A Novel Isoflavone, ME-344, Enhances Venetoclax Antileukemic Activity Against AML Via Suppression of Oxidative Phosphorylation and Purine Biosynthesis
Authors: Hege Hurrish Katie , Su Yongwei , Wiley Sandra , Hou Zhanjun , Carter Jenna , Kalpage Hasini , Hüttemann Maik , Edwards Holly , Polin Lisa , Li Jing , Yang Jay , Matherly Larry , Taub Jeffrey W. , Ge Yubin 
Published: November 2021

Abstract
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&lt;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.