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FMS-like Tyrosine Kinase 3 (FLT3) mutation is associated with poor survival in acute myeloid leukemia (AML). The specific Anexelekto/MER Tyrosine Kinase (AXL) inhibitor, ONO-7475, kills FLT3-mutant AML cells with targets including Extracellular- signal Regulated Kinase (ERK) and Myeloid Cell Leukemia 1 (MCL1). ERK and MCL1 are known resistance factors for Venetoclax (ABT-199), a popular drug for AML therapy, prompting the investigation of the efficacy of ONO-7475 in combination with ABT-199 in vitro and in vivo. ONO-7475 synergizes with ABT-199 to potently kill FLT3-mutant acute myeloid leukemia cell lines and primary cells. ONO-7475 is effective against ABT-199-resistant cells including cells that overexpress MCL1. Proteomic analyses revealed that ABT-199-resistant cells expressed elevated levels of pro-growth and anti-apoptotic proteins compared to parental cells, and that ONO-7475 reduced the expression of these proteins in both the parental and ABT-199-resistant cells. ONO-7475 treatment significantly extended survival as a single in vivo agent using acute myeloid leukemia cell lines and PDX models. Compared to ONO-7474 monotherapy, the combination of ONO-7475/ABT-199 was even more potent in reducing leukemic burden and prolonging the survival of mice in both model systems. These results suggest that the ONO-7475/ABT-199 combination may be effective for AML therapy.

MCL-1 and BCL-2 are both frequently overexpressed in acute myeloid leukemia and critical for the survival of acute myeloid leukemia cells and acute myeloid leukemia stem cells. MCL-1 is a key factor in venetoclax resistance. Using genetic and pharmacological approaches, we discovered that MCL-1 regulates leukemia cell bioenergetics and carbohydrate metabolisms, including the TCA cycle, glycolysis and pentose phosphate pathway and modulates cell adhesion proteins and leukemia-stromal interactions. Inhibition of MCL-1 sensitizes to BCL-2 inhibition in acute myeloid leukemia cells and acute myeloid leukemia stem/progenitor cells, including those with intrinsic and acquired resistance to venetoclax through cooperative release of pro-apoptotic BIM, BAX, and BAK from binding to anti-apoptotic BCL-2 proteins and inhibition of cell metabolism and key stromal microenvironmental mechanisms. The combined inhibition of MCL-1 by MCL-1 inhibitor AZD5991 or CDK9 inhibitor AZD4573 and BCL-2 by venetoclax greatly extended survival of mice bearing patient-derived xenografts established from an acute myeloid leukemia patient who acquired resistance to venetoclax/decitabine. These results demonstrate that co-targeting MCL-1 and BCL-2 improves the efficacy of and overcomes preexisting and acquired resistance to BCL-2 inhibition. Activation of metabolomic pathways and leukemia-stroma interactions are newly discovered functions of MCL-1 in acute myeloid leukemia, which are independent from canonical regulation of apoptosis by MCL-1. Our data provide new mechanisms of synergy and rationale for co-targeting MCL-1 and BCL-2 clinically in patients with acute myeloid leukemia and potentially other cancers.

Focal adhesion kinase (FAK) promotes cancer cell growth and metastasis. We previously reported that FAK inhibition by the selective inhibitor VS-4718 exerted antileukemia activities in acute myeloid leukemia (AML). The mechanisms involved, and whether VS-4718 potentiates efficacy of other therapeutic agents, have not been investigated. Resistance to apoptosis inducted by the BCL-2 inhibitor ABT-199 (venetoclax) in AML is mediated by preexisting and ABT-199–induced overexpression of MCL-1 and BCL-XL. We observed that VS-4718 or silencing FAK with siRNA decreased MCL-1 and BCL-XL levels. Importantly, VS-4718 antagonized ABT-199–induced MCL-1 and BCL-XL. VS-4718 markedly synergized with ABT-199 to induce apoptosis in AML cells, including primary AML CD34+ cells and AML cells overexpressing MCL-1 or BCL-XL. In a patient-derived xenograft (PDX) model derived from a patient sample with NPM1/FLT3-ITD/TET2/DNMT3A/WT1 mutations and complex karyotype, VS-4718 statistically significantly reduced leukemia tissue infiltration and extended survival (72 vs. control 36 days, P = 0.0002), and only its combination with ABT-199 effectively decreased systemic leukemia tissue infiltration and circulating blasts, and prolonged survival (65.5 vs. control 36 days, P = 0.0119). Furthermore, the combination decreased NFκB signaling and induced the expression of IFN genes in vivo. The combination also markedly extended survival of a second PDX model developed from an aggressive, TP53-mutated complex karyotype AML sample. The data suggest that the combined inhibition of FAK and BCL-2 enhances antileukemia activity in AML at least in part by suppressing MCL-1 and BCL-XL and that this combination may be effective in AML with TP53 and other mutations, and thus benefit patients with high-risk AML.

Although highly effective, BCR-ABL1 tyrosine kinase inhibitors do not target chronic myeloid leukemia (CML) stem cells. Most patients relapse upon tyrosine kinase inhibitor therapy cessation. We reported previously that combined BCR-ABL1 and BCL-2 inhibition synergistically targets CML stem/progenitor cells. p53 induces apoptosis mainly by modulating BCL-2 family proteins. Although infrequently mutated in CML, p53 is antagonized by MDM2, which is regulated by BCR-ABL1 signaling. We hypothesized that MDM2 inhibition could sensitize CML cells to tyrosine kinase inhibitors. Using an inducible transgenic Scl-tTa-BCR-ABL1 murine CML model, we found, by RT-PCR and CyTOF proteomics increased p53 signaling in CML bone marrow (BM) cells compared with controls in CD45+ and linage-SCA-1+C-KIT+ populations. CML BM cells were more sensitive to exogenous BH3 peptides than controls. Combined inhibition of BCR-ABL1 with imatinib and MDM2 with DS-5272 increased NOXA level, markedly reduced leukemic linage-SCA-1+C-KIT+ cells and hematopoiesis, decreased leukemia burden, significantly prolonged the survival of mice engrafted with BM cells from Scl-tTa-BCR-ABL1 mice, and significantly decreased CML stem cell frequency in secondary transplantations. Our results suggest that CML stem/progenitor cells have increased p53 signaling and a propensity for apoptosis. Combined MDM2 and BCR-ABL1 inhibition targets CML stem/progenitor cells and has the potential to improve cure rates for CML.

Context: MLL1-menin interaction in NPM1-mutated (NPM1c) AML shares a common HOX gene signature and dependencies as that of MLL-rearrangements (MLL1-r) with menin. Menin inhibition has demonstrated anti-leukemia activity in both NPM1c and MLL-r AML. NPM1c frequently occurs in AML with FLT3-ITD/FLT3-TKD mutations. Co-inhibition of menin and FLT3 has shown enhanced anti-leukemia activity in MLL-r/FLT3- and NPM1c/FLT3-mutated AML. Targeting Bcl-2 has emerged as a promising therapeutic option for AML patients. However, despite the major improvement of combining the Bcl-2 inhibitor venetoclax with hypomethylating agents, most patients develop resistance and ultimately relapse. Objective: Investigate anti-leukemic activities, potential synergism, and mechanisms of menin-MLL1 inhibitor SDNX-50469, an equipotent surrogate of the clinical compound SNDX-5613 and venetoclax combination in vivo in an NPM1c/FLT3-ITD/TKD PDX model. Design: The PDX cell-engrafted NSG mice were treated with 0.05 or 0.1% SNDX-50469-spiked chow, venetoclax (50 mg/kg), or 0.1% SNDX-50469 plus venetoclax (one month). Engraftment and disease progression were assessed by flow cytometric measurement of circulating human CD45+ cells. The treatment effects on leukemia cell populations and protein levels were determined by CyTOF mass cytometry. Results: Menin inhibition exhibited strong anti-leukemia activity, which was enhanced by venetoclax, while venetoclax alone had minimal effects. The combination most effectively extended survival (143 d for 0.1% SNDX-50469 plus venetoclax, P=0.0003; 131 and 125 d for 0.1% or 0.05% SNDX-50469, respectively, both P=0.0001; 69 d for venetoclax, P=0.025; vs 61 d for controls). At the end of treatment, CyTOF analysis of bone marrow cells demonstrated that menin inhibition preferentially targeted CD34+CD38+ cells, while venetoclax targeted CD34+CD38- cells. Only the combination effectively eliminated bulk and CD34+CD38+/CD34+CD38-stem/progenitor cells. Menin inhibition increased the percentage of CD11b+ myeloid cells. Mechanistically, menin inhibition decreased Bcl-2 and Bcl-xL and concomitantly increased Bax and Bim, which seemingly enhanced venetoclax activity. However, we observed increased p-FLT3 in the surviving leukemia cells, particularly in the combination group, which may contribute to the regrowth of leukemia cells. Conclusions: Our study validated menin as a therapeutic target and demonstrated that its inhibition synergizes with venetoclax in NPM1/FLT3-mutated AML, which warrants clinical evaluation. Inhibition of FLT3 may further enhance menin and Bcl-2 co-targeting efficacy in NPM1- and FLT3-mutated AML.

Purpose: Wnt/β-catenin signaling is required for leukemic stem cell function. FLT3 mutations are frequently observed in acute myeloid leukemia (AML). Anomalous FLT3 signaling increases β-catenin nuclear localization and transcriptional activity. FLT3 tyrosine kinase inhibitors (TKI) are used clinically to treat FLT3-mutated AML patients, but with limited efficacy. We investigated the antileukemia activity of combined Wnt/β-catenin and FLT3 inhibition in FLT3-mutant AML. Experimental Design: Wnt/β-catenin signaling was inhibited by the β-catenin/CBP antagonist C-82/PRI-724 or siRNAs, and FLT3 signaling by sorafenib or quizartinib. Treatments on apoptosis, cell growth, and cell signaling were assessed in cell lines, patient samples, and in vivo in immunodeficient mice by flow cytometry, Western blot, RT-PCR, and CyTOF. Results: We found significantly higher β-catenin expression in cytogenetically unfavorable and relapsed AML patient samples and in the bone marrow–resident leukemic cells compared with circulating blasts. Disrupting Wnt/β-catenin signaling suppressed AML cell growth, induced apoptosis, abrogated stromal protection, and synergized with TKIs in FLT3-mutated AML cells and stem/progenitor cells in vitro. The aforementioned combinatorial treatment improved survival of AML-xenografted mice in two in vivo models and impaired leukemia cell engraftment. Mechanistically, the combined inhibition of Wnt/β-catenin and FLT3 cooperatively decreased nuclear β-catenin and the levels of c-Myc and other Wnt/β-catenin and FLT3 signaling proteins. Importantly, β-catenin inhibition abrogated the microenvironmental protection afforded the leukemic stem/progenitor cells. Conclusions: Disrupting Wnt/β-catenin signaling exerts potent activities against AML stem/progenitor cells and synergizes with FLT3 inhibition in FLT3-mutant AML. These findings provide a rationale for clinical development of this strategy for treating FLT3-mutated AML patients. Clin Cancer Res; 24(10); 2417–29. ©2018 AACR.

