Targeting Mcl-1 By Proteasome Inhibitors Enhances the Anti-Tumor Activity of Venetoclax in Mantle Cell Lymphoma Pre-Clinical Models

Background: Mantle cell lymphoma (MCL) accounts for 6% of all non-Hodgkin lymphomas and is a therapeutic challenge. At the molecular level, MCL is characterized by the deregulation of Bcl-2 family members (Mcl-1, BIM) altering apoptosis, as well as, cell cycle (cyclin D1) partially regulated by the ubiquitin-proteasome system (UPS). Inhibitors of the UPS, also known as proteasome inhibitors (PI), have been tested against MCL. The lethality by PIs has been associated with pronounced up-regulation of the pro-apoptotic protein Noxa, which prevented the upregulation of Mcl-1. Changes in the ratio of Mcl-1 and Bak/Bax result in the release of Bak triggering apoptosis particularly in cells with functional p53. Venetoclax is a highly selective Bcl-2 inhibitor, BH-3 mimetic with potent activity against MCL, follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) cell lines, as well as in a t(14;18)-carrying xenograft model with an established tolerability and safety in a recent phase I trial. We sought to look at further enhancing the pre-clinical activity of venetoclax using PIs in MCL models. We hypothesized that dual inhibition of anti-apoptotic proteins Bcl-2 and Mcl-1 could result in synergistic activity that could be translated clinically. Materials and Methods: We performed in vitro studies on a panel cytarabine sensitive (Rec-1, Jeko, Granta, HBL-2, Z-138 and Mino) and resistant (araC) cell line clones (Jeko araC, HBL-2 araC, and Mino araC). MCL cell lines were exposed to venetoclax, carfilzomib, ixazomib and bortezomib for 24, 48 and 72 hours. Cell viability was calculated measuring the ATP content. IC₅₀ drug concentrations were calculated for each agent. Subsequently, MCL cell lines were exposed to escalating doses of venetoclax (0.001uM-5uM) and carfilzomib, an irreversible PI (1.5625nM-50nM); or two clinically available PI: bortezomib (3.125nM-100nM) or ixazomib (3.125nM-100nM). Cell viability was determined by Cell Titerglo. Coefficient of synergy were calculated using CalcuSyn. Primary tumor cells isolated from B-cell lymphoma patients (N= 15) including MCL were exposed ex vivo to venetoclax +/- carfilzomib or bortezomib. Results: In vitro exposure of MCL cell lines to either venetoclax, carfilzomib, bortezomib and ixazomib induced cell death in a dose- and time-dependent manner. Anti-tumor activity was observed in cytarabine sensitive or resistant cell lines. Significant synergistic activity was observed by combining both venetoclax with carfilzomib or ixazomib at known sub-therapeutic and therapeutic doses of individual agents. Similar findings were observed in primary tumor cells isolated from B-cell lymphoma patients. Conclusion: Our data suggests that venetoclax exhibits strong synergistic activity with carfilzomib and ixazomib in vitro and ex vivo MCL models. This pharmacological interaction could be related to the effects of proteasome inhibition on Mcl-1 in MCL cells, further enhancing the activity of venetoclax. In vivo experiments will further confirm the synergistic activity observed between BH3 mimetics and PIs.(Supported by a NCI SPORE Lymphoma grant: 5 P50 CA130805-04, an NIH grant R01 CA136907-01A1 and a grant from The Roswell Park Cancer Institute Alliance Foundation)