Cyclin-Dependent Kinase 4/6 Inhibitor Palbociclib Synergizes with BH3-Mimetics in Experimental Models of Relapsed/Refractory Mantle Cell Lymphoma

Introduction: Mantle cell lymphoma (MCL) is an aggressive subtype of B-cell non-Hodkin lymphomas characterized by cyclin D1 upregulation. The complex of upregulated cyclin D1 and cyclin-dependent kinases (CDK) 4 and 6 deregulates G1/S cell cycle transition of malignant lymphocytes, which represents a rationale druggable target. Palbociclib, a CDK 4/6 inhibitor approved for the therapy of patients with breast cancer, demonstrated single-agent clinical activity in relapsed/refractory (R/R) MCL. BH3-mimetics bind to an anti-apoptotic BCL-2 proteins (Bcl-2, Bcl-xL or Mcl-1) and thereby block their anti-apoptotic function. Specific Bcl-2 inhibitor venetoclax, the only FDA approved member of BH3-mimetics, is currently being tested in numerous clinical trials in hematological malignancies. Interestingly, the combination of palbociclib and venetoclax showed promising results in patients with breast cancer but has never been tested in patients with MCL.

Methods: Experimental therapy aimed at inhibition of CDK by palbociclib and anti-apoptotic BCL-2 proteins using venetoclax, S63845 and A1155463 was performed on nine MCL cell lines and four patient-derived xenograft (PDX) models from patients with R/R MCL. Mitochondrial priming to apoptosis was assessed by BH3 profiling. Expression of selected cell cycle regulators, pro-, and anti-apoptotic proteins was evaluated before and after exposure to palbociclib by western blotting both in vitro and in vivo. Spectrum of BCL-2-bound BH3-only proteins were detected by immunoprecipitation of MCL tumors ex vivo 24 and 48 hours after start of palbociclib therapy (compared to tumors obtained from untreated controls). Impact of palbociclib on energo-metabolic pathways in MCL cells was measured by SeaHorse assay. MCL clones with knockdown or overexpression of CDKN2A, MYC, CDK4 and RB1 were used to evaluate impact of these aberrations on the sensitivity to palbociclib (and venetoclax).

Results: Synergistic effect of palbociclib was observed in combination with all BH3-mimetics (venetoclax, S63845 and A1155463) in vitro, and confirmed in vivo on PDX R/R MCL models using venetoclax, as the only FDA approved member. Exposure of MCL-bearing mice to palbociclib induced downregulation of D-type cyclins, cyclin A, Rb1 and Mcl-1 proteins. Even though single-agent palbociclib had limited cytotoxic (proapoptotic) effect, it increased pro-apoptotic mitochondrial priming. Immunoprecipitation experiments revealed that the level of Bim bounded to Bcl-2 was not significantly changed, and the level of Bim bounded to Bcl-xL was increased 24 hours after exposure to palbociclib. Curiously, palbociclib significantly increased level of basal and maximal oxidative phosphorylation after cultivation with MCL cells for 72 hours (but not 24 hours). Pre-treatment of MCL cells with palbociclib showed even greater synergy than simultaneous palbociclib and BH3-mimetic application. MCL cell clones with RB1 knockdown became resistant to palbociclib. Susceptibility of MCL clones with knockout of CDKN2A, overexpression of MYC, and overexpression of CDK4 to palbociclib and venetoclax was not significantly changed.

Conclusion: Our data strongly support investigation of palbociclib in combination with venetoclax as an innovative treatment strategy for chemoresistant MCL patients without RB1 deletion. Although we demonstrated that palbociclib increases pro-apoptotic mitochondrial priming and induces metabolic stress, the detailed contribution of these changes to the observed synergy are under investigation.