Pre-Clinical Development of Targeted Therapies for Double Hit (DH) Diffuse Large B-Cell Lymphoma (DLBCL)

Background: Gene expression profile classifies DLBCL into three subtypes with distinct pathogenesis and clinical outcomes: activated B-cell (ABC), germinal center B-cell (GCB) and primary mediastinal B-cell lymphoma (PMBL). DH-DLBCL is a distinct subgroup, with a poor clinical outcome, harboring concurrent gene translocations involving the C-MYC and either/both BCL2 or BCL6 proto-oncogenes. DH-DLBCL has inferior response rates to rituximab-based chemotherapy, and a shorter progression-free survival (PFS)/overall survival (OS). In addition, pts with DH-DLBCL who are refractory to frontline chemoimmunotherapy fare dismally; a retrospective review at our institute yielded survival rates of zero and median OS of 6 months in these patients, highlighting the need for novel therapies. DH-DLBCL is characterized by de-regulation of apoptosis and cell cycle progression, leading to rapid proliferation of neoplastic cells, and resistance to apoptotic stimuli. Upregulation of the anti-apoptotic protein Mcl-1 is implicated in chemotherapy resistance and poor clinical outcomes in ABC-DLBCL. Transcriptional regulation of MCL1 by c-MYC might further potentiate Mcl-1 induced resistance to chemotherapy in DH-DLBCL. Specific inhibitors targeting cell pro-survival proteins might provide a novel and alternative therapeutic approach in rituximab-chemotherapy resistant DH-DLBCL. We hypothesize that dual inhibition of anti-apoptotic proteins Bcl-2 and Mcl-1 using readily available agents (Venetoclax and Proteasome inhibitors) is an effective strategy in inducing lymphoma cell death. Materials and Methods: We conducted pre-clinical in vitro and in vivo models targeting Bcl-2 with Venetoclax and Mcl-1 with either a c-Myc inhibitor (JQ-1) or Proteasome inhibitor (Carfilzomib) in DHL. Val, DOHH-2, and ROS-50 cells were exposed to Venetoclax (0-10 uM), JQ-1 (0-100 uM) and Carfilzomib (CFZ) (0-50 nM) for 24, 48 and 72 hours. IC₅₀ drug concentrations were calculated for each agent. Subsequently, DHL cells were exposed to Venetoclax in combination with JQ-1 or CFZ for 48 hours. Cell viability was determined by Cell Titerglo. Coefficient of synergy was calculated using CalcuSyn. In addition, induction of apoptosis was evaluated by flow cytometry (Annexin V/PI staining) and changes in Bcl-2 family members and Caspase activation were determined by Western blotting. Primary tumor cells isolated from B-cell lymphoma patients (N= 15) including DHL were exposed ex vivo to Venetoclax +/- Carfilzomib. For in vivo experiments 6-8 weeks old severe combined immunodeficiency (SCID) mice were inoculated with 10x10⁶ ROS-50 cells via tail vein injection (IV). Subsequently, animals were treated with Venetoclax (100mg/kg/dose via gastric lavage on days 3-7 and 10-14); Carfilzomib (2mg/kg/dose IV days 3, 4, 10, 11), or both agents. A group of untreated animals was used as a control. Differences in survival were evaluated between treatment groups. Results: In vitro, Venetoclax, JQ-1, and CFZ induced cell death in a dose- and time-dependent manner. Significant synergistic activity was observed by combining both Venetoclax and CFZ and to a lesser degree with JQ-1. Similar findings were observed in primary tumor cells isolated from B-cell lymphoma patients. Annexin/PI staining with the combination of Venetoclax and CFZ resulted in caspase dependent apoptosis. Western blotting confirmed changes in Bcl-2 family members favoring an apoptotic state as well as PARP cleavage following in vitro exposure to Venetoclax and CFZ. In vivo the combination of Venetoclax with Carfilzomib resulted in significant increased survival increase (P<0.05) when compared to single agent Venetoclax or CFZ. Conclusion: Our data suggests that Venetoclax exhibits strong synergistic activity with Carfilzomib in vitro and in vivo . Perhaps related to the synergy observed between Venetoclax and CFZ, is the effects of proteasome inhibition on Mcl-1 levels. Combination therapy targeting the proteasome inhibition and BCL-2 may provide an alternative therapeutic approach in DH-DLBCL.