Although overexpression/activation of focal adhesion kinase (FAK) is widely known in solid tumors to control cell growth, survival, invasion, metastasis, gene expression, and stem cell self-renewal, its expression and function in myeloid leukemia are not well investigated. Using reverse-phase protein arrays in large cohorts of newly diagnosed acute myeloid leukemia (AML) and myeloid dysplastic syndrome (MDS) samples, we found that high FAK expression was associated with unfavorable cytogenetics (P = 2 × 10−4) and relapse (P = 0.02) in AML. FAK expression was significantly lower in patients with FLT3-ITD (P = 0.0024) or RAS (P = 0.05) mutations and strongly correlated with p-SRC and integrinβ3 levels. FAK protein levels were significantly higher in CD34+ (P = 5.42 × 10−20) and CD34+CD38− MDS (P = 7.62 × 10−9) cells compared with normal CD34+ cells. MDS patients with higher FAK in CD34+ cells tended to have better overall survival (P = 0.05). FAK expression was significantly higher in MDS patients who later transformed to compared with those who did not transform to AML and in AML patients who transformed from MDS compared with those with de novo AML. Coculture with mesenchymal stromal cells (MSC) increased FAK expression in AML cells. Inhibition of FAK decreased MSC-mediated adhesion/migration and viability of AML cells and prolonged survival in an AML xenograft murine model. Our results suggest that FAK regulates leukemia–stromal interactions and supports leukemia cell survival; hence, FAK is a potential therapeutic target in myeloid leukemia. Mol Cancer Ther; 16(6); 1133–44. ©2017 AACR.

Tyrosine kinase inhibitor (TKI) resistance and progression to blast crisis (BC), both related to persistent β-catenin activation, remain formidable challenges for chronic myeloid leukemia (CML). We observed overexpression of β-catenin in BC-CML stem/progenitor cells, particularly in granulocyte–macrophage progenitors, and highest among a novel CD34+CD38+CD123hiTim-3hi subset as determined by CyTOF analysis. Co-culture with mesenchymal stromal cells (MSCs) induced the expression of β-catenin and its target CD44 in CML cells. A novel Wnt/β-catenin signaling modulator, C82, and nilotinib synergistically killed KBM5T315I and TKI-resistant primary BC-CML cells with or without BCR–ABL kinase mutations even under leukemia/MSC co-culture conditions. Silencing of β-catenin by short interfering RNA restored sensitivity of primary BCR–ABLT315I/E255V BC-CML cells to nilotinib. Combining the C82 pro-drug, PRI-724, with nilotinib significantly prolonged the survival of NOD/SCID/IL2Rγ null mice injected with primary BCR–ABLT315I/E255V BC-CML cells. The combined treatment selectively targeted CML progenitors and inhibited CD44, c-Myc, survivin, p-CRKL and p-STAT5 expression. In addition, pretreating primary BC-CML cells with C82, or the combination, but not with nilotinib alone, significantly impaired their engraftment potential in NOD/SCID/IL2Rγ-null-3/GM/SF mice and significantly prolonged survival. Our data suggest potential benefit of concomitant β-catenin and Bcr–Abl inhibition to prevent or overcome Bcr–Abl kinase-dependent or -independent TKI resistance in BC-CML.

Background: Outcomes in patients (pts) with relapsed/refractory (R/R) myelofibrosis (MF) post JAK inhibitor are poor [overall survival (OS) 13-14 months] (Newberry K, Blood 2017; Kuykendall A, Ann Hematol 2018). There are no approved therapies beyond JAK inhibitors for pts with MF. In certain clinical situations, such as severe thrombocytopenia, administration of JAK inhibitors may be difficult or even contraindicated. SMAC mimetics (or IAP antagonists), a novel class of anti-cancer therapeutics, lead to apoptotic cancer cell death through targeting of IAPs. Because these agents are hypothesized to be particularly effective in a TNFα-rich micro-environment, such as in pts with MF, a group known to have markedly increased cytokines including TNFα (Fleischman A Blood 2011; Heaton W, Leukemia 2018; Tefferi A, JCO 2011), we conducted an investigator-initiated phase 2 clinical trial with LCL161 in pts with intermediate to high risk PMF or post-ET/PV MF. Objectives: Primary: overall response rate (ORR) by IWG-MRT 2013; secondary and exploratory: time to response, response duration, safety, improvement in symptom burden by MPN-SAF TSS, measurement of cIAP1/2, XIAP, and mutational profiling via next-generation sequencing. Clinical Trial Design: LCL161 is an oral monovalent SMAC mimetic, administered once weekly, starting dose 1500 mg with 2 dose level reductions (DL-1, 1200mg; DL-2, 900 mg) in pts with MF. One cycle was 28 days. Pts age ≥18, PS 0-2, who were not candidates for, intolerant to, or R/R to JAK inhibitors were eligible. After 3 cycles, bone marrow biopsy/response evaluations were performed. There was no minimum platelet count for eligibility, and prior allogeneic stem cell transplant (SCT) was allowed. Results: The study has completed enrollment. A total of 47 pts were treated with a median age of 72 years [56-85]. The median baseline platelet count was 51 x 109/L [6-1365]. The median baseline spleen size was 12 cm among 28 pts with enlarged spleen at baseline. 70% of the pts had ≥2 prior therapies; 55% had a prior JAK inhibitor, 2 pts had prior SCT. 75% were IPSS high-risk. Driver mutations: 64% JAK2 V167F; 13% CALR; 11% MPL W515L; 5 pts had triple negative MF. Mutation analysis revealed the three most common additional mutations: ASXL1 (23%); TET2 (15%); DNMT3a (11%). ORR was 32% (15/47 pts); 15 pts exhibited a total of 19 objective responses (4 pts met criteria for 2 separate IWG 2013 responses): Clinical improvement (CI) symptoms (n=11); CI anemia (n=6); CI spleen (n=1), cytogenetic response (n=1). At a median follow-up of 21.1 months (mo) [3.9-49.7+], the median number of cycles received was 5 [1-52+], the median time to response was 1.4 mo [0.9-9.1] and median response duration (mo) was 31.5 [3.6-48.8+]. Long-term responders: n=8 for ≥1 year; n=4 for ≥2 years/ongoing. Importantly, median OS is not yet reached on this study (Figure). The most common non-hematologic toxicities (grade 1/2): nausea/vomiting (60%); fatigue syndrome (49%), dizziness/vertigo (32%); non-hematologic Grade 3/4: syncope (n=2); nausea/vomiting (n=1). Hematologic toxicities (grade 3/4): thrombocytopenia n=3 (6%); anemia n=2 (4%). 36% pts had a dose reduction; the most common cause was fatigue (n=10). Overall, 81% pts are now off study [Stable disease/no objective response (n=16); progressive disease (n=9); toxicity (n=5), pt's choice/other (n=6), proceeded to SCT (n=2)]. Correlative analysis: Preliminary analysis demonstrates on-target inhibition of cIAP1 observed in all responding pts analyzed; high levels of XIAP and/or XIAP increases were observed in many pts with resistance/disease progression. Anemia responders: 6 pts achieved CI anemia (n=4 hemoglobin responses, n=2 achievement of transfusion independence). Among these 6 pts: median time to response (mo): 3.1 [0.9-9.1]; median response duration (mo): 8.4 [5.5-28.6]. Conclusion: In an older group of pts with 70% with ≥2 prior therapies (55% JAK inhibitor-exposed), median baseline platelet count of 51, ASXL1-mutated in 23%, we observed a 32% ORR. The median OS has not yet been reached. Oral SMAC mimetics may represent a possible option for older pts, those who have failed prior JAK inhibitor, and those with thrombocytopenia limiting entry onto other trials. Future directions include combination studies with hypomethylating agents and JAK inhibitors for pts with myeloid malignancies. This clinical trial is registered at ClinicalTrials.gov as NCT02098161. Figure Disclosures Pemmaraju: sagerstrong: Research Funding; plexxikon: Research Funding; Daiichi-Sankyo: Research Funding; novartis: Consultancy, Research Funding; samus: Research Funding; abbvie: Consultancy, Honoraria, Research Funding; cellectis: Research Funding; celgene: Consultancy, Honoraria; incyte: Consultancy, Research Funding; affymetrix: Research Funding; Stemline Therapeutics: Consultancy, Honoraria, Research Funding; mustangbio: Consultancy, Research Funding. Carter:Amgen: Research Funding; AstraZeneca: Research Funding; Ascentage: Research Funding. Kantarjian:Amgen: Honoraria, Research Funding; Astex: Research Funding; Cyclacel: Research Funding; Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Ariad: Research Funding; BMS: Research Funding; Takeda: Honoraria; Agios: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding; Jazz Pharma: Research Funding; Immunogen: Research Funding; Daiichi-Sankyo: Research Funding; Novartis: Research Funding. Cortes:Sun Pharma: Research Funding; Forma Therapeutics: Consultancy, Honoraria, Research Funding; Daiichi Sankyo: Consultancy, Honoraria, Research Funding; BiolineRx: Consultancy; Jazz Pharmaceuticals: Consultancy, Research Funding; Biopath Holdings: Consultancy, Honoraria; Astellas Pharma: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Merus: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Research Funding; Immunogen: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding. Bose:CTI BioPharma: Research Funding; Promedior: Research Funding; NS Pharma: Research Funding; Astellas: Research Funding; Pfizer: Research Funding; Incyte Corporation: Consultancy, Research Funding, Speakers Bureau; Celgene Corporation: Consultancy, Research Funding; Blueprint Medicine Corporation: Consultancy, Research Funding; Kartos: Consultancy, Research Funding; Constellation: Research Funding. Kadia:Amgen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bioline RX: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Research Funding; Celgene: Research Funding; Genentech: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; AbbVie: Consultancy, Research Funding. Garcia-Manero:Merck: Research Funding; Novartis: Research Funding; AbbVie: Research Funding; Celgene: Consultancy, Research Funding; Astex: Consultancy, Research Funding; Onconova: Research Funding; H3 Biomedicine: Research Funding; Amphivena: Consultancy, Research Funding; Helsinn: Research Funding. Bueso-Ramos:Incyte: Consultancy. DiNardo:jazz: Honoraria; celgene: Consultancy, Honoraria; abbvie: Consultancy, Honoraria; agios: Consultancy, Honoraria; medimmune: Honoraria; syros: Honoraria; notable labs: Membership on an entity's Board of Directors or advisory committees; daiichi sankyo: Honoraria. Popat:Bayer: Research Funding; Incyte: Research Funding; Jazz: Consultancy. Konopleva:Genentech: Honoraria, Research Funding; Ascentage: Research Funding; Kisoji: Consultancy, Honoraria; Reata Pharmaceuticals: Equity Ownership, Patents & Royalties; Ablynx: Research Funding; Astra Zeneca: Research Funding; F. Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria; Agios: Research Funding; Cellectis: Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Eli Lilly: Research Funding; Forty-Seven: Consultancy, Honoraria; Stemline Therapeutics: Consultancy, Honoraria, Research Funding; Calithera: Research Funding. Borthakur:Oncoceutics, Inc.: Research Funding; PTC Therapeutics: Consultancy; BioLine Rx: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Strategia Therapeutics: Research Funding; Xbiotech USA: Research Funding; Tetralogic Pharmaceuticals: Research Funding; GSK: Research Funding; Argenx: Membership on an entity's Board of Directors or advisory committees; Arvinas: Research Funding; BioTheryX: Membership on an entity's Board of Directors or advisory committees; Eisai: Research Funding; Cantargia AB: Research Funding; AbbVie: Research Funding; FTC Therapeutics: Membership on an entity's Board of Directors or advisory committees; Incyte: Research Funding; Cyclacel: Research Funding; Polaris: Research Funding; NKarta: Consultancy; Agensys: Research Funding; Merck: Research Funding; Bayer Healthcare AG: Research Funding; AstraZeneca: Research Funding; Janssen: Research Funding; Novartis: Research Funding; Oncoceutics: Research Funding; BMS: Research Funding; Eli Lilly and Co.: Research Funding. Jain:Precision Biosciences: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Adaptive Biotechnologies: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Cellectis: Research Funding; Verastem: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Servier: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AstraZeneca: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen Pharmaceuticals, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Genentech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Research Funding; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; ADC Therapeutics: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics, an AbbVie company: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Jabbour:BMS: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; Adaptive: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Cyclacel LTD: Research Funding; Takeda: Consultancy, Research Funding. Verstovsek:Incyte: Research Funding; Roche: Research Funding; NS Pharma: Research Funding; Celgene: Consultancy, Research Funding; Gilead: Research Funding; Promedior: Research Funding; CTI BioPharma Corp: Research Funding; Genetech: Research Funding; Blueprint Medicines Corp: Research Funding; Novartis: Consultancy, Research Funding; Sierra Oncology: Research Funding; Pharma Essentia: Research Funding; Astrazeneca: Research Funding; Ital Pharma: Research Funding; Protaganist Therapeutics: Research Funding; Constellation: Consultancy; Pragmatist: Consultancy. OffLabel Disclosure: LCL161- not yet FDA approved drug; investigational agent = SMAC mimetic /IAP antagonist

