Mechanistic dissection and inhibitor targeting of autophagy in RAS driven cancers

RAS 驱动的癌症中自噬的机制剖析和抑制剂靶向

基本信息

批准号：
10200721
负责人：
Kirsten L Bryant
金额：
$ 44.69万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-07-01 至 2025-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10200721
关键词：
APC gene Acute Address Apoptotic Autophagocytosis BRAF gene Biological Biological Markers CRISPR/Cas technology Cancer Etiology Cell Cycle Cell Cycle Progression Cell Cycle Regulation Cells Cessation of life Clinical Clinical Trials Colorectal Cancer Combined Modality Therapy DNA Damage DNA Repair Dependence Development Dissection Drug Combinations Eating Frequencies Future Genes Genetic Genome Growth Hydroxychloroquine Hypersensitivity Impairment In Vitro KRAS2 gene Knowledge Label Large Intestine Carcinoma Lead Light Lysosomes MAPK3 gene MEKs Malignant Neoplasms Malignant neoplasm of pancreas Mediator of activation protein Metabolic Metabolism Mitogen-Activated Protein Kinases Mutate Mutation Nutrient Oncoproteins Organelles Organoids Pancreatic Ductal Adenocarcinoma Pathway interactions Patients Pharmacology Pharmacotherapy Process Protein Kinase Protein-Serine-Threonine Kinases Proteomics Publishing Regulation Reporting Resistance Role Source Starvation Therapeutic Up-Regulation base cancer cell cancer therapy cancer type clinical candidate clinical efficacy effectiveness evaluation improved inhibition of autophagy inhibitor/antagonist loss of function macromolecule member mouse model mutant new combination therapies novel pancreatic ductal adenocarcinoma cell pancreatic ductal adenocarcinoma model protein kinase inhibitor research clinical testing resistance gene response standard of care targeted treatment tumor tumor progression tumorigenic

项目摘要

Autophagy is a self-degradation process whereby cells can orderly clear defective organelles and recycle macromolecules as a nutrient source. Autophagy is elevated and essential for the tumorigenic growth of KRAS- mutant pancreatic ductal adenocarcinoma (PDAC), providing the rationale for clinical evaluation of the autophagy inhibitor hydroxychloroquine (HCQ) for PDAC. Disappointingly, when used as monotherapy or in combination with standard of care, HCQ has shown limited to no clinical efficacy for PDAC. We recently determined that the treatment of PDAC with inhibitors of the key KRAS effector pathway, the RAF-MEK-ERK mitogenic activated protein kinase (MAPK) cascade, unexpectedly caused further elevation of autophagy, rendering PDAC acutely dependent on this process, and hypersensitive to autophagy inhibition. We determined that ERK inhibition impaired other critical processes that then led to compensatory upregulation of autophagy. Our findings, together with essentially identical conclusions by another independent co-published study, have led to the initiation of clinical trials evaluating either MEK (trametinib, binimetinib) or ERK (LY3214996) inhibitor in combination with HCQ for metastatic KRAS-mutant PDAC. While early observations from compassionate use of this combination support a significant clinical impact, our preliminary studies support our premise that we can improve upon this therapy. We propose three aims to further advance autophagy inhibition as an anti-RAS therapeutic approach. First, we will determine if the ERK MAPK + HCQ combination will be similarly effective in KRAS/NRAS/BRAF- mutant CRC (Aim 1). HCQ is a lysosome inhibitor and consequently not selective for autophagy. We hypothesize that inhibitors of the ULK1/2 serine/threonine protein kinases, key initiators of starvation-induced autophagy, will act as more specific autophagy inhibitors. However, as with all protein kinase inhibitors, inhibitor-induced compensatory mechanisms will promote resistance to ULK inhibitor efficacy. Additionally, a comprehensive determination of ULK1/2 substrates remains to be completed. Thus, we will determine the direct and compensatory effects of ULK inhibition on the phosphoproteome and kinome to critically evaluate ULK inhibitors as autophagy inhibitors (Aim 2). Our Aim 3 studies are based on our application of a 2,500-gene druggable genome CRISPR/Cas9 genetic-loss-of-function screen to identify genes that modulate CQ anti-tumor activity. The identified hits that either enhance or reduce CQ growth inhibition activity represent candidate combinations or biomarkers for CQ resistance, respectively. We have identified mediators of the DNA damage response and cell cycle regulators as two major classes of resistance-promoting genes. We will mechanistically dissect these relationships and determine how inhibition of members of these pathways influences autophagic flux. In summary, our studies will enhance our understanding of autophagy regulation in cancer and aid in the development of novel combination therapies to target autophagy for the treatment of KRAS-mutant cancers.

自噬是一个自降解过程，细胞可以有序清除有缺陷的细胞器并回收大分子作为营养来源。自噬升高，对于Kras-的致瘤性生长至关重要突变的胰腺导管腺癌（PDAC），为自噬的临床评估提供了理由 PDAC的抑制剂羟氯喹（HCQ）。令人失望的是，当用作单一疗法或组合时有了护理标准，HCQ已显示出PDAC的临床疗效不限。我们最近确定用关键KRAS效应途径的PDAC处理PDAC，RAF-MEK-ERK有丝分裂激活了蛋白激酶（MAPK）级联反应出乎意料地导致自噬的进一步升高，使PDAC急性取决于此过程，并且对自噬抑制过敏。我们确定ERK抑制作用损害了其他关键过程，然后导致自噬的补偿性上调。我们的发现，一起通过另一项独立共同发表的研究基本相同的结论，导致了评估MEK（Trametinib，Binimetinib）或ERK（LY3214996）抑制剂的临床试验 HCQ用于转移性Kras突变PDAC。而这种组合的富有同情使用的早期观察支持重大的临床影响，我们的初步研究支持我们可以改进的前提治疗。我们提出三个目标，旨在进一步推进自噬作为一种抗RAS治疗方法。首先，我们将确定ERK MAPK + HCQ组合是否在KRAS/NRAS/BRAF-中类似地有效突变CRC（AIM 1）。 HCQ是一种溶酶体抑制剂，因此对自噬不是选择性。我们假设 ULK1/2丝氨酸/苏氨酸蛋白激酶的抑制剂，饥饿诱导的自噬的主要启动者将充当更具体的自噬抑制剂。但是，与所有蛋白激酶抑制剂一样，抑制剂诱导补偿机制将促进对ULK抑制剂功效的抵抗力。另外，这是一个全面的 ULK1/2底物的确定仍有待完成。因此，我们将确定直接和 ULK抑制对磷酸蛋白质组和Kinome的补偿性影响，以评估ULK抑制剂作为自噬抑制剂（AIM 2）。我们的目标3研究基于我们的2500基因可药品的应用基因组CRISPR/CAS9遗传功能筛选，以鉴定调节CQ抗肿瘤活性的基因。确定的命中可以增强或减少CQ生长抑制活性代表候选组合或分别用于CQ抗性的生物标志物。我们已经确定了DNA损伤反应的介体和细胞周期调节因子作为两种主要的抗性促进基因。我们将机械地剖析这些关系并确定这些途径成员的抑制如何影响自噬通量。在总而言之，我们的研究将增强我们对癌症自噬调节的理解，并有助于开发新型组合疗法以靶向自噬来治疗KRAS突变癌。