Abstract 3959: Targeting BCL2 family protein phosphorylation in venetoclax resistant lymphoid malignancies
Authors: Chong Stephen J. , Zhu Fen , Lai Jolin X. , Hackett Liam , Collins Mary C. , Pervaiz Shazib , Coppe Jean-Philippe , Davids Matthew S. 
Published: June 2022

Abstract
Introduction: BCL2 family protein dysfunction is an important mediator of chemoresistance in lymphoid malignancies. Venetoclax (VEN), a BH3 mimetic that selectively targets the anti-apoptotic protein BCL2, is FDA approved for treatment of chronic lymphocytic leukemia (CLL), with clinical trials underway for other lymphoid malignancies. Despite the clinical efficacy of VEN monotherapy, complete remission and progression free rates are still relatively low, thereby indicating the existence of inherent or acquired mechanisms of resistance. Preclinically, increases in anti-apoptotic BCL2 and MCL1 phosphorylation (BCL2-P/MCL1-P) and MCL1 protein level have been implicated in chemoresistance. We therefore question if phosphorylation of these proteins may drive VEN resistance and if removing this modification may re-sensitize resistant cells to VEN induced cell death.
Methodology: BH3 profiling (a functional assay that assesses mitochondrial apoptotic priming and anti-apoptotic protein dependence), western blot and cell death assays were performed.
Result: We report that VEN acquired resistant DLBCL cell line (OCI-Ly1R) has increased BCL2-P, MCL1-P and MCL1 protein levels as compared to sensitive parental cell line (OCI-Ly1S), while inherently resistant DHL cell line (Su-DHL4) possesses higher MCL1-P and MCL1 protein levels as compared to OCI-Ly1S. We further show that this increase in phosphorylation status contributes to the induction of VEN resistance. Using BH3 profiling, we demonstrate that anti-apoptotic protein phosphorylation-driven VEN resistance involves a change in sensitivity to pro-apoptotic proteins, with an increase in sensitivity to MCL1 inhibition and reciprocal decrease in sensitivity to BCL2 inhibition.
We further show that BCL2 and MCL1 dephosphorylation by PP2A activating drugs (PADs) re-sensitizes resistant cells to VEN treatment. Mechanistically, PADs such as the fingolimod (FTY720) not only dephosphorylate BCL2-P and MCL1-P, but also dissociate BAX from BCL2 and destabilize MCL1 protein. These changes lead to the rewiring of cellular sensitivity to BCL2 inhibition, thereby re-sensitizing both VEN acquired and inherent resistant cells to VEN induced cell death. Importantly, the PP2A activating effects of FTY720 were recapitulated in primary cells from the peripheral blood of 16 CLL patients, where FTY720 treatment also reduced BCL2-P, MCL1-P and MCL1 protein levels and dissociated BAX from BCL2. This led to an increased sensitivity to BCL2 inhibition and enhanced cell death upon combined treatment with FTY720 and VEN.
Conclusion: Our work defines a new targetable mechanism of VEN resistance in lymphoid malignancies due to the phosphorylation of anti-apoptotic BCL2 family proteins. PADs re-sensitize resistant malignant cells to VEN treatment via dephosphorylation of anti-apoptotic proteins, suggesting a promising combination approach to explore further.
Citation Format: Stephen J. Chong, Fen Zhu, Jolin X. Lai, Liam Hackett, Mary C. Collins, Shazib Pervaiz, Jean-Philippe Coppe, Matthew S. Davids. Targeting BCL2 family protein phosphorylation in venetoclax resistant lymphoid malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3959.