A Pathway for Necrotic Cell Death

与其他死亡途径不同，药物诱导的坏死性细胞死亡的蛋白质介体的定义不明确。 坏死激活免疫细胞，诱导免疫原性细胞死亡。因此，认识坏死 为加强药物开发和癌症免疫治疗提供了新的途径。我们的抗癌药物 BHPI和ERSO通过雌激素受体α(ERα)诱导致死性坏死诱导的细胞过度激活 预期未折叠蛋白反应(a-UPR)。在异种原位移植和PDX中，ERSO被根除 原发和转移耐药ERα+乳腺癌，导致致命性近乎完全消退 脑部乳腺癌，子宫内膜癌和卵巢癌，并杀死大多数卵巢癌细胞在 病人恶性腹水。从抗BHPI和ERSO的CRISPR筛选中，我们鉴定了钙激活的， 质膜Na+通道TRPM4作为BHPI和ERSO诱导的执行者蛋白 坏死和可能的膜弹性调节剂FGD3。BHPI和ERSO诱导的钙离子开放 TRPM4通道，引发外部Na+、Cl-和伴随水的快速内流。这扩大了 细胞，导致渗透应激，从而过度激活UPR，导致ATP耗竭，FGD3增强 胎膜破裂和坏死细胞死亡。TRPM4基因敲除消除了ATP耗竭，持续了UPR 过度活跃，细胞肿胀和死亡。值得注意的是，TRPM4基因敲除也抑制了由 无关的抗癌治疗，线粒体靶向溶瘤多肽LTX-315，钙通道 靶向剂、恩格列菌素A和钙离子电穿孔(CAEP)。目标1.确定并确定功能特征 TRPM4途径的已知和其他共享组件。我们将合并来自Complete的数据 CRISPR屏幕，使用LTX-315、Engerin A和CAEP以及来自我们最近的RNA-SEQ数据的新屏幕 开发了耐ERSO的细胞系。目标2.使用细胞和肿瘤研究，检验不同的假设 诱导坏死的抗癌治疗，其中钙离子水平通过短暂的a-UPR激活或 其他机制共享一个共同的通路，该通路汇聚在UPR-TRPM4-FGD3通路上。至 将UPR激活疗法扩展到ER-α癌症，测试临床上有希望的，机械性的 晦涩难懂的坏死诱导疗法，钙离子电穿孔，部分通过UPR-TRPM4-FGD3发挥作用 坏死途径。目的3.利用同基因小鼠模型建立坏死诱导剂 将免疫治疗的范围扩大到迅速致命的乳腺癌，这种乳腺癌已经转移到脑内，并 不表达新抗原。目的4.抗坏死致癌药物的机制主要是 未被开发的。利用我们的Myc下调的可逆静止细胞，我们将鉴定ERSO抗性 静止期细胞Myc丢失的机制及检测是否与a-UPR介导的ATP耗竭有关 激活AMPK，从而抑制通过eEF2的蛋白质合成。这些研究将建立一个新的 免疫原性抗癌治疗通过UPR诱导坏死细胞死亡的途径。

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 α (ERα) 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 ERα+ 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 ERα- 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.