Background. BCL-2 inhibition is a novel and highly effective treatment modality in acute myeloid leukemias (AML). AML patients with IDH1/2 mutations are highly sensitive to BCL-2 inhibition by venetoclax (VEN) (Chen et al Nat Med 2015). High expression levels of the BCL-2 family proteins MCL-1 or BCL-XL, or knockout of TP53 have been reported to confer resistance to BCL-2 inhibition (Pan et al. Cancer Cell 2017, Nechiporuk et al. Cancer Discov 2019). p73 is one of the p53 family transcription factors and generates two isoforms, transactivation p73 (TAp73) and the N-terminally truncated ΔNp73. TAp73 shares a homologous N-terminal activation domain with p53 and has pro-apoptotic function similar to p53. ΔNp73 lacks an activation domain and has a dominant negative effect on the DNA binding of TAp73 and more importantly, of p53.TP73 is expressed in AML except in acute promyelocytic leukemias. However, the associations of TP73 isoforms with clinical and genetic characteristics or sensitivity to BCL-2 inhibition in AML have not been explored. Results. We determined copy numbers of TAp73 and ΔNp73 mRNA levels in AML samples (N = 78) and normal CD34+ hematopoietic cells (HPC) using droplet digital PCR and investigated their clinical and biological relevance. Both TP73 isoforms were expressed in AML, with TAp73 expression being 50-fold higher in AML than in CD34+ HPC (P = 0.027); no difference seen for ΔNp73 (P = 0.80), suggesting that TAp73 is aberrantly expressed in AML cells. ΔNp73 and TAp73 mRNA levels were highly correlated (R2 = 0.72, P < 0.0001). AML samples had 10-fold more abundant TAp73 than ΔNp73 mRNA levels (P = 0.0017) and isoforms were not associated with disease status (de novo vs relapsed/refractory) or cytogenetic groups, and were mutation-agnostic, except for IDH1/2. IDH1/2 mutant AML showed lower levels of TAp73 and ΔNp73 than those with wild-type IDH1/2 (P = 0.06 and P = 0.007 for TAp73 and ΔNp73, respectively). In a separate dataset, we observed repressed TP73 in IDH1/2 mutant vs. wild-type AML samples (P = 0.073) by RNAseq analysis (N = 47). Mechanistically, treatment with cell permeable octyl-(R)-2HG, the oncometabolite of mutant IDH1/2, reduced both TAp73 and ΔNp73 and increased susceptibility to VEN. Lentiviral knockdown of p73 in OCI-AML3 cells resulted in enhanced sensitivity to VEN with no significant changes in MCL-1 and p53 protein levels, or TP53 targets (MDM2, CDKN1A, FAS and BBC3). VEN resistant AML cells (MOLM-13 and MV4;11) generated through long-term culture with VEN expressed highly elevated TP73 mRNA and protein levels without significant changes in p53 or TP53 target changes, suggesting that elevated p73 could confer resistance to VEN independent of p53 function (Figure). Knockdown of TP73 showed increased protein levels of SDHB, UQCRC2 and ATP5A, components of mitochondrial respiratory chain complex II, III and V, indicating increased dependency on oxdative phosphorylation by depleting p73. Overexpression of TAp73α by lentiviral gene transfer minimally increased VEN-induced apoptosis, while ΔNp73γ overexpression conferred striking resistance to VEN in MOLM-13 cells, suggesting p73 isoform-specific dependency of VEN sensitivity/resistance. The combination of 5'-azacitidine (5'-aza) and VEN decreased ΔNp73 level by 50%. Conclusion. Repression of TP73 in IDH1/2 mutant AML, and downregulation of TP73 by the oncometabolite 2-HG were associated with enhanced sensitivity to VEN, suggesting that TP73 determines AML susceptibility to BCL-2 inhibition. VEN resistant cells massively overexpressed TP73, and TP73 knockdown restored sensitivity to VEN. Specifically, overexpression of the ΔNp73γ isoform resulted in induced VEN resistance. ΔNp73 levels were also reduced by combining VEN with 5'-aza. Results may explain the high sensitivity of IDH1/2 mutant AML to VEN as consequence of downregulation of TP73 by 2-HG, and establish the mechanism of synergistic effect by VEN + 5'-aza combination and overexpression of p73 as a novel resistance mechanism to BCL-2 inhibition. Disclosures Ishizawa: Daiichi Sankyo: Patents & Royalties: Joint submission with Daiichi Sankyo for a PTC patent titled "Predictive Gene Signature in Acute Myeloid Leukemia for Therapy with the MDM2 Inhibitor DS-3032b," United States, 62/245667, 10/23/2015, Filed. Takahashi:Symbio Pharmaceuticals: Consultancy. Carter:Amgen: Research Funding; AstraZeneca: Research Funding; Ascentage: Research Funding. Andreeff:BiolineRx: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; Jazz Pharmaceuticals: Consultancy; Celgene: Consultancy; Amgen: Consultancy; AstaZeneca: Consultancy; 6 Dimensions Capital: Consultancy; Reata: Equity Ownership; Aptose: Equity Ownership; Leukemia Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; German Research Council: Membership on an entity's Board of Directors or advisory committees; NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; Cancer UK: Membership on an entity's Board of Directors or advisory committees; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; NCI-RDCRN (Rare Disease Cliln Network): Membership on an entity's Board of Directors or advisory committees; Oncoceutics: Equity Ownership; Breast Cancer Research Foundation: Research Funding; CPRIT: Research Funding; NIH/NCI: Research Funding; Oncolyze: Equity Ownership; Eutropics: Equity Ownership; Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Bcl-2 and Mcl-1 play critical roles in AML stem/progenitor cell survival. Venetoclax (VEN), a highly selective Bcl-2 inhibitor, showed limited clinical efficacy in AML as a single agent. FLT3 is the most frequently mutated gene in AML, resulting in constitutive activation of FLT3 tyrosine kinase and its downstream signaling pathways, which can be targeted by FLT3 tyrosine kinase inhibitors (TKIs). However, patients can adapt to TKI treatment by reactivating the MEK signaling pathway (Bruner JK et al., Cancer Res 2017), which is known to stabilize Mcl-1 levels. Furthermore, deregulated Mcl-1 expression was identified as a novel mechanism of primary TKI resistance in a subset of FLT3-ITD mutated AML patients (Breitenbuecher F et al., Blood 2009). Importantly, Mcl-1 can be induced by VEN treatment and could be a major resistance factor to VEN (Pan R et al., Cancer Discover 2014; Carter BZ et al., ASH 2018). Hence, Mcl-1 inhibition may enhance the efficacy of TKIs in FLT3 mutated AML and synergize with VEN, targeting AML cells and stem/progenitor cells. We treated FLT3-ITD positive AML cells with a selective inhibitor of Mcl-1 (AMG 176) and FLT3 TKIs and found that inhibition of Mcl-1 induced cell death and significantly enhanced the activity of sorafenib or gilteritinib in cell lines including cells acquired resistance to VEN (CI We previously showed that overexpression/knockdown of Mcl-1 greatly protected/sensitized AML cells from VEN induced cell death (Carter BZ, ASH 2018) supporting Mcl-1 as a key VEN resistance factor. We treated primary AML cells (n=5) with VEN (10 nM) or AMG 176 (250 nM) alone, or in combination and found that VEN+AMG 176 synergistically induced cell death in AML blasts and AML stem/progenitor cells even in samples clinically resistant to or relapsed after VEN containing regimen (CI To investigate the antileukemia activity in vivo, we tested combined inhibition of Mcl-1 and Bcl-2 using two PDX models in NSG mice. The first model was developed from a resistant/relapsed patient with FLT3-ITD mutation and complex karyotype. The combination showed the most significant antileukemic activity and extension of survival, followed by AMG 176 and VEN treatment alone (median survival for the combination, 146 d, p=0.004; AMG 176, 137 d, p=0.032; VEN, 102 d, p>0.05 vs. control, 85.5 d; respectively). The second PDX model was developed from a patient who first responded and then became resistant to the combination of VEN and decitabine and harbors FLT3-ITD, NRAS, and GATA2 mutations and complex karyotype. VEN or AMG 176 monotherapies marginally prolonged survival (median survival 127 or 129 vs. control 124 d). The combination was highly effective in this model and greatly decreased circulating blasts (Fig. 1) and leukemia tissue infiltration, measured by flow cytometry and spleen size. CyTOF analysis demonstrated that only the combination strongly reduced blasts as well as the AML stem/progenitor cell populations. Median survival for the combination group currently has not been reached (>325 d) (Fig. 2). Collectively, these data demonstrate that inhibition of Mcl-1 enhances the efficacy of TKIs in FLT3 mutated AML. Furthermore, it synergizes with VEN, targeting not only AML blasts but also AML stem/progenitor cells, both in vitro and in vivo in PDX models with the potential of significantly improving treatment outcome, which warrants clinical evaluation. Disclosures Carter: Amgen: Research Funding; AstraZeneca: Research Funding; Ascentage: Research Funding. Zhang:The University of Texas M.D.Anderson Cancer Center: Employment. Kuruvilla:The University of Texas M.D.Anderson Cancer Center: Employment. Konopleva:Kisoji: Consultancy, Honoraria; Eli Lilly: Research Funding; Forty-Seven: Consultancy, Honoraria; Calithera: Research Funding; Stemline Therapeutics: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Cellectis: Research Funding; Reata Pharmaceuticals: Equity Ownership, Patents & Royalties; Amgen: Consultancy, Honoraria; F. Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Genentech: Honoraria, Research Funding; Ascentage: Research Funding; Ablynx: Research Funding; Agios: Research Funding; Astra Zeneca: Research Funding. Caenepeel:Amgen Inc.: Employment. Canon:Amgen Inc.: Employment. Hughes:Amgen Inc.: Employment. Morrow:Amgen Inc.: Employment. Andreeff:Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; Celgene: Consultancy; Jazz Pharmaceuticals: Consultancy; Amgen: Consultancy; AstaZeneca: Consultancy; 6 Dimensions Capital: Consultancy; Reata: Equity Ownership; Aptose: Equity Ownership; Eutropics: Equity Ownership; Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oncoceutics: Equity Ownership; Oncolyze: Equity Ownership; Breast Cancer Research Foundation: Research Funding; CPRIT: Research Funding; NIH/NCI: Research Funding; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees; Cancer UK: Membership on an entity's Board of Directors or advisory committees; NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; German Research Council: 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; NCI-RDCRN (Rare Disease Cliln Network): Membership on an entity's Board of Directors or advisory committees; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; BiolineRx: Membership on an entity's Board of Directors or advisory committees.

