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A Pathway for Necrotic Cell Death

坏死细胞死亡的途径

基本信息

批准号：
10680459
负责人：
DAVID J SHAPIRO
金额：
$ 37.89万
依托单位：
UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-08-09 至 2027-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10680459
关键词：
Antineoplastic Agents Apoptosis Apoptosis Promoter Autophagocytosis Brain Breast Cancer Cell Breast cancer metastasis CRISPR screen Calcium Cell Culture Techniques Cell Death Cell Death Induction Cell Death Inhibition Cell Line Cell membrane Cells Cessation of life Clinical Clinical Trials Colon Colon Carcinoma Data Development Diapause Disseminated Malignant Neoplasm Drug Targeting Electroporation Embryo Endometrial Carcinoma Estrogen Receptor alpha Estrogens Goals Immune Immune checkpoint inhibitor Immunocompromised Host Immunotherapeutic agent Immunotherapy Knock-out Knowledge Link MYC Family Protein Malignant Neoplasms Malignant neoplasm of ovary Mammary Neoplasms Mediating Mediator Membrane Metastatic Neoplasm to the Bone Metastatic Neoplasm to the Liver Metastatic Neoplasm to the Lung Metastatic malignant neoplasm to brain Mitochondria Mus Necrosis Necrosis Induction Neoplasm Metastasis Oncolytic Osmosis Pathway interactions Patients Peptides Pharmaceutical Preparations Protein Synthesis Inhibition Proteins Recurrence Regulatory Pathway Resistance Retreatment Rupture Sodium Channel Stress Swelling Testing Therapeutic Tumor Markers Uterus Water Work Xenograft Model Xenograft procedure cancer cell cancer immunotherapy cancer therapy cancer type drug development endoplasmic reticulum stress flexibility genome-wide immunogenic immunogenic cell death malignant ascites malignant breast neoplasm mouse model neoantigens novel anticancer drug novel therapeutics protein biomarkers protein folding proteostasis receptor resistance mechanism response sensor success targeted agent therapeutic target therapy resistant transcriptome sequencing triple-negative invasive breast carcinoma tumor virtual

项目摘要

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.

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。