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Acute myeloid leukemia (AML) is a hematopoietic malignancy characterized by the accumulation of malignant myeloid cells that have arrested maturation. Most therapeutic regimens approved or under development are cytotoxics. An alternate, but less explored therapeutic approach, is to induce terminal differentiation of AML cells. Upon differentiation, AML cells cease to proliferate or die. Phosphatidylserine decarboxylase (PISD) is a mitochondrial enzyme that converts phosphatidylserine (PS) to phosphatidylethanolamine (PE). Here, we explored the effects of inhibiting PISD on AML growth, stemness and differentiation. Knockout of PISD by CRISPR reduced the growth and clonogenic growth of OCI-AML2 cells. The reported chemical PISD inhibitor, 7-chloro-N-(4-ethoxyphenyl)-4-quinolinamine (aka: MMV007285), reduced growth and viability of OCI-AML2 cells (IC50 = 4.741 μM) and TEX cells (IC50 = 4.868 μM). Using the 8227 primary AML cell culture model, we showed that inhibiting PISD induced cell death in the functionally defined stem cell fraction (CD34+CD38-). MMV007285 also preferentially inhibited the clonogenic growth of primary AML cells (n = 7) over normal hematopoietic cells (n= 3). Moreover, MMV007285 induced AML cell differentiation as evidenced by increased CD11b expression and staining for non-specific esterase. Using high-performance thin layer chromatography (HPTLC), we found that inhibition of PISD with MMV007285 increased intracellular PS. To determine whether increased PS was functionally important, OCI-AML2 cells were treated with PS, resulting in reduced growth and clonogenic growth. Furthermore, PS supplementation targeted AML progenitor cells as it decreased engraftment of TEX cells in mice. Mechanistically, inhibiting PISD induced differentiation and decreased stemness in AML by activating Toll-like receptor (TLR) signaling. Specifically, inhibiting PISD upregulated TLR4 and 8 expression and increased expression of cytokines downstream of TLR activation. We also showed that TLR activation was functionally important to induce AML differentiation. Finally, we evaluated the effects of PISD inhibition in AML mouse models. MMV007285 (300 mg/kg/5 of 7 days orally for 10 days) decreased the growth of OCI-AML2 cells in SCID mice. Moreover, MMV007285 (150 mg/kg/5 of 7 days orally for 5 weeks) impeded the leukemic engraftment of primary AML cell in NOD/SCID mice without toxicity. Using secondary transplants, we showed that MMV007285 also targeted the leukemic stem cells. Taken together, inhibition of PISD altered phospholipid metabolism, inhibited growth and stemness, and increased differentiation in AML cells. Our findings reveal a previously undescribed link between mitochondrial phospholipid metabolism and AML stemness and differentiation, highlighting a potential new therapeutic strategy for AML. Citation Format: Mingjing Xu, Ayesh Seneviratne, Val A. Fajardo, Geethu E. Thomas, G. Wei Xu, Rose Hurren, S. Kim, Neil MacLean, Xiaoming Wang, Marcela Gronda, Danny Jeyaraju, Yulia Jitkova, David Sharon, Ahmed Aman, Rima Al-awar, Steven Chan, Mark D. Minden, Paul LeBlanc, Aaron D. Schimmer. Inhibiting the mitochondrial enzyme phosphatidylserine decarboxylase (PISD) reduces stemness and increases differentiation in acute myeloid leukemia (AML) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3003.

Abstract 61 To identify new therapeutic strategies for AML, we compiled and screened an in-house library of on-patent and off-patent drugs to identify agents cytotoxic to leukemia cells. From this screen, we identified mefloquine, an off-patent drug indicated for the treatment and prophylaxis of malaria. In secondary assays, mefloquine decreased the viability of 9/10 human and murine leukemia cell lines (EC50 3.25–8.0 μM). Moreover, it reduced the viability of 4/5 primary AML samples, but was not cytotoxic to normal hematopoietic cells (EC50>31 μM). Importantly, mefloquine reduced the clonogenic growth of primary AML samples, but not normal hematopoietic cells, and completely inhibited engraftment of primary AML cells into immune deficient mice. Finally, systemic treatment with oral mefloquine (50 mg/kg/day) decreased leukemic burden without evidence of toxicity in 4 mouse models of leukemia, including mice engrafted with primary AML cells. Thus, mefloquine effectively targets leukemic cells, including leukemia stem cells, at concentrations that appear pharmacologically achievable and are not toxic to normal hematopoietic cells. To identify the mechanisms of mefloquine-mediated cell death in AML cells, we performed a binary drug combination screen, hypothesizing that drugs that synergized with mefloquine may share overlapping mechanism of action. From this combination screen of 550 drugs, we identified 18 that reproducibly synergized with mefloquine as measured by the Excess over Bliss additivism score, including 3 members of the artemisinin class of anti-malarials: artemisinin, artesunate and artenimol. Strikingly, 10/18 synergistic compounds, including the artemisinins, were known generators of reactive oxygen species (ROS). Therefore we tested mefloquine's ability to increase ROS in leukemic cells. Mefloquine increased ROS production in leukemia cells in a dose- and time-dependent manner. Co-treatment with ROS scavengers α-tocopherol and N-acetyl-cysteine abrogated mefloquine-induced ROS production and cell death, indicating that ROS production was functionally important for mefloquine-mediated cell death. Moreover, the artemisinins induced ROS as single agents, and synergistically increased ROS when combined with mefloquine. To identify cellular target(s) of mefloquine's anti-leukemic effects, we performed a yeast genome-wide functional screen to identify heterozygous gene deletions that rendered yeast more sensitive to mefloquine. 