Bcl-2 and Mcl-1 play critical roles in AML stem/progenitor cell survival. Venetoclax (VEN), a highly selective Bcl-2 inhibitor, showed potent preclinical activity but limited clinical efficacy in AML. Expression of Mcl-1, a major VEN resistance factor is induced by VEN. Non-apoptotic activity of Mcl-1 has been reported but is not well understood in AML. We previously demonstrated that inhibition of Mcl-1 enhances VEN apoptogenic activity and overcomes intrinsic and acquired VEN resistance in vitro and in vivo in a PDX murine model of AML (Carter BZ et al., ASH 2018). Interestingly, CyTOF analysis of bone marrow PDX cells collected from mice treated with Bcl-2 inhibitor VEN, Mcl-1 inhibitor AZD5991, and the combination showed that AZD5991 or AZD5991+VEN, but not VEN alone greatly decreased CXCR4 in leukemia cells and stem/progenitor cell populations. Mcl-1 expression is to a large degree regulated by microenvironment clues, including CXCR4-CXCL12 axis. We found that inhibition of Mcl-1 also suppressed cell metabolic activities in AML cells suggesting that Mcl-1 may regulate leukemia-microenvironment interaction and cell metabolic functions, which may contribute to the efficacy of this combination against AML stem cells in the bone marrow microenvironment. To further investigate the roles of Mcl-1 in leukemia-stromal interactions, we determined the expression of proteins involved in cell migration and adhesion and found that Mcl-1 overexpressing (OE) or knockdown (KD) OCI-AML3 cells have increased/decreased cell surface expression of CXCR4 and CD44, both critical for leukemia-MSC (mesenchymal stroma cell) interactions. Mcl-1 OE or KD AML cells showed increased/decreased migration towards and adhesion to MSCs compared to their respective controls. Consistent with this observation, pharmacological inhibition of Mcl-1 with AZD5991 also decreased surface CXCR4 and CD44 levels in OCI-AML3 cells and decreased the interactions between leukemia and MSCs. Under the same conditions, Mcl-1 seems to exert a more profound effect on leukemia cell adhesion than migration to MSCs. Interestingly, inhibition of Bcl-2 with VEN did not significantly decrease CD44 or migration and adhesion of AML cells to MSCs, although a decrease in surface levels of CXCR4 was observed. To gain additional insight into the effects of Mcl-1 on cellular energetics and metabolism, we performed metabolomic analysis, employing a previously established method using ion chromatography-mass spectrometry (IC-MS) to trace 13C labels from 13C2-1,2-glucose and 13C5-glutamine in Mcl-1 genetically and pharmacologically modulated AML cells. Metabolomic analysis showed that overall 13C enrichment into key TCA cycle intermediates including citrate, fumarate, and malate was significantly lower in cells with Mcl-1 inhibition, by either AZD5991 or Mcl-1 KD, compared to the respective controls, both for 13C coming from glucose and glutamine, the two main carbon sources entering the TCA cycle. We observed a decrease in the total amount of secreted lactate in both the 13C2-1,2-glucose and 13C5-glutamine tracing experiments. Enrichment of 13C label in secreted lactate was mostly observed during 13C2-1,2-glucose tracing, but not during 13C5-glutamine tracing, indicating that lactate secretion is largely derived from glucose, and not from glutamine, suggesting a reduction in flux through glycolysis and/or pentose phosphate pathway (PPP). Flux through the oxidative PPP (OxPPP) is a rate limiting pathway in the generation of reductive equivalents of NADPH for ROS neutralization. Relative OxPPP flux calculation showed a decrease in OxPPP flux for both AZD5991 treatment and Mcl-1 KD. Levels of 6-Phospho-Gluconic acid (6PG) were also greatly reduced in Mcl-1 inhibited cells, in accordance with differential regulation of the OxPPP. Mcl-1 inhibition by AZD5991 or KD also decreased ATP levels. Collectively, inhibition of Mcl-1 results in a broad downregulation in cellular energetics and metabolism. Conclusion: data demonstrate that in addition to regulating apoptosis, Mcl-1 regulates leukemia-stromal interactions and metabolic activity in leukemia cells and that inhibition of Mcl-1 has anti-leukemia activity and enhance VEN activity through not only apoptosis induction, but also modulation of leukemia-stromal interactions and metabolic functions. Disclosures Carter: Ascentage: Research Funding; Amgen: Research Funding; AstraZeneca: Research Funding. Cidado:AstraZeneca: Employment. Drew:AstraZeneca: Employment. Andreeff:Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; Jazz Pharmaceuticals: Consultancy; Oncoceutics: Equity Ownership; Oncolyze: Equity Ownership; Breast Cancer Research Foundation: Research Funding; CPRIT: Research Funding; NIH/NCI: Research Funding; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees; Cancer UK: Membership on an entity's Board of Directors or advisory committees; NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; German Research Council: 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; NCI-RDCRN (Rare Disease Cliln Network): Membership on an entity's Board of Directors or advisory committees; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; BiolineRx: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy; AstaZeneca: Consultancy; 6 Dimensions Capital: Consultancy; Reata: Equity Ownership; Aptose: Equity Ownership; Eutropics: Equity Ownership.

The apoptosis repressor with caspase recruitment domain (ARC) protein is a strong independent adverse prognostic marker in acute myeloid leukemia (AML). We previously reported that ARC regulates leukemia–microenvironment interactions through the NFκB/IL1β signaling network. Malignant cells have been reported to release IL1β, which induces PGE2 synthesis in mesenchymal stromal cells (MSC), in turn activating β-catenin signaling and inducing the cancer stem cell phenotype. Although Cox-2 and its enzymatic product PGE2 play major roles in inflammation and cancer, the regulation and role of PGE2 in AML are largely unknown. Here, we report that AML–MSC cocultures greatly increase Cox-2 expression in MSC and PGE2 production in an ARC/IL1β–dependent manner. PGE2 induced the expression of β-catenin, which regulated ARC and augmented chemoresistance in AML cells; inhibition of β-catenin decreased ARC and sensitized AML cells to chemotherapy. NOD/SCIDIL2RγNull-3/GM/SF mice transplanted with ARC-knockdown AML cells had significantly lower leukemia burden, lower serum levels of IL1β/PGE2, and lower tissue human ARC and β-catenin levels, prolonged survival, and increased sensitivity to chemotherapy than controls. Collectively, we present a new mechanism of action of antiapoptotic ARC by which ARC regulates PGE2 production in the tumor microenvironment and microenvironment-mediated chemoresistance in AML. Significance: The antiapoptotic protein ARC promotes AML aggressiveness by enabling detrimental cross-talk with bone marrow mesenchymal stromal cells.

The apoptosis repressor with caspase recruitment domain (ARC) protein is known to suppress both intrinsic and extrinsic apoptosis. We previously reported that ARC expression is a strong, independent adverse prognostic factor in acute myeloid leukemia (AML). Here, we investigated the regulation and role of ARC in AML. ARC expression is upregulated in AML cells co-cultured with bone marrow-derived mesenchymal stromal cells (MSCs) and suppressed by inhibition of MAPK and PI3K signaling. AML patient samples with RAS mutations (N = 64) expressed significantly higher levels of ARC than samples without RAS mutations (N = 371) (P = 0.016). ARC overexpression protected and ARC knockdown sensitized AML cells to cytarabine and to agents that selectively induce intrinsic (ABT-737) or extrinsic (TNF-related apoptosis inducing ligand) apoptosis. NOD-SCID mice harboring ARC-overexpressing KG-1 cells had significantly shorter survival than mice injected with control cells (median 84 versus 111 days) and significantly fewer leukemia cells were present when NOD/SCID IL2R null mice were injected with ARC knockdown as compared to control Molm13 cells (P = 0.005 and 0.03 at 2 and 3 weeks, respectively). Together, these findings demonstrate that MSCs regulate ARC in AML through activation of MAPK and PI3K signaling pathways. ARC confers drug resistance and survival advantage to AML in vitro and in vivo, suggesting ARC as a novel target in AML therapy.

Mcl-1 and Bcl-2 are two major anti-apoptotic Bcl-2 proteins frequently overexpressed in malignant cells. They cooperatively support cell survival and are associated with therapy resistance. ABT-199 (venetoclax), a highly selective Bcl-2 inhibitor, showed potent preclinical activity but limited clinical efficacy in AML as a single agent. Mcl-1 is induced by and a major resistance factor to ABT-199. Mcl-1 was recently found to also positively regulate mitochondrial oxidative phosphorylation that induces cancer stem cells and promotes chemoresistance. Mcl-1 is essential for the development of AML and for the survival of AML cells and stem cells. Increased mitochondrial oxidative phosphorylation has been demonstrated in these cells. First we found that Mcl-1 overexpressing (OE)/knockdown (KD) AML cells were markedly more resistant/sensitive to ABT-199 than were corresponding control cells, supporting the notion of Mcl-1 as a resistance factor to ABT-199. Inhibition of Mcl-1 by the selective Mcl-1 inhibitor AZD5991 or the CDK9 inhibitor AZD4573, which down-regulates short-lived proteins such as Mcl-1, induced apoptosis and showed strong synergy when combined with ABT-199 in AML cell lines, primary AML blasts, and stem/progenitor cells from patients. Importantly, combinations of AZD5991 or AZD4573 with ABT-199 synergistically induced apoptosis in OCI-AML3 and Mcl-1 OE cells intrinsically resistant to ABT-199 and in AML cell lines and primary patient cells with acquired resistance to ABT-199. Although OE/KD Mcl-1 in AML cells did not show obvious alterations in baseline cell viability, NSGS mice harboring Mcl-1 OE/KD OCI-AML3 cells survived significantly shorter/longer than those transplanted with control cells, supporting additional, non-apoptogenic functions of Mcl-1 in AML. We observed that genetic modulation of Mcl-1 alters cellular mitochondrial respiration and ROS levels. AML cells with Mcl-1 OE/KD increased/decreased O2 consumption and mitochondrial ATP and ROS generation. Consistent with this finding, inhibition of Mcl-1 by AZD5991 or AZD4573 decreased O2 consumption and ATP generation in AML cells and also in MV4-11 cells with acquired ABT-199 resistance. Mass spectrometry-based stable isotope tracing experiments using 1,2-13C-glucose showed that both genetic and pharmacological inhibition of Mcl-1 decreased flux of glucose carbon through glycolysis, the TCA cycle, and the pentose phosphate pathway, suggesting a role for Mcl-1 in cellular respiration and redox metabolism. To further assess the efficacy of combined Mcl-1 and Bcl-2 inhibition in primary AML cells resistant to ABT-199, we developed a PDX model using cells from an AML patient who initially responded to ABT-199/demethylating agent and then relapsed. NSGS mice engrafted with these PDX cells were treated with ABT-199 (50 mg/kg, oval gavage qd), AZD5991 (60 mg/kg, i.v. weekly), AZD4573 (15 mg/kg, i.p. bid with 2 h interval for two consequent days/week), ABT-199+AZD5991, or ABT-199+AZD4573 for 6 wks. Flow cytometric analysis of circulating human CD45+ cells on day 18 of therapy showed that each agent significantly decreased leukemia burden and that the combinations were significantly more effective (P Conclusion: we demonstrate that Mcl-1 has metabolic functions in AML and that inhibition of Mcl-1 enhances ABT-199 apoptogenic activity and overcomes intrinsic and acquired ABT-199 resistance in vitro and in vivo in a PDX murine model of AML, suggesting that inhibition of Mcl-1 improves the efficacy of ABT-199, and overcomes established resistance to Bcl-2 inhibition. Suppressing metabolic activity and CXCR4 inhibition may also contribute to the efficacy of this combination against AML stem cells in the BM microenvironment. Figure. Figure. Disclosures Carter: AstraZeneca: Research Funding; novartis: Research Funding. Lorenzi:Erytech Pharma: Consultancy; NIH: Patents & Royalties. Cidado:AstraZeneca: Employment, Equity Ownership. Drew:AstraZeneca: Employment. Andreeff:AstraZeneca: Research Funding.

