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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.

The Bcl-2 inhibitor venetoclax (VEN)/hypomethylation agent (HMA) combination achieves high response rates, has improved outcomes for many patients with AML and is now considered standard of care for patients who are older or unfit to receive intensive chemotherapy. However, the median overall survival is only 14.7 months on this regimen and only 2.5 months post relapse. Molecular analysis demonstrates that mutations in TP53 and oncogenic kinases are key determinants of lower response rates and early relapse. Preclinical studies also show that increased kinase signaling in AML stem/progenitor cells in TP53 mutant AML. The heat shock protein 90 (HSP90) chaperone, a key regulator of proteostasis, is responsible for the correct folding of kinases and transcription factors. HSP90-associated-epichaperomes, formed in malignant cells, are complexes consisting of HSP90, co-chaperones, and associated proteins that support the maturation, activity, and stability of many cancer-associated kinases and transcription factors including mutated TP53. Hence, HSP90 epichaperome inhibition has the potential of targeting TP53 mutant AML. In contrast to other HSP90 ATP inhibitors, PU-H71 (zelavespib) is a competitive inhibitor specific for the ATP binding site of HSP90 epichaperomes. We here investigate the therapeutic potentials of targeting HSP90 epitherachorme with PU-H71 in TP53 mutated AML. Western blot analysis found increased HSP90 and several signaling proteins in TP53 knockout and mutant Molm13 cells generated by CRISPR/cas-9 or by exposing to idasanutlin, compared to the isogeneic wild-type controls. Using a fluorochrome-labelled PU-H71 and flow cytometry, we demonstrate the presence of HSP90 epichaperomes in AML cells and AML stem/progenitor cells with TP53 mutations, but not in normal bone marrow and bone marrow stem/progenitor cells. PU-H71 effectively kills AML cells and AML stem/progenitor cells with various TP53 mutations, and prolongs survival in TP53-mutant AML xenograft mice with minimal effects on normal CD34+ bone marrow cells and hematopoiesis. PU-H71 increased Bim expression and enhanced VEN activity in AML cells and AML stem/progenitor cells with TP53 mutations. Importantly, in a mixture of TP53 wild-type/R248W Molm13 cells (1000:1), nutlin3a selectively killed TP53 wild-type but enriched TP53 mutant Molm13 cells; VEN treatment favored the outgrowth of TP53-mutant cells, while PU-H71 effectively killed TP53 wild-type and mutant cells. Furthermore, PU-H71 exhibited anti-leukemia activity against both TP53 WT and mutant AML cells, which was further enhanced by VEN in vivo in a xenograft model of mixed TP53 WT and mutated Molm13 cells (10:1). Our data support that the HSP90 epichaperome is essential for the growth and survival of AML and AML stem/progenitor cells harboring mutant TP53. Inhibition of HSP90 by PU-H71 targets AML cells/stem/progenitor cells enhances VEN activity and prevents outgrowth of VEN-resistant TP53 mutant AML cells. This concept warrants clinical evaluations. Citation Format: Bing Carter, Po Yee Mak, Wenjing Tao, Lauren B Ostermann, Yuki Nishida, Steffen Boettcher, Michael Andreeff. Targeting HSP90 epichaperome in TP53 mutant AML [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A08.

The oncoprotein c-MYC, a major regulator of the epigenome and transcriptome, is dysregulated in 70% of all human cancers. MYC is highly expressed in Burkitt lymphoma and TP53 mutant and venetoclax (ven) resistant AML (Sallman, Blood 2021, Nishida, ASH 2021). However, targeting c-Myc or the MYC pathway has not been successful and remains a major unmet clinical need. We developed the first cereblon E3 ligase modulators (CELMoDs) for c-MYC: GT19630 and GT19715 (salt form of GT19630). C-MYC was one of the top decreased proteins in chromatin-enriched proteomics, was pulled down by biotinylated GT19630 in in vitro affinity purification assay and was degraded with IC50 of 0.33 nM in MYC-amplified HL-60 cells. Proteasome inhibitor ixazomib completely blocked c-MYC reduction, suggesting a CRL4CRBN-dependent degradation. Blood cancer cell lines responded to GT19715 greater than other cancer cell lines such as lung, breast and brain tumors in a broad cell line panel, providing rationale to develop the degrader in hematologic malignancies. In agreement with other CELMoDs, proteomic analyses revealed degradation of translation termination factor GSPT1 (G1 to S phase transition proteins 1), an important factor in LSC survival Whereas a selective GSPT1 degrader CC-90009 reduced GSPT1 protein levels but not c-MYC, GT19715 reduced both c-MYC and GSPT1 and exerted a 20x higher cytoreduction than CC-90009 (IC50 of 1.8 nM vs 40.4 nM for GT19715 and CC-90009, respectively) in HL-60 cells. GT19630 degraded c-MYC and GSPT1 and inhibited tumor growth in a xenograft model with HL-60 cells. GT19715 eliminated circulating blasts and prolonged survival in the systemic Burkitt lymphoma model (Daudi). GT19715 significantly reduced human CD45+ AML blasts in peripheral blood, bone marrow (BM) and spleens compared to vehicle controls in vivo in a chemotherapy-resistant AML PDX model. MV4;11 venetoclax resistant (VR) cells demonstrated elevated protein levels of c-MYC and GSPT1 and GT19715 induced 4log10 cytoreduction in BM and prolonged survival of mice with MV4;11 VR cells. Baseline c-Myc protein levels associated with sensitivity to GT19715 in MOLM-13 cells with CRISPR engineered knockout/mutations of TP53 (R2 = 0.86, P = 0.02). GT19715 induced comparable cell death in primary AML samples with wild-type or mutant TP53 (95.4% and 91.7% cytoreduction, P = 0.48 for wild-type and mutant TP53 samples at 64 nM of GT19715, respectively). CD34+ AML cells were more susceptible to GT19715 than CD34- AML cells, suggesting a greater efficacy in AML stem/progenitor than in more mature AML cells. Notably, single cell mass cytometry revealed that CD34+ AML cells had higher c-MYC protein levels than CD34+ normal BM cells and GT19715 reduced c-Myc levels in CD34+ AML but not normal BM cells. GT19715 induced greater cytoreduction in CD34+ AML than in normal BM cells, suggesting a therapeutic window. Conclusions: The novel dual c-MYC/GSPT1 degrader GT19715 exerts TP53 independent preclinical anti-lymphoma and -leukemia efficacy, providing rationale for its clinical development. Citation Format: Yuki Nishida, Darah A Scruggs, Edward Ayoub, Lauren B Ostermann, Tallie Patsilevas, Vivian R Ruvolo, Po Yee Mak, Bing Z. Carter, Steffen Boettcher, Abhishek Maiti, Koji Sasaki, Qianxiang Zhou, Zhaohui Yang, Honghua Yan, Liandong Ma, Michael Andreeff. C-MYC Targeting by Degradation: Novel Dual c-MYC/GSPT1 Degrader GT19715 Induces TP53-independent Cell Death in MYC-amplified Acute Myeloid Leukemia and Lymphomas [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A31.

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.

Peposertib (M3814) is a potent and selective DNA-PK inhibitor in early clinical development. It effectively blocks non-homologous end-joining repair of DNA double-strand breaks (DSB) and strongly potentiates the antitumor effect of ionizing radiation (IR) and topoisomerase II inhibitors. By suppressing DNA-PK catalytic activity in the presence of DNA DSB, M3814 potentiates ATM/p53 signaling leading to enhanced p53-dependent antitumor activity in tumor cells. Here, we investigated the therapeutic potential of M3814 in combination with DSB-inducing agents in leukemia cells and a patient-derived tumor. We show that in the presence of IR or topoisomerase II inhibitors, M3814 boosts the ATM/p53 response in acute leukemia cells leading to the elevation of p53 protein levels as well as its transcriptional activity. M3814 synergistically sensitized p53 wild-type, but not p53-deficient, AML cells to killing by DSB-inducing agents via p53-dependent apoptosis involving both intrinsic and extrinsic effector pathways. The antileukemic effect was further potentiated by enhancing daunorubicin-induced myeloid cell differentiation. Further, combined with the fixed-ratio liposomal formulation of daunorubicin and cytarabine, CPX-351, M3814 enhanced the efficacy against leukemia cells in vitro and in vivo without increasing hematopoietic toxicity, suggesting that DNA-PK inhibition could offer a novel clinical strategy for harnessing the anticancer potential of p53 in AML therapy.

