ALBERT

Description: Notch1-mutated T-ALL is an aggressive hematologic malignancy lacking targeted therapeutic options. Genomic alterations in Notch1-gene and its activated downstream pathways are associated with metabolic stress response and heightened glutamine (Gln) utilization to fuel oxidative phosphorylation (OxPhos) (Kishton at al., Cell Metabolism 2016, 23:649, Herranz at al., Nat Med, 2015, 21(10): 1182-1189). Hence, targeting NOTCH1-associated OxPhos and/or Gln dependency could constitute a plausible therapeutic strategy for T-ALL. In this study we examined metabolic vulnerabilities of NOTCH1-driven T-ALL and tested pre-clinical efficacy of novel mitochondrial complex I (OxPhosi) IACS-010759 and of glutaminase inhibitor CB-839 (GLSi) in T-ALL models including Notch1-mutated T-ALL cell lines, patient-derived xenograft (PDX) and primary T-ALL cells. We have previously reported and confirmed in this expanded study the anti-leukemia efficacy of IACS-010759 (EC50s 0.1-15 nM) (Molina at al., Nat Med, 2018, 24: 1036; Baran at al., Blood, 2018, 132:4020). Metabolic characterization demonstrated that OxPhosi caused striking dose-dependent decrease in basal and maximal oxygen consumption rate (OCR), ATP and NADH generation in T-ALL cell lines and primary T-ALL samples (p

Description: Acute myeloid leukemia (AML) cells highly depend on oxidative phosphorylation (OxPhos) to satisfy their heightened demands for energy, and the complex I OxPhos inhibitor IACS-010759 (Molina, Nat. Med. 2018) is currently in Phase 1 clinical trial in AML. In this study, we investigated how the bone marrow (BM) microenvironment affects the response to OxPhos inhibition in AML. To characterize the molecular mechanisms of sensitivity to OxPhos inhibition, we performed Cap Analysis of Gene Expression analysis (CAGE) on 31 genetically diverse primary AML samples (20 were defined as sensitive and 11 as resistant to IACS-010759; cut off 〉3.0 fold annexin V(+) by 100 nM IACS-010759/DMSO at 72 hours). CAGE identified higher expression of transcription start sites (TSS) for 17 genes in IACS-010759 resistant AML samples compared to sensitive (fold change 〉2.0, FDR 〈 0.05, EdgeR), which were related to cell adhesion, integrin and/or Rho GTPase family genes that modulate intracellular actin dynamics. We next investigated the interactions between IACS-010759 sensitive OCI-AML3 cells and BM-derived mesenchymal stem cells (MSC). Under conditions mimicking the BM microenvironment, IACS-010759 upregulated the pathways of focal adhesion and ECM-receptor interaction in OCI-AML3 cells (KEGG analysis based on CAGE). In turn, MSC co-culture increased oxygen consumption by AML, induced generation of mitochondrial ROS (control 4.4% vs IACS 44.4%), increased mtDNA (2-fold by q-PCR) and upregulation of mitochondrial proteins VDAC and cytochrome C, translating into dampened growth-inhibitory effects of IACS-010759. We further demonstrated that OCI-AML3 cells adhering to MSCs were fully protected from IACS-010759 induced apoptosis (IACS-induced specific apoptosis: non-adherent cells 16.2% ± 1.6% vs adherent cells 1.6% ± 0.7%, p=0.008, 30nM, 72hours). Similarly, adherent cells were fully protected from apoptosis induced by combination of IACS and AraC. These findings indicate that direct interactions with MSC trigger compensatory activation of mitochondrial respiration, increase in mitochondrial mass and resistance to OxPhos inhibition in AML. We next hypothesized that the trafficking of mitochondria from BM stroma cells to AML cells could represent a putative mechanism of an acquired resistance to OxPhos inhibition. To visualize mitochondria, OCI-AML3 and MSC were stably transfected with mitochondria-targeted PDHA1-GFP and -dsRed, respectively. We discovered that IACS-010759 induced transfer of MSC-derived mitochondria to OCI-AML3 cells (% of GFP/dsRed double-positive OCI-AML, control 4.1 ± 1.7 vs IACS 26.2 ± 13.4, p=0.002) via tunneling nanotubes (TNTs) detected by confocal and electron microscopy (Fig.1). Mitochondria transfer was only observed in the direct contact but not in the transwell co-cultures, and was abrogated by ICAM-1 neutralizing antibody and TNT blockade with Cytochalasin B. Likewise, combination of IACS with AraC increased mitochondrial transfer. We further found that IACS-010759 induced autophagy in OCI-AML3 cells co-cultured with MSC, as noted by increased conversion of LC3-I to LC3-II, which was further enhanced by the lysosome inhibitor Bafilomycin. Additionally, we observed autophagosome formation enwrapping MSC-derived mitochondria (Fig.1F), along with the degradation of an outer mitochondrial membrane protein Tom20. Finally, IACS-010759-induced transfer of mtDNA in BM-resident AML cells was confirmed in vivo in humanized AML PDX models (n=2). Daily oral treatment of mice harboring