Purpose: Carfilzomib, while active in B-cell neoplasms displayed heterogeneous response in chronic lymphocytic leukemia (CLL) samples from patients and showed interpatient variability to carfilzomib-induced cell death. To understand this variability and predict patients that would respond to carfilzomib, we investigated the mechanism by which carfilzomib induces CLL cell death. Experimental Design: Using CLL patient samples and cell lines, complementary knockdown and knockout cells, and carfilzomib-resistant cell lines, we evaluated changes in intracellular networks to identify molecules responsible for carfilzomib's cytotoxic activity. Carfilzomib-treated cells were immunoblotted for molecules involved in ubiquitin, apoptotic, and endoplasmic reticulum (ER) stress response pathways and results correlated with carfilzomib cytotoxic activity. Co-immunoprecipitation and pull down assays were performed to identify complex interactions among MCL-1, Noxa, and Bak. Results: Carfilzomib triggered ER stress and activation of both the intrinsic and extrinsic apoptotic pathways through alteration of the ubiquitin proteasome pathway. Consequently, the transcription factor CCAAT/enhancer-binding protein homology protein (CHOP) accumulated in response to carfilzomib, and CHOP depletion conferred protection against cytotoxicity. Carfilzomib also induced accumulation of MCL-1 and Noxa, whereby MCL-1 preferentially formed a complex with Noxa and consequently relieved MCL-1's protective effect on sequestering Bak. Accordingly, depletion of Noxa or both Bak and Bax conferred protection against carfilzomib-induced cell death. Conclusions: Collectively, carfilzomib induced ER stress culminating in activation of intrinsic and extrinsic caspase pathways, and we identified the CHOP protein level as a biomarker that could predict sensitivity to carfilzomib in CLL.
- Received October 18, 2015.
- Revision received March 1, 2016.
- Accepted March 16, 2016.
- Copyright ©2016, American Association for Cancer Research.