Pitavastatin Potentiates Mitochondrial Dysfunction and Venetoclax Cytotoxicity in Acute Myeloid Leukemia Cells

Introduction

Venetoclax, a selective BCL2 inhibitor, is a standard-of-care agent for patients with acute myeloid leukemia (AML) in combination with hypomethylating agents (HMAs); however, resistance to venetoclax-based regimens is common in AML and most responders will ultimately relapse. TP53 loss or mutation and changes in mitochondrial dynamics are among the predominantly reported drivers of venetoclax resistance. Statins are a commonly prescribed class of mevalonate synthesis inhibitors targeting HMG-CoA reductase that we reported can sensitize AML cell lines to venetoclax. We have since identified pitavastatin as an optimal statin for oncology clinical trials and a phase 1 study (NCT04512105) showed that pitavastatin added to venetoclax regimens in AML and chronic lymphocytic leukemia (CLL) is well tolerated with preliminary signs of efficacy (Blood Neoplasia, in press). Inhibition of mevalonate production by statins promotes apoptosis in AML cells largely due to the depletion of cellular pools of geranylgeranylpyrophosphate (GGPP). The objective of this study was to understand the GGPP-dependent mechanisms leading to the pro-apoptotic effect of statins in AML cells to circumvent the mechanisms of resistance yielding clinical advantage.

Methods

Established statin-sensitive and resistant AML cell lines and AML patient myeloblasts were treated with pitavastatin (PIT) and assessed for gene expression (bulk RNA sequencing), protein expression, mitochondrial mass (TOMM20 staining by flow cytometry), basal oxygen consumption rate (OCR measured by Seahorse flux analyzer), mitochondrial priming (by BCL2-Homology 3 domain (BH3) profiling) and cell viability (flow cytometry). For experiments using established cell lines, assays performed on three independent cell cultures for each line were considered biological replicates. For patient-derived myeloblast cultures, the response of each individual specimen was reported as an independent data point.

Results

We observed potent cytotoxic activity of PIT in primary AML myeloblasts and sensitization to venetoclax in wildtype and isogenic TP53-mutant AML cell lines via geranylgeranyl pyrophosphate (GGPP) dependent upregulation of PUMA, a BH3-only apoptotic sensitizer that binds to all BCL2 family members. Addition of GGPP significantly reversed the cytotoxic effect of PIT alone or with venetoclax in AML cell lines. GGPP supplementation was sufficient to prevent PUMA upregulation. BH3 profiling assay showed that PIT treatment increased mitochondrial priming in AML cell lines. Transcriptome analysis of PIT treated AML samples revealed upregulation of apoptotic gene signatures including BBC3 (PUMA), downregulation of pyrimidine and purine metabolism, regulation of select transcription factor (Myc, FOXO, ERR) target genes, and broad changes in mitochondrial gene expression. Nearly half (42%) of the differentially expressed genes in the PIT vs vehicle comparison were dependent on GGPP depletion. These included many of the FOXO target genes including BBC3, TSC22D3, and BMF. Among gene ontology (GO) terms, apoptosis was enriched and nuclear-encoded mitochondrial genes were downregulated. Consistent with a reduction in mitochondrial gene expression, OCI-AML3 cells treated with PIT had reduced mitochondrial mass and OCR. These changes were not observed in MOLM13 cells that are resistant to PIT alone, including no significant changes in mitochondrial gene expression. Interestingly, the combination of PIT and venetoclax caused the greatest decrease in OCR. In line with this finding, the venetoclax and PIT combination also led to depolarized mitochondria and ROS accumulation in AML cell lines.

Conclusion

Mevalonate pathway inhibition by PIT can overcome resistance to venetoclax in AML cell lines and primary cells. The mechanism is p53-independent and is associated with downregulation of nuclear-encoded mitochondrional gene expression, a likely advantage for overcoming resistance to venetoclax. Notably, PIT sensitizes AML cells to venetoclax by upregulating PUMA via a p53-independent mechanism and by reducing mitochondrial fitness. These results demonstrate that targeting the mevalonate pathway with PIT in combination with venetoclax might be effective to overcome resistance, increase survival and improve clinical outcome.

No relevant conflicts of interest to declare.