Abstract 4606: Heat shock proteins in immunosuppressive tumor microenvironment
Authors: Alkan Fulya Koksalar , Wilson Justin , Benson Elayne , Patel Tulshi , McEvoy Virginia , Francois Nate , Nguyen Emma , Alkan Hilmi Kaan , Chadli Ahmed , Gestwicki Jason , Korkaya Hasan 
Published: April 2023

Abstract
We previously demonstrated that upregulation of A20 in TNBC subtype in response to TNFα protects these cells from cytotoxic cell death by upregulating HSP70 protein and maintaining EMT/CSC phenotype. In contrast, luminal MCF7 or ZR75-1 cells display approximately 70% apoptosis when treated with TNFα. Overexpression of A20 in luminal cells not only protected them from TNFα-induced cytotoxicity by upregulating HSP70 and EMT/CSC phenotype, but also exhibited aggressive metastatic properties in mouse xenograft models.
We determined that TNFα-induced HSP70 upregulation in TNBC cell lines was dependent on A20 de-ubiquitinase activity that protected its degradation. Interestingly, our preliminary findings also suggested that A20 protein upregulation may be dependent on HSP70 chaperone activity. We show significant overexpression of HSP70 and A20 proteins in 4T1 cell line when treated with TNFα or chemotherapeutic agents. However, A20 expression is significantly reduced when we block HSP70 activity in cells treated with TNFα or chemotherapeutic agents (Docetaxel-DTX or Doxorubicin-DOX). We proposed that A20 transcriptional upregulation upon TNFα stimulation leads to suppression of E3-ligase and accumulation of HSP70 which then stabilizes A20 with chaperone activity. Based on our reasoning, we performed the LIMBO chaperone binding assay which predicted A20 being the potential HSP70 client protein.
Furthermore, we show that A20/HSP70 pathway attracts tumor-infiltrating lymphocytes (TILs) while inducing the accumulation of immunosuppressive MDSCs in syngeneic mouse models. Interestingly, pulmonary DTCs as well as the immune infiltrates from 4T1 tumor-bearing mice exhibited significantly higher HSP70 expression. Therefore, targeting HSP70 will have a dual activity on tumors and MDSCs and thus it may potentiate the efficacy of immunotherapy in preclinical models of breast cancer. As previously reported, murine 4T1 tumors fail to respond to check point inhibitors. We reasoned that this may be an appropriate model to test the efficacy of HSP70 inhibitor, JG-231. Expectedly, there was no difference in tumor growth and metastasis between control and anti-PDL1 treated animals, however, combination of anti-PDL1 antibody with JG-231 and chemotherapy (cyclophosphamide-CTX) significantly reduced primary tumor growth (&gt;10 fold) and eliminated metastasis. Collectively, our pilot experiments provide a strong rationale for testing our hypothesis and may lead to a rapid translation into the clinical utility.
Citation Format: Fulya Koksalar Alkan, Justin Wilson, Elayne Benson, Tulshi Patel, Virginia McEvoy, Nate Francois, Emma Nguyen, Hilmi Kaan Alkan, Ahmed Chadli, Jason Gestwicki, Hasan Korkaya. Heat shock proteins in immunosuppressive tumor microenvironment. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4606.