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.

Bcl-2 family protein-regulated intrinsic and IAP-family protein-regulated extrinsic pathways are two major apoptotic cell death mechanisms. Components of both pathways are regulated by the tumor suppressor p53. Although combinations of Bcl-2 inhibitor venetoclax (VEN) and a hypomethylating agent induce high response rates in AML, most patients ultimately relapse. In addition, pre-clinical and clinical studies have shown that TP53-mutant AML cells are less sensitive to VEN (Carter BZ, ASH 2019 and 2020; DiNardo CD, Blood 2020). We investigated if simultaneous inhibition of Bcl-2 and IAPs and activation of p53, via MDM2 inhibition could maximize apoptosis induction in AML cells with acquired resistance to VEN-based therapy or in those carrying TP53 mutations. We treated MV4-11 cells with acquired resistance to VEN (VEN-R) with the Bcl-2 inhibitor APG2575, IAP inhibitor APG1387, or MDM2 inhibitor APG115, and with their combinations. As expected, VEN-R cells were more resistant to APG2575 compared to control cells. APG1387 alone had limited activity in both VEN-R and in control cells and the combination of APG2575 and APG1387 enhanced cell killing (P < 0.05 compared to each single agent). APG115 was active in both VEN-R and control cells and its activity was only increased by APG2575 in the control cells, but minimally affected by either APG2575 or APG1387 in VEN-R cells. However, maximal apoptosis induction was observed in both VEN-R and control cells when all three compounds were combined (P < 0.05 compared to any of the double combinations or single agent treatments). We next treated NSG mice harboring PDX cells derived from an AML patient who relapsed on the VEN/decitabine therapy with APG2575 (50 mg/kg, p.o., daily), APG1387 (10 mg/kg, i.v., once/wk), APG115 (50 mg/kg, p.o., daily at wk 1 and 5), or combinations. At the end of a 5-wk treatment, significant reductions of human CD45 + cells were observed in all treatment groups. At 4 wks post treatment, decreased circulating leukemia cells were found in the triple and APG115+APG2575 combination groups. APG115 (139 d, p=0.009), APG1387 (130 d, p=0.004), or APG2575 (132 d, p=0.004) significantly extended mouse survival compared to controls (116 d). Among two drug combinations, APG115 plus APG1387 did not further prolong survival, APG2575 plus APG1387 (144 d, p Finally, we treated Molm13 cells lacking TP53 or carrying TP53 mutations (R248W/R213*, R248Q, R175H, R282W, Y220C) with the three agents and their combinations. All mutant cells were insensitive to single drugs. Enhanced activity was observed when any of two agents were combined and combined inhibition of Bcl-2, IAPs, and MDM2 most effectively induced cell death in TP53 knockout and all TP53 mutant cells (P < 0.05 for the triple combination compared to any of the double combinations or single agent treatments, and double combinations compared to their respective single agent treatments). Western blot analysis showed that decreased cIAP1, cIAP2, XIAP, or p21 was observed in single agent or combination-treated cells. Only in the triple combination group, cIAP1, cIAP2, and XIAP as well as MDM2 were largely diminished and p21 was marked decreased. In conclusion, our study demonstrates that co-targeting intrinsic and extrinsic apoptosis maximizes cell death induction in AML cells with acquired resistance to VEN or with TP53 deletion/mutations by antagonizing Bcl-2, eliminating cIAPs and XIAP, as well as MDM2 and p21, a finding that needs to be validated clinically. Disclosures Carter: Syndax: Research Funding; Ascentage: Research Funding. Zhai: Ascentage Pharma Group Inc.: Current Employment, Current equity holder in publicly-traded company, Other: Leadership and other ownership interests, Patents & Royalties, Research Funding; Ascentage Pharma (Suzhou) Co., Ltd.: Current Employment, Current equity holder in publicly-traded company, Other: Leadership and other ownership interests, Patents & Royalties, Research Funding. Yang: Ascentage Pharma (Suzhou) Co., Ltd.: Current Employment, Current equity holder in publicly-traded company, Other: Leadership and other ownership interests, Patents & Royalties, Research Funding. Andreeff: Reata, Aptose, Eutropics, SentiBio; Chimerix, Oncolyze: Current holder of individual stocks in a privately-held company; Karyopharm: Research Funding; Oxford Biomedica UK: Research Funding; AstraZeneca: Research Funding; Syndax: Consultancy; Daiichi-Sankyo: Consultancy, Research Funding; Breast Cancer Research Foundation: Research Funding; Glycomimetics: Consultancy; Senti-Bio: Consultancy; Aptose: Consultancy; ONO Pharmaceuticals: Research Funding; Amgen: Research Funding; Medicxi: Consultancy; Novartis, Cancer UK; Leukemia & Lymphoma Society (LLS), German Research Council; NCI-RDCRN (Rare Disease Clin Network), CLL Foundation; Novartis: Membership on an entity's Board of Directors or advisory committees.

Background: Targeting apoptosis pathways in cancer has been extensively studied including the recent breakthrough therapy using venetoclax in acute myeloid leukemia (AML). However, certain therapy-resistant subsets of AML still pose a clinical challenge, leading us to investigate therapeutic strategies to bypass apoptosis pathways. Recently, non-apoptotic regulated cell death modes have attracted attention. Ferroptosis is a form of regulated cell death characterized by reactive oxygen species (ROS) induction and iron-dependent lipid peroxidation. Leukemia cells exhibit increased oxidative stress, which is further enhanced by iron overload, but only limited studies have explored therapeutic potential of targeting ferroptosis in AML. With mitochondria being the center for ROS production and iron metabolism containing 20-50% of cellular iron, mitochondrial (mito-) regulation of ferroptosis remains unclear. Incorporating our recent knowledge on mito-protease ClpP, hyperactivation of which selectively kills cancer cells through mito-proteolysis and oxidative stress (Ishizawa et al. 2019 Cancer Cell), we here aim to study the therapeutic targeting of the ferroptosis pathway and potential combinatorial targeting of mitochondria in AML. Results: We first utilized the TCGA dataset to analyze gene expression of key components in one of the major anti-ferroptosis pathways cysteine-glutathione-GPX4 axis. AML patients with higher mRNA expression of SLC7A11 (cystine importer), GCLM (a subunit of rate-limiting glutathione synthetase) or GPX4 have significantly shorter overall survival (p < 0.05), suggesting potential prognostic impact of this pathway. Here we focused on GPX4, the most downstream molecule that reduces lipid hydroperoxide to block ferroptosis. Although GPX4 is essential for embryonic development in mice, acquired depletion with conditional knock out was reported to have no significant effect on the number and function of hematopoietic stem cells, suggesting the tolerability of GPX4-targeted therapy. We demonstrate that GPX4 inhibition with sub-micromolar concentrations of ML210, a covalent GPX4 inhibitor with proteome-wide specificity, or with shRNA-mediated knockdown induces prominent cell death in various AML cell lines. The anti-leukemia effects were associated with lipid peroxidation and were almost completely abrogated by a lipophilic antioxidant Liproxstatin-1 (Lip1). The effect of ML210 was also blocked by the iron chelator deferoxamine, indicating ferroptosis. We also demonstrate that GPX4 knockdown increases ROS prominently in mitochondria before cell death is induced, suggesting that mitochondria is involved in the anti-leukemia effects. Meanwhile, GPX4 is among the top 15 sensitization hits in a previously published genome-wide CRISPR screening of leukemia cells treated with potent ClpP agonists ONC201 and ONC212 (Jacques et al. 2020 Genetics), indicating the protective function of GPX4 against mito-oxidative stress. We then found that genetic or pharmacologic hyperactivation of ClpP upregulated GPX4 protein expression predominantly in mitochondria in AML cells, collectively suggesting that upregulation of mito-GPX4 is cell-protective against ClpP-mediated cell death. Indeed, combinatorial ClpP hyperactivation enhanced the anti-leukemia effects of GPX4 inhibition through increased induction of mito-ROS and lipid peroxidation. We further demonstrate that the synergistic cell killing is inhibited by Lip1, indicating that the dual targeting of ClpP and GPX4 induces lipid peroxidation-mediated cell death. Furthermore, the inhibition of lipid peroxidation simultaneously resulted in over 80% reduction of mito-ROS, unexpectedly. This suggests that most of the increased mito-ROS by this combination is associated with lipid peroxidation. Mechanisms of induction of mitochondrial lipid peroxides jointly engaged by ClpP and mito-GPX4, as well as its involvement in the induced oxidative cell death, have yet to be explored. Conclusion: Our data suggests the potential involvement of mitochondrial lipid peroxidation in the anti-leukemia effects of GPX4 inhibition, along with its therapeutic potential in conjunction with mito-oxidative stress induction though instability in mito-proteome. Further investigations are in progress to assess the molecular mechanisms and the in vivo efficacy of the combinatorial treatment. Disclosures Andreeff: Oxford Biomedica UK: Research Funding; Aptose: Consultancy; Medicxi: Consultancy; Syndax: Consultancy; Glycomimetics: Consultancy; Senti-Bio: Consultancy; Novartis, Cancer UK; Leukemia & Lymphoma Society (LLS), German Research Council; NCI-RDCRN (Rare Disease Clin Network), CLL Foundation; Novartis: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Research Funding; ONO Pharmaceuticals: Research Funding; Amgen: Research Funding; Reata, Aptose, Eutropics, SentiBio; Chimerix, Oncolyze: Current holder of individual stocks in a privately-held company; Karyopharm: Research Funding; Breast Cancer Research Foundation: Research Funding; AstraZeneca: Research Funding.

