Abstract 937: The PP2A activation using a small molecule agonist triggers apoptosis by releasing mitochondrial permeability transition pores in multi-drug resistant leukemic B cells
Authors: Jayappa Kallesh D. , Farrington Caroline , Gordon Vicki L. , Saha Shekhar , Morris Christopher , Isaac Krista M. , Bender Timothy P. , Williams Michael E. , Portell Craig A. , Narla Goutham , Weber Michael J. 
Published: July 2021

Abstract
The Bcl-2 inhibitor venetoclax (VEN) shows clinical activity in Chronic Lymphocytic Leukemia (CLL), and the response is potentiated when combined with the BTK inhibitor ibrutinib (IBR) (Portell et al. 2019; Tam et al. 2018). However, many VEN or VEN+IBR responses are incomplete and drug resistance develops in most cases. We previously noted that CLL cells exposed to microenvironmental agonists ex vivo exhibit resistance to VEN+IBR due to upregulation of multiple anti-apoptotic proteins (Jayappa et al. 2017). Here, we find in the circulation of CLL patients that activated (CD69Pos) leukemic B cells recently emigrated from the lymph node exhibit resistance to inhibitors of Bcl-2, Mcl-1, or Bcl-xL due to impaired activation of Bax/Bak proteins. Our molecular analysis suggests that multiple upregulated anti-apoptotic proteins (Mcl-1, Bcl-xL, and Bcl-2) swap for pro-apoptotic proteins (e.g. Bim) in these cells when subjected to single-drug treatments, leading to defective Bax/Bak activation resulting in resistance to drug induced apoptosis. A large-scale cell-based screen using small molecule agonist of Protein Phosphatase 2A (PP2A) (SMAP, TRC-382) revealed that blood cancer cell lines are highly sensitive to SMAP mediated activation of PP2A, a serine/threonine phosphatase known to regulate cell survival. A further pharmacologically optimized SMAP compound (DT061) showed activity even in leukemia cell lines or patient-derived CD69Pos CLL cells resistant to inhibitors of Bcl-2, Mcl-1, or Bcl-xL, suggesting PP2A activation can overcome apoptosis resistance. The PP2A activator DT061 induced apoptosis in these cells in the absence of Bax/Bak activation and was equally effective in an isogenic Bax/Bak double knockout cells, suggesting PP2A activation overcomes multi-drug resistance via induction of Bax/Bak-independent apoptosis. We next examined various Bax/Bak-independent apoptosis mechanisms using inhibitors, and found that inhibitors of mitochondrial permeability transition pores (mPTP) (NIM811 and cyclopsorin-A) blocked the DT061-induced apoptosis in CLL cells. Using the CalceinAM/CoCl2 assay, we noted that DT061 triggers mPTP opening in primary CLL cells, which was blocked by mPTP inhibitors, suggesting DT061 induces apoptosis via mPTP activation. In summary, microenvironmentally activated cancer cells displaying resistance to apoptosis due to defective Bax/Bak activation are found de novo in CLL patients. Reactivation of the tumor suppressive serine/threonine phosphatase by a series of small molecules (SMAPs) overcomes this resistance by inducing mPTP dependent apoptosis. Collectively, these findings demonstrate the existence of an anti-apoptotic multi-drug resistant pool of cancer cells in CLL patients, and validates a novel pharmaceutically tractable pathway to deplete this reservoir.
Citation Format: Kallesh D. Jayappa, Caroline Farrington, Vicki L. Gordon, Shekhar Saha, Christopher Morris, Krista M. Isaac, Timothy P. Bender, Michael E. Williams, Craig A. Portell, Goutham Narla, Michael J. Weber. The PP2A activation using a small molecule agonist triggers apoptosis by releasing mitochondrial permeability transition pores in multi-drug resistant leukemic B cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 937.