Synergistic Activity of Adefovir Dipivoxil and Venetoclax to Target the Bioenergetic Metabolism of Acute Myeloid Leukemia

Abstract

Adefovir dipivoxil (ADV) is an oral prodrug of the nucleotide analogue adefovir that inhibits viral DNA polymerase and is an FDA-approved drug for the treatment of hepatitis B virus. Preliminary evidence of the antileukemic activity of ADV was recently reported in a patient with acute promyelocytic leukemia (APL; Gong, X., et al. Exp Hematol Oncol, 2022). Herein we report on the antileukemic activity of ADV, alone and in combination with venetoclax (VEN), an oral FDA-approved BCL-2 inhibitor for the treatment of acute myeloid leukemia (AML). Firstly, a significant dose-dependent reduction of cell growth and increase in apoptosis were observed in FLT3-ITD+ MV4-11 (IC50: 97nΜ) and Molm-13 (IC50: 0.816μM) cells, and in primary CD34+ blasts from refractory/relapsed (R/R) AML patients (n=5; IC50: 1.285-3.703μM), exposed to serial concentrations of ADV for 48 hours (hrs). In contrast, no significant changes were observed in ADV-treated CD34+ cells from healthy donors (n=5). Combination of ADV and VEN were synergistic (combination index < 1.0) in reducing cell viability of AML cells (i.e., MV-4-11, U937, KG1 and THP-1) and CD34+ blasts from R/R AML patients (n=3). To gain insights into the molecular mechanisms underlying the antileukemic activity of ADV alone and in combination with VEN, we performed RNA-Seq on AML CD34+ blasts from R/R AML patients (n=6) treated with vehicle, ADV (5 μΜ), VEN (100 nΜ), or ADV+VEN for 24 hrs. Gene set enrichment analysis (GSEA) highlighted upregulation of p53 pathway and apoptosis signaling, and downregulation of oxidative phosphorylation (OXPHOS), glycolysis, and MYC target hallmark gene sets in ADV- and ADV+VEN-treated cells compared with vehicle-treated controls. These results were confirmed by western blot analysis showing a dose-dependent reduction in c-MYC, anti-apoptotic proteins (i.e., BCL-2 and MCL-1), and carnitine palmitoyltransferase-1a [CPT1A, a key enzyme for fatty acid oxidation (FAO), a process that feeds into OXPHOS], and an increase in pro-apoptotic proteins (i.e., BAX and BAK) in ADV- and ADV+VEN-treated MV4-11 and primary AML CD34+ cells, compared with vehicle-treated control cells. Seahorse analysis confirmed a significant reduction of OXPHOS [measured by oxygen consumption rate (OCR)], associated with decreased mitochondrial numbers and sizes evaluated by electron microscopy imaging of AML CD34+ blasts treated with ADV or ADV+VEN compared with vehicle-treated controls. To test in vivo the antileukemic activity of ADV+VEN, we treated a cohort of CD45.1 recipient mice transplanted with BM cells from congenic CD45.2 Mll PTD/WT/Flt3 ITD/ITD AML mice with vehicle, ADV (100mg/kg, oral gavage), VEN (100mg/kg, oral gavage), or ADV+VEN (n=10 per group) for 4 weeks. ADV+VEN-treated mice lived longer (median survival: 60 days after treatment completion) than ADV (20 days, p=0.0001), VEN (20 days, p=0.0001), or vehicle (14 days, p=0.0002) -treated controls (Figure 1). We then performed secondary transplants by engrafting 10 6 BM cells from treated donors into CD45.1 recipient mice (n=10 per group). Recipients of BM from ADV+VEN-treated donors survived longer (median survival: 83 days) than recipients of BM from ADV (60.5 days, p<0.01), VEN (45.5 days, p<0.0001), and vehicle (42.5 days, p<0.0001) -treated donors (Figure 2), confirming a decrease in leukemic stem cell (LSC) burden by ADV+VEN. Next, we examined the antileukemic activity of ADV+VEN in human AML models. We first treated a cohort of NSG mice (n=10 per group) transplanted with luciferase-expressing Molm-13 cells. ADV+VEN-treated mice survived longer (median survival: 59.5 days) than ADV (52 days, p=0.04), VEN (40 days, p<0.0001), and vehicle (40.5 days, p<0.0001) -treated mice. We then treated a cohort of NSG mice (n=8 per group) transplanted with FLT3-ITD+ blasts from a patient with R/R AML. ADV+VEN-treated mice survived longer (median: 38.5 days) than ADV (34.5 days, p=0.035), VEN (25 days, p<0.0001), and vehicle (18.5 days, p<0.0001) -treated mice. In summary, ADV has in vivo anti-leukemic activity in AML likely by interfering with the bioenergetic metabolism of leukemic cells and decreasing their apoptotic threshold. The combination of ADV and VEN is seemingly synergistic and prolonged survival of murine and human AML models, providing a rationale of translating this “all oral” regimen to the clinic for treatment of AML patients, especially those requiring low intensity therapy.