Novel Genetic Mechanism of Venetoclax Resistance in AML: BAX Deletion

Venetoclax is a highly selective B-cell lymphoma 2 (BCL-2) inhibitor which recently received accelerated US FDA approval for use in combination with hypomethylating agents or with low-dose cytarabine in elderly or unfit acute myeloid leukemia (AML) patients. Venetoclax was also approved by the US FDA for the treatment of chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL).

Venetoclax-based AML treatment showed tolerable safety and favorable overall response rate in elderly patients with AML, particularly among high-risk groups, such as patients older than 75 yrs, adverse cytogenetics, and secondary AML. Acquisition of resistance to venetoclax in AML is the main cause of treatment failure in venetoclax-based AML treatment approaches. Intrinsic resistance in AML cells is attributed to increased levels of MCL-1 and/or BCL-XL, which lead to sequestration of pro-apoptotic proteins that are released from BCL-2 upon venetoclax treatment. Few reports of acquired resistance to venetoclax in other tumors describes acquisition of mutations in BCL-2 gene, for example: G101V in CLL and P104I in follicular lymphoma. With the increasing use of venetoclax in AML more reports of acquired and intrinsic resistance are expected, which in turn will shape future use of venetoclax.

In order to study the mechanisms of acquired venetoclax resistance, we created venetoclax resistant MV4-11 cells by treating MV4-11 cells with incremental doses of venetoclax starting from 1nM to 100nM for a period of 8 weeks. Colonies of resistant MV4-11 clones were then isolated after culturing the cells in methylcellulose based medium. Five clones of venetoclax resistant cells (MV4-11 ABT-199R 1 through 5) were studied in detail. The MV4-11 ABT-199R clones demonstrated 300-700-fold higher resistance to venetoclax than the parental MV4-11 cells. The MV4-11 ABT-199R clones also demonstrated co-resistance to ABT-737 (BCL-2 and BCL-XL inhibitor), S63845 (MCL-1 inhibitor), and S55746 (BCL-2 inhibitor). Protein analysis by western blot demonstrated no differences in MCL-1 levels between resistant clones and the parental MV4-11 cells. Flow cytometry analysis demonstrated no significant change in the expression of BCL-XL in the resistant clones as compared to parental cells. Real-time PCR and western blotting showed loss of BAX expression. We subsequently performed whole-genome high density Cytoscan arrays for parental MV4-11 and one resistant clone. This revealed a micro-deletion in the resistant clone that included the promoter region and the first 3 exons of the BAX gene. These deletions were further confirmed by performing genomic DNA PCR. This was then further validated in the other 4 venetoclax resistant MV4-11 clones, showing similar genomic deletions of the region upstream of BAX and the first three exons of BAX gene.

In summary, we are reporting a novel mechanism of venetoclax resistance by genomic deletion of BAX in an AML cell line. BAX is an essential gateway to mitochondrial apoptosis. As a result of BAX gene deletion, the inhibitors of BCL-2, BCL-XL, and MCL-1 are rendered ineffective in inducing apoptosis. Alternative mechanisms of apoptosis induction need to be explored to overcome BAX deletion-induced resistance.