The BCL-2 inhibitor venetoclax (ven) in combination with hypomethylating agents (HMA) has revolutionized acute myeloid leukemia (AML) therapy. However, the majority of patients with AML who received ven/HMA eventually relapse and cures are still elusive. We previously generated ven-resistant MV4;11 (MV4;11 VR) cells and found them to express elevated c-Myc protein levels. The dual inhibition of MDM2 and XPO1 significantly increased nuclear p53 protein and dramatically upregulated p53 target genes. On the other hand, dual inhibition of MDM2 and XPO1 profoundly decreased c-Myc protein levels in a p53-dependent manner, resulting in the profound downregulation of MYC transcriptional program. Clinical grade MDM2 and XPO1 inhibitors milademetan (mil) and selinexor (sel) significantly reduced leukemia burden and prolonged survival in the xenograft model injected with MV4;11 VR cells. However, the treatment effect did not result in very long relapse free responses. To investigate potential resistance mechanisms to dual inhibition of MDM2 and XPO1, we utilized our previously reported (Muftuoglu, ASH 2020) multiparameter flow cytometry to assess multiple stress responses pathways including p21, ATF4, PARP, LC3B, Ki-67, active caspase-3 and amine-reactive viability dye at the single cell level in ven-resistant AML cells. We found that residual cells, after combined MDM2 and XPO1 inhibition, expressed high levels of p21, ATF4 and LC3B and low Ki-67 levels, suggesting that the resistant population is in a cell kinetic quiescent state with activation of autophagy and ER stress. Interestingly, ven-resistant MV4;11 cells with in vivo acquired resistance to dual inhibition of MDM2 and XPO1 demonstrated elevated protein levels of respiratory chain complex proteins (NDUFB8, MTCO1, UQCRC2 and ATP5A), suggesting increased OXPHOS dependency. Intriguingly, these MV4;11VR cells with subsequently acquired resistance to MDM2 and XPO1 inhibition in vivo have restored sensitivity to ven. We previously reported that the combination of MDM2 and BCL-2 inhibitors induces synergistic apoptosis through the elimination of dormant p21 high residual AML cell (Pan, Cancer Cell 2017). In a clinical trial of Idasanutlin + Venetoclax we observed over 45% response rates in relapsed/refractory AML patients (Daver, ASH 2020). Therefore, we hypothesized that combining MDM2/XPO1 inhibition with BCL-2 inhibition further induces cell killing in ven/HMA resistant AML cells. To address this, we treated ven-resistant AML cells with triple combination of mil, sel and ven, resulting in a profound cytoreduction in vitro compared with other single and doublet treatments. Next, we treated NSG mice injected with PDX AML cells with FLT3-ITD, GATA2 and NRAS mutations obtained from a patient who relapsed after ven/decitabine treatment. The doublet combinations especially of mil + ven reduced circulating blasts and significantly prolonged survival. Of note, no mice have died at day 180 after treatment in the group receiving triple combination of mil, ven and sel, with profound and sustained cytoreduction (more than 2 log 10 difference) and significantly prolonged survival compared with any other treatment (Fig. A, B). The triple combination was well-tolerated and did not decrease mouse CD45+ cells, platelets and hemoglobin. In conclusion, the concomitant inhibition of MDM2, XPO1 and BCL-2 was feasible and exerted dramatic and sustained anti-leukemia activity in ven/HMA resistant AML cell in vitro and in vivo. Milademetan (RAIN-32) is currently being developed by Rain Therapeutics. Figure 1 Figure 1. Disclosures Carter: Syndax: Research Funding; Ascentage: Research Funding. Daver: Pfizer: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy, Other: Data Monitoring Committee member; Novartis: Consultancy; Gilead Sciences, Inc.: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; ImmunoGen: Consultancy, Research Funding; Trillium: Consultancy, Research Funding; Glycomimetics: Research Funding; Sevier: Consultancy, Research Funding; Astellas: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Abbvie: Consultancy, Research Funding; Novimmune: Research Funding; Hanmi: Research Funding; Amgen: Consultancy, Research Funding; Trovagene: Consultancy, Research Funding; FATE Therapeutics: Research Funding; Dava Oncology (Arog): Consultancy; Celgene: Consultancy; Syndax: Consultancy; Shattuck Labs: Consultancy; Agios: Consultancy; Kite Pharmaceuticals: Consultancy; SOBI: Consultancy; STAR Therapeutics: Consultancy; Karyopharm: Research Funding; Newave: Research Funding. Lesegretain: Daiichi-Sankyo Inc.: Current Employment. Seki: Daiichi-Sankyo Inc.: Current Employment. Shacham: Karyopharm: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties: (8999996, 9079865, 9714226, PCT/US12/048319, and I574957) on hydrazide containing nuclear transport modulators and uses, and pending patents PCT/US12/048319, 499/2012, PI20102724, and 2012000928) . Konopleva: Ascentage: Other: grant support, Research Funding; Reata Pharmaceuticals: Current holder of stock options in a privately-held company, Patents & Royalties: intellectual property rights; Eli Lilly: Patents & Royalties: intellectual property rights, Research Funding; Novartis: Other: research funding pending, Patents & Royalties: intellectual property rights; AstraZeneca: Other: grant support, Research Funding; Rafael Pharmaceuticals: Other: grant support, Research Funding; F. Hoffmann-La Roche: Consultancy, Honoraria, Other: grant support; AbbVie: Consultancy, Honoraria, Other: Grant Support, Research Funding; Genentech: Consultancy, Honoraria, Other: grant support, Research Funding; Forty Seven: Other: grant support, Research Funding; Cellectis: Other: grant support; Calithera: Other: grant support, Research Funding; Agios: Other: grant support, Research Funding; KisoJi: Research Funding; Ablynx: Other: grant support, Research Funding; Stemline Therapeutics: Research Funding; Sanofi: Other: grant support, Research Funding. Andreeff: Medicxi: Consultancy; Glycomimetics: Consultancy; AstraZeneca: Research Funding; Syndax: Consultancy; Amgen: Research Funding; Oxford Biomedica UK: Research Funding; Karyopharm: Research Funding; Daiichi-Sankyo: Consultancy, Research Funding; Breast Cancer Research Foundation: Research Funding; Novartis, Cancer UK; Leukemia & Lymphoma Society (LLS), German Research Council; NCI-RDCRN (Rare Disease Clin Network), CLL Foundation; Novartis: Membership on an entity's Board of Directors or advisory committees; Senti-Bio: Consultancy; Reata, Aptose, Eutropics, SentiBio; Chimerix, Oncolyze: Current holder of individual stocks in a privately-held company; ONO Pharmaceuticals: Research Funding; Aptose: Consultancy.

