P-191: Acquisition of venetoclax resistance is characterized by higher expression of anti-apoptotic regulators, less mitochondrial priming, and broader resistance to anti-MM agents.

Introduction: In multiple myeloma (MM), venetoclax represents the first example of personalized medicine, as it has meaningful clinical activity in patients harboring the t(11;14) translocation, which is present in 15% to 20% of the patient population. This subgroup of patients generally displays upregulated BCL-2 expression and a higher BCL-2/BCL-XL ratio. The Bellini study demonstrated that combination therapy of BH3-mimetic venetoclax with velcade improves progression-free survival (PFS) and response rates in this subgroup of MM patients. However, the overall survival in the arm with venetoclax was inferior. Part of the decrease in overall survival could be from more infections, but overall survival is also impacted by subsequent therapies. Here, we hypothesized that acquired resistance to venetoclax leads to a global resistance to subsequent therapeutic interventions impacting outcome.

Methods: To answer this question and delineate the mechanisms that contribute to the development of an acquired drug-tolerant/resistant phenotype, we exposed venetoclax-sensitive myeloma cells to high dose treatment and generated monoclonal drug-tolerant expanded persister (DTEP) clones with a three- to ten-fold increase in IC50 compared to the parental cells. These cells were subjected to an integrated analysis including WGS, RNA-seq and proteomics, as well as drug screening.

Results: Parental and resistant cells showed no marked differences in mutational frequencies, including the absence of genomic alterations in the BCL-2 gene such as the Gly101Val BCL-2 mutation. Instead, these clones exhibited reduced mitochondrial priming and had increased protein expression of anti-apoptotic regulators, including MCL-1, BCL-XL, and BCL-W, resulting in the sequestration of the pro-apoptotic ligand BIM. Since these data suggested a functional substitution between anti-apoptotic 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 next evaluated if the altered mitochondrial priming observed in the resistant cells was also responsible for a broader resistance to anti-cancer agents. Indeed, we found that most targeted agents, regardless of the mechanism of action, demonstrated a reduced ability to induce cell death in venetoclax-resistant cells compared with parental cells, suggesting a broad resistance to anti-cancer agents due to general selection for reduced apoptotic signaling.

Conclusions: In conclusion, we report that resistance to Venetoclax in in vitro models of MM evolves from the outgrowth of persister clones with a shift in mitochondrial dependency that confers broad resistance to all anti-tumor agents, including standard-of-care myeloma drugs.