human AML with IACS-010759 (5.0 mg/kg/day, 21 days) increased the ratio of murine/human mtDNA in human AML cells isolated from BM, in 5 days on/2 days off PDX models tested (2.1 ± 0.3 fold, n=2). In conclusion, the findings of this study indicate an important role of mitochondria trafficking from BM stromal cells to AML cells in a compensatory adaptation to OxPhos inhibition in BM microenvironment. We propose that blocking of mitochondrial transfer could enhance the anti-AML efficacy of OxPhos targeting agents. Disclosures Zhang: The University of Texas M.D.Anderson Cancer Center: Employment. Kuruvilla:The University of Texas M.D.Anderson Cancer Center: Employment. Andreeff:BiolineRx: Membership on an entity's Board of Directors or advisory committees; Breast Cancer Research Foundation: Research Funding; Oncolyze: Equity Ownership; Oncoceutics: Equity Ownership; Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Eutropics: Equity Ownership; Reata: Equity Ownership; Aptose: Equity Ownership; 6 Dimensions Capital: Consultancy; Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; Jazz Pharmaceuticals: Consultancy; Celgene: Consultancy; Amgen: Consultancy; AstaZeneca: Consultancy; 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. Konopleva:Astra Zeneca: Research Funding; Agios: Research Funding; Eli Lilly: Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Cellectis: Research Funding; Amgen: Consultancy, Honoraria; F. Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Genentech: Honoraria, Research Funding; Ascentage: Research Funding; Kisoji: Consultancy, Honoraria; Reata Pharmaceuticals: Equity Ownership, Patents & Royalties; Ablynx: Research Funding; Forty-Seven: Consultancy, Honoraria; Calithera: Research Funding; Stemline Therapeutics: Consultancy, Honoraria, Research Funding.

Description: Separation of leukemic stem cells (LSC) and residual hematopoietic stem cells (HSC) from the same individual patient with acute myeloid leukemia (AML) is essential for a proper understanding of the leukemic driving mechanisms. We have studied the role of aldehyde dehydrogenase (ALDH) for this purpose and have defined the functional properties of ALDHbright cells in specific subgroups of AML. We have examined the ALDH activity by flow cytometry in bone marrow samples (BM) from 14 healthy donors and 73 patients with de novo AML. The median frequency of cells with high ALDH activity (ALDHbright cells) in the healthy subjects was 1.92% with a range from 0.58 to 3.16%. For patients with AML, the median number of ALDHbright cells was 0.25% with a broad range from 0.004 to 33.57%. Whereas the majority of patients with AML (n = 56) had low frequencies of ALDHbright cells (median 0.11%; range 0.004 – 1.77%; defined as ALDH-low AML), 17 patients had relatively numerous ALDHbright cells (median 9.01; range 3.54 – 33.57%; defined as ALDH-numerous AML). In both groups, ALDHbright cell populations were highly enriched for CD34+CD38- cells. The ALDHbright cells derived from ALDH-low AML did not contain chromosomal and molecular aberrations characteristic of the original leukemia, and were able to induce multi-lineage hematopoiesis in NSG mouse models. Thus, genetically and functionally normal HSC could be successfully isolated in the ALDHbright subset, whereas LSC were enriched in ALDHdimCD34+CD38- subset for patients with ALDH-low AML. For 17 patients with ALDH-numerous AML, the ALDHbright subset was consistently contaminated with LSC. In clinical follow-ups, patients with ALDH-numerous AML showed resistance to induction chemotherapy and were characterized by a very poor long-term outcome that was comparable to patients with high-risk cytogenetic or molecular genetic markers. In four patients with ALDH-numerous AML we demonstrated that the ALDHbrightCD34+CD38- subset contained chemotherapy-resistant clones with repopulating ability. Furthermore, such ALDHbright cells were characterized by a lower cell-cycle activity and an increased resistance to cytarabine in comparison with ALDHdim blasts in in vitro assays. Our data have provided evidence that LSC and residual HSC can be separated using ALDH in patients with low frequencies of ALDHbright cells. In patients with ALDH-numerous AML, the ALDHbright subset is associated with leukemic features both in vitro and in animal models. Thus our data demonstrated the feasibility of appropriate comparisons of LSC versus HSC from the same patient with specific subtypes of AML and the impact of LSC properties on clinical outcome. Disclosures: Buss: Novartis: Travel support Other; Micromet/Amgen: Reimbursements for participation in a clinical study , Reimbursements for participation in a clinical study Other. Ho:Sanofi-Aventis: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Genzyme: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees.

