Venetoclax (VEN), in combination with low dose cytarabine (AraC) or a hypomethylating agent, is FDA approved to treat acute myeloid leukemia (AML) in patients who are over the age of 75 or cannot tolerate standard chemotherapy. Despite high response rates to these combination therapies, most patients succumb to the disease due to relapse and/or drug resistance, providing an unmet clinical need for novel therapies to improve AML patient survival. ME-344 is a potent isoflavone with demonstrated inhibitory activity toward oxidative phosphorylation (OXPHOS) and clinical activity in solid tumors. Given that OXPHOS inhibition enhances VEN antileukemic activity against AML, we hypothesized that ME-344 could enhance the anti-AML activity of VEN. Here we report that ME-344 synergized with VEN to target AML cell lines and primary patient samples while sparing normal hematopoietic cells. Cooperative suppression of OXPHOS was detected in a subset of AML cell lines and primary patient samples. Metabolomics analysis revealed a significant reduction of purine biosynthesis metabolites by ME-344. Further, lometrexol, an inhibitor of purine biosynthesis, synergistically enhanced VEN-induced apoptosis in AML cell lines. Interestingly, AML cells with acquired resistance to AraC showed significantly increased purine biosynthesis metabolites and sensitivities to ME-344. Furthermore, synergy between ME-344 and VEN was preserved in these AraC-resistant AML cells. These results translated into significantly prolonged survival upon combination of ME-344 and VEN in NSGS mice bearing parental or AraC-resistant MV4-11 leukemia. This study demonstrates that ME-344 enhances VEN antileukemic activity against preclinical models of AML by suppressing OXPHOS and/or purine biosynthesis.
Acute myeloid Leukemia (AML) is an aggressive hematologic malignancy with poor prognosis. Despite chromosomal and genetic heterogeneity, AML are uniformly characterized by increased reliance on oxidative phosphorylation (OXPHOS). This key metabolic hallmark of leukemia was recently reported as a feature of resistance to Cytarabine (AraC)-based therapy. Also, an aggressive phenotype and poor response to chemotherapy is associated with increased levels of Bcl-2. Despite the introduction of the Bcl-2 inhibitor venetoclax (VEN), the overall survival, particularly in older patients, remains poor. Thus, approaches to improve the sensitivity of leukemic cells to AraC-based or Bcl-2 based therapies are urgently needed.Here, we investigated the preclinical activity of ME-344, a novel isoflavone OXPHOS inhibitor, on AML cell lines and relapsed/refractory (R/R) patient samples in vitro and examined the efficacy of ME-344 in combination with VEN in Ara-C sensitive and resistant AML cell lines and patient-derived xenografts (PDX) both in vitro and in vivo.ME-344 (0-300 nM, 24 hr) significantly reduced viability of AML cell lines with EC50 of 75-100 nM and R/R AML patient samples with EC50 of 200-300 nM respectively. The cytotoxic response in AML was enhanced when ME-344 was combined with VEN, producing strong synergistic viability reduction and induction of apoptosis, as evidenced by Annexin V assay and an increased level of cleaved caspase 3 and PARP (immunoblotting). The dual inhibition of OXPHOS/Bcl-2 reduced Mcl-1 levels and showed efficacy in Mcl-1 overexpressingand Ara-C resistant AML models.Functional metabolic characterization of AML by transcriptomics and mass spectrometry demonstrated that ME-344 effectively inhibited biosynthetic pathways of nucleotides uncovering the purine biosynthesis pathway as crucial for therapeutic efficacy. ME-344 induced a dose-dependent decrease in the oxygen consumption rate (by Seahorse assay), in both AraC-sensitive and -resistant AML cell lines, and in R/R AML patient samples, which was further significantly potentiated by combination with VEN.Finally, in an aggressive AML xenograft model, ME-344 (200 mpk, i.v.) combined with subtherapeutic doses of VEN (25 mpk) reduced circulating leukemia burden and extended survival. Ongoing in vivo studies in AML PDX models will address the impact of ME-344 in the context of acquired AraC- and Bcl-2- resistance. In summary, our findings indicate ME-344 alone or in combination with Bcl-2 inhibition constitutes an important therapeutic modality that targets a unique metabolic vulnerability of AML. Citation Format: Katie H. Hurrish, Yongwei Su, Shraddha Patel, Sandra E. Wiley, Zhanjun Hou, Jenna Carter, Hasini Kalpage, Maik Hüttemann, Connie Weng, Holly Edwards, Lisa Polin, Jing Li, Jay Yang, Larry H. Matherly, Sergej Naumovich Konoplev, Jeffrey W. Taub, Marina Konopleva, Yubin Ge, Natalia Baran. ME-344, a novel isoflavone mitochondrial inhibitor, in combination with venetoclax constitutes a new metabolism-targeted approach to overcome resistance to Bcl-2 inhibition and standard of care treatment in AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3785.