21/37 genes whose depletion conferred >4-fold sensitivity to mefloquine were associated with function of the yeast vacuole, equivalent to the mammalian lysosome. Consistent with these data, fluorescent confocal microscopy demonstrated that mefloquine and artesunate disrupted lysosomes. Cell death after mefloquine and artesunate treatment was caspase-independent and associated with increased incorporation of monodancylcadaverin in autophagosomes, consistent with the effect of these drugs on the lysosomes. To further explore the anti-leukemic activity of lysosomal disruption, we evaluated the anti-leukemic effects of the known lysosomal disrupter L-leucine-leucine methyl ether (LeuLeuOMe). Similar to mefloquine and artesunate, LeuLeuOMe induced cell death in leukemia cells, increased ROS production, and disrupted the lysosomes. Highlighting the potential clinical utility of lysosomal disrupters for the treatment of leukemia, a patient with relapsed/refractory juvenile myelomonocytic leukemia self-administered artemisinin. The artemisinin cleared the circulating blasts from the circulating blasts and the patient proceeded to allotransplant. Finally, to investigate the basis of leukemic cell hypersensitivity to lysosomal disruption, we assessed lysosomal characteristics of primary AML and normal hematopoietic cells. By gene expression analysis, AML patient samples had higher mRNA levels of the lysosomal cathepsins A, B, C, D, H, L, S and Z, compared to CD34+ normal hematopoietic cells, and cathepsins C, D and Z were significantly over-expressed in the LSC compartment, compared to normal HSCs. In summary, our data demonstrate that lysosomal disruption preferentially targets AML cells and AML stem cells through a mechanism related to increased ROS production. Thus, this work highlights lysosomal disruption as a novel therapeutic strategy for AML. Disclosures: Off Label Use: This study includes a case report of off-label use of the anti-malarial artemisinin in the treatment of a case of juvenile myelomonocytic leukemia.

Abstract 290 Known drugs with previously unrecognized anti-cancer activity can be rapidly repurposed for this new indication, given their prior safety and toxicity testing. To identify such compounds, we compiled and screened an in-house library of on-patent and off-patent drugs and screened them to identify agents cytotoxic to hematologic malignancies. From this screen, we identified mefloquine, a quinoline licensed for malaria treatment and prophylaxis. In secondary assays, leukemia and myeloma cell lines were treated with mefloquine for 72 hours and cell viability measured by MTS. Mefloquine decreased the viability of 10/10 human and murine leukemia (LD50 10-fold up-regulation of its downstream targets, including OAS1, IFIT3 and TRIM22, by 24 hr after treatment. Upregulation of additional TLR targets IRF1, IRF7 and IL-8 was also noted by 8 hours after treatment. Mefloquine also induced early activation of NF-κB with a 2.5± 0.2-fold increase noted after 1 hr, using an ELISA-based DNA binding assay. In contrast to TLR activation in malignant cells, changes in TLR targets were not detected in mefloquine-resistant normal dendritic cells, suggesting that mefloquine's effects on TLR signaling were specific to malignant cells. We next investigated whether TLR activation was functionally important for mefloquine's cytotoxicity in malignant cells. STAT1 activity was required for mefloquine-mediated cell death, as U4A bladder sarcoma cells lacking JAK1 were resistant to mefloquine (LD50 14.6± 4.9 μM), compared to the mefloquine sensitive parental line (LD50 2.3± 0.4 μM). TLR signaling requires the immediate downstream adapter proteins MyD88 and TRIF1. To assess the functional importance of TLR activation for mefloquine induced cell death, we knocked down MyD88 and TRIF1 with siRNA. Double knockdown of MyD88 and TRIF1 completely abrogated mefloquine-induced cell death in K562 leukemia cells at concentrations where control cells exhibited up to 80% loss of viability. TLR signaling and up-regulation of STAT1 can increase reactive oxygen species (ROS) generation. Therefore, we measured ROS generation in leukemia cells after mefloquine treatment. Mefloquine increased ROS production in leukemia cells in a dose-dependent manner within 24 hr. Co-treatment with the ROS scavenger N-Acetyl-L-Cysteine abrogated mefloquine-induced ROS production and cell death. Mefloquine-induced ROS production was also abrogated in MyD88 and TRIF1 double knockdown cells. Our data suggest that the known anti-malarial mefloquine displays preclinical activity in leukemia and myeloma through a mechanism related to TLR activation. Thus, these results highlight TLR activation as a novel therapeutic strategy for the treatment of leukemia and myeloma. Moreover, given its prior toxicology and pharmacology testing, mefloquine could be rapidly advanced into clinical trial for patients with leukemia and myeloma. Disclosures: No relevant conflicts of interest to declare.