Changes in Bcl-2 members after ibrutinib or venetoclax uncover functional hierarchy in determining resistance to venetoclax in CLL

Abstract

Chronic lymphocytic leukemia (CLL) cells cycle between lymph node (LN) and peripheral blood (PB) and display major shifts in Bcl-2 family members between those compartments. Specifically, Bcl-XL and Mcl-1, which are not targeted by the Bcl-2 inhibitor venetoclax, are increased in the LN. Since ibrutinib forces CLL cells out of the LN, we hypothesized that ibrutinib may thereby affect expression of Bcl-XL and Mcl-1 and sensitize CLL cells to venetoclax. We investigated expression of Bcl-2 family members in patients under ibrutinib or venetoclax treatment combined with dissecting functional interactions of Bcl-2 family members in an in vitro model for venetoclax resistance. In the PB, recent LN emigrants had higher Bcl-XL and Mcl-1 expression than cells immigrating back to the LN. Under ibrutinib treatment, this distinction collapsed, and significantly, the pre-treatment profile reappeared in patients who relapsed on ibrutinib. In response to venetoclax however, Bcl-2 members displayed an early increase, underlining the different modes of action of these two drugs. Profiling by BH3 mimetics was performed in CLL cells fully resistant to venetoclax due to CD40-mediated induction of Bcl-XL, Mcl-1 and Bfl-1. Several dual or triple combinations of BH3 mimetics were highly synergistic in restoring killing of CLL cells. Lastly, we demonstrated that pro-apoptotic Bim interacts with anti-apoptotic Bcl-2 members in a sequential manner: Bcl-2>Bcl-XL>Mcl-1>Bfl-1. Combined, the data indicate that Bcl-XL is more important in venetoclax resistance than Mcl-1 and provide biological rationale for potential synergy between ibrutinib and venetoclax.

Novel Agents in Chronic Lymphocytic Leukemia: New Combination Therapies and Strategies to Overcome Resistance

“…As the sensitivity to BH3 mimetics depends largely on the ratio of the expression of proapoptotic and antiapoptotic proteins, it was hypothesized early on that an increase in anti-apoptotic proteins following venetoclax exposure would confer resistance to Bcl-2 inhibition [ 71 , 72 ]. Various studies have demonstrated that venetoclax-associated overexpression of Mcl-1 and Bcl-X L can confer resistance to the drug in vitro and in vivo [ 23 , 24 , 72 , 73 , 74 , 75 , 76 ]. Recently it has been postulated that among the Bcl-2 family members, Bcl-X L is more relevant for the development of venetoclax resistance, as functional analyses have shown that proapoptotic proteins preferably interact with Bcl-X L when both anti-apoptotic proteins are present [ 24 ].…”

“…Various studies have demonstrated that venetoclax-associated overexpression of Mcl-1 and Bcl-X L can confer resistance to the drug in vitro and in vivo [ 23 , 24 , 72 , 73 , 74 , 75 , 76 ]. Recently it has been postulated that among the Bcl-2 family members, Bcl-X L is more relevant for the development of venetoclax resistance, as functional analyses have shown that proapoptotic proteins preferably interact with Bcl-X L when both anti-apoptotic proteins are present [ 24 ]. Guièze and colleagues have performed an extensive analysis including genome-scale screens in a Bcl-2-driven lymphoma cell line and integrated expression profiling and identified Mcl-1 overexpression and BIM sequestration as a resistance-conferring mechanism to venetoclax treatment [ 23 ].…”

“…Combination therapies would possibly also reduce the selection of BTK - or BCL2 -mutated clones, as e.g., BTK C481S-mutated cells could still be eliminated by concomitant venetoclax or an anti-CD20 antibody while they would likely outgrow under ibrutinib monotherapy. On the other hand, it has been shown that the combination of venetoclax with a BTKi was able to reprogram apoptotic dependencies and venetoclax resistance could be overcome in malignant B cells [ 24 , 80 , 81 ]. Also, resistance-mediating upregulation of Bcl-2 under ibrutinib monotherapy can increase the sensitivity towards venetoclax [ 82 , 83 ].…”

“…Based on the observations that venetoclax resistance appears to be mediated by an upregulation of the anti-apoptotic proteins Mcl-1 and to a lesser extent Bcl-X L these proteins are considered as promising targets to overcome resistance to Bcl-2 inhibition [ 23 , 24 , 73 , 75 ]. AMG-176, a direct Mcl-1 antagonist has shown activity in a preclinical setting by effectively killing CLL cells while sparing normal blood cells and showing synergy with venetoclax; however, a phase 1 study of the drug had to be suspended due to safety concerns [ 118 ].…”

“…The mechanisms behind these clinical manifestations of resistance have been studied, and are in part explained by genetic resistance mechanisms [ 18 , 19 , 20 ]. Besides mutations in the drug-targeting proteins, several non-genetic mechanisms rendering CLL cells resistant to treatment have been described [ 21 , 22 , 23 , 24 ]. In this article, we will review the current state of research on resistance to novel agents, propose strategies to avoid resistance and provide guidance on treating patients who relapse or progress on novel agents.…”