Venetoclax Resistance in B-Cell Precursor Acute Lymphoblastic Leukemia Is Characterized By Increased Mitochondrial Metabolism and Can be Overcome By Co-Targeting Oxidative Phosphorylation

Abstract

Deregulated cell death pathways are a hallmark of many cancers and contribute to leukemogenesis and treatment failure in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Apoptosis is controlled by different pro- and anti-apoptotic molecules either inducing or counteracting cell death induction. Inhibition of anti-apoptotic molecules like B-cell lymphoma 2 (BCL-2) has been developed as therapeutic strategy. Venetoclax (VEN) binds selectively to anti-apoptotic BCL-2 resulting in apoptosis induction and has shown clinical activity in lymphoid malignancies, but insensitivity and resistance have been described. In BCP-ALL, we and others have shown preclinical activity of VEN and VEN is evaluated in first clinical trials. In this study, we have addressed and modeled VEN resistance in BCP-ALL and investigated underlying mechanisms in cell lines and patient-derived xenograft (PDX) samples in order to identify potential strategies to overcome VEN insensitivity. Starting from the BCP-ALL cell line RS4;11, five VEN insensitive lines were generated in parallel by exposure to increasing concentrations of VEN over time (49 passages, 8 months of continuous treatment) and five control lines were exposed to corresponding concentrations of solvent (DMSO). Measuring half maximal effective concentrations (EC50) showed increasing EC50 values from 4 nM to 26.2 µM over time in all VEN treated lines, indicating acquired resistance in our model. VEN insensitive leukemia lines displayed unchanged proliferation and same sensitivities to the prototypic apoptosis inducer staurosporine, the induction therapy drugs vincristine/dexamethasone/asparaginase or the anthracycline daunorubicin as compared to controls, indicating acquisition of VEN specific resistance. In order to further characterize the mechanism of VEN insensitivity, we performed RNA-Seq analysis comparing five RS4;11 VEN insensitive to five control lines. Interestingly, gene set enrichment analysis on significantly regulated genes showed a significant upregulation of gene sets annotated to the citric/tricarboxylic acid cycle and the respiratory electron transport chain, pointing to increased mitochondrial metabolism in VEN resistant cells. We addressed mitochondrial activity and carried out metabolic profiling (Agilent Seahorse XF Cell Mito Stress Test) of VEN resistant and control lines (N=5+5). Most interestingly, in contrast to significant reduction of oxygen consumption rates (OCR) in VEN treated control lines, we found sustained high mitochondrial metabolism indicated by persistent high OCR in VEN resistant lines. To further validate our findings, we investigated a series of BCP-ALL PDX samples (N=30) and titrated VEN EC50 values identifying heterogenous VEN activities. Metabolic profiling (Mito Stress test) was performed on sensitive as compared to highly resistant leukemias (N=3+3). Most interestingly and in line with our findings in cells with acquired resistance, PDX samples with intrinsic VEN insensitivity showed higher oxygen consumption and ATP production rates, thus further highlighting that increased mitochondrial activity is a characteristic feature of VEN resistant ALL. Along this line, significant higher mitochondrial DNA content was found in highly insensitive PDX leukemias when compared to VEN sensitive samples. Moreover, we analyzed mitochondrial morphology by electron microscopy. Although numbers of mitochondria did not differ between VEN resistant and sensitive leukemias, the overall mitochondrial area and structure was found to be significantly larger and elongated, further corroborating our finding of augmented mitochondrial metabolism in VEN resistant ALL. Finally, we addressed whether VEN resistance in ALL can be overcome by directly targeting oxidative phosphorylation (OxPhos). Using oligomycin, an inhibitor of the complex V/ATPase subunit, we found synergistic induction of apoptosis together with VEN in BCP-ALL cell lines and PDX samples, showing that acquired and intrinsic VEN insensitivity can be overcome by co-targeting BCL-2 and the OxPhos pathway. Taken together, we show that VEN resistance in BCP-ALL is characterized by high mitochondrial metabolic activity. Co-targeting of BCL-2 and oxidative phosphorylation results in synergistic anti-leukemia activity setting the basis for further preclinical and potential clinical evaluation.