The 5-year survival rate for adult patients with acute myeloid leukemia (AML) treated with cytarabine-based chemotherapy remains less than 30%, due to drug resistance and disease relapse. Recently, a selective inhibitor of anti-apoptotic Bcl-2, venetoclax, was approved by the FDA in combination with low dose cytarabine or hypomethylating agents for treating newly diagnosed AML patients who are 75 years of age or older or for those who are unfit for standard chemotherapy, providing more treatment options for this group of patients. Although the response rate to these newly approved combination therapies is reported to be 70%, the median overall survival is only 10-18 months showing that the duration of response is limited. Therefore, novel therapeutic agents are in demand to enhance venetoclax activity against AML and to combat AML resistant to cytarabine-based chemotherapy. Cytarabine-resistant AML cells lead to relapse and rely on oxidative phosphorylation (OXPHOS) for survival. In addition, it has been reported that targeting OXPHOS can enhance venetoclax activity against preclinical models of AML. Thus, we hypothesize that OXPHOS suppressing agents would be good candidates to combine with and enhance venetoclax antileukemic activity against newly diagnosed AML and those with resistance to cytarabine. A novel isoflavone, ME-344, has been shown to suppress OXPHOS in cell lines derived from solid tumors by inhibiting Complex I of the electron transport chain. We hypothesized that combining ME-344 with venetoclax would result in synergistic antileukemic activity against AML. Consistent with our hypothesis, combining ME-344 with venetoclax resulted in synergistic induction of apoptosis in AML cell lines, including those with acquired cytarabine resistance. The combination of these two agents also resulted in synergistic antileukemic activity in one primary AML patient sample, as determined by MTT assay. The combination of ME-344 and venetoclax prolonged the median survival of MV4-11 leukemia- bearing NSGS mice by 37% (median survival of 48 days compared to 35 days for vehicle control treated mice, n=5 per arm, p<0.0001). This is in contrast to the venetoclax combination with cytarabine, which prolonged median survival of the same xenograft model by 7.5% (Luedtke et al., Signal Transduction and Targeted Therapy, 2020; 5:17). ME-344 alone (9-hour treatment) reduced basal mitochondrial respiration in AML cells by 10% prior to induction of apoptosis. When treated with ME-344 for 8-hours followed by combined ME-344 and venetoclax for an additional 1-hour, basal mitochondrial respiration was reduced by 18% (again prior to detection of apoptosis initiation). This sequential combination regimen also decreased the mitochondrial membrane potential (by JC-1 staining and flow cytometry analysis) when compared to untreated control and single treatment. Additionally, apoptosis induction by the combination of ME-344 and venetoclax or ME-344 alone was significantly enhanced when AML cells were forced to utilize OXPHOS by replacing glucose with galactose in the culture medium. Further investigation revealed that apoptosis induced by ME-344 was partially attenuated when Mcl-1 was overexpressed, Bak was knocked down, or caspase activation was inhibited. This suggests a mechanism that involves components of the intrinsic apoptosis pathway. Targeted metabolomics analyses of MV4-11 cells treated with ME-344 for 8 h revealed a significant reduction of essential metabolites involved in the de novo purine biosynthesis pathway, specifically AICAR (p=0.001) and IMP (p=0.004). Given the critical role of purine in cell proliferation and survival, suppression of purine biosynthesis by ME-344 may represent a novel mechanism underlying its enhancement on the antileukemic activity of venetoclax against AML. Interestingly, inhibition of this pathway by the purine biosynthesis inhibitor lometrexol, also synergistically enhanced apoptosis in AML cells induced by venetoclax. Taken together, these results suggest that ME-344 suppresses OXPHOS and the purine biosynthesis pathway to enhance the antileukemic activity of venetoclax against AML. Further in-depth mechanistic studies into the suppression of purine biosynthesis and OXPHOS, as well as studies of ME-344 and venetoclax against cytarabine-resistant AML in a mouse model are warranted. Disclosures Wiley: MEIPharma: Current Employment.
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