Venetoclax Resistance Results in Broad Resistance to Majority of Anti-MM Agents Due to the Suppression of Apoptosis but Can be Overcome By BCMA-Targeted Immunotherapy

Venetoclax represents the first example of personalized medicine in multiple myeloma (MM), as it has meaningful clinical activity in patients harboring the t(11;14) translocation. This subgroup of patients generally displays upregulated BCL-2 expression and/or a higher BCL-2/BCL-X _L ratio, reflecting an imbalance between cell survival (promoted by BCL-2) and cell death (promoted by BCL-X _L). The Bellini study demonstrated significantly superior response rate and progression-free survival (PFS) with combination of Venetoclax and Bortezomib in relapsed myeloma, however, the overall survival in the arm with venetoclax was inferior. Part of the decrease in overall survival (OS) was considered to be related with increased incidence of infections, however, OS is also significantly impacted by response to subsequent therapies. Here, we investigated whether acquired venetoclax resistance could lead to global resistance to multiple subsequent anti-MM therapies and in turn can explain the decreased OS.

We exposed venetoclax-sensitive myeloma cells (KMS27 and KMS12PE) to high-dose venetoclax treatment and generated monoclonal drug-tolerant expanded persister (DTEP) clones from single cell with a three- to ten-fold increase in IC50 compared to the parental cells (4 single cell clones for each cell line). Interestingly, the venetoclax resistance evolved without genomic alterations, as the parental and resistant cells did not show marked differences in mutational frequencies or copy number variations, including the absence of genomic alterations in the BCL-2 gene, such as the Gly101Val mutation, 1q21 gain or 8p loss. Consistent with previous literature in other disease models, these resistant clones also exhibited reduced mitochondrial priming and had increased protein expression of anti-apoptotic regulators, including MCL-1, BCL-X _L, and BCL-W, thus overcoming the venetoclax-induced inhibition of the anti-apoptotic regulator BCL-2, resulting in the sequestration of the pro-apoptotic ligand BIM. We next evaluated if the altered mitochondrial priming observed in the resistant cells was also responsible for a broader resistance to anti-cancer agents. We observed that venetoclax-resistant clones were resistant to most standard-of-care anti-MM agents including alkylating agents (Melphalan, cyclophosphamide, bendamustine), proteasome inhibitors (bortezomib and carfilzomib) or immunomodulatory drugs (lenalidomide and pomalidomide) than parental cells, suggesting a broad resistance to anti-cancer agents possibly due to reduced apoptotic signaling. This indicates that switching to these agents after the acquisition of venetoclax resistance may not be efficacious. Since these data suggested a functional substitution between BCL-2 family members, we next evaluated if MCL1 or BCL-XL represent a co-dependency in resistant cells that can be therapeutically targeted to overcome resistance. Simultaneous inhibition of MCL1 (via S63845) or BCL-XL (via A155463) and BCL2 (via venetoclax) increased BIM release and enhanced cell death in the resistant clones compared to single agents, with combination index (CI) values < 0.3 in all doses tested.

We further investigated the efficacy of immunotherapeutic approaches to overcome venetoclax resistance as these immunotherapies do not rely exclusively on traditional apoptotic signaling to mediate cell death. We observed that both antibody-dependent cellular cytotoxicity (ADCC) induced by Daratumumab and BCMA targeting CAR-T cells induced comparable cell death in venetoclax-resistant clones and parental cells. Moreover, myeloma cells from a patient with t(11;14) MM progressing on venetoclax, showed significant cytotoxicity to anti-BCMA CAR T cells in vitro and achieved a deep response subsequently when treated with Cilta-cel.

In conclusion, we report that resistance to venetoclax confers broad resistance to standard-of-care myeloma drugs and that immunotherapies, especially CAR-T cells and CD38 monoclonal antibody, may remain effective, thus conferring potential benefits in managing acquired resistance to venetoclax.