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质定义不清。 坏死激活免疫细胞，诱导免疫原性细胞死亡。因此，了解坏死 为增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超激活， 预期未折叠蛋白反应（a-UPR）。在原位异种移植物和PDX中，ErSO根除 原发性和转移性治疗耐药ERα+乳腺癌，诱导致死性肿瘤几乎完全消退， 乳腺癌，子宫内膜癌和卵巢癌，并杀死大多数卵巢癌细胞， 患者恶性腹水。从针对BHPI和ErSO的CRISPR筛选中，我们鉴定了Ca 2+激活， 质膜Na+通道TRPM 4作为BHPI和ErSO用于诱导 坏死和可能的膜柔性调节剂FGD 3。BHPI和ErSO诱导的升高的Ca 2+开放 TRPM 4通道，引发外部Na+，Cl-和伴随的水的快速流入。这使 细胞，引起渗透压，过度激活UPR，导致ATP耗尽，FGD 3增强 膜破裂和坏死细胞死亡。TRPM 4敲除消除ATP耗竭，持续UPR 过度活化细胞肿胀和死亡值得注意的是，TRPM 4敲除还抑制了由细胞凋亡诱导的坏死。 不相关的抗癌疗法，线粒体靶向溶瘤肽，LTX-315，Ca 2+通道 靶向剂、Englerin A和Ca 2+电穿孔（CaEP）。目标1.识别和功能表征 TRPM 4途径的已知和额外的共享组分。我们将联合收割机的数据从完成 CRISPR筛选，使用LTX-315，Englerin A和CaEP和RNA-seq数据的新筛选，来自我们最近的 开发了ErSO抗性细胞系。目标2.利用细胞和肿瘤研究，测试不同的假设， - 诱导坏死的抗癌疗法，其中Ca 2+水平通过瞬时a-UPR活化而增加，或 其他机制，共享一个共同的途径，收敛于UPR-TRPM 4-FGD 3途径。到 将UPR激活疗法扩展到ERα-癌症，测试临床上有前途的， 一种模糊的坏死诱导疗法，Ca 2+电穿孔，部分通过UPR-TRPM 4-FGD 3起作用。 坏死途径目标3.使用同系小鼠模型确定坏死诱导剂是否 将免疫治疗的范围扩大到快速致命的乳腺癌，这些乳腺癌已经转移到大脑， 不表达新抗原。目标4。对坏死诱导癌症药物的抗性机制主要是 未开发的使用我们的Myc下调可逆性静止细胞，我们将鉴定ErSO抗性 机制，并测试静止细胞中Myc的丢失是否是由于a-UPR介导的ATP消耗 激活AMPK，从而通过eEF 2抑制蛋白质合成。这些研究将建立新的 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.