Inhibition of Cyclin Dependent Kinase 9 Synergistically Enhanced Venetoclax Activity in Mantle Cell Lymphoma Cells

Better therapeutic strategies are needed for patients with mantle cell lymphoma (MCL), an aggressive and largely incurable subtype of Non-Hodgkin Lymphoma. Concurrent expression of anti-apoptotic BCL2 family proteins in lymphoma cells contribute to their evasion of apoptosis. Therefore, targeting only one anti-apoptotic protein may lead to or uncover resistance associated with activity of other anti-apoptotic BCL2 family members. A variety of cyclin-dependent kinase (CDK) inhibitors are undergoing clinical trials either as a single agent or in combination with other approved drugs. CDK9, a portion of the elongation factor P-TEFb, phosphorylates Ser-2 in the C-terminal domain of RNA Polymerase II, which is required for transcript elongation. The effect of CDK9 inhibition is observed most immediately on those proteins with rapid turnover rates such as the BCL2 family protein MCL1, which is associated with both intrinsic and acquired resistance to venetoclax in B-cell malignancies. Here we report the responses of 4 MCL cell lines (Mino, Jeko-1, CCMCL1 and JVM2) and 5 primary MCL samples (representing de novo and relapsed cases, including two relapsed cases after ibrutinib failure and a relapsed case harbor Myc rearrangement) to venetoclax and a novelCDK9 inhibitor A-1467729.

Exposure of Mino and Jeko-1 cells to venetoclax rapidly induced apoptosis (IC50 at 5 hours were 235 and 955 nM, respectively). In contrast, CCMCL1 and JVM2 cells were not sensitive to venetoclax with IC50s > 3000 nM. However, CCMCL1 cells were more responsive to A-1467729 alone than the other 3 lines, while JVM2 cells were much less sensitive to A-1467729. All primary samples were sensitive to venetoclax, ex vivo, at the doses between 1 - 100 nM, although their IC50s were variable (range: 2-90 nM). A-1467729 at doses of 1-20 nM had modest single agent effects on the primary samples; however, its combination with venetoclax synergistically induced apoptosis and decreased the IC50 of venetoclax by 2-10 times in all cell lines and primary samples. The strongest synergy was observed in Jeko-1 cells with all combined indexes < 0.1. Studies on mechanisms through immunoblotting and immunohistochemical staining demonstrated that A-1467729 quickly down-regulated phospho-RNA Polymerase II (Ser2) and MCL1 protein levels. CCMCL1 cells lack BCL2 expression, while JVM2 displayed higher expression of MCL1 than other cells. The expression levels of BCL2 and MCL1 in primary samples were case-dependent as well. The expression pattern and level of anti-apoptotic BCL2 family proteins in cell lines and primary cases may be responsible for their variable reactions to these two agents. To further confirm that CDK9 inhibition was affecting cell viability at least partially through its function on MCL1, A-1210477, a MCL1 inhibitor, was applied to the same study. Strong synergistic apoptotic induction was also observed when A-1210477 was combined with venetoclax, especially in MCL1-"dependent" CCMCL1 cells as evidenced by flow cytometry based apoptotic assay and PARP cleavage. Further mechanism studies aiming the effects of CDK9 inhibitor/venetoclax on MCL1/BIM association is being under investigation. MCL mouse xenograft study for such a combined effect has been planned. In summary, the combination of a CDK9 inhibitor and venetoclax showed synergistic induction of apoptosis in both MCL cell lines and primary patient samples. These findings support further evaluation of the efficacy of such a combination in MCL, including ibrutinib-resistant MCL.