PP2A modulation overcomes multidrug resistance in chronic lymphocytic leukemia via mPTP-dependent apoptosis
Kallesh D. Jayappa, … , Michael J. Weber, Goutham Narla
Kallesh D. Jayappa, … , Michael J. Weber, Goutham Narla
Published May 11, 2023
Citation Information: J Clin Invest. 2023;133(13):e155938. https://doi.org/10.1172/JCI155938.
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PP2A modulation overcomes multidrug resistance in chronic lymphocytic leukemia via mPTP-dependent apoptosis

Abstract

Targeted therapies such as venetoclax (VEN) (Bcl-2 inhibitor) have revolutionized the treatment of chronic lymphocytic leukemia (CLL). We previously reported that persister CLL cells in treated patients overexpress multiple antiapoptotic proteins and display resistance to proapoptotic agents. Here, we demonstrated that multidrug-resistant CLL cells in vivo exhibited apoptosis restriction at a pre-mitochondrial level due to insufficient activation of the Bax and Bak (Bax/Bak) proteins. Co-immunoprecipitation analyses with selective BH domain antagonists revealed that the pleiotropic proapoptotic protein (Bim) was prevented from activating Bax/Bak by “switching” interactions to other upregulated antiapoptotic proteins (Mcl-1, Bcl-xL, Bcl-2). Hence, treatments that bypass Bax/Bak restriction are required to deplete these resistant cells in patients. Protein phosphatase 2A (PP2A) contributes to oncogenesis and treatment resistance. We observed that small-molecule activator of PP2A (SMAP) induced cytotoxicity in multiple cancer cell lines and CLL samples, including multidrug-resistant leukemia and lymphoma cells. The SMAP (DT-061) activated apoptosis in multidrug-resistant CLL cells through induction of mitochondrial permeability transition pores, independent of Bax/Bak. DT-061 inhibited the growth of wild-type and Bax/Bak double-knockout, multidrug-resistant CLL cells in a xenograft mouse model. Collectively, we discovered multidrug-resistant CLL cells in patients and validated a pharmacologically tractable pathway to deplete this reservoir.

Authors

Kallesh D. Jayappa, Brian Tran, Vicki L. Gordon, Christopher Morris, Shekhar Saha, Caroline C. Farrington, Caitlin M. O’Connor, Kaitlin P. Zawacki, Krista M. Isaac, Mark Kester, Timothy P. Bender, Michael E. Williams, Craig A. Portell, Michael J. Weber, Goutham Narla

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Figure 4

PP2A activation by the small-molecule agonist DT-061 induces Bax/Bak-independent apoptosis in CLL cells.

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PP2A activation by the small-molecule agonist DT-061 induces Bax/Bak-ind...
(A) PBMCs from patients with CLL were preincubated with the agonist mix for 12 hours. Samples were treated with a second dose of the agonist mix as well as DT-061 (12, 16, and 20 μM) for 18 hours. Apoptosis induction (Bax activation and cleaved caspase 9 and cleaved PARP) and viability dye staining in CLL (CD5+CD19+) cells were analyzed by flow cytometry. (B) PBMCs from patients with CLL were screened by flow cytometry for Bax activation as well as cleaved PARP following incubation with DT-061 (9, 12, 15, and 18 μM) for 9 hours without added agonists. Data show the percentage CD69Pos or CD69Neg CLL (CD5+CD19+) cells positive for active Bax or cleaved PARP, after subtraction of the spontaneous apoptosis values from the DMSO-treated controls. (C) The Bax/Bak-DKO CLL cell line MEC1 was developed using the CRISPR/Cas9 system, as described in Methods. WB data show the expression of Bax and Bak proteins in WT and Bax/Bak-DKO clones. (D) The parent MEC1 cell line as well as WT and Bax/Bak-DKO clones were treated with DT-061 (12, 16, and 20 μM) or a combination of VEN (0.2 μM), S63845 (2 μM), and A1155463 (1.6 μM) (SVA) for 12 hours. Cleaved PARP was analyzed by flow cytometry. The average data from 3 independent experiments are presented as a bar graph, which shows the percentage of MEC1 cells positive for cleaved PARP. Statistical significance was determined by ANOVA with Šidák’s post hoc test for multiple comparisons. ***P < 0.001 and ****P < 0.0001. Data are presented as the mean ± SD.