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：
10432054
负责人：
Kirsten L Bryant
金额：
$ 31.54万
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/10432054
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 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.

自噬是一种自我降解过程，细胞可以有序地清除有缺陷的细胞器并循环利用大分子作为营养源。自噬水平升高，对于 KRAS 的致瘤性生长至关重要突变型胰腺导管腺癌（PDAC），为自噬的临床评估提供依据 PDAC 抑制剂羟氯喹 (HCQ)。令人失望的是，当用作单一疗法或联合疗法时按照护理标准，HCQ 对 PDAC 的临床疗效有限甚至没有。我们最近确定使用关键 KRAS 效应通路抑制剂治疗 PDAC，激活 RAF-MEK-ERK 有丝分裂蛋白激酶 (MAPK) 级联意外地导致自噬进一步升高，使 PDAC 急剧恶化依赖于这个过程，并且对自噬抑制高度敏感。我们确定 ERK 抑制损害其他关键过程，然后导致自噬的补偿性上调。我们的发现共同与另一项共同发表的独立研究得出的结论基本相同，导致启动了评估 MEK（曲美替尼、binimetinib）或 ERK（LY3214996）抑制剂与针对转移性 KRAS 突变 PDAC 的 HCQ。虽然早期观察到富有同情心地使用这种组合支持重大的临床影响，我们的初步研究支持我们的前提，即我们可以对此进行改进治疗。我们提出了三个目标，以进一步推进自噬抑制作为抗 RAS 治疗方法。首先，我们将确定 ERK MAPK + HCQ 组合在 KRAS/NRAS/BRAF 中是否同样有效- 突变 CRC（目标 1）。 HCQ 是一种溶酶体抑制剂，因此对自噬没有选择性。我们假设 ULK1/2 丝氨酸/苏氨酸蛋白激酶抑制剂（饥饿诱导自噬的关键启动子）将作为更特异性的自噬抑制剂。然而，与所有蛋白激酶抑制剂一样，抑制剂诱导的补偿机制将促进对 ULK 抑制剂功效的抵抗。此外，还提供全面的 ULK1/2底物的测定仍有待完成。因此，我们将确定直接和 ULK 抑制对磷酸蛋白质组和激酶组的补偿作用，以严格评估 ULK 抑制剂作为自噬抑制剂（目标 2）。我们的目标 3 研究基于我们对 2,500 个基因可成药药物的应用基因组 CRISPR/Cas9 遗传功能丧失筛选，以鉴定调节 CQ 抗肿瘤活性的基因。所确定的增强或降低 CQ 生长抑制活性的命中代表候选组合或 CQ 抗性的生物标志物。我们已经确定了 DNA 损伤反应的介质细胞周期调节因子作为两大类抗性促进基因。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，并有助于开发针对自噬的新型联合疗法来治疗 KRAS 突变癌症。