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，药物诱导的坏死细胞死亡的蛋白质介体尚不清楚。坏死会激活免疫细胞，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新途径。 BHPI 和 ErSO 通过雌激素受体 α (ERα) 起作用，诱导致死性坏死诱导的过度激活 ErSO 可消除原位异种移植物和 PDX 中的预期未折叠蛋白反应 (a-UPR)。原发性和转移性治疗耐药的 ERα+ 乳腺癌，诱导致死性几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通过针对 BHPI 和 ErSO 的 CRISPR 筛选，我们鉴定出 Ca2+ 激活，质膜 Na+ 通道 TRPM4 作为 BHPI 和 ErSO 用来诱导的刽子手蛋白坏死以及可能的膜灵活性调节剂 FGD3 和 ErSO 诱导的 Ca2+ 升高。 TRPM4 通道，引起外部 Na+、Cl- 和伴随的水快速流入。细胞，引起渗透应激，从而过度激活 UPR，导致 ATP 耗尽，FGD3 增强膜破裂和坏死细胞死亡消除了 ATP 耗尽，持续的 UPR。值得注意的是，TRPM4 敲除也抑制了由过度激活、细胞肿胀和死亡引起的坏死。不相关的抗癌疗法、线粒体靶向溶瘤肽、LTX-315、Ca2+通道靶向剂、Englerin A 和 Ca2+ 电穿孔 (CaEP) 目标 1. 鉴定和功能表征。我们将结合已完成的数据。 CRISPR 筛选、使用 LTX-315、Englerin A 和 CaEP 的新筛选以及我们最近的 RNA-seq 数据开发 ErSO 抗性细胞系目标 2。利用细胞和肿瘤研究，检验多样化的假设。诱导坏死的抗癌疗法，其中 Ca2+ 水平通过短暂的 a-UPR 激活或其他机制共享一个共同的途径，汇聚于 UPR-TRPM4-FGD3 途径。将 UPR 激活疗法扩展到 ERα-癌症，测试临床上有希望的、机制上的想法不起眼的坏死诱导疗法 Ca2+ 电穿孔部分通过 UPR-TRPM4-FGD3 发挥作用目标 3. 使用同基因小鼠模型确定是否存在坏死诱导剂。将免疫疗法的范围扩大到已转移至脑部并确实会导致快速致命的乳腺癌不表达新抗原目标 4. 对诱导坏死的癌症药物的耐药机制主要是使用我们的 Myc 下调可逆静止细胞，我们将鉴定 ErSO 耐药性。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 耗竭所致激活 AMPK，通过 eEF2 抑制蛋白质合成，这些研究将由此建立一个新的方法。免疫原性抗癌治疗通过 UPR 诱导坏死细胞死亡的途径。