Venetoclax resistance in acute lymphoblastic leukemia is characterized by increased mitochondrial activity and can be overcome by co-targeting oxidative phosphorylation

Deregulated apoptosis signaling is characteristic for many cancers and contributes to leukemogenesis and treatment failure in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Apoptosis is controlled by different pro- and anti-apoptotic molecules. Inhibition of anti-apoptotic molecules like B-cell lymphoma 2 (BCL-2) has been developed as therapeutic strategy. Venetoclax (VEN), a selective BCL-2 inhibitor has shown clinical activity in different lymphoid malignancies and is currently evaluated in first clinical trials in BCP-ALL. However, insensitivity to VEN has been described constituting a major clinical concern. Here, we addressed and modeled VEN-resistance in BCP-ALL, investigated the underlying mechanisms in cell lines and patient-derived xenograft (PDX) samples and identified potential strategies to overcome VEN-insensitivity. Leukemia lines with VEN-specific resistance were generated in vitro and further characterized using RNA-seq analysis. Interestingly, gene sets annotated to the citric/tricarboxylic acid cycle and the respiratory electron transport chain were significantly enriched and upregulated, indicating increased mitochondrial metabolism in VEN-resistant ALL. Metabolic profiling showed sustained high mitochondrial metabolism in VEN-resistant lines as compared to control lines. Accordingly, primary PDX-ALL samples with intrinsic VEN-insensitivity showed higher oxygen consumption and ATP production rates, further highlighting that increased mitochondrial activity is a characteristic feature of VEN-resistant ALL. VEN-resistant PDX-ALL showed significant higher mitochondrial DNA content and differed in mitochondria morphology with significantly larger and elongated structures, further corroborating our finding of augmented mitochondrial metabolism upon VEN-resistance. Using Oligomycin, an inhibitor of the complex V/ATPase subunit, we found synergistic activity and apoptosis induction in VEN-resistant BCP-ALL cell lines and PDX samples, demonstrating that acquired and intrinsic VEN-insensitivity can be overcome by co-targeting BCL-2 and the OxPhos pathway. These findings of reprogrammed, high mitochondrial metabolism in VEN-resistance and synergistic activity upon co-targeting BCL-2 and oxidative phosphorylation strongly suggest further preclinical and potential clinical evaluation in VEN-resistant BCP-ALL.

increase of concentrations, half maximal effective concentrations (EC50) were determined (forward/side scatter criteria, Attune NxT Flow Cytometer).VEN exposed lines showed increasing EC50 values from 15.67 to 35.2 µM over time, indicating acquired resistance development in these cells.

BCP-ALL Patient-Derived Xenograft Samples
Primary leukemia samples of BCP-ALL patients were collected after written informed consent in accordance with the institution's ethical review board.Patient-derived xenograft samples were generated by intravenous transplantation of ALL cells into female NOD/SCID mice (NOD.CB17-Prkdcscid, Charles River Laboratories, Wilmington, Massachusetts, USA) as previously described 1 .Immunophenotyping of PDX samples were done according to standard protocols using an LSR-II flow cytometer (BD Biosciences).Genetic alterations were assessed by Multiplex Ligation-dependent Probe Amplification (MLPA) and RT-PCR as previously described Cytometer, Thermo Fisher Scientific).

Immunoprecipitation and Immunoblotting
Proteins were isolated using a lysis buffer (30 mM Tris-HCl pH 7.

CellTiterGlo Staining
Cells were incubated with indicated drugs for 24 h in a total volume of 50 µl per well in an opaque 384 well plate.After incubation, treated cells were equilibrated to room temperature and 50 µl of CellTiterGlo® reagent was added to each well.Contents were mixed on an orbital shaker for 2min and incubated for 10min at room temperature.Luminescence was recorded with a Tecan plate reader and signals were normalized to control wells.
2. PDX cells were cultured in RPMI (Gibco Life Technologies, Carlsbad, (kindly provided by the Pharmacy, Ulm University Medical Center) or indicated combinations and cell death was analyzed either according to forward/side scatter criteria (cell lines) or propidium iodide positivity (PDX samples).Cell death rates and half maximal effective concentration values (EC50) were analyzed upon exposure to inhibitors for 72 hours in cell lines and for 24 hours in PDX samples (Attune NxT Flow Maryland, USA).Expression values of fusion and fission factors were normalized to B2M and log-transformed and 2 (-ΔΔ)CT values are shown.Statistical analyses were performed with GraphPad Prism software (version 9).Data obtained from replicate analyses with numbers of biological and/or technical replicates as indicated in the corresponding figure legends were analyzed by twosided T-test assuming equal variances, Spearman correlation or Chi-square test as indicated.Combination effects and synergy scores were analyzed (Synergyfinder, https://synergyfinder.fimm.fi/synergy/20240318114830462623/)using the Bliss independence model.