The Novel Proteasome Inhibitor Carfilzomib (CFZ) Potentiates the Anti-Tumor Activity of Chemotherapeutic Agents in Rituximab-Chemotherapy Resistant Lymphoma Through Inducing G2/M Cell Cycle Arrest and Cell Death

Abstract 4970

The concept of proteasome inhibition (PI) is an attractive therapeutic approach for resistant/refractory lymphoma. While bortezomib has modest activity against certain types of lymphomas, a significant number of patients develop resistance or experience dose-limiting toxicity. Carfilzomib (CFZ), a novel second-generation irreversible proteasome inhibitor, has demonstrated significant and well-tolerated anti-tumor activity in relapsed/refractory (r/r) myeloma patients. Here we evaluated CFZ's mechanism(s)-of-action and anti-tumor activity in several lymphoma pre-clinical models. Rituximab-chemotherapy sensitive cell lines (RSCL), rituximab-chemotherapy resistant cell lines (RRCL), as well as primary tumor cells (n=25) were exposed in vitro and/or ex vivo to escalating doses of CFZ or BTZ (0.1-10nM) +/− caspase inhibitors (zVAD-fmk or Q-VD-OPh) for 24, 48 and 72hrs. Changes in ATP content (apoptosis) were determined using the Cell Titer Glow assay and in cell cycle were analyzed by FACScan DNA methodology. Patient-derived lymphoma cells were isolated from fresh biopsy tissue via negative selection using magnetic beads. Western blots were performed using cell lysates from control versus treated (i.e. CFZ or BTZ) cells to detect PARP-cleavage and changes in Bcl-2 family members or cell cycle regulators. CFZ was found to be 10 times more potent than BTZ and exhibited dose- and time- dependent cytotoxicity against RSCL, RRCL, and primary tumor cells. CFZ has potent anti-tumor activity in tumor cells derived from patients with follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), Hodgkin lymphoma (HL) and other histologies. CFZ exposure induced apoptosis by up-regulation of Bak, Mcl-1 levels and subsequent PARP cleavage in RSCL and (to a lesser degree) RRCL. Co-incubation of RSCL with either BTZ or CFZ and pan-caspase inhibitor led to a significant decrease in BTZ- or CFZ-induced cell death. In contrast, zVAD-fmk and Q-VD-OPh were associated with only partial blocking of BTZ- or CFZ-induced cell death of RRCL. In addition, In vitro exposure of RRCL cells to CFZ (and to a much lesser degree BTZ) reduced RRCL S-phase and induced arrest at G2/M phase. CFZ stabilized G2/M cell cycle regulators cdc2 and cyclinB only in RRCL. Finally, CFZ demonstrated the ability to overcome resistance to chemotherapy in RRCL and potentiated the anti-tumor activity of paclitaxel and vincristine in B-cell lymphoma cell lines and in primary tumor samples. In summary, our data strongly suggest that CFZ is a novel and potent proteasome inhibitor which is has the potential to: overcome resistance to some conventional chemotherapeutic agents, upregulate proapoptotic proteins to enhance cell death, and induce G2/M cell cycle arrest in lymphoma cells. Our preclinical data supports future clinical evaluation of CFZ in patients with refractory B-cell lymphoma. Research, supported in part as a subproject of NIH grant R01 CA136907-01A1 awarded to Roswell Park Cancer Institute.