OR16-06 A Novel Pathway Controlling Anticancer Drug Induced Immunogenic Necrosis In ERα Positive Cancer Cells
Authors: Zhu Junyao , Ghosh Santanu , Duraki Darjan , Boudreau Matthew , Jabeen Musarrat , Mao Chengjian , Park Ben Ho , Cheng Georgina , Nelson Erik Russell , Hergenrother Paul J , Shapiro David J 
Published: October 2023

Abstract
Disclosure: J. Zhu: None. S. Ghosh: None. D. Duraki: None. M. Boudreau: Other; Self; Patent on ErSO. M. Jabeen: None. C. Mao: None. B. Park: None. G. Cheng: None. E.R. Nelson: None. P.J. Hergenrother: Advisory Board Member; Self; System Oncology. Other; Self; Patent on ErSO. D.J. Shapiro: Other; Self; Patent on ErSO.
Acting through estrogen receptor alpha (ER), in orthotopic mouse xenograft models and a PDX, our non-competitive anticancer drug ErSO induces complete or near complete regression of primary and metastatic therapy-resistant ERα-positive breast, ovarian and endometrial tumors. ErSO is also highly effective in ovarian cancer PDOs from patient ascites. Unlike most anticancer agents which inhibit cancer cell proliferation or induce apoptosis, ErSO induces immune-cell-activating necrosis through ERα dependent sustained hyperactivation of the anticipatory unfolded protein response (a-UPR) pathway resulting in ATP depletion and cell swelling followed by membrane rupture and necrotic cell death. The medium from cancer cells killed by ErSO not only robustly activates mouse and human macrophages, but also dramatically increases monocyte migration. ErSO therefore has the potential to help extend the reach of immunotherapy to many solid tumors that do not express neoantigens. However, unlike apoptosis which has an established signaling pathway, how ErSO-induced necrosis was elusive. From a genome-wide CRISPR-Cas9 screen in MCF7 cells with negative selection against ErSO, we identified a guanine exchange factor (GEF) protein as a top target. Consistent with the screen, through subsequent knockout and overexpression of this protein in human breast cancer cells, we found that the knockout cells have significant resistance to ErSO while the overexpressing cells exhibit increased sensitivity to killing by ErSO. Our data indicates that this protein is not an upstream regulator of the ErSO-induced necrosis pathway. Instead, it is a regulator of the last stage of necrosis - cell membrane rupture. Thus, we identified a cytoskeleton-modifying signaling pathway with multiple components that plays a pivotal role in whether the breast cancer cell responds to a-UPR activation and swelling by membrane rupture and necrotic cell death or reorganizes its cytoskeleton enabling the cancer cell to survive. RNA-seq and other studies indicate that by regulating cytoskeleton organization, this axis is important in ErSO-persister breast cancer cells exhibiting long-term survival in ErSO. Unlike wild type and overexpressing cells, the knockout cells and persister cells display the persister cell phenotype - rapid re-growth of the cancer cells once ErSO is removed. This work enables identification of breast cancer patients whose elevated levels of components of this signaling pathway make them most likely to benefit from this novel therapy. Here, we describe a therapy that induces necrosis and immunogenic cell death in ERα-positive breast, ovarian and endometrial cancer cells. We show that, as cancer cells respond to this unique therapeutic approach, a cytoskeleton regulating pathway plays a critical role in life-death decisions.
Presentation: Friday, June 16, 2023