AML patients with TP53 mutations have extremely poor clinical outcomes. This is due primarily to limited responses to available therapies including the highly promising FDA-approved combination of Bcl-2 inhibition by venetoclax (VEN) with hypomethylating agents (DiNardo CD et al., Blood 2020), which resulted in CR/CRi rates of 70-95% and good tolerability in elderly patients (DiNardo CD et al., Lancet Oncol 2018 and Blood 2019). Apoptosis is regulated by anti- and pro-apoptotic proteins. While p53 does not directly regulate anti-apoptotic Bcl-2 proteins that are resistance factors for VEN, p53 transcriptionally up-regulates pro-apoptotic Bcl-2 proteins. Reverse phase protein array analysis of samples from newly-diagnosed AML patients found that pro-apoptotic Bax was significantly decreased in patients with TP53 mutations (Carter BZ, ASH 2019), which, as expected, diminished the effectiveness of Bcl-2 inhibition. Thus, strategies to target additional anti-apoptotic proteins, or increase pro-apoptotic proteins, are needed to enhance the efficacy of Bcl-2 inhibition in these patients. We determined protein levels of Bcl-2 family members in isogeneic Molm13 cells with TP53-knockout (KO), or with various hotspot TP53 mutations including R175H, Y220C, M237I, R248Q, R273H, and R282W. We observed markedly decreased Bax expression, to a less degree Bak decrease, and variable alterations in other Bcl-2 proteins in these cells compared to TP53-wild-type (WT) controls. We treated the aforementioned cells with VEN or the Mcl-1 inhibitor AMG 176 and found that TP53-KO or mutant cells were more resistant to both VEN and AMG 176 compared to WT controls. However, the combination of two inhibitors was highly synergistic in both settings, controls (CI = 0.2) and TP53-KO and mutant cells (CI < 0.1). To demonstrate that the decreased sensitivity to BH3 mimetics was, at least in part, mediated through Bax reduction in the TP53-mutant cells, we treated Bax knockdown (KD) Molm13 cells with VEN, AMG 176, or both. The Bax KD cells were resistant to VEN and AMG 176, while the combination of the two agents synergistically induced cell death. To establish potential clinical relevance of co-targeting Bcl-2 and Mcl-1 in TP53-mutant AML, we co-cultured cells from various TP53-mutant AML patients (n = 8) with mesenchymal stromal cells and treated them with VEN, AMG 176, or both. The combination synergistically induced cell death in both CD45 + leukemia blasts (CI values between 0.04 ± 0.04 to 0.34 ± 0.10) and CD34 + AML stem/progenitor cells (CI values between 0.07 ± 0.08 to 0.28 ± 0.14). RNA-sequencing of mononuclear and MRD cells of clinical samples (Issa G, ASH 2019) collected after induction therapy revealed that Mcl-1 expression was significantly higher in the TP53-mutated mononuclear and MRD cells compared to their WT counterparts (Fig. 1), which suggests that Mcl-1 contributes to treatment resistance and disease relapse. This further suggests that Mcl-1 inhibition should be incorporated in AML treatment, including VEN-based therapies, for patients with TP53 mutations. Finally, we treated NSG mice inoculated with isogeneic TP53-WT luciferase/GFP-labeled Molm13 and BFP-labeled TP53 R248W/R213* Molm13 cells (10:1) with VEN, AMG 176, or their combination. Only the combination treatment markedly decreased the number of GFP- and BFP-labeled cells in circulation and significantly prolonged mouse survival (median 23 d, 25 d, 24.5 d for control, VEN, AMG 176, respectively; and 45 d for VEN + AMG 176: P = 0.0007, 0.0009, and 0.0011 of combination vs. control, VEN, and AMG 176, respectively) (Fig. 2). Collectively, we demonstrate that decreased Bax contributes to resistance of TP53-mutant AML to BH3 mimetics. Mcl-1 expression positively impacts therapy resistance and disease reoccurrence in TP53-mutant AML. Thus, targeting Bcl-2 or Mcl-1 individually is insufficient and inhibition of both proteins is needed to shift cell fate from survival to death and circumvents resistance of TP53 deficient/mutant AML and AML stem/progenitor cells to BH3 mimetics. The concept warrants further clinical evaluation. Figure 1 Figure 1. Disclosures Carter: Syndax: Research Funding; Ascentage: Research Funding. Jabbour: Amgen, AbbVie, Spectrum, BMS, Takeda, Pfizer, Adaptive, Genentech: Research Funding. Andreeff: Medicxi: Consultancy; Daiichi-Sankyo: Consultancy, Research Funding; Breast Cancer Research Foundation: Research Funding; Novartis, Cancer UK; Leukemia & Lymphoma Society (LLS), German Research Council; NCI-RDCRN (Rare Disease Clin Network), CLL Foundation; Novartis: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Research Funding; Amgen: Research Funding; ONO Pharmaceuticals: Research Funding; Karyopharm: Research Funding; Syndax: Consultancy; Senti-Bio: Consultancy; Aptose: Consultancy; Glycomimetics: Consultancy; Oxford Biomedica UK: Research Funding; Reata, Aptose, Eutropics, SentiBio; Chimerix, Oncolyze: Current holder of individual stocks in a privately-held company.

