Hyperphosphorylation of BCL-2 family proteins underlies functional resistance to venetoclax in lymphoid malignancies
Stephen Jun Fei Chong, … , Constantine S. Mitsiades, Matthew S. Davids
Stephen Jun Fei Chong, … , Constantine S. Mitsiades, Matthew S. Davids
Published September 26, 2023
Citation Information: J Clin Invest. 2023;133(22):e170169. https://doi.org/10.1172/JCI170169.
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Research Article Hematology Oncology

Hyperphosphorylation of BCL-2 family proteins underlies functional resistance to venetoclax in lymphoid malignancies

Abstract

The B cell leukemia/lymphoma 2 (BCL-2) inhibitor venetoclax is effective in chronic lymphocytic leukemia (CLL); however, resistance may develop over time. Other lymphoid malignancies such as diffuse large B cell lymphoma (DLBCL) are frequently intrinsically resistant to venetoclax. Although genomic resistance mechanisms such as BCL2 mutations have been described, this probably only explains a subset of resistant cases. Using 2 complementary functional precision medicine techniques — BH3 profiling and high-throughput kinase activity mapping — we found that hyperphosphorylation of BCL-2 family proteins, including antiapoptotic myeloid leukemia 1 (MCL-1) and BCL-2 and proapoptotic BCL-2 agonist of cell death (BAD) and BCL-2 associated X, apoptosis regulator (BAX), underlies functional mechanisms of both intrinsic and acquired resistance to venetoclax in CLL and DLBCL. Additionally, we provide evidence that antiapoptotic BCL-2 family protein phosphorylation altered the apoptotic protein interactome, thereby changing the profile of functional dependence on these prosurvival proteins. Targeting BCL-2 family protein phosphorylation with phosphatase-activating drugs rewired these dependencies, thus restoring sensitivity to venetoclax in a panel of venetoclax-resistant lymphoid cell lines, a resistant mouse model, and in paired patient samples before venetoclax treatment and at the time of progression.

Authors

Stephen Jun Fei Chong, Fen Zhu, Olga Dashevsky, Rin Mizuno, Jolin X.H. Lai, Liam Hackett, Christine E. Ryan, Mary C. Collins, J. Bryan Iorgulescu, Romain Guièze, Johany Penailillo, Ruben Carrasco, Yeonjoo C. Hwang, Denise P. Muñoz, Mehdi Bouhaddou, Yaw Chyn Lim, Catherine J. Wu, John N. Allan, Richard R. Furman, Boon Cher Goh, Shazib Pervaiz, Jean-Philippe Coppé, Constantine S. Mitsiades, Matthew S. Davids

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Figure 12

FTY720-induced PP2A activation displays similar cytotoxic effects on treatment-naive primary CLL cells.

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FTY720-induced PP2A activation displays similar cytotoxic effects on tre...
(A) Ficoll isolation of PBMCs from 28 treatment-naive patients with CLL. Samples were divided for DBP and Western blotting following ex vivo FTY720 treatment or were cocultured with NKTert stromal cells for cell viability measurement following FTY720 and ABT199/venetoclax cotreatments. The number of experiments performed was based on patient sample availability. The diagram was generated using BioRender software. (B) Western blots showing decreased T163pMCL-1 (n = 12), S70pBCL-2 (n = 18), and MCL-1 (n = 18) in treatment-naive primary CLL cells following increasing treatment concentrations of FY720 (1μM and 2.5 μM) for 4 hours. S112pBAD and S184pBAX were not evaluated because of insufficient samples. Quantification is displayed in Supplemental Figure 8A. (C) Δ Changes in the percentage of Cytc loss between ex vivo FTY720 (2.5 μM) and DMSO treatments for 4 hours in treatment-naive primary CLL cells (n = 20) indicated increased BCL-2 dependence. Šidák’s multiple-comparison test was used. (D) Viability of treatment-naive primary CLL cells following an ex vivo 4-hour pretreatment with 2.5 μM (n = 22) FTY720 and a subsequent 24-hour cotreatment with 10 nM venetoclax, as measured by annexin V/Hoechst assay. Šidák’s multiple-comparison test was used. (E) Viability of treatment-naive primary CLL cells (n = 10) following ex vivo pretreatment with OA (5 nM) for 2 hours followed by FTY720 (2.5 μM) for 4 hours and ABT199/venetoclax (10 nM) for 48 hours of cotreatment, as measured by annexin V/Hoechst assay. Šidák’s multiple-comparison test was used. (F) Western blots showing reversal of T163pMCL-1, S70pBCL-2, and MCL-1 reductions in treatment-naive primary CLL cells (n = 8) following ex vivo pretreatment with OA (5 nM) for 2 hours followed by FTY720 (2.5 μM) cotreatment for 4 hours. Quantification is displayed in Supplemental Figure 10A. (G) Co-IP of BCL-2 and immunoblotting of BAX and BCL-2 in treatment-naive primary CLL cells (n = 3) following ex vivo 4-hour pretreatment with FTY720 (2.5 μM) and 1-hour cotreatment with ABT199/venetoclax (10 nM). Quantification is displayed in Supplemental Figure 10B. **P < 0.01, ***P < 0.001, and ****P < 0.0001.