Description: Adult T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy characterized by limited therapeutic options and a high rate of treatment failure due to chemoresistance. T-ALL is largely driven by activating NOTCH1 mutations, where oncogenic NOTCH1 facilitates glutamine oxidation, induces metabolic stress, and facilitates reliance on oxidative phosphorylation (OXPHOS)1. In other malignancies, the shift toward OXPHOS-dependent high-energy status is associated with acquired chemoresistance. In this study, we found that the novel inhibitor of mitochondrial complex I (OXPHOSi) IACS-0107592 has preclinical activity in NOTCH1-mutated T-ALL; we also characterize the cellular and metabolic responses to OXPHOS inhibition and propose that an OXPHOSi be incorporated into standard-of-care therapy to improve outcomes in patients harboring NOTCH1-mutated T-ALL. Exposure to IACS-010759 (0-370 nM) in vitro drastically reduced T-ALL viability, with EC50 ranging from 0.1-10 nM for cell lines (n=7) and from 13-60 nM for patient-derived xenograft (PDX)-derived and primary T-ALL cells (n=10) (Fig.1). Oral administration of IACS-010759 (7.5 mg/kg/day) significantly reduced leukemia burden and extended overall survival (p

Description: Immune checkpoint-based therapies, which target the regulatory pathways of immunocompetent cells to enhance anti-tumor responses, have been at the heart of many recent clinical advances and have led to long-term remissions and possible cures. Most of this success was achieved with T-cells, but there are compelling reasons to predict that checkpoint ablation could modify NK cells in ways that would facilitate their antitumor activity. The suppressor of cytokine signaling (SOCS) family of proteins plays an important role in NK cell biology by attenuating cytokine signaling and effector function against cancer. One of its members, cytokine inducible SH2 containing protein (CIS), encoded by the CISH gene, is as an important checkpoint molecule in NK cells and is upregulated in response to IL-15. We hypothesized that CIS may act as a potent checkpoint in our iC9/CAR19/IL15 NK cells given the fact that they continuously produce IL-15, and that targeting this pathway would enhance their potency against B cell malignancies. In a series of in vitro studies, we showed that CISH is induced in iC9/CAR19/IL15 NK cells in a time dependent manner. To examine the functional consequences of CISH deletion in our CAR-NK cells, we developed a protocol for combined Cas9 ribonucleoprotein (Cas9 RNP)-mediated gene editing to silence CISH and retroviral transduction with the iC9/CAR19/IL15 construct. On day 7 we nucleofected the CAR transduced NK cells with Cas9 alone (Cas9 control) or Cas9 pre-loaded with crRNA:tracrRNA duplex targeting CISH exon 4. Gene editing efficiency was 〉90% as quantified by PCR and western blot. CISH knockout induced a phenotype characterized by the increased expression of markers of activation and cytotoxicity. These included granzyme-b, perforin, TRAIL and CD3z; transcription factors such as eomesodermin and T-bet; adaptor molecules such as DAP12; and activating coreceptors/proliferation markers such as DNAM, CD25 and Ki67. CISH knockout resulted in significantly enhanced function of iC9/CAR19/IL15 NK cells against Raji lymphoma evident by increased cytokine production (TNFa p=0.007, IFNg p=0.033) and degranulation (CD107a p=0.003) compared to Cas9 control cells. Moreover, CISH KO iC9/CAR.19-IL15 NK cells killed Raji lymphoma more efficiently than Cas9 control cells and formed a stronger immunologic synapse (p=0.037). RNA sequencing with gene set enrichment analysis (GSEA) confirmed enrichment of JAK/STAT signaling, TNFα and IFN-γ inflammatory response, mTORC1, and MYC hallmark pathways in CISH KO iC9/CAR19/IL15 NK cells compared to Cas9 control counterparts, providing a molecular mechanism for their enhanced effector function. Moreover, in an in-vivo NSG mouse model of Raji lymphoma, the antitumor activity of a single dose of CISH KO iC9/CAR19/IL15 transduced CB NK cells was significantly better than that of Cas9 control cells leading to a significant survival advantage (p=0.003) without evidence of increased toxicity. Thus, we demonstrate for the first time, that silencing a critical checkpoint in CAR-NK cells improves their potency, permitting greater cytotoxic effector function than seen with unmodified CAR-NK cells. Our data support the merging of CAR-engineering and immune checkpoint gene editing to enhance the therapeutic potential of NK cells. We are in the process of scaling up this approach in our GMP facility for translation to the clinic for the treatment of relapsed/refractory B cell hematologic malignancies. Disclosures Konopleva: Calithera: Research Funding; Stemline Therapeutics: Consultancy, Honoraria, Research Funding; Forty-Seven: Consultancy, Honoraria; Eli Lilly: Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Cellectis: Research Funding; Amgen: Consultancy, Honoraria; F. Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Genentech: Honoraria, Research Funding; Ascentage: Research Funding; Kisoji: Consultancy, Honoraria; Reata Pharmaceuticals: Equity Ownership, Patents & Royalties; Ablynx: Research Funding; Astra Zeneca: Research Funding; Agios: Research Funding. Champlin:Sanofi-Genzyme: Research Funding; Actinium: Consultancy; Johnson and Johnson: Consultancy.