FLT3 inhibitors (FLT3i) have had transient success treating FLT3-mutant acute myeloid leukemia (AML) patients, especially those with FLT3 internal tandem duplication (ITD) mutations that account for one-third of adult AML cases (Daver et al., 2021). However, FLT3i are typically ineffective in eliminating leukemia stem cells in the protective bone marrow (BM) microenvironmental "niche" (Borthakur et al., 2011; Cortes et al., 2013; Zhang et al., 2008). Cytokines and chemokines such as CXCR4 and E-selectin ligands play a critical role in leukemia cell protection in the BM niche. Indeed, interactions of leukemic cells with their vicinal support cells, including mesenchymal stem cells (MSCs) and endothelial cells (ECs), in the BM niche is mediated mainly through the CXCR4/SDF-1 and E-selectin/HECA-452/CD44 axes (Erbani et al., 2020; Peled and Tavor, 2013). Therefore, evaluation of the effects of FLT3i on CXCR4 and E-selectin signaling in leukemia cells could enhance our understanding of AML FLT3i resistance mechanisms. To this end, we investigated the levels of CXCR4 and E-selectin ligands on FLT3-ITD-mutated AML cells in vitro and in vivo during FLT3i treatment (e.g., quizartinib or sorafenib), and evaluated the anti-leukemia effects of CXCR4/E-selectin blockade with the dual inhibitor GMI-1359. We first checked the effects of FLT3i on the levels of CXCR4 and E-selectin ligands, as well as CD44, in vitro in human MOLM14 AML cells, which harbor FLT3-ITD mutations. All of these were upregulated, as measured by flow cytometry, following exposure to quizartinib (p < 0.001) or sorafenib (p < 0.01) for 96 h. The mRNA levels were also increased roughly 2-fold, as measured by qPCR, suggesting transcriptional regulation was involved in the upregulation. Further, the upregulation of CXCR4 and E-selectin ligands and CD44 was time dependent (from 2 to 96 h). FLT3i profoundly suppresses activation of ERK, AKT, and Stat5 (Zhang et al., 2008). Therefore, we tested if the suppression of each signaling pathways individually could upregulate of CXCR4. Unexpectedly, 72-h suppression of MEK/ERK signaling with selumetinib or pimasertib also upregulated CXCR4 in MOLM14 cells. No effects in this regard were observed by suppressing AKT/mTOR or Stat5 with AZD8055 or STAT5-IN-1, respectively. Additionally, in Dox-inducible NRAS (G12D)-mutated MOLM13 AML cells which also harbor FLT3 ITD mutations, ERK activation by doxycycline downregulated CXCR4 levels implying the MEK/ERK signaling pathway was associated with the suppression of CXCR4. Furthermore, under BM microenvironment-mimicking, co-culture using human MSCs/ECs and MOLM14 cells, blockade of CXCR4 and/or E-selectin signaling using the CXCR4 antagonist plerixafor, the E-selectin antagonist GMI-1271, or the CXCR4 and E-selectin dual inhibitor GMI-1359 showed that GMI-1359 markedly abrogated BM protection and sensitized MOLM14 cells to quizartinib-induced apoptosis. We further validated the effect of GMI-1359 in a PDX model of AML which were from a patient who relapsed from sorafenib+E6201+DAC in clinic and showed resistant to quizartinib ex vivo. The combination of GMI-1359 with quizartinib profoundly reduced leukemia burden and extended survival of the PDX mice compared to the vehicle or the single-agent treatments (median survival was 158 days vs. 82.5, 79 and 128 days, respectively, in combination group vs. vehicle, quizartinib and GMI-1359; p < 0.0001) [Figure 1,2]. Our results suggest that FLT3i can upregulate CXCR4 and E-selectin ligands and CD44 in FLT3-ITD leukemia cells, which is mediated, at least in part, via suppression of MEK/ERK signaling. GMI-1359 sensitized AML cells to quizartinib-induced apoptosis in vitro and statistically significantly extended AML PDX mouse survival in vivo. These findings provide a pre-clinical rational for using GMI-1359 to prevent or overcome FLT3i resistance when treating FLT3-mutant AML patients. Figure 1 Figure 1. Disclosures Fogler: GlycoMimetics Inc.: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Magnani: GlycoMimetics Inc.: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Konopleva: Eli Lilly: Patents & Royalties: intellectual property rights, Research Funding; Forty Seven: Other: grant support, Research Funding; KisoJi: Research Funding; AstraZeneca: Other: grant support, Research Funding; AbbVie: Consultancy, Honoraria, Other: Grant Support, Research Funding; Ablynx: Other: grant support, Research Funding; Genentech: Consultancy, Honoraria, Other: grant support, Research Funding; Reata Pharmaceuticals: Current holder of stock options in a privately-held company, Patents & Royalties: intellectual property rights; Rafael Pharmaceuticals: Other: grant support, Research Funding; F. Hoffmann-La Roche: Consultancy, Honoraria, Other: grant support; Calithera: Other: grant support, Research Funding; Sanofi: Other: grant support, Research Funding; Novartis: Other: research funding pending, Patents & Royalties: intellectual property rights; Agios: Other: grant support, Research Funding; Cellectis: Other: grant support; Stemline Therapeutics: Research Funding; Ascentage: Other: grant support, Research Funding. Andreeff: Senti-Bio: Consultancy; Daiichi-Sankyo: Consultancy, Research Funding; AstraZeneca: Research Funding; Karyopharm: Research Funding; Glycomimetics: Consultancy; Aptose: Consultancy; Novartis, Cancer UK; Leukemia & Lymphoma Society (LLS), German Research Council; NCI-RDCRN (Rare Disease Clin Network), CLL Foundation; Novartis: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; ONO Pharmaceuticals: Research Funding; Reata, Aptose, Eutropics, SentiBio; Chimerix, Oncolyze: Current holder of individual stocks in a privately-held company; Medicxi: Consultancy; Oxford Biomedica UK: Research Funding; Breast Cancer Research Foundation: Research Funding; Syndax: Consultancy.

Cancer cells depend on aberrant antioxidant mechanisms to survive increased oxidative stress, which is particularly critical for cancer stem cell maintenance and its therapy resistance phenotype. Mitochondria is the primary site of oxygen consumption and reactive oxygen species (ROS) production, thus is essential for regulating cellular redox homeostasis. We have recently reported that hyperactivation of mitochondrial (mito-) protease CLPP by imipridones (ONC201/212) exerts cancer selective lethality through degradation of mito-proteins including respiratory chain complexes and induction of oxidative stress (Ishizawa et al., Cancer Cell 2019). However, the cell death mechanisms remain to be fully determined. We hypothesized that deregulation of mito-redox homeostasis induces a distinct mode of cell death in CLPP-hyperactivated cells and that concomitant inhibition of both mito- and cytosolic antioxidant mechanisms could enhance the anti-leukemia effects. We first tested if inhibition of glutathione (GSH) synthesis enhances the anti-leukemia effects by CLPP hyperactivation by using buthionine sulfoximine (BSO), a glutamate-cysteine ligase inhibitor. As hypothesized, BSO highly sensitized AML cells to genetic or pharmacologic hyperactivation of CLPP by overexpressing CLPP-Y118A (an activating mutation) or ONC201 treatment. We next investigated if a downstream target of GSH depletion is causing the synergy, and re-analysis of recently published CRISPR-knockout screens using leukemic cells treated with imipridones identified GPX4 as one of the 15 top-hit genes whose knockout sensitized cells to CLPP hyperactivation. GPX4 is a selenoprotein that is known for its GSH-dependent reduction of lipid hydroperoxides to protect cells from oxidative cell death including ferroptosis. We found that CLPP hyperactivation increased GPX4 protein levels in AML cells, prominently in mitochondria. Consistently, the GPX4 inhibitors ML210 and JKE1674 sensitized cells to ONC201 in multiple AML cell lines. Overexpression of the activating CLPP-Y118A was also synthetically lethal to GPX4 inhibition, confirming the on-target effect of ONC201. Mechanistically, GPX4 inhibition induced iron-dependent lipid peroxidation and AML cell killing, thus defining ferroptosis as the operational mechanism of cell death. Dual targeting of CLPP and GPX4 induced enhanced lipid peroxidation and mito-ROS but ferroptosis inhibitors only partially blocked cell death, indicating that other modes of oxidative cell death are also involved in the combinatorial synergism. Collectively, our data indicate therapeutic potential of targeting GPX4 and CLPP in AML and potential identification of a novel mode of cancer cell death. Investigations are ongoing to elucidate the underlying molecular mechanisms and to assess in vivo efficacy of the combinatorial treatment. Citation Format: Hiroki Akiyama, Ran Zhao, Lauren B. Ostermann, Jo Ishizawa, Michael Andreeff. Dual targeting of glutathione-GPX4 nexus and mitochondrial protease CLPP causes synergistic anti-leukemia effects in acute myeloid leukemia (AML) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2017.

