PF358 FUNCTIONAL HIERARCHY AMONG PRO-SURVIVAL BCL-2 MEMBERS IN DETERMINING RESISTANCE TO VENETOCLAX IN CLL
Authors: M. Haselager, H. Ter Burg, K. Kielbassa, J. Borst, A. Kater, E. Eldering
Journal: HemaSphere (2019)

Background: The Bcl-2 inhibitor Venetoclax rapidly induces apoptosis in chronic lymphocytic leukemia (CLL) cells in peripheral blood (PB) whereas lymph node (LN) responses are less complete. This may be linked to pro-survival signals in the LN, where micro-environmental signals strongly induce Bcl-XL, Mcl-1 and Bfl-1 which are not targeted by Venetoclax. In some patients resistance occurs over time which sometimes can be explained by acquired mutations, but in most cases the mechanism(s) remain unknown. Aims: We hypothesize that changes in expression or shifts in functional interactions among Bcl-2 family members are key to clinical response and refractory disease. Methods: Intracellular FACS staining for Bcl-2, Mcl-1 and Bcl-XL in circulating recent LN emigrants (CD19+/CXCR4high/CD5dim) versus returning (CD19+/CXCR4llow/CD5high) lymphocytes in patient samples treated with Venetoclax. To investigate the functional impact of expression of Bcl-2 family members, profiling with additional BH3 mimetics against Bcl-XL (A1331852) and Mcl-1 (S63845) was applied in a CLL LN model. Primary CLL cells were co-cultured with fibroblasts expressing CD40L in order to mimic T cell help in the LN. Results: Under Venetoclax treatment, levels of Bcl-2 itself and other family members may increase in some patients, early in clinical response. During short-term in vitro incubation with Venetoclax or other BH3 mimetics, we established that Mcl-1 and Bfl-1 can increase but not Bcl-2 or Bcl-XL. The effect on Mcl-1 and Bfl-1 can be explained by a stabilization of these normally short-lived proteins. In vitro CD40 activation renders CLL cells fully resistant to single Venetoclax treatment. Using this model, we find that dual or triple BH3 mimetic combinations targeting Bcl-2, Bcl-XL and Mcl-1 restored killing. Specifically, whereas the IC50 of dual Bcl-2+Mcl-1 targeting is >1uM, combined inhibition of Bcl-2+Bcl-XL has an IC50 around 0.3uM. Bcl-2 and Bcl-XL thus seem to be the most dominant players. However, even when Bcl-2, Bcl-XL and Mcl-1 were inhibited using triple BH3 mimetic treatment, Venetoclax resistance was not fully reversed to unstimulated control (IC50 1 versus 10 nM), highlighting a potential role for Bfl-1. To further dissect the functional roles, Bim co-immunoprecipitation was performed. In PB CLL cells, Bim only binds to Bcl-2 and upon Venetoclax treatment it is released and targets Bax/Bak to kills cells. After CD40 stimulation, Bim shifts predominantly to Bcl-XL and less to Mcl-1. Only when applying triple BH3 mimetic combinations targeting Bcl-2, Bcl-XL and Mcl-1, Bim starts to bind Bfl-1. These observations suggest a hierarchical model in which Bcl-2 is most important and where upon Venetoclax treatment, Bcl-XL and Mcl-1 come into play. When all of these are targeted by triple BH3 mimetics, Bfl-1 is engaged to intercept apoptosis. Summary/Conclusion: We applied BH3 mimetics in primary CLL and showed that combined targeting of Bcl-2 members can abrogate resistance to Venetoclax. Moreover, we dissected the roles of the different Bcl-2 family members and constructed a hierarchical model of Bcl-2 family members, providing new insights into Venetoclax resistance which may have clinical utility.