Unlike other death pathways, protein mediators of drug-induced necrotic cell death were poorly defined.
Necrosis activates immune cells, inducing immunogenic cell death. Therefore, understanding necrosis
provides new avenues for enhancing drug development and cancer immunotherapy. Our anticancer drugs
BHPI and ErSO act via estrogen receptor a (ERa) to induce lethal necrosis-inducing hyperactivation of the
anticipatory Unfolded Protein Response (a-UPR). In orthotopic xenografts and a PDX, ErSO eradicates
primary and metastatic therapy-resistant ERa+ breast cancer, induces near complete regression of lethal
breast cancer in brain, and of endometrial cancer and ovarian cancer, and kills most ovarian cancer cells in
patient malignant ascites. From CRISPR screens against BHPI and ErSO, we identified the Ca2+ activated,
plasma membrane Na+ channel TRPM4 as the executioner protein that BHPI and ErSO use to induce
necrosis and the likely membrane flexibility modulator FGD3. BHPI and ErSO-induced elevated Ca2+ opens
the TRPM4 channel, eliciting a rapid influx of external Na+, Cl- and accompanying water. This swells the
cells, causing osmotic stress, which hyperactivates the UPR, leading to ATP depletion, FGD3 enhanced
membrane rupture and necrotic cell death. TRPM4 knockout abolished ATP depletion, sustained UPR
hyperactivation, cell swelling and death. Notably, TRPM4 knockout also inhibited necrosis induced by
unrelated anticancer therapies, the mitochondrial targeting oncolytic peptide, LTX-315, the Ca2+ channel
targeting agent, Englerin A and Ca2+ electroporation (CaEP). Aim 1. Identify and functionally characterize
known and additional shared components of the TRPM4 pathway. We will combine data from completed
CRISPR screens, new screens using LTX-315, Englerin A, and CaEP and RNA-seq data from our recently
developed ErSO resistant cell lines. Aim 2. Using cell and tumor studies, test the hypothesis that diverse
necrosis-inducing anticancer therapies, in which Ca2+ levels are increased by transient a-UPR activation or
other mechanisms, share a common pathway that converges on the UPR-TRPM4-FGD3 pathway. To
extend UPR activation therapies to ERa- cancers, test the idea that the clinically promising, mechanistically
obscure, necrosis-inducing therapy, Ca2+ electroporation, works in part through the UPR-TRPM4-FGD3
necrosis pathway. Aim 3. Using syngeneic mouse models establish whether necrosis-inducing agents
extend the reach of immunotherapy to rapidly lethal breast cancer that has metastasized to brain and does
not express neoantigens. Aim 4. Mechanisms of resistance to necrosis inducing cancer drugs are largely
unexplored. Using our Myc down-regulated reversibly quiescent cells, we will identify ErSO resistance
mechanisms and test whether loss of Myc in the quiescent cells is due to a-UPR mediated ATP depletion
activating AMPK, thereby inhibiting protein synthesis via eEF2. These studies will establish a new
pathway of immunogenic anticancer therapy-induced necrotic cell death through the UPR.