Acute myelogenous leukemia (AML) is characterized by an accumulation of abnormal white blood cells. Internal tandem duplications in the fms-like tyrosine kinase 3 (FLT3-ITD) account for 30% of adult AML cases and confer poor prognosis (Nakao et al., Leukemia 1996). FLT3 inhibitors like sorafenib efficiently eliminate circulating leukemia blasts, but frequently not in the bone marrow (BM), which suggests a protective effect of the BM niche for leukemic stem cell survival (Zhang et al., JNCI 2008). The homing of AML cells in BM is mediated chiefly by the adhesion to E-selectin on endothelial cells (ECs) and by CXCR4-directed cellular migration to stromal CXCL12 (SDF1) sources (Chien et al., Blood 2013; Peled and Tavor, Theranostics 2013). In many respects, BM homing signals are shared between leukemia and hematopoietic stem cells (HSCs). Our previous study demonstrated that targeting E-selectin/CXCR4 with the dual E-selectin/CXCR4 antagonist GMI-1359 markedly reduced leukemia cell adhesion to ECs and mesenchymal stem cells, reduced the BM-mediated protection of leukemic cells during FLT3-targeted therapy in vitro, and effectively reduced leukemia cellularity in the BM in vivo (Zhang et al., Can Res suppl 2016). Further, GMI-1359 combined with cytarabine/daunorubicin provided a profound survival benefit in mice with FLT3-mutated leukemia (Zhang et al., Blood suppl 2015). In the present study, we sought to evaluate dual E-selectin/CXCR4 blockage in the context of FLT3 inhibition by sorafenib in vivo, and to better understand the underlying mechanism. We compared expression levels of E-selectin ligands and CXCR4 in FLT3 inhibitor-sensitive Ba/F3-FLT3-ITD cells and their inhibitor-resistant counterparts Ba/F3-FLT3-ITD+D835Y and Ba/F3-FLT3-ITD+F691L. Resistant cells expressed 1.7 to ~5.6-fold higher levels of total E-selectin ligand detected by a soluble E-selectin reagent, and 10-fold higher levels of CXCR4. In addition, BM-mimetic hypoxia culture profoundly upregulated the cell surface expression of E-selectin ligands and CXCR4 on leukemia cells. We evaluated anti-leukemia effects of co-targeting E-selectin/CXCR4 and FLT3 with GMI-1359 and sorafenib in a patient-derived AML xenograft (PDX) model harboring FLT3-ITD and WT1 mutations. We observed that addition of GMI-1359 to sorafenib greatly reduced leukemia cellularity compared to sorafenib alone, and as much as by 92%, 82%, 69% and 45% in, respectively, liver, lung, spleen and BM (Fig. 1) as compared with vehicle-treated mice (p < 0.05). As expected, the number of circulating leukemia cells transiently increased. The GMI-1359/sorafenib combination improved mouse survival (median survival 138.5 versus 109, 87 and 126 days for the GMI-1359/sorafenib versus vehicle, GMI-1359 and sorafenib, respectively, p < 0.001). Using intravital 2-photon microscopy, we observed AML cell behavior in calvarial BM and their response to acute GMI-1359 bolus infusion. Remarkably, AML cell mobility began to increase in the BM microenvironment as soon as 20 min after treatment (Fig. 2), followed by intravasation and cellular outflow through the BM capillary vasculature over the next 2-4 hours. Moreover, although BM homing signals are thought to be shared between leukemia and HSCs, the combination therapy improved hematopoiesis parameters compared to sorafenib alone. In particular, this important effect was associated with increased numbers of megakaryocytes (2.1-fold), myelocytes (2.1-fold), and erythrocytes (7.1-fold) in BM (p < 0.01). The underlying mechanism(s) of hematopoiesis protection by GMI-1359 are under investigation. Conclusion: Co-inhibition of E-selectin/CXCR4 enhances the anti-leukemia efficacy of FLT3 inhibition and preserves hematopoiesis in the BM in a PDX model of AML. Disclosures Fogler: GlycoMimetics: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Magnani:GlycoMimetics, Inc.: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Zal:Daiichi-Sankyo: Research Funding; Moleculin Biotech, Inc.: Research Funding. Andreeff:Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy.

Acute myeloid leukemia (AML) is an aggressive heterogeneous hematologic disease with high mortality in patients older than 60 years. Clinical studies have proven that combinations of FDA-approved Bcl-2 inhibitor, venetoclax and hypomethylating agents (Ven/HMA) are highly effective in elderly patients with AML (DiNardo et al., 2019). Despite improved remission rates, the duration of response is still inadequate. Adhesion to the bone marrow (BM) niche is critical for AML initiation, progression and leukemic stem cell (LSC) survival after induction therapy. The vascular adhesion molecule, E-selectin (E-sel) is responsible for the tethering and rolling of leukocytes on perivascular endothelial BM niche cells (EC). In leukemia, E-sel has crucial roles in BM homing and engraftment (Krause et al, 2006). Through patient-derived AML xenograft (PDX) models and single cell proteomics, we have elucidated the roles of E-sel in AML survival and drug resistance. A PDX model derived from a patient who had developed resistance to Ven/HMA were treated with the E-sel antagonist, GMI-1271 (uproleselan; GlycoMimetic, Inc). We found that targeting E-sel mobilized human AML cells and sensitized them to Ven/HMA. The number of circulating leukemic cells was significantly reduced by combinatorial treatment of GMI-1271 with Ven/HMA in comparison to Ven/HMA alone (p < 0.05). The synergistic effects of the combinatorial treatment on AML-PDX mouse survival were determined by Kaplan-Meier analysis. The combination of GMI-1271 and Ven/HMA significantly prolonged the survival of mice compared to vehicle control (p = 0.015) as well as the Ven/HMA (p = 0.0009) and GMI-1271 groups (p = 0.03). The median survival of the vehicle control, GMI-1271, Ven/HMA, and combination-treated groups of mice was 86, 91, 81.5, and 106.5 days, respectively. Histological analysis of BM, spleen, lung and liver demonstrated differences in leukemia cell infiltration, confirming enhanced anti-leukemia efficacy of the combination treatment. To delineate the mechanism of E-sel at the onset of drug mediated changes in AML signaling signatures, we employed another PDX model (Flt3-ITD and WT1 mutations, sorafenib-resistant). PDX mice with advanced AML (more than 20% human AML cells circulation in peripheral blood) were administered Ven/HMA, GMI-1271, or combination for 2 days. Single cell proteomics analysis by CyTOF determined that combinatorial treatment diminished levels of Ki67, IDU, and pRb compared to vehicle control or Ven/HMA alone, resulting in decreased proliferation of AML blasts. Activation of eNOS to produce nitric oxide (NO) through PI3K/AKT kinase, maintains clonogenic cell growth in malignant cells. A recent publication has demonstrated that introduction of NOS blockers in combination with chemotherapy led to slower leukemia progression and longer remissions in contrast to chemotherapy alone (Passaro et al, 2017). Interestingly, we observed reduced activation of PI3K and AKT in AML blasts as well as in BM CD31+EC cells in the GMI-1271 treated PDX model. eNOS phosphorylation was subsequently decreased in EC, suggesting that inhibition of E-sel may protect BM vasculature by blocking the production of NO. In addition, targeting E-sel showed signaling alterations in AML MSC. Administration of E-sel antagonist increased mTOR expression in MSC from AML-PDX. Combination treatment induced higher Ki67 positivity, and hyperactivation of pRb and p-S6 in MSC in vivo. Our group and others have recently reported that Ven-resistant AML cells exhibit an increased dependence on alternate anti-apoptotic proteins, Mcl-1 and Bcl-xl (Konopleva et al., 2016). We found that concomitant treatment in vivo with GMI-1271 and Ven/HMA further decreased the expression of Bcl-xl and Mcl-1 in AML blasts compared to Ven/HMA alone, suggesting a critical role for E-sel antagonists in overcoming drug resistance. E-selectin binding potential and focal adhesion kinase activity in AML blasts were decreased upon acute administration of pharmacological E-sel inhibitor. Other oncogenic signaling pathways including MAPK, p-S6, and STAT3, were all inhibited by the addition of GMI-1271 to Ven/HMA. Collectively, our results provide first evidence that an E-sel targeting strategy with GMI-1271 can overcome microenvironmental resistance to Ven/HMA-based therapy in AML by cancer cell autonomous and non-cell autonomous mechanisms in the BM vascular niche. Figure Disclosures Fogler: GlycoMimetics: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Magnani:GlycoMimetics, Inc.: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Andreeff:Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy.

