Abstract
BackgroundBCL‐2 inhibition through venetoclax (VEN) targets acute myeloid leukemia (AML) blast cells and leukemic stem cells (LSCs). Although VEN-containing regimens yield 60–70% clinical response rates, the vast majority of patients inevitably suffer disease relapse, likely because of the persistence of drug-resistant LSCs. We previously reported preclinical activity of the ribonucleoside analog 8-chloro-adenosine (8-Cl-Ado) against AML blast cells and LSCs. Moreover, our ongoing phase I clinical trial of 8-Cl-Ado in patients with refractory/relapsed AML demonstrates encouraging clinical benefit. Of note, LSCs uniquely depend on amino acid-driven and/or fatty acid oxidation (FAO)-driven oxidative phosphorylation (OXPHOS) for survival. VEN inhibits OXPHOS in LSCs, which eventually may escape the antileukemic activity of this drug. FAO is activated in LSCs isolated from patients with relapsed AML.MethodsUsing AML cell lines and LSC-enriched blast cells from pre-treatment AML patients, we evaluated the effects of 8-Cl-Ado, VEN and the 8-Cl-Ado/VEN combination on fatty acid metabolism, glycolysis and OXPHOS using liquid scintillation counting, a Seahorse XF Analyzer and gene set enrichment analysis (GSEA). Western blotting was used to validate results from GSEA. HPLC was used to measure intracellular accumulation of 8-Cl-ATP, the cytotoxic metabolite of 8-Cl-Ado. To quantify drug synergy, we created combination index plots using CompuSyn software. The log-rank Kaplan–Meier survival test was used to compare the survival distributions of the different treatment groups in a xenograft mouse model of AML.ResultsWe here report that VEN and 8-Cl-Ado synergistically inhibited in vitro growth of AML cells. Furthermore, immunodeficient mice engrafted with MV4-11-Luc AML cells and treated with the combination of VEN plus 8-Cl-Ado had a significantly longer survival than mice treated with either drugs alone (p ≤ 0.006). We show here that 8-Cl-Ado in the LSC-enriched population suppressed FAO by downregulating gene expression of proteins involved in this pathway and significantly inhibited the oxygen consumption rate (OCR), an indicator of OXPHOS. By combining 8-Cl-Ado with VEN, we observed complete inhibition of OCR, suggesting this drug combination cooperates in targeting OXPHOS and the metabolic homeostasis of AML cells.ConclusionTaken together, the results suggest that 8-Cl-Ado enhances the antileukemic activity of VEN and that this combination represents a promising therapeutic regimen for treatment of AML.
Highlights
BCL‐2 inhibition through venetoclax (VEN) targets acute myeloid leukemia (AML) blast cells and leukemic stem cells (LSCs)
Venetoclax and 8‐chloro‐adenosine synergize in the inhibition of oxidative phosphorylation in AML Having demonstrated an 8-Cl-Ado-mediated inhibition of fatty acid oxidation (FAO) and OXPHOS, we examined whether addition of 8-Cl-Ado to VEN would augment the antileukemic activity in LSC-enriched AML blasts
When we performed Gene set enrichment analysis (GSEA) on MV4-11 AML cells treated with VEN/8-Cl-Ado, we found that VEN treatment alone significantly increased expression of the fatty acid metabolism gene set at 12 h (Fig. 3a), whereas the VEN/8-Cl-Ado combination decreased the expression of this gene set, as indicated by the negative enrichment scores ES and NES (Fig. 3b)
Summary
BCL‐2 inhibition through venetoclax (VEN) targets acute myeloid leukemia (AML) blast cells and leukemic stem cells (LSCs). Venetoclax (VEN) is a selective BCL-2 inhibitor that has been shown to be highly effective in combination with low dose cytarabine (LDAC) or hypomethylating agents (HMA; i.e., azacytidine or decitabine) in inducing disease remission, with an overall response rate of approximately 60–70% in older and unfit AML patients [5,6,7]. Despite these encouraging results, the majority of AML patients treated with VEN-based regimens eventually relapse. These treatment failures are often attributed to the persistence of leukemia stem cells (LSCs), a subpopulation of primitive AML cells with self-renewal and leukemia-initiating capacities, which have been shown to be highly refractory to conventional antileukemic treatments [8]
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