Background: Second mitochondria-derived activator of caspases (Smac) mimetics cause increased apoptotic cancer cell death via degradation of cellular IAP1 (cIAP1) (Infante JCO 2014). Significantly increased levels of cytokines, including TNFα, have been observed in patients (pts) with MF (Tefferi JCO 2011; Fleischman Blood 2011). Baseline cIAP protein expression is increased in CD34+ MF cells, and cIAP/TNF- induced NFkB activation is higher in MF as compared to non-pathologic CD34+ bone marrow cells (Heaton/Deininger Leukemia 2018). On this basis, we hypothesized that treatment with Smac mimetics for pts with MF would have clinical benefit. Aims: Primary objective: determine efficacy of LCL161 as single-agent therapy for pts with MF (IWG-MRT, Blood 2013). Secondary objectives: safety, durability of response, and symptom burden assessment (MPN-TSS). Exploratory objectives: assess JAK2V617F, CALR allele burden; 28-gene panel for molecular mutations via next-generation sequencing; and markers of IAP pathway degradation. Methods: An investigator-initiated phase 2 study of LCL161 for pts with MF is being conducted at our institution. Pts age ≥18, PS=0-2, intermediate to high risk MF, who were intolerant to, ineligible for, or relapsed/refractory to JAK inhibitors were eligible. There was no baseline cut-off requirement for spleen size or platelet (PLT) count, and pts with prior allogeneic stem cell transplant (SCT) were eligible. LCL161 was given at starting dose 1500mg po once weekly. Each cycle=28 days. After 3 cycles, bone marrow exam and response assessments were performed. Results: 43 pts have been enrolled From January 2015 to July 2018. Baseline pt characteristics are listed in Table 1. JAK2V617F mutations were present in 29(67%), CALR mutations in 5(12%), and MPLW515L mutation in 5(12%) pts. The most common non-driver molecular mutations were: ASXL1(n=11); TET2(n=5);DNMT3A(n=5); RAS(n=4); EZH2(n=4). 29 (67%) had ≥2 prior therapies. 2 pts had prior SCT. By IPSS, 32(74%) were high risk MF. Median number of cycles received=5 [1-44+], median treatment duration=5.1 months [0.3 - 39.4+]. Median follow-up time: 13.7 months [0.3-39.4+]. 13 pts had 17 objective responses (4 pts: 2 separate IWG-MRT 2013 responses): Clinical improvement (CI) (anemia) was observed in 5 pts; CI (Symptom) in 10 pts; CI (Spleen) in 1 pt; CCyR in 1pt. Grade 3/4 non-hematologic adverse events: syncope, n=2. Most common grade 1/2 non-hematologic toxicities: fatigue (n=22), nausea (n=20), dizziness/vertigo (n=12). Dose reductions: 17 pts, to dose -1 level (1200 mg po once weekly); subsequently, 5 of 17 down to dose -2 level (900 mg); most common reason: grade 2 fatigue (n=10). 34 pts are now off study [n=20 no IWG response; n=4 progressive disease/relapse; n=4 AML; n=2 concurrent co-morbidity; n=2 proceeded to SCT; n=1 toxicity; n=1 off for patient choice]. Thus far, preliminary, ongoing correlative studies demonstrate among samples fully collected from 5 responders, all have demonstrated marked reduction of cIAP1 levels during therapy. Interestingly, cIAP1 remained low in 3 of them who were longer-term responders and then cIAP1 levels rebounded in 2 who later relapsed. The 2 relapsed pts also had relatively high baseline XIAP levels. Among non-responders, 3 of them also showed marked reduction of cIAP1 levels (>80%) at some point of the therapy, and notably 2 of them had more than 10 fold increases in XIAP levels and one had relatively high baseline XIAP. Conclusions: In this study of pts with MF with median age of 72 years, 74% IPSS high-risk, median baseline PLT count of 49, 67% with ≥2 prior therapies, and 26% with ASXL1 mutations, with median follow-up time now exceeding one year, we observed an objective response rate of 30% (13/43 pts) including 5 anemia-responders. LCL161 represents a novel target for pts with MPNs, and is able to be administered to pts who have failed or intolerant/ineligible for JAK inhibitor therapy. Notably, grade 2 fatigue was common, and continues to represent the most common reason for dose reduction and study discontinuation. Our ongoing translational studies demonstrate that reduction of cIAP1 is associated with responses and high baseline XIAP and/or its increase during therapy may contribute to lack of responses or relapses, suggesting upregulated levels of XIAP as a potential, novel mechanism of escape/resistance. This clinical trial is registered at ClinicalTrials.gov as NCT02098161. Table 1. Table 1. Disclosures Pemmaraju: abbvie: Research Funding; stemline: Consultancy, Honoraria, Research Funding; daiichi sankyo: Research Funding; celgene: Consultancy, Honoraria; SagerStrong Foundation: Research Funding; Affymetrix: Research Funding; plexxikon: Research Funding; samus: Research Funding; cellectis: Research Funding; novartis: Research Funding. Carter:AstraZeneca: Research Funding; novartis: Research Funding. Cortes:novartis: Research Funding. Kadia:Novartis: Consultancy; BMS: Research Funding; Jazz: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Jazz: Consultancy, Research Funding; Takeda: Consultancy; Amgen: Consultancy, Research Funding; Celgene: Research Funding; Abbvie: Consultancy; Pfizer: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Takeda: Consultancy; Abbvie: Consultancy; BMS: Research Funding; Celgene: Research Funding; Novartis: Consultancy. DiNardo:Celgene: Honoraria; Bayer: Honoraria; Medimmune: Honoraria; Agios: Consultancy; Karyopharm: Honoraria; Abbvie: Honoraria. Bose:Pfizer, Inc.: Research Funding; Incyte Corporation: Honoraria, Research Funding; Astellas Pharmaceuticals: Research Funding; Celgene Corporation: Honoraria, Research Funding; Constellation Pharmaceuticals: Research Funding; Blueprint Medicines Corporation: Research Funding; CTI BioPharma: Research Funding. Daver:Pfizer: Consultancy; Karyopharm: Consultancy; Sunesis: Consultancy; ImmunoGen: Consultancy; Pfizer: Research Funding; Alexion: Consultancy; Karyopharm: Research Funding; Sunesis: Research Funding; Incyte: Research Funding; Novartis: Research Funding; Otsuka: Consultancy; Incyte: Consultancy; ARIAD: Research Funding; Daiichi-Sankyo: Research Funding; Novartis: Consultancy; Kiromic: Research Funding; BMS: Research Funding. Konopleva:cellectis: Research Funding; abbvie: Research Funding; Immunogen: Research Funding; Stemline Therapeutics: Research Funding. Jain:Novimmune: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding; Pharmacyclics: Research Funding; Verastem: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Research Funding; Incyte: Research Funding; Genentech: Research Funding; Servier: Research Funding; BMS: Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Research Funding; Adaptive Biotechnologioes: Research Funding; Celgene: Research Funding; Astra Zeneca: Research Funding; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Verastem: Research Funding; Seattle Genetics: Research Funding; Cellectis: Research Funding; Infinity: Research Funding; ADC Therapeutics: Research Funding; Seattle Genetics: Research Funding; ADC Therapeutics: Research Funding; Incyte: Research Funding; Pfizer: Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; ADC Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding; Infinity: Research Funding; Pharmacyclics: Research Funding; Servier: Honoraria, Membership on an entity's Board of Directors or advisory committees; Genentech: Research Funding; Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Research Funding; Astra Zeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees; Verastem: Honoraria, Membership on an entity's Board of Directors or advisory committees; Servier: Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; ADC Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Verastem: Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Cellectis: Research Funding; Servier: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novimmune: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologioes: Research Funding; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees. Jabbour:novartis: Research Funding. Verstovsek:Italfarmaco: Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees.

Wnt/β-catenin signaling regulates self-renewal and proliferation of AML cells and is critical in AML initiation and progression. Overexpression of β-catenin is associated with poor prognosis. We previously reported that inhibition of Wnt/β-catenin signaling by C-82, a selective inhibitor of β-catenin/CBP, exerts anti-leukemia activity and synergistically potentiates FLT3 inhibitors in FLT3-mutated AML cells and stem/progenitor cells in vitro and in vivo (Jiang X et al., Clin Cancer Res, 2018, 24:2417). BCL-2 is a critical survival factor for AML cells and stem/progenitor cells and ABT-199 (Venetoclax), a selective BCL-2 inhibitor, has shown clinical activity in various hematological malignancies. However, when used alone, its efficacy in AML is limited. We and others have reported that ABT-199 can induce drug resistance by upregulating MCL-1, another key survival protein for AML stem/progenitor cells (Pan R et al., Cancer Cell 2017, 32:748; Lin KH et al, Sci Rep. 2016, 6:27696). We performed RNA Microarrays in OCI-AML3 cells treated with C-82, ABT-199, or the combination and found that both C-82 and the combination downregulated multiple genes, including Rac1. It was recently reported that inhibition of Rac1 by the pharmacological Rac1 inhibitor ZINC69391 decreased MCL-1 expression in AML cell line HL-60 cells (Cabrera M et al, Oncotarget. 2017, 8:98509). We therefore hypothesized that inhibiting β-catenin by C-82 may potentiate BCL-2 inhibitor ABT-199 via downregulating Rac1/MCL-1. To investigate the effects of simultaneously targeting β-catenin and BCL-2, we treated AML cell lines and primary patient samples with C-82 and ABT-199 and found that inhibition of Wnt/β-catenin signaling significantly enhanced the potency of ABT-199 in AML cell lines, even when AML cells were co-cultured with mesenchymal stromal cells (MSCs). The combination of C-82 and ABT-199 also synergistically killed primary AML cells (P To examine the effect of C-82 and ABT-199 combination in vivo, we generated a patient-derived xenograft (PDX) model from an AML patient who had mutations in NPM1, FLT3 (FLT3-ITD), TET2, DNMT3A, and WT1 genes and a complex karyotype. The combination synergistically killed the PDX cells in vitro even under MSC co-culture conditions. After PDX cells had engrafted in NSG (NOD-SCID IL2Rgnull) mice, the mice were randomized into 4 groups (n=10/group) and treated with vehicle, C-82 (80 mg/kg, daily i.p injection), ABT-199 (100 mg/kg, daily oral gavage), or the combination for 30 days. Results showed that all treatments decreased circulating blasts (P=0.009 for C-82, P These results suggest that targeting Wnt/β-catenin and BCL-2, both essential for AML cell and stem cell survival, has synergistic activity via Rac1-mediated MCL-1 inhibition and could be developed into a novel combinatorial therapy for AML. Disclosures Andreeff: SentiBio: Equity Ownership; Oncolyze: Equity Ownership; Oncoceutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Consultancy; Amgen: Consultancy, Research Funding; Eutropics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Patents & Royalties: MDM2 inhibitor activity patent, Research Funding; Aptose: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Reata: Equity Ownership; Astra Zeneca: Research Funding; Celgene: Consultancy; United Therapeutics: Patents & Royalties: GD2 inhibition in breast cancer . Carter:novartis: Research Funding; AstraZeneca: Research Funding.