TP53 mutations are observed in 5 to10% of de novo AML cases with dismal disease prognosis and response to therapy. We found that MDM2 loss of heterozygosity (MDM2 LOH) in AML was always concomitant with TP53 missense mutations (log2 odds ratio>3, p Disclosures Andreeff: Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy; Amgen: Research Funding; Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees.

Background: CD123 is frequently expressed in hematologic malignancies including AML. CD123 has been a potential immunotherapeutic target in AML due to its association with leukemic stem cells that play an essential role in disease progression and relapse. Our previous study using T-cells secreting CD123/CD3-bispecific T-cell engagers (BiTEs) (CD123-ENG T-cells) has shown activity in preclinical studies, recognizing and killing acute myeloid leukemia (AML) blasts in vitro and in vivo. CD123-ENG T-cells secrete bispecific molecules that recognize CD3 (T-cells) and CD123 (AML blasts), and are able to direct transduced T-cells and recruit bystander T-cells to kill CD123-positive blasts. Venetoclax is a BCL-2 inhibitor that can restore functional apoptosis signaling in AML cells, and has been FDA approved for the treatment of AML patients in combination with hypomethylating agents. To improve the efficacy of CD123-ENG T-cells we explored efficacy in AML by combining targeted immunotherapy (CD123-ENG T cells) with targeted inhibition of anti-apoptotic BCL-2 (venetoclax) in vitro and in vivo models of AML. Methods : CD123-ENG T-cells were generated by retroviral transduction and in vitro expansion. Non-transduced (NT) T-cells served as control. In vitro, GFP+ MOLM-13 AML cells were pretreated with venetoclax (0, 10µM, and 20µM) for 24 hours prior to co-culture with CD123-ENG or NT T-cells at an effector/target ratio of 1:10. After 16 hours, MOLM-13 AML cells were analyzed by flow cytometry and quantitated using counting beads; cytotoxicity was calculated relative to untreated MOLM-13 control. The anti-AML activity of the combination was further evaluated in a MOLM-13-luciferase xenograft AML mouse model. Leukemia progression was assessed by bioluminescence imaging. The frequency of MOLM13 AML and human T cells in periphera blod (PB) was determined by flow cytometry. Results: In vitro, we demonstrated that pretreatment of Molm13 AML cells with venetoclax enhanced the cytolytic activity of CD123-ENG T-cells compared to NT- or no T-cell controls. Interestingly, venetoclax sensitized Molm13 to CD123-ENG T-cell killing in a dose-dependent manner (Fig.1; 50%/31% killing by CD123-ENG T-cells versus 27%/14% of killing by NT T cells post pretreatment with 10µM or 20µM ventoclax, p Conclusion: Our data support a concept of combining pro-apoptotic targeted and immune therapy using venetoclax and CD123-ENG T-cells in AML. While it has been reported that venetoclax does not impair T-cell functionality, more in-depth analysis of the effect of Bcl-2 inhibition on T-cell function and survival appears warranted, as it could diminish survival not only of AML blasts but also of immune cells. Disclosures Bonifant: Patents filed in the field of engineered cellular therapies: Patents & Royalties: Patents filed in the field of engineered cellular therapies. Gottschalk:Patents and patent applications in the fields of T-cell & Gene therapy for cancer: Patents & Royalties; Inmatics and Tidal: Membership on an entity's Board of Directors or advisory committees; Merck and ViraCyte: Consultancy; TESSA Therapeutics: Other: research collaboration. Velasquez:Rally! Foundation: Membership on an entity's Board of Directors or advisory committees; St. Jude: Patents & Royalties. Andreeff:Amgen: Research Funding; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy; Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees.

Acute myeloid leukemia (AML) is an aggressive heterogeneous hematologic disease with high mortality in patients older than 60 years. Clinical studies have proven that combinations of FDA-approved Bcl-2 inhibitor, venetoclax and hypomethylating agents (HMA) are highly effective in elderly patients with AML (DiNardo et al., 2019). Adhesion to the BM niche is critical for AML initiation, progression and LSC survival after induction therapy. The vascular adhesion molecule, E-selectin (E-sel) has crucial roles in BM homing and engraftment in leukemia (Krause et al, 2006). Here we elucidated the roles of E-sel in AML survival using human AML cell lines and patient-derived AML xenograft (PDX) models. In our experiments, E-sel decreased CDK4/6 expression and increased dormancy of AML cells in vitro. Pharmacological inhibition of E-sel by GMI-1271 increased expression of cell cycle regulating proteins including CDK4/6 and CyclinD1/2 in HUVEC co-cultured AML. To determine if E-sel has indispensable effects in BM niche component cells, we exposed healthy donor derived-mesenchymal stroma cells (MSC) to increasing concentrations of E-sel: E-sel upregulated surface expression of the most potent E-sel ligand, CD44 in human MSC and abrogation of E-sel binding by GMI-1271 diminished CD44 expression in vitro. Targeting E-sel with GMI-1271 attenuated eNOS phosphorylation in HUVEC co-cultured with AML cells, suggesting that E-sel inhibition may protect disruption of BM vasculatures during AML progression. Further, we found that targeting E-sel mobilized human AML cells and sensitized them to venetoclax/HMA in a PDX model derived from a patient who had developed resistance to venetoclax/HMA. The number of circulating leukemic cells was significantly reduced by combinatorial treatment of GMI-1271 with venetoclax/HMA compared to venetoclax/HMA alone (p

Bone marrow mesenchymal stem cells (BM-MSCs) are multipotent stromal cells that can differentiate into a variety of lineages and play a critical role in tissue homeostasis upon injury and repair. Previous studies using Osx-Cre transgenic mice have demonstrated that the expression of Sp7 (Osterix) marks a population of mesenchymal progenitor cells that can differentiate to osteoblasts as well as bone marrow stromal cells (Mizoguchi et al., 2014). Using a lineage-tracing system, we show that in addition to marking mesenchymal progenitor cells in the bone marrow, Osx-Cre also marks a population of BM-MSCs that circulate throughout the body and home in different tissues such as lung, spleen, intestine and muscle. Osx-Cre mice crossed with R26-mTmG reporter mice were analyzed at E14.5 and adulthood by fluorescence microscopy and flow cytometry. At E14.5, GFP+ cells were exclusively located in bone tissues. At two months of age, GFP+ cells were detectable in blood and many other tissues such as lung, liver, spleen and intestine. Flow cytometric profiling indicated that the GFP+ cells were positive for BM-MSC markers CD105, CD73 and CD140a. In order to exclude the possibility of non-specific recombination of the reporter in the non-osteoblast-lineage, as previously reported, we performed a pulse chase experiment utilizing Osx-CreER;mTmG mice. Fluorescence microscopy of the bone marrow upon Tamoxifen injection revealed that Cre activity was primarily limited to the osteoblast-lineage and bone tissue, whereas GFP+ cells were undetectable in lung and spleen, indicating that the GFP+ cells in the lung migrated from the bone marrow. Given that previous reports identified p53 as a negative regulator of osteoblast differentiation (Lengner et al., 2006), we further sought to determine the effect of p53 on circulatory BM-MSCs. Flow cytometric analysis of Osx-Cre;p53Fx/Fx;mTmG peripheral blood cells revealed a significant reduction of circulatory GFP+ cells as compared to p53 wild type mice (p Lengner, C.J., Steinman, H.A., Gagnon, J., Smith, T.W., Henderson, J.E., Kream, B.E., Stein, G.S., Lian, J.B., and Jones, S.N. (2006). Osteoblast differentiation and skeletal development are regulated by Mdm2-p53 signaling. J Cell Biol 172, 909-921. Mizoguchi, T., Pinho, S., Ahmed, J., Kunisaki, Y., Hanoun, M., Mendelson, A., Ono, N., Kronenberg, H.M., and Frenette, P.S. (2014). Osterix marks distinct waves of primitive and definitive stromal progenitors during bone marrow development. Dev Cell 29, 340-349. Figure Disclosures Andreeff: Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy; Amgen: Research Funding.

