SAT-302 Acquired Resistance to Breast Cancer Therapy is a Dynamic Multi-step Evolutionary Process

Abstract Disclosure: M. Jabeen: None. X. Dai: None. J. Zhu: None. P. Hergenrother: SAB Systems Oncology. D.J. Shapiro: None. Classically, the development of resistance to therapies for estrogen receptor (ER) positivebreast cancer, is thought to often involve a single mutational change in ER. In contrast,detailed time course analysis of the process by which ER positive breast cancer cellsbecome resistant to the promising small molecule anticancer drug, ErSO, shows that theacquisition of resistance is a dynamic multi-step process in which cells initially acquireresistance through relatively high frequency events that result in poor growth and cellfitness. These cells are rapidly and progressively replaced by resistant cells with other,apparently lower frequency, changes until resistant cells acquire near wild-type growthrates. We identified the ErSO compounds that work by inducing lethal hyperactivation ofthe normally tumor protective anticipatory unfolded protein response (a-UPR). In ERpositive breast cancer cells key steps in ErSO action are initial activation through anER:Src:PLC complex, resulting in calcium release from endoplasmic reticulum stores,activation of the TRPM4 sodium channel, leading to cell swelling, plasma membranerupture and necrotic cell death. In orthotopic breast and ovarian cancer mousexenografts, ErSO often induced complete regression without recurrence of large primarytumors and most metastases and was effective in PDOs and fresh patient tumor samples.In cell culture and PDOs, we generated more than 10 clonal ErSO-resistant lines.Unexpectedly the resistant clones did not arise from a large pool of non-dividing persistercells and instead derived from a few single survivor cells with initial very slow proliferation,leading to clonal outgrowth. Surprisingly, the initial resistance event in most of the cloneswas massive overexpression of progesterone receptor (PR). PR was known to bind Srctightly, enabling it to sequester Src away from the ErSO activated ER:Src:PLC complex.But overexpression of PR is toxic to cells, and resulted in very slow growing resistantcells. We found that within weeks, these slow-growing clones lost overexpression of PRand engaged other mechanisms that enabled both ErSO-resistance and more robustproliferation. From multiple cycles of RNA-seq done on more than 10 clonal lines asresistance progressed, we obtained snapshots of the evolution of resistance. We did notobserve down-regulator of the intensively studied regulator of plasma membrane rupture,NINJ1. Instead, down-regulation of the TRPM4 sodium channel, other transporters andsystems that might enhance cell survival progressively evolved. These data suggest thatthe evolution of resistance to anticancer agents may often be a dynamic multi-stepprocess rather than a single rare event and that there may be different, potentiallytargetable, vulnerabilities in cancer cells as they progressively evolve resistance toanticancer therapies. Presentation: Saturday, July 12, 2025