BCR-ABL tyrosine kinase inhibitors (TKIs) are effective against chronic myeloid leukemia (CML), but they rarely eliminate CML stem cells. Disease relapse is common upon therapy cessation, even in patients with complete molecular responses. Furthermore, once CML progresses to blast crisis (BC), treatment outcomes are dismal. We hypothesized that concomitant targeting of BCL-2 and BCR-ABL tyrosine kinase could overcome these limitations. We demonstrate increased BCL-2 expression at the protein level in bone marrow cells, particularly in Lin(-)Sca-1(+)cKit(+) cells of inducible CML in mice, as determined by CyTOF mass cytometry. Further, selective inhibition of BCL-2, aided by TKI-mediated MCL-1 and BCL-XL inhibition, markedly decreased leukemic Lin(-)Sca-1(+)cKit(+) cell numbers and long-term stem cell frequency and prolonged survival in a murine CML model. Additionally, this combination effectively eradicated CD34(+)CD38(-), CD34(+)CD38(+), and quiescent stem/progenitor CD34(+) cells from BC CML patient samples. Our results suggest that BCL-2 is a key survival factor for CML stem/progenitor cells and that combined inhibition of BCL-2 and BCR-ABL tyrosine kinase has the potential to significantly improve depth of response and cure rates of chronic-phase and BC CML.

Leukemia cells are protected from chemotherapy-induced apoptosis by their interactions with bone marrow mesenchymal stromal cells (BM-MSCs). Yet the underlying mechanisms associated with this protective effect remain unclear. Genome-wide gene expression profiling of BM-MSCs revealed that coculture with leukemia cells upregulated the transcription of genes associated with nuclear factor (NF)-κB signaling. Moreover, primary BM-MSCs from leukemia patients expressed NF-κB target genes at higher levels than their normal BM-MSC counterparts. The blockade of NF-κB activation via chemical agents or the overexpression of the mutant form of inhibitor κB-α (IκBα) in BM-MSCs markedly reduced the stromal-mediated drug resistance in leukemia cells in vitro and in vivo. In particular, our unique in vivo model of human leukemia BM microenvironment illustrated a direct link between NF-κB activation and stromal-associated chemoprotection. Mechanistic in vitro studies revealed that the interaction between vascular cell adhesion molecule 1 (VCAM-1) and very late antigen-4 (VLA-4) played an integral role in the activation of NF-κB in the stromal and tumor cell compartments. Together, these results suggest that reciprocal NF-κB activation in BM-MSCs and leukemia cells is essential for promoting chemoresistance in the transformed cells, and targeting NF-κB or VLA-4/VCAM-1 signaling could be a clinically relevant mechanism to overcome stroma-mediated chemoresistance in BM-resident leukemia cells.

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that regulates cell adhesion, proliferation, stem cell functions, and cell-microenvironment communications. It is activated and/or overexpressed in many malignant cells and promotes tumor progression and metastasis. Several small molecule FAK inhibitors have been developed and some of them have reached clinical trials in solid tumors. High FAK expression was found to be associated with enhanced blast migration, increased cellularity, and poor prognosis in AML, indicating that FAK could be a potential therapeutic target in AML. We showed previously that VS-4718, a potent and selective FAK inhibitor, effectively decreased viable cell number, and also induced cell death in leukemia cell lines with variable potencies in vitro, even in AML cells co-cultured with mesenchymal stromal cells (MSCs) (ASH 2015). To further examine the effect of VS-4718 in vivo, we transplanted Molm14-GFP/Luc cells into NSGS (NOD-SCID IL2Rgnull-3/GM/SF, NSG-SGM3) mice, and treated the mice with VS-4718 (75 mg/kg) twice a day via oral gavage. We found that VS-4718 as a single agent exerted anti-leukemia activity as assessed by in vivo imaging for leukemia burden, human CD45 positivity in mouse peripheral blood, and histological staining of mouse tissues. VS-4718 treated mice survived significantly longer than the untreated controls (medium survival 27 vs 20 days, P = 0.0003). FAK activates multiple signaling pathways and supports tumor cell survival. We found that inhibition of FAK with VS-4718 in Molm14 cells reduced the expression of MCL-1. The BCL-2 antagonist ABT-199 is being tested clinically for the treatment of hematological malignancies. However, as a single agent, ABT-199-treated cells can acquire drug resistance by upregulating MCL-1 and BCL-XL after treatment. We therefore hypothesized that combination of VS-4718 and ABT-199 would be more effective in inducing cell death and reversing the resistance of AML cells exposed to ABT-199 alone. In vitro studies showed that VS-4718 significantly improved the potency of ABT-199 in AML cell lines (ABT-199 EC50 at 24 h: 880.3 nM and 14.5 nM in the presence of 0.4 mM VS-4718, respectively, in Molm14 cells), and the combination of VS-4718 and ABT-199 also synergistically killed primary AML cells even when co-cultured with MSCs in the majority of samples examined, while largely sparing normal BM CD34+ cells. Furthermore, the upregulation of MCL-1 in ABT-199-treated AML cells was antagonized by combining ABT-199 with VS-4718. BCL-XL is known to be regulated by STAT5. The activation of STAT5, which can be regulated by FAK, is considered to be significant in maintaining MCL-1 expression in FLT3-ITD AML cells. We observed that treatment with VS-4718 decreased the level of p-STAT5 as well as MCL-1 and BCL-XL in Molm14 cells harboring FLT3-ITD mutation. These results suggest a novel therapeutic strategy for targeting FAK and BCL-2 family proteins for the treatment of AML. Disclosures Pachter: Verastem, Inc: Employment. Weaver:Verastem, Inc: Employment. Carter:PRISM Pharma/Eisai: Research Funding.

Wnt/β-catenin signaling is associated with pathogenesis of AML and required for establishment of leukemic stem cells. FLT3 mutations are frequently observed in AML and predict poor clinic outcomes. Aberrant activation of FLT3 signaling stabilizes β-catenin and increases its nuclear localization and transcriptional activity. FLT3 tyrosine kinase inhibitors (TKIs) are used to treat FLT3-mutated AML, but their effects are limited due to primary or secondary resistance to TKIs. We previously reported (ASH 2015) that disrupting Wnt/b-catenin signaling by C-82, a selective β-catenin/cAMP binding protein antagonist in combination with FLT3 inhibitors had a synergistic cytotoxicity in vitro in FLT3-mutated AML cells and AML stem/progenitor cells through inhibiting nuclear localization of β-catenin and suppressing the expression of β-catenin target proteins including survivin, CD44, c-Myc, and cyclin D1; several frizzled family receptors/co-receptors; Wnt ligands; and FLT3 downstream signaling proteins. The synergy was also observed in TKI-resistant FLT3 mutated cells. In this study, we evaluated the antileukemia effect of combined inhibition of β-catenin and FLT3 signaling in vivo in immunodeficient mice xenografted with FLT3-ITD mutated cells either from a cell line or from an AML patient. Molm13-GFP/Luc cells were injected into NOD-SCID IL2RγNull (NSG) mice. Once engraftment was confirmed by in vivo imaging, mice were treated with PRI-724 (C-82 pro-drug), sorafenib, or both. Treatment with PRI-724 or sorafenib decreased leukemia burden, and the combination was the most effective as assessed by in vivo imaging, flow cytometric measurement of human CD45+ cells in blood, and bone marrow (BM) and spleen H&E staining. The mice in PRI-724 (19 days, P = 0.025) or sorafenib (28 days, P = 0.0002) treated group had significantly longer median survival time than the control group (17 days), and the combined treatment further prolonged the survival time (30.5 days) (P = 0.0005, combination vs RPI-724; P = 0.0056, combination vs sorafenib). CyTOF and SPADE tree analysis showed a great reduction of human CD45+ cells and decreased expression of β-catenin, CD44, c-Myc, survivin, p-FLT3, p-ERK, p-AKT, and p-STAT5 in BM cells of the combination treated mice. Cells from a FLT3-ITD mutated AML patient sample collected from patient-derived xenograft (spleen) were injected into NOD-SCID IL2RγNull-3/GM/SF (NSGS) mice. After engraftment was confirmed by flow cytometry, mice were treated as above. Leukemia burden was decreased by sorafenib or PRI-724 treatment, as determined by flow cytometry measurement of human CD45+ cells in blood, BM, and spleen samples, but did not reach statistical significance. The combination significantly enhanced the antileukemia effect. PRI-724 (31 days, P = 0.008) or sorafenib (48 days, P = 0.0003) significantly prolonged median survival time compared with control (29 days), and the combination further extended the survival time (54 days) (P = 0.0005, combination vs RPI-724; P = 0.0067, combination vs sorafenib). Our in vivo study further demonstrates that disruption of Wnt/b-catenin signaling exerts antileukemia activity and sensitizes with TKIs in FLT3 mutated AML. These findings provide a rationale for clinic development of combined inhibition of Wnt/β-catenin and FLT3 signaling to overcome resistance and improve outcomes in AML patients with FLT3 mutations. Disclosures Konopleva: Cellectis: Research Funding; Calithera: Research Funding. Carter:PRISM Pharma/Eisai: Research Funding.

