Resistance Mechanisms Underlying Venetoclax Resistance in Mantle Cell Lymphoma

Background: BCL-2 is highly expressed in mantle cell lymphoma (MCL), and targeting BCL-2 with venetoclax (ABT-199) has resulted in positive clinical outcomes in MCL patients. However, resistance to various targeted agents often emerges; therefore, we generated venetoclax-resistant MCL cell lines to uncover the mechanisms mediating acquired venetoclax resistance and performed high-throughput drug screening to identify novel anti-MCL agents that can overcome venetoclax resistance.

Methods: We generated 3 venetoclax-resistant cell lines (Mino, Rec-1 and Granta519) by long-term venetoclax exposure, and the anti-MCL activity of venetoclax against these acquired resistance lines was measured with dose-dependent cell viability and apoptosis assays compared with the parental lines. Aberrant protein expression between the parental and resistant cell lines was detected using Reverse Phase Protein Array (RPPA) analysis. A drug library (> 1000 drugs; Selleck Chemicals Ltd) was screened to identify agents that inhibit the cell growth of both the parental and resistant cells. The candidate drugs that showed anti-MCL activity were confirmed by dose-dependent cell viability and Annexin V apoptosis assays. To determine the mechanisms underlying the anti-MCL activity of the candidate agents in the parent and venetoclax-resistant MCL cell lines, we conducted RPPA analysis before and after drug treatment.

Results: Chronic exposure to venetoclax resulted in markedly less sensitivity to venetoclax. The IC₅₀ values of the venetoclax-resistant cell lines (Mino-Re, Rec-1-Re and Granta519-Re) were determined to be 603 nM, 676 nM and 456 nM, respectively, which are 600-fold, 500-fold and 25-fold higher than the IC₅₀ values of the parental cells lines. RPPA analysis showed that proteins such as PTEN, PDCD4, AKT, ATM, BCL-2, BAX and BAK were down-regulated and p-AKT, p21, STAT3, Cyclin B1 and MCL-1 were up-regulated in the venetoclax-resistant MCL cell lines. The results were confirmed by Western blotting, and further Western blotting experiments revealed expression and modification differences among anti-apoptotic and pro-apoptotic protein family members between the parental and venetoclax-resistant MCL cell lines. Specifically, both MCL-1 and BCL-xL were elevated; however, BIM and BAX were absent in the venetoclax-resistant cells. High-throughput drug screening showed striking anti-MCL activity with two PI3K inhibitors, PIK-75 (PI3K α inhibitor) and GSK1059615 (pan PI3K inhibitor), in dose-dependent cell viability and apoptosis assays. RPPA analysis of the parental and venetoclax-resistant MCL cell lines with and without PI3K inhibitor treatment suggested that both drugs inhibited the cell viability of the MCL cell lines by blocking the activated PI3K/AKT pathway.

Conclusion:The PI3K/AKT pathway is activated in venetoclax-resistant MCL cell lines, and targeting this pathway may be a potential approach to overcoming venetoclax resistance.