Acute myelogenous leukemia (AML) is characterized by an accumulation of abnormal white blood cells. Internal tandem duplications in the Fms-like tyrosine kinase 3 (FLT3-ITD) account for 30% of adult AML cases and confer poor prognosis (Kottaridis et al., 2003; Thiede et al., 2002). FLT3 inhibitors like sorafenib efficiently eliminate leukemia blast in the peripheral blood (PB), but frequently not in the bone marrow (BM) (Zhang et al., 2008). This suggests a protective effect of the BM on leukemic stem cells, which is mediated by E-selectin and CXCL12 expression in endothelial cells (ECs) and mesenchymal stem cells (MSCs) in the BM vascular and endosteal niches (Horacek et al., 2013; Peled and Tavor, 2013). Our previous study demonstrated that targeting E-selectin/CXCR4 with the dual E-selectin/CXCR4 antagonist GMI-1359 markedly reduced leukemia cell adhesion to ECs and MSCs and eliminated the BM-mediated protection of leukemic cells during FLT3-targeted therapy in vitro, and effectively reduced leukemia cell in the BM in vivo (Zhang et al., 2016). Further, GMI-1359 combined with cytarabine/daunorubicin provided a profound survival benefit in FLT3-mutated leukemia cell MV4-11-bearing mice (Zhang et al., 2015). In the preset study, we compared expression levels of E-selectin ligands and CXCR4 in FLT3 inhibitor-sensitive Ba/F3-FLT3-ITD cells and their inhibitor-resistant counterparts Ba/F3-FLT3-ITD+D835Y and Ba/F3-FLT3-ITD+F691L. The resistant cells expressed 1.7 to ~5.6-fold higher E-selectin ligand and 10-fold higher CXCR4 levels. In addition, BM-mimetic hypoxia culture profoundly upregulated P-selectin glycoprotein ligand-1 (PSGL-1) and CXCR4 levels on leukemia cells. PSGL-1 is also a high-efficiency ligand for E-selectin. We then evaluated anti-leukemia effects of co-targeting E-selectin/CXCR4 and FLT3 with GMI-1359 and sorafenib in a patient-derived AML xenograft model (harboring FLT3-ITD and WT1 mutations). Leukemia cell engraftment was evaluated using FACS and immunohistochemistry by hCD45 staining. We observed that GMI-1359 efficiently mobilized leukemic cells into PB and in combination with sorafenib enhanced the anti-leukemia activity and significantly reduced leukemia cell infiltration by 92%, 82%, 69% and 45% in liver, lung, spleen and BM, respectively, as compared with vehicle-treated mice (p < 0.05). The GMI-1359/sorafenib combination improved mouse survival (median survival were 109, 87, 126 and 138.5 days for the vehicle, GMI-1359, sorafenib and combination groups, respectively). Importantly, we observed that the combination protected normal hematopoiesis by increasing the number of BM megakaryocytes, myelocytes, and erythrocytes. The underlying mechanism(s) for this effect is under investigation. Our findings suggest that co-targeting E-selectin/CXCR4/FLT3 exerts remarkable protection of normal hematopoiesis in addition to more efficiently reducing leukemia cells. Citation Format: Weiguo Zhang, Kyung Hee Chang, Mahesh Basyal, Yannan Jia, Lauren Ostermann, William E. Fogler, John L. Magnani, Michael Andreeff. Combined targeting of E-selectin/CXCR4 and FLT3 by GMI-1359 and sorafenib effectively reduces leukemia cell burden and protects normal hematopoiesis in a patient-derived AML xenograft model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6038.

Acute myeloid leukemia (AML) is a heterogeneous disease that develops within a complex microenvironment. Reciprocal interactions between the bone marrow mesenchymal stem/stromal cells (BM-MSCs) and AML cells can promote AML progression and resistance to chemotherapy (Jacamo et al., 2014). We have recently reported that BM-MSCs derived from AML patients (n=103) highly express p53 and p21 compared to their normal counterparts (n=73 p Disclosures Khoury: Angle: Research Funding; Stemline Therapeutics: Research Funding; Kiromic: Research Funding. Bueso-Ramos:Incyte: Consultancy. Andreeff:BiolineRx: 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; 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; NIH/NCI: Research Funding; CPRIT: Research Funding; 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; Aptose: Equity Ownership; Reata: Equity Ownership; 6 Dimensions Capital: Consultancy; AstaZeneca: Consultancy; Amgen: Consultancy; Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; Jazz Pharmaceuticals: Consultancy; Celgene: Consultancy. OffLabel Disclosure: Mdm2 inhibitor-DS 5272

Background: CD123 is frequently expressed on hematologic malignancies including 96-98% of AML. CD123 has been a potential immunotherapeutic target in AML due to its association with leukemic stem cells that play an essential role in disease progression and relapse. Our previous study using T-cells secreting CD123/CD3-bispecific T-cell engagers (BiTEs) (CD123-ENG T-cells) showed a promising approach anti-AML activity, however T-cell persistence was limited. Interleukin-15 (IL15) has emerged as a candidate immunomodulator as it enhances T-cell expansion and persistence, and induces long-lasting memory T-cells. To improve the efficacy and persistence of CD123-ENG T-cells we developed IL15 expressing CD123-ENG T-cells. Here, we report on the characterization and efficacy of IL15-secreting CD123-ENG T cells in vitro and in vivo models of adult AML. Methods/Results: A cDNA encoding IL15 was cloned into retroviral vectors encoding CD123-ENG or CD19-ENG (CD123-ENG.IL15; CD19-ENG.IL15). ENG T-cells were generated from human peripheral blood mononuclear cells (PBMCs) from normal donors or T-cells from AML patients by retroviral transduction and in vitro expansion. Non-transduced (NT) T-cells and T-cells expressing CD123 (CD123-ENG T-cells) served as controls. IL15 production of CD19-ENG.IL15 and CD123-ENG.IL15 T cells was confirmed by ELISA (144-159 pg/ml vs 38 and 46 pg/ml of NT and CD123-ENG T cells, p Conclusion: We here demonstrated that transgenic expression of IL15 in CD123-ENG T-cells results in improved expansion and persistence, and anti-AML activity. These results warrant further exploration of IL15-modified CD123-targeted T-cells as immunotherapy for AML. Disclosures Bonifant: Patents filed in the field of engineered cellular therapies: Patents & Royalties: Patents filed in the field of engineered cellular therapies. Gottschalk:EMD Serono: Honoraria; Inmatics: Membership on an entity's Board of Directors or advisory committees; ASSISI fundation of Memphis: Research Funding; TESSA Therapeutics: Other: Research Collaboration; ViraCyte: Consultancy; NHLBI: Research Funding; America Lebanese Syrian Associated Charities: Research Funding; California Institute for Regenerative Medicine: Research Funding; Patents and patent applications in the fields of T-cell & Gene therapy for cancer: Patents & Royalties; MBIO: Other: St. Jude Children's Research Hospital has an existing exclusive license and ongoing partnership with Mustang Bio for the further clinical development and commercialization of this XSCID gene therapy; Sanofi: Honoraria; Tidal: Membership on an entity's Board of Directors or advisory committees; Merck: Consultancy. Velasquez:St. Jude: Patents & Royalties: Patent Applications in the Fields of Cell and Gene Therapy ; Rally! Foundation: Membership on an entity's Board of Directors or advisory committees. Andreeff:Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; AstaZeneca: Consultancy; Amgen: Consultancy; Eutropics: Equity Ownership; Aptose: Equity Ownership; Reata: Equity Ownership; 6 Dimensions Capital: Consultancy; Celgene: Consultancy; Jazz Pharmaceuticals: Consultancy; Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; 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; NIH/NCI: Research Funding; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees; CPRIT: Research Funding; Oncoceutics: Equity Ownership; Oncolyze: Equity Ownership; Breast Cancer Research Foundation: Research Funding.