Background: A role for the Chemokine (C-C motif) ligand 2 (CCL2) in attracting tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSC) and infiltrating monocytes has been described for many solid tumors in which they play an essential role in modifying the adaptive immune response, ultimately favoring tumor progression. Unfortunately, little is known about the importance of this mechanism for the progression of AML. We recently identified CCL2 as the most prominent chemokine produced by bone marrow (BM) mesenchymal stromal cells (BM-MSC) in response to the interaction with myeloid leukemia cells (PMID: 24599548). In addition, elevated CCL2 plasma levels have been reported in patients of AML (PMID: 17822317), ALL (PMID: 21298741) and CLL (PMID: 22397722) when compared to normal controls. In this study we assessed the effects of blocking the CCR2-CCL2 axis on the migration and signaling of hematopoietic cells as well as on the infiltration of immune-suppressive cells in leukemia-bearing mice. Results: We first studied the efficacy and potency of agents at inhibiting CCL2-mediated migration, using the human monocytic leukemia cell line THP-1. Migration towards human recombinant CCL2 (5 ng/ml) was significantly inhibited by as little as 1 nM of NOX-E36, a human-specific CCL2 Spiegelmer (NOXXON Pharma, Berlin). Spiegelmers are RNA-like molecules built from L-ribose units that are able to bind molecules such as peptides and proteins with an affinity in the pico-to nanomolar range. Similar results were obtained with a CCR2 antagonist (100 ng/ml; Santa Cruz). In anticipation of in vivo studies in mice, we next confirmed the ability of a mouse-specific CCL2 Spiegelmer (mNOX-E36) to inhibit migration and signaling pathway activation in murine hematopoietic cells. For this purpose, we cloned and overexpressed via lentiviral transduction the murine CCL2 receptor (CCR2) in Ba/F3 cells (a murine pro-B cell line). Stimulation of Ba/F3-CCR2 cells with 5 ng/ml of mouse recombinant CCL2 induced a ~2000 fold increase in migration of Ba/F3-CCR2 cells and was successfully blocked with mNOX-E36 in a concentration-dependent manner. Western blot analysis of protein lysates from mCCL2-stimulated cells (30 minutes treatment) indicated activation of AKT, ERK and p38-MAPK. The CCL2-induced phosphorylation of these molecules was completely abrogated by pre-treatment with mNOX-E36. Subsequently, we determined whether the expression of CCL2 by stromal cells in leukemia-resident organs triggers the infiltration of TAMs and possibly other immune-suppressive cells into those organs. We conducted preliminary in vivo studies in non-irradiated immunocompetent C57BL/6 mice (n=5 per group) injected with syngeneic AML1/ETO9a-expressing primary murine leukemia cells (PMID: 19339691). After confirmation of leukemia engraftment by IVIS imaging, mice were treated with mNOX-E36 (14.4 mg/kg, s.c., three times per week) or vehicle control for 3 weeks. At this point, all animals were sacrificed and their tissues (spleens and BM from femurs) were collected for analysis. Although we did not observe differences in leukemia burden by imaging between vehicle and mNOX-E36 treated groups, flow cytometry analysis revealed an increase in the frequency of CD11b+ Ly6Clow MHC IIlow macrophages (2 to 7 fold increase) in spleens of mice engrafted with leukemia (vehicle-treated group) when compared to spleens collected from healthy mice. These MHC IIlow macrophages were previously identified as immunosuppressive M2-like macrophages as opposed to MHC IIhi macrophages which show a pro-inflammatory M1-like phenotype (PMID: 20570887). Importantly, CCL2 inhibitor mNOX-E36 abrogated this macrophage infiltration within the leukemia microenvironment. Conclusions: Our results indicate that blockade of the CCR2-CCL2 axis not only affects migration and signaling of treated cells in vitro, but also interferes with the infiltration of M2-like macrophages into spleens of leukemia-bearing mice. Current in vivo experiments using a combination of standard chemotherapy with mNOX-E36 in AML immunocompetent models are undergoing. We expect that in vivo modulation of CCL2 will improve response to chemotherapy of AML by reducing the marrow infiltration of infiltrating monocytes and tumor-associated macrophages, which would facilitate translation of this novel concept into clinical trials in AML. Disclosures Zuber: Boehringer Ingelheim: Research Funding; Mirimus Inc.: Consultancy, Other: Stock holder. Eulberg:NOXXON Pharma AG: Employment. Kruschinski:NOXXON Pharma AG: Employment.

Focal adhesion kinase (FAK), a non-receptor tyrosine kinase is overexpressed/activated in several solid cancers. It controls cell growth, survival, invasion, metastasis, cell movement, gene expression, as well as stem cell self-renewal. Integrin ligation and growth factor/receptor interaction activate FAK leading to SRC phosphorylation and subsequent FAK/SRC phosphorylation at multiple sites which relays the external signal into cells by activating multiple cell proliferating/survival pathways. Although it is extensively studies in solid tumors, the expression and function of FAK in myeloid leukemia are not well investigated. We determined FAK expression by reversed-phase protein array in samples from a large cohort of newly diagnosed AML (n = 511) and MDS (n = 279). We found that in AML, FAK expression is associated with unfavorable cytogenetic group (P = 2x10-4). The lowest expression was seen in patients with inv16 (n = 19), t8;21 (n = 15) and t15;17 (n = 20) chromosome translocations. Patients with -5, -7, +8 (n = 100) expressed relatively higher FAK. FAK expression is higher in relapsed compared to paired newly diagnosed samples (n = 47, P = 0.02). FAK expression was significantly lower in patients with FLT-ITD (n=83, P = 0.0024) or RAS mutation (n = 64, P = 0.05) suggesting functional redundancy of these signaling pathways and therefore mutually exclusive expression patterns. FAK expression is highly correlated with SRC/p-SRC and ITGb3/ITGa2 levels among over 200 proteins analyzed suggesting a role of intergrin/FAK/SRC signal in AML cells. We also observed overexpression of FAK in MDS. FAK expression levels were significantly higher in both CD34+ (P = 5.42e-20) and CD34+ CD38- MDS cells (P = 7.62e-9) than in normal CD34+ cells (n = 16). Patients with higher expression of FAK in CD34+ cells had a trend towards better overall survival (P = 0.05) in newly diagnosed MDS. Furthermore, we determined the FAK mRNA expression levels in bone marrow CD34+ cells from 63 patients (57 MDS and 6 AML). In this cohort of patients, fifty (79%) were previously untreated with chemotherapy (46 MDS and 4 AML). No significant difference in FAK gene expression was observed when comparing patients with or without prior chemotherapy in either MDS or AML patients. Aberrant up-regulation (≥ 2 fold) was observed in 23% and 50% of the patients respectively compared to normal CD34+ cells (n = 5). In patients who received no prior chemotherapy (n = 50), aberrant up-regulation (≥2 fold) was observed in 22% and 50% of the patients respectively. FAK protein levels by immunohistochemical analysis were correlated with mRNA levels. Growth factor GM-CSF and co-culture with mesenchymal stem cells (MSCs) increased the expression of FAK in leukemia cells suggesting that the microenvironment modulates leukemia cell function in part via activating FAK signaling. Inhibition of FAK with VS-4718, a potent selective FAK inhibitor decreased viable cells and induced apoptosis in various AML cell lines and in co-cultures with MSCs. Inhibition of FAK decreased adhesion and migration of OCI-AML3 to MSCs suggesting FAK signaling promotes leukemia/stromal interactions. Furthermore, VS-4718 induced cell death in bulk, CD34+, and CD34+ CD38- cells from patients with AML even in co-cultures with MSCs. In conclusion, our results demonstrate that FAK is expressed in AML patient samples and that high expressions associate with unfavorable patient characteristics. Overexpression of FAK in MDS suggests that FAK signaling may be involved in pathogenesis of the disease. FAK, activated by tumor microenvironment, supports the survival of leukemia cells and in turn promotes leukemia/stromal interaction. FAK inhibition can induce apoptosis in leukemia cells. FAK may hence be a potential therapeutic target in AML and MDS. Disclosures Carter: PrismBiolab: Research Funding. Andreeff:Oncoceutics, Inc.: Membership on an entity's Board of Directors or advisory committees.

Tyrosine kinase inhibitors (TKIs) have dramatically improved the outcome of CML. However, these agents are not able to eliminate leukemia stem cells (LSC). Additionally, TKIs have limited activity in CML blast crisis (BC). Accumulating evidence demonstrates that activation of β-catenin plays a critical role in the clonal evolution from CML chronic phase to BC and development/maintenance of CML-BC LSC. We previously demonstrated that combinations of β-catenin/CBP modulator C-82 with TKIs eradicated CD34+ CD38-/+ and quiescent TKI-resistant CML-BC stem/progenitor cells and diminished mesenchymal stem cell-mediated microenvironmental protection in vitro (Blood, 2014, 124:401). In this study, aided with single-cell mass cytometry (CyTOF) analysis and data quantification/visualization with SPADE and viSNE, we investigated the effects of combining C82 or PRI-724 (a C82 pro-drug and first-in-class β-catenin/CBP modulator) with nilotinib in various phenotypically defined CML stem/progenitor cells in vitro or in vivo in human CML-BC xenografted NOD/SCID/IL2γnull (NSG) mice. Cells from heavily-treated TKI-resistant CML-BC patients were stained with multiple cell surface markers including those defining typical LSC phenotype and intracellular survival signaling molecules and subjected to CyTOF analysis. β-catenin expression was found to be increased in various CML-BC stem/progenitor cell compartments and was highest in the CD34+ CD38+ CD123+ Tim3+ (Tim-3+ GMP) stem/progenitor subset. viSNE visualization demonstrated that overexpression of β-catenin was associated with high levels of p-CRKL, c-Myc, p-AKT, p-Tyr, p-STAT3, and p-STAT5 in CML-BC progenitor/stem cells. Cells from TKI-resistant primary CML-BC samples were then treated with C82, nilotinib, or both for 48 hours in vitro and analyzed by CyTOF. Nilotinib, as expected, had no effect on CML stem/progenitor cells in these samples. viSNE 3D visualization and quantified SPADE tree analysis revealed that C82 and more significantly the combined regimen decreased various stem/progenitor cells, especially Tim-3+ GMP cells, a subset representing the candidate for CML-BC LSC. Next, NSG mice engrafted with human CML-BCT315I/E255V cells were treated daily with PRI-724 (ALZET pump, 30 mg/kg), nilotinib (oral gavage, 50 mg/kg), or combination. At the end of a 4-week treatment, bone marrow cells were analyzed by CyTOF. viSNE 4D visualization revealed that PRI-724 and the combination strikingly reduced various CML-BC stem/progenitor subsets. SPADE tree quantification analysis demonstrated that the combination, more profoundly than PRI-724 alone, reduced the leukemia burden, CD34+ CD38-, CD34+ CD38+ ,CD34+ CD38+ CD123+, and especially Tim-3+ GMP stem/progenitor cells in NSG mice, which was further confirmed by conventional flow cytometry. Furthermore, PRI-724 alone and the combination with nilotinib induced CD11b expression in various CML-BC stem/progenitor subsets, suggesting CML-BC stem/progenitor cell differentiation. Notably, these treatments significantly inhibited CD44, a β-catenin downstream target gene and an important component in the leukemia/bone marrow niche interaction in CD34+ CD38+ CD123high and CD34+ CD38+ CD123high Tim-3high subsets; and Tim-3, a novel marker for myeloid LSC in the CD34+ CD38+ CD123high Tim-3high subset; respectively. Importantly, PRI-724 (P Collectively, results show that inhibition of β-catenin effectively targets CML-BC stem/progenitor cells and improves survival of NSG mice engrafted with human CML-BC cells. The combination of β-catenin and tyrosine kinase inhibitors markedly enhanced the effect even in cells resistant to TKIs. Furthermore, the combination induces differentiation, and may also interrupt the interaction of CML-BC LSC with the bone marrow niche. This study suggests that combined inhibition of β-catenin and Bcr-Abl represents a novel potential strategy for targeting CML-BC stem/progenitor cells and improving patient outcomes in CML-BC that merits further investigation in preclinical and clinical settings. Disclosures Carter: PrismBiolab: Research Funding.