Description: Introduction Anti-apoptotic Bcl2 family members mediate resistance to therapies in acute myeloid leukemia (AML)1. The small molecule Bcl2 inhibitor ABT-199 (venetoclax) promotes mitochondria driven intrinsic apoptosis, and in combination with hypomethylating agents or chemotherapy, has been highly promising in the clinic as treatment of AML2-4. The response rate to ABT-199 is very impressive, but acquired resistance is a major problem. Compensatory upregulation of Mcl1 is an important mechanism of such acquired resistance to mitochondrial apoptosis5. Autophagy is vital for mitochondrial health, mediates resistance to apoptosis and is induced by Bcl2 inhibition6. We performed mechanistic studies to address our hypothesis that disabling autophagy by targeting the apical autophagy kinase ULK1 can reverse resistance to ABT-199. Methods ULK1 was genetically modified in OCIAML3 (human AML cell line), by shRNA knockdown (KD) or CRISPR-Cas9 knockout (KO). In addition, AML cell lines (including ABT-199 resistant) and patient samples were treated with ABT-199 and ULK1 inhibitor SBI-02069657. Combination index (CI) for drug synergy was calculated based on Chou-Talalay method8. Drug-treated or genetically manipulated cells were profiled by reverse phase protein array (RPPA), mass cytometry (CyTOF) and gene expression profiling (GEP). Autophagy was detected by LC3 quantification by western blot (WB) and flow cytometry, and monodansylcadaverine assay. Mitochondrial functions were analyzed by Seahorse Cell Mito Stress test, and MTG, TMRE and ROS assays (flow cytometry). For in vivo studies ULK1 KO and corresponding control cells were injected in NSG mice and monitored by bioluminescent imaging (BLI) and quantification of human CD45 cells. Results ABT-199 induced autophagy in OCIAML3 (increase by 175±27%, p=0.01 - LC3 flow; 4X increase in LC3 II/I ratio - WB). Apoptosis induction by ABT-199 was enhanced by ULK1 KD (36±1.9% over control, p

Description: Background: Acute myeloid leukemia (AML) is a devastating hematopoietic malignancy caused by differentiation arrest and suppression of apoptosis of immature myeloid cells. The long-term survival of AML under the established therapies remains poor. Differentiation therapy has been developed to promote the normal process of hematopoietic maturation from self-renewing progenitors to terminally differentiated effector cells. The recent discovery of a novel target, the enzyme dihydroorotate dehydrogenase (DHODH), offers differentiation-promoting therapy for the majority of AML (Sykes, D.B., et al.Cell, 2016). DHODH is the rate-limiting enzyme in the pyrimidine biosynthesis pathway. DHODH inhibition was reported to efficiently relieve the differentiation block caused by HoxA9 overexpression in 70% of AML, making this discovery potentially universally applicable for AML patients with diverse genomic alterations. AG-636 is a novel, potent, selective DHODH inhibitor developed by Agios Pharmaceuticals. This small molecule inhibitor has favorable pharmacokinetic properties and is in dose-finding Phase I clinical trials in lymphoma patients (NCT03834584) and is ready to enter a Phase I study in acute leukemia and myeloid dysplasia syndrome. Here, we investigated single agent activity of AG-636 in pre-clinical AML models. Results: AG-636 inhibited cell proliferation, induced apoptosis in AML cell lines, primary blasts and CD34+ leukemic stem/progenitor cells from AML patients with various genomic alterations cultured under physiologic conditions of stromal support (Fig. 1A). Flow cytometry and multi-parametric mass cytometry (CyTOF) analysis demonstrated that AG-636 reduced bulk AML and facilitated emergence of the differentiated myelo-monocytic cell subset co-expressing CD11b, CD11c and CD14 (Fig. 1B). Both cytotoxic and differentiating effects were rescued by supplementing the DHO downstream metabolite uridine, supporting on-target activity