Dual Targeting Bcl-2 By Venetoclax and Mcl-1 By AZD5991 to Overcome Therapeutic Resistance in Aggressive R/R Mantle Cell Lymphoma

Introduction

Mantle cell lymphoma (MCL) is an aggressive subtype of non-Hodgkin's lymphoma. Frequent relapse from prior therapies remains a major medical challenge. BTK inhibitors (BTKi), such as ibrutinib and acalabrutinib, have demonstrated clinical benefit in MCL patients, however, resistance to BTKi is acquired by most MCL patients following initial response in the clinic. Unbalanced pro- and anti-apoptotic proteins have been shown to contribute to therapeutic resistance. Bcl-2 inhibitor venetoclax was approved by FDA to treat chronic lymphocytic leukemia, small lymphocytic lymphoma, or acute myeloid leukemia, and is currently under investigation in MCL patients. Venetoclax is efficacious with ORR up to 75% in treating BTKi-naive MCL patients and 53% BTKi-R/R patients. Interestingly, increased expression of Mcl-1 and Bcl-x _L highly correlates with venetoclax resistance in MCL. AZD5991 is a highly selective Mcl-1 inhibitor and our preliminary data showed it is potent in targeting MCL cells (IC ₅₀ =76-600 nM). Therefore, we hypothesize that dual targeting Bcl-2 by venetoclax and Mcl-1 by AZD5991 will achieve synergistic effect and significantly improve treatment outcome for aggressive R/R MCL patients. In this study, we assessed the synergistic efficacy of AZD5991 in combination with venetoclax in MCL preclinical models with R/R phenotype to ibrutinib, venetoclax or CD19 CAR T therapies.

Methods

Cell viability assay was performed to assess the in vitro efficacy of AZD5991 and venetoclax alone or in combination in a panel of ibrutinib/venetoclax-sensitive and -resistant MCL cell lines. Cell apoptosis assay was also performed to determine if AZD5991 and venetoclax induce cell death by cell apoptosis in MCL cell lines. Protein expression profiles of a panel of pro- and anti-apoptotic proteins and other relevant proteins were detected by western blots. In vivo efficacy of AZD5991 (30/30 mg/kg, intravenously, weekly) and venetoclax (5 mg/kg, oral, daily) alone or in combination was evaluated using PDX models derived from an ibrutinib-resistant patient or an ibrutinib-CAR-T dual-resistant MCL patient.

Results

AZD5991 and venetoclax combination potently and synergistically inhibited cell viability (combination index = 0.16-0.88) and enhanced cell apoptosis in vitro in both ibrutinib/venetoclax sensitive and resistant cell lines. Consistently, pro-apoptotic markers cleaved caspase 3 and cleaved PARP were increased. In an ibrutinib-resistant PDX mouse model, the combination of AZD5991 and venetoclax resulted in anti-MCL synergistic response. Consistently, the amount of β2M in the mouse plasma from the combo group was much lower than vehicle group (n =5, p = 0.010) and the two single agent groups (n =5, p = 0.005 and 0.013, respectively). In an ibrutinib-CAR-T dual-resistant PDX mouse model, co-treatment of AZD5991 and venetoclax inhibited tumor growth significantly and prolonged mouse survival for at least 100 days compared to vehicle or either single agent. All mice tolerated the treatment dose without any weight loss.

Conclusion

Significant anti-MCL synergy was observed for AZD5991 and venetoclax combo in vitro in both pairs of Jeko-1/JeKo-ibrutinib-R and Mino/Mino-venetoclax-R. This was further validated in aggressive R/R PDX models including an ibrutinib-resistant PDX model and an ibrutinib-CD19 CAR-T dual-resistant PDX model. Altogether, these findings suggest that dual targeting Bcl-2 and Mcl-1 is synergistic and has the potential in overcoming multiple acquired resistance phenotypes, including CD19 CAR T-cell therapy. These data provide insights on therapeutic development to improve patient outcome by overcoming therapeutic resistance.