Bcr-Abl tyrosine kinase supports CML cell survival in part by regulating antiapoptotic Bcl-2 proteins such as Bcl-xL and Mcl-1. Tyrosine kinase inhibition, the front-line therapy for patients with chronic phase CML, is less effective in blast crisis (BC) patients and inactive against quiescent CML stem/progenitor cells. We reported that ABT-737, a dual Bcl-2/Bcl-xL inhibitor, induces apoptosis in BC CML cells including CD34+quiescent CML cells. ABT-199, a potent Bcl-2 specific inhibitor, has entered clinical trials for various hematological malignancies. We hypothesized that cooperative targeting of antiapoptotic Bcl-2 proteins using a combination of ABT-199 and tyrosine kinase inhibitors (TKIs) would exert enhanced activity against BC CML and CML stem/progenitor cells. Cells from patients (n=4) with TKI-resistant BC CML were treated with ABT-199, TKIs, and combinations. Although exerting low activity by itself, ABT-199 in combination with TKIs synergistically induced apoptosis (CI To evaluate the effect of these combinations on TKI-insensitive quiescent stem/progenitor CML cells, BC CML patient cells were stained with the cell division-tracking dye carboxyfluorescein succinimidyl ester (CFSE) and then co-cultured with human bone marrow (BM)-derived mesenchymal stromal cells (MSCs). Once proliferating and quiescent cells were distinguishable by flow cytometry, cells were treated with ABT-199, TKIs, and their combinations for 48 hours with or without MSC co-culture. Apoptosis was measured in proliferating and quiescent progenitor cells, defined as the percentage of annexin V positivity in CD34+CFSEdim and CD34+CFSEbright cells, respectively. ABT-199 as a single agent decreased viability of CML cells cultured alone or co-cultured with MSCs in both proliferating (IC50=191±103nM and 194±64nM, respectively) and quiescent (IC50=221±75nM and 205±123nM, respectively) CD34+ CML cells. Combinations of ABT-199 with TKIs, including imatinib, nilotinib, dasatinib, or ponatinib, synergistically induced death (CI To further test the ability of ABT-199 and TKI combinations to eradicate CML stem cells, we used an inducible transgenic CML mouse model in which the BCR-ABL gene is expressed under control of a tet-regulated enhancer of the murine stem cell leukemia (Scl) gene, allowing targeted BCR-ABL expression in stem/progenitor cells. Once BM cells from transgenic Scl-tTa-BCR-ABL/GFP mice were engrafted in wild type recipient mice, the mice were treated with ABT-199, nilotinib, or both. At the end of a 3-week treatment period, each single agent alone, and even more so with the combinations, significantly decreased blood total GFP+ WBC (12.9±1.4, 5.2±0.3, 6.1±0.4, and 1.6±0.3 x106/ml in controls, ABT-199, nilotinib, and combination, respectively) and neutrophils (1.43±0.03, 0.49±0.06, 0.32±0.03, and 0.25±0.05 x106/ml in the respective groups). ABT-199 (P=0.02), and more so with the combination (P Conclusions: ABT-199 and TKIs cooperatively target antiapoptotic Bcl-2 family proteins. This combination is highly effective in killing bulk and CD34+38- CML cells and quiescent CD34+ CML stem/progenitor cells from BC CML patients in vitro and in suppressing leukemia and leukemia stem cells in vivo. This strategy has the potential to eradicate BC CML cells and CML stem/progenitor cells, neither of which are effectively targeted by TKIs alone. Disclosures Carter: AbbVie, Inc.: Research Funding. Leverson:AbbVie, Inc.: Employment. Konopleva:AbbVie, Inc: clinic trial Other.

S376 Background: Individuals with congenital Trisomy 21 (+21) have an increased risk of developing acutemyeloid leukemia (AML). AMLwith +21 has traditionally been regarded as an intermediate risk prognosis. This study focuses on the biological and clinical features of patients (pts) with +21 AML. Methods: We analyzed pts treated at University of Texas MD Anderson Cancer Center (UTMDACC) between 1995 and 2011. Only pts that presented to UTMDACC at initial diagnosis with no prior therapy were included. Four cytogenetic groups were defined: +21 alone, +21 plus favorable cytogenetics, +21 plus intermediate cytogenetics and +21 plus unfavorable cytogenetics. Time to Progression (TTP) was defined as time from diagnosis to relapse or last follow up. Survival curves were calculated using Kaplan-Meier estimates. Results: A total of 90 pts harboring +21 aberrations at diagnosis were identified. Median age was 59 years (range, 18-88), 58% were male and 72 (80%) were of white race. At diagnosis, median white cell count was 4.6 (0.6-190) x 10/L, neutrophils 14 (0-86)%, hemoglobin 8.6 (3.3-13.4) g/dL, platelets 53 (4-395) x 10/L , peripheral blasts 17% (0-96), and bone marrow blasts 48% (0-97). FAB classification: M0-2 64%, M3 1%, M4-5 20%, M6 10%, M7 4%. Cytogenetics: +21 alone 11 (12%), plus favourable 7 (8%), plus intermediate 7 (85), plus unfavourable 65 (72%). Molecular mutations: NPM1 1/25 (4%), NRAS 4/53 (7%), CKIT 0/26 (0%), FLT3 4/49 (8%). Induction regimens included: Idarubicin+AraC-based 36%, fludarabine-based 24%, clofarabine-based 11%, topotecan-based 10%, hypomethylating-based 11%, and miscellaneous 8%. Overall Response Rate was 54%. Median TTP was 11 (2-130) months. Conclusions: Pts with AML harboring +21 have unique biological and clinical features. It rarely presents as a solely aberration and mostrly associates with complex cytoenetics. Further investigation is needed to determine its specific prognostic role.

We have reported that “apoptosis repressor with caspase recruitment domain” (ARC), an antiapoptotic protein promotes leukemia-stromal interactions in part by increasing the expression of CXCR4 in AML cells and of CXCL12 (SDF-1) in mesenchymal stromal cells (MSCs) (Carter et al., ASH 2012). To further understand the mechanisms of action of ARC in mediating leukemia-stromal interactions, we examined the expression of multiple chemokines by quantitative cytokine PCR array in ARC knockdown (KD) and control MSCs. We found that among 6 C-X-C motif and 22 C-C motif chemokines, CXCL12, CCL2 and CCL4 are highly expressed and upregulated by ARC in MSCs. We then determined the protein levels of CCR2 and CCR5, the respective receptors for CCL2 and CCL4, in bone marrow samples from 8 AML patients and found that CCR2 was highly expressed in all samples and CCR5 was detectable in 75 % suggesting that the CCR2/CCL2 and CCR5/CCL4 axes may also contribute to leukemia-stromal interactions in AML. Trans-well migration assays showed that CCL2 and CCL4 induced the migration of AML cells which was antagonized by blocking antibodies or small molecule inhibitors. AML cells migrated less to ARC KD than to control MSCs. Primary leukemia cells from AML patient samples also migrated towards CCL2 and CCL4, and this migration correlated with the expression of the respective receptors in the leukemia cells. Co-culture of MSCs with leukemia cells greatly increased the levels of CXCL12, CCL2, and CCL4 in MSCs and the increase was diminished when co-cultured with ARC KD and increased when co-cultured with ARC overexpressing (OE) cells was compared to their respective controls, suggesting that chemokine expression in MSCs is in part regulated by ARC-mediated signaling from AML cells. Cytokine PCR array revealed that IL1β expression is increased in ARC OE and decreased in ARC KD cells. Conversely, the level of the IL1 receptor antagonist IL1RN is lower in ARC OE and higher in ARC KD. ARC OE cells also secreted more IL1β while ARC KD cells secreted less. Furthermore, co-cultures with MSCs increased the expression of IL1β in AML cells. IL1β greatly increased the levels of CCL2, CCL4, and CXCL12 in MSCs and the increase was largely diminished by co-treating the MSCs with IL1β antagonist IL1βRA. Similarly, treating MSCs with conditioned media from AML cells increased the expression of the chemokines in MSCs, and the increase was reduced with conditioned medium from ARC KD cells; this increase was diminished by IL1βRA. Furthermore, IL1βRA blocked the migration of these cells toward CCL2 and CCL4. Using a 3-D model in which cancellous bone was covered with MSCs, we found fewer OCI-AML3 cells attached to MSCs when ARC was knocked down in these cells further supporting the role of ARC in leukemia-stromal interactions. ARC is a member of caspase recruitment domain (CARD) containing proteins that have diverse functions such as antiapoptosis and regulation of NFκB activity. ARC participation in cell death suppression and leukemia-stromal interactions suggests that ARC may interact not only with apoptosis regulators but also with signaling proteins to regulate multiple cellular functions in AML. Conclusions Our findings suggest that multiple ARC-regulated receptor/ligand pairs play important roles in leukemia-stromal interactions, and that there is reciprocal crosstalk between malignant cells and microenvironment cells that is in part mediated through ARC-regulated inflammatory cytokine IL1β. ARC is therefore a novel target not only for direct apoptosis induction in leukemia cells but also for disruption of protective leukemia-stromal interactions to further sensitize leukemia cells to their elimination by chemotherapy. Disclosures: No relevant conflicts of interest to declare.

Abstract 2626 Hematopoietic cells express a wide range of adhesion molecules and bone marrow (BM) stroma cells produce their corresponding ligands. Through these ligand-receptor pairs, hematopoietic cells interact with their BM microenvironment. The same system is hijacked by AML and often adhesion molecules in leukemia cells and/or their ligands in stroma cells are upregulated, promote leukemia-stroma interactions, and protect leukemia cells from therapeutic agents. Understanding the underlying mechanisms is critical for therapeutic strategies aimed at disrupting leukemia-stroma interactions. For example, pharmacological blockage of the CXCR4-SDF1a axis has been shown to result in chemosensitization of AML cells in vitro, in vivo and in clinical trials (Zeng Z et al., Blood 2009; Uy GL et al., Blood 2012; Andreeff M et al., ASCO 2012). ARC (Apoptosis repressor with caspase recruitment domain) is an antiapoptotic protein. We recently determined ARC expression in samples from 511 newly diagnosed AML patients by reverse-phase protein array and reported that ARC is one of the strongest adverse prognostic markers in AML (Carter BZ et al., Blood 2011). In the same sample set, we also probed the expression of more than 200 additional proteins enabling us to correlate ARC expression with the expressions of other proteins. Surprisingly, ARC expression was correlated with multiple proteins involved in cell adhesion and migration. We generated stable ARC overexpressing (O/E) KG-1 cells, ARC knock down (K/D) OCI-AML3 and Molm13 cells, and ARC K/D BM derived mesenchymal stroma cells (MSCs) to investigate ARC's roles in leukemia-stroma interactions. Expression levels of FAK, integrinb3, fibronectin, and VLA4 were increased in ARC O/E and decreased in ARC K/D cells, compared to controls. CXCR4 mRNA and cell surface CXCR4 protein were higher in ARC O/E KG-1 (3.80- and 1.53-fold, P Migration of leukemic cells towards MSCs was significantly higher for ARC O/E and lower for ARC K/D AML cells (P The role of ARC in leukemia-stroma interactions was further assessed using a novel human extramedullary BM model in mice recently developed by our group (Chen Y et al., Blood 2012). Luciferase/GFP Molm13 cells were injected into Nod/ScidIL2Rg−/− mice after extramedullary human BM containing either ARC K/D or control MSCs was established in the flanks of mice. Significantly fewer AML cells engrafted in the human extramedullary BM developed from ARC K/D than from control MSCs as determined by immunohistochemistry staining for human CD45+ cells and quantitative image analysis (P In conclusion, we here demonstrate that antiapoptotic ARC regulates migration and adhesion of leukemia cells to BM stroma in vitro and in vivo via activation of multiple mechanisms, not only in AML cells but also in MSCs, thus enhancing its anti-apoptotic activity. Given the fact that both intrinsic apoptosis resistance and extrinsic environmental factors contribute to drug resistance and relapse in AML, ARC may play a central role in AML and be an excellent therapeutic target: ARC inactivation may disrupt leukemia-stroma interactions and increase leukemia cell sensitivity to chemotherapy by increasing susceptibility to apoptosis. We speculate that ARC, a CARD containing protein, acts at least in part via activating NF-kB signaling, which is known to be regulated by CARD proteins. Disclosures: No relevant conflicts of interest to declare.