (PD) biomarkers and characterize the PK/PD relationship. Key inclusion criteria include participants with pathologically confirmed AML or intermediate/higher-risk MDS relapsed and/or refractory to standard therapies, ECOG ≤2, and adequate kidney and liver functions. Key exclusion criteria include pathologically confirmed acute promyelocytic leukemia, central nervous system involvement, immediately life-threatening/severe complications of AML/MDS such as uncontrolled bleeding, uncontrolled active infection, pneumonia with hypoxia or shock, and/or disseminated intravascular coagulation, SCT within 100 days of first dose, active GVHD, or post-SCT systemic immunosuppressive therapy, concomitant active malignancy requiring chemotherapy and adverse cardiac history. Up to 60 participants (20 in dose escalation and 40 in dose expansion) will be enrolled at approximately 10 sites in the U.S and Australia. Recruitment is ongoing and this trial is registered with clinicaltrials.gov: NCT04402541. Disclosures Benajiba: Gilead Foundation: Research Funding. Carraway:BMS: Consultancy, Other: Research support, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Jazz: Consultancy, Speakers Bureau; Stemline: Consultancy, Speakers Bureau; Takeda: Other: Independent Advisory Committe (IRC); ASTEX: Other: Independent Advisory Committe (IRC); Abbvie: Other: Independent Advisory Committe (IRC). Hamad:Novartis: Honoraria; Abbvie: Honoraria. Stein:Biotheryx: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; PTC Therapeutics: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees; Syndax: Consultancy, Research Funding; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bayer: Research Funding; Seattle Genetics: Consultancy; Daiichi-Sankyo: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Consultancy; Astellas Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Consultancy. Burroughs:Chiasma: Current equity holder in publicly-traded company; Synthetic Genomics: Current equity holder in private company; Cleave Therapeutics: Current Employment, Current equity holder in private company; Crinetics Pharmaceuticals: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Harris:Cleave Therapeutics: Current Employment, Current equity holder in private company. Lane:Cleave Therapeutics: Consultancy; Regimmune Corporation: Consultancy; Star Therapeutics: Consultancy; Nanoscope Therapeutics: Consultancy; Viewpoint Therapeutics: Ended employment in the past 24 months; Valitor: Consultancy; Revolution Medicines: Consultancy. Nguyen:Cleave Therapeutics, Inc: Current Employment, Current equity holder in private company. Stuart:Cleave Therapeutics: Consultancy; Triphase Accelerator U.S Corporation: Consultancy; Revolution Medicines Inc: Consultancy; RegImmune Corp: Consultancy; IgM Biosciences: Consultancy; Portola Pharmaceuticals Inc: Consultancy; Artiva Biotherapeutics: Consultancy; Gilead Sciences Inc: Consultancy; Theravance Biopharma: Consultancy; Integral Medicines Inc: Consultancy. Vargas:Cytomx Therapeutics, Inc.: Current equity holder in publicly-traded company; Sangamo Therapeutics: Current equity holder in publicly-traded company; Cleave Therapeutics, Inc.: Current Employment, Current equity holder in private company. Stegmaier:Novartis: Research Funding; Auron Therapeutics: Consultancy. DiNardo:ImmuneOnc: Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Daiichi Sankyo: Consultancy, Honoraria, Research Funding; Takeda: Honoraria; Notable Labs: Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Honoraria, Research Funding; MedImmune: Honoraria; Agios: Consultancy, Honoraria, Research Funding; Jazz: Honoraria; Novartis: Consultancy; Calithera: Research Funding; Syros: Honoraria.

Abstract: Bromodomain inhibition comprises a promising therapeutic strategy in cancer, particularly for hematologic malignancies. To date, however, genomic biomarkers to direct clinical translation have been lacking. We conducted a cell-based screen of genetically defined cancer cell lines using a prototypical inhibitor of BET bromodomains. Integration of genetic features with chemosensitivity data revealed a robust correlation between MYCN amplification and sensitivity to bromodomain inhibition. We characterized the mechanistic and translational significance of this finding in neuroblastoma, a childhood cancer with frequent amplification of MYCN. Genome-wide expression analysis showed downregulation of the MYCN transcriptional program accompanied by suppression of MYCN transcription. Functionally, bromodomain-mediated inhibition of MYCN impaired growth and induced apoptosis in neuroblastoma. BRD4 knockdown phenocopied these effects, establishing BET bromodomains as transcriptional regulators of MYCN. BET inhibition conferred a significant survival advantage in 3 in vivo neuroblastoma models, providing a compelling rationale for developing BET bromodomain inhibitors in patients with neuroblastoma. Significance: Biomarkers of response to small-molecule inhibitors of BET bromodomains, a new compound class with promising anticancer activity, have been lacking. Here, we reveal MYCN amplification as a strong genetic predictor of sensitivity to BET bromodomain inhibitors, show a mechanistic rationale for this finding, and provide a translational framework for clinical trial development of BET bromodomain inhibitors for pediatric patients with MYCN-amplified neuroblastoma. Cancer Discov; 3(3); 308–23. ©2012 AACR. See related commentary by Schnepp and Maris, p. 255 This article is highlighted in the In This Issue feature, p. 239