of AG-636 through DHODH inhibition and dependency of AML survival and stemness on the pyrimidine biosynthesis (Fig 1A top). Mass spectrometric analysis of 166 metabolites confirmed that targeting DHODH by AG-636 resulted in accumulation of the upstream L-dihydroorotic acid and ureidosuccinic acid, and depletion of the downstream metabolites, such as uridine 5'-diphsophate, uridine 5'-monophsophate, CDP and dCMP in pyrimidine biosynthesis pathway. Metabolic profiling further demonstrated the depletion of 5'-phosphoribosyl-N-formylglycinamide in treated cells, indicating the sequential effect of AG-636 on purine biosynthesis and metabolism. Seahorse-based metabolic assay showed inhibition of basal oxygen consumption and ATP generation in AG-636-treated cells, suggesting a contribution of DHODH in coupling of the mitochondria function. Proteomic profiling and immunoblots analysis revealed that AG-636 triggered AMPK activation in response to metabolic stress, and upregulated the expression of TP53, PUMA and NOXA known to regulate mitochondrial integrity. A role for DHODH inhibition in impairment of mitochondria function is of note given the key metabolic dependence of AML cells on OXPHOS/mitochondria function as shown by us and others (Molina J.R. et al. Nat Med, 2018). In vivo, twice daily administration of AG-636 significantly extended survival in a xenograft MOLM13-GFP-luciferase mouse model (Fig. 1C). Flow cytometry and CyTOF analysis demonstrated that AG-636 induced differentiation of CD11b+CD14+ and CD11b+CD11c+CD14+ monocytes in the bone marrow of treated mice (Fig. 1D). Administration of AG-636 significantly reduced tumor burden, induced differentiation and delayed leukemia progression in two AML patient-derived xenograft mouse models, one harboring mutations EZH2, NRAS and TET3, the other with mutations in ASXL1, BCOR and U2AF1. Daily treatment of AG-636 was well tolerated in all xenograft AML models tested, with a minimal effect on body weight and no significant toxicity recorded over the course of regimen. Conclusions: Our preliminary findings demonstrate that AG-636 is highly active against ex-vivo stroma-supported AML and AML stem/progenitor cells and in the in vivo AML xenograft models with diverse genetic subtypes. The significant monotherapy efficacy observed in pre-clinical studies provides the strong rationale for a clinical evaluation of AG-636 in myeloid malignancies. Disclosures Kuruvilla: The University of Texas M.D.Anderson Cancer Center: Employment. Kantarjian:Daiichi-Sankyo: Research Funding; Agios: Honoraria, Research Funding; Astex: Research Funding; Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding; BMS: Research Funding; Takeda: Honoraria; Ariad: Research Funding; Cyclacel: Research Funding; Pfizer: Honoraria, Research Funding; Immunogen: Research Funding; Jazz Pharma: Research Funding; Novartis: Research Funding. Andreeff: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; 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. DiNardo:celgene: Consultancy, Honoraria; daiichi sankyo: Honoraria; jazz: Honoraria; medimmune: Honoraria; syros: Honoraria; notable labs: Membership on an entity's Board of Directors or advisory committees; abbvie: Consultancy, Honoraria; agios: Consultancy, Honoraria. Murtie:Agios Pharmaceuticals, Inc.: Employment. Ulanet:Agios: Employment, Equity Ownership. Konopleva:Calithera: Research Funding; Stemline Therapeutics: Consultancy, Honoraria, Research Funding; Forty-Seven: Consultancy, Honoraria; Eli Lilly: Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Cellectis: Research Funding; Amgen: Consultancy, Honoraria; F. Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Genentech: Honoraria, Research Funding; Ascentage: Research Funding; Kisoji: Consultancy, Honoraria; Reata Pharmaceuticals: Equity Ownership, Patents & Royalties; Ablynx: Research Funding; Astra Zeneca: Research Funding; Agios: Research Funding.