ABCC1 and glutathione metabolism limit the efficacy of BCL-2 inhibitors in acute myeloid leukemia

The BCL-2 inhibitor Venetoclax is a promising agent for the treatment of acute myeloid leukemia (AML). However, many patients are refractory to Venetoclax, and resistance develops quickly. ATP-binding cassette (ABC) transporters mediate chemotherapy resistance but their role in modulating the activity of targeted small-molecule inhibitors is unclear. Using CRISPR/Cas9 screening, we find that loss of ABCC1 strongly increases the sensitivity of AML cells to Venetoclax. Genetic and pharmacologic ABCC1 inactivation potentiates the anti-leukemic effects of BCL-2 inhibitors and efficiently re-sensitizes Venetoclax-resistant leukemia cells. Conversely, ABCC1 overexpression induces resistance to BCL-2 inhibitors by reducing intracellular drug levels, and high ABCC1 levels predicts poor response to Venetoclax therapy in patients. Consistent with ABCC1-specific export of glutathionylated substrates, inhibition of glutathione metabolism increases the potency of BCL-2 inhibitors. These results identify ABCC1 and glutathione metabolism as mechanisms limiting efficacy of BCL-2 inhibitors, which may pave the way to development of more effective therapies.


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Replication Randomization performed using TraceFinder Version 5.0 (Thermo Fisher Scientific) The Prism 6.0.1 software (Graphpad, USA) and Microsoft Excel 2016 were used for data compilation and for statistical analyses. BLISS score was calculated using the SynergyFinder online tool, Version 3.
All data generated in this study are provided in the Source Data file. Publicly available datasets used are not in the Source Data file, but is listed. Gene expression data for ABCC1 were extracted from the Haferlach Leukemia, Valk Leukemia or TCGA Leukemia datasets (reporter: 202804_at) using the Oncomine™ Research Premium Edition database (Thermo Fisher, USA) (88), accessed in July, 2021. Gene expression analysis of ABC transporters in AML patients in the BeatAML dataset (47) was accessed in January, 2021 from the NCI Genomic Data Commons: https://gdc.cancer.gov/about-data/publications/BEATAML1-0-COHORT-2018. The Ordino database was used to extract gene expression data in human AML cell lines (KG-1, THP-1, PL-21, MV4-11, HL-60, MOLM-13). The Ordino database contains data from: The Cancer Genome Atlas (TCGA), the Cancer Cell Line Encyclopedia (CCLE), two depletion screen data sets (89,90); data extracted from August, 2021.
We did not distinguish between sex in our analysis, but we made sure to include both female and male patients, based on availability.
not applicable Primary AML samples were obtained by bone marrow puncture or venipuncture during routine investigations at the time of diagnosis. Cells were stored in a local biobank until used. All patients gave written informed consent before bone marrow or blood was obtained.
Samples of AML patients prior to Venetoclax treatment were selected to evaluate if ABC transporter expression is predictive of treatment response. Venetoclax treatment response was evaluated by the medical doctors based on the patient's remission.
Primary AML samples were obtained by bone marrow puncture or venipuncture during routine investigations at the time of diagnosis. Cells were stored in a local biobank until used. All patients gave written informed consent before bone marrow or blood was obtained. The study was approved by the ethics committee of the Medical University of Vienna (EK-No: 1355/2021).
An appropriate sample size was chosen based on the magnitude and consistency of measurable differences between groups. All sample sizes are indicated in the figure legends. Sample was choosen based on availability and practical reasons and costs.
Non-responders were excluded from the analysis in Figure 6C and Suppl. Figure 4C.
Numbers of biological and/or technical replicates are indicated in the figure legends.
Age-matched mice (16-20 weeks old) were randomly distributed into two groups of equal size and transplanted with equal cell numbers of either MOLM-13 Cas9 AAVS1.1 or ABCC1 KO cells. Both cohorts were treated with 10 mg/kg AZD-4320 or vehicle.
Cell culture experiments were not randomized as the experimental groups needed to be known for subsequent analysis.

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Investigators were not blinded to the group allocation of mice since the experimental setup defined an equal endpoint for both groups and the kowledge of the genotype of transplanted cells needed to be known for subsequent analysis.
Anti MRP1  Cell lines were fingerprinted using STR analysis.
All cell lines were routinely tested for mycoplasma contamination and confirmed negative.
No commonly misidentified cell lines were used.
Male and female mice were distributed in the different groups. We didn't distinguish between the sex in our study because according to the literature we didn't expect any difference and also could not observe any differenceb in leukemia onset or treatment response.
No field collected samples were used in the study.