Mechanistic dissection and inhibitor targeting of autophagy in RAS driven cancers

自噬是一种自我降解的过程，细胞可以有序地清除有缺陷的细胞器并循环利用 作为营养源的大分子。自噬升高，对KRAS的致瘤生长至关重要- 突变型胰腺导管腺癌(PDAC)，为临床评估自噬提供了理论基础 PDAC的抑制剂羟基氯喹(HCQ)。令人失望的是，当作为单一疗法或联合使用时 有了标准的护理，HCQ对PDAC的临床疗效有限。我们最近确定， 用关键KRAS效应通路抑制剂治疗PDAC，激活RAF-MEK-ERK有丝分裂原 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 依赖于这一过程，并对自噬抑制过敏。我们确定ERK的抑制作用 损害了其他关键过程，从而导致自噬的补偿性上调。我们的发现加在一起 与另一项独立的联合发表的研究得出的基本相同的结论，导致了 评估MEK(曲美替尼，比尼美替尼)或ERK(LY3214996)抑制剂与 转移性KRAS突变体PDAC的HCQ。虽然早期对这一组合的同情使用观察到 支持显著的临床影响，我们的初步研究支持我们可以在此基础上改进的前提 心理治疗。我们提出了三个目标，以进一步推进自噬抑制作为抗RAS的治疗方法。 首先，我们将确定ERK MAPK+HCQ组合是否在KRAS/NRAS/BRAF中同样有效- 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/苏氨酸蛋白激酶的抑制剂，饥饿诱导自噬的关键启动者，将 作为更具体的自噬抑制剂。然而，与所有的蛋白激酶抑制剂一样，抑制剂诱导 代偿机制将促进对ULK抑制剂疗效的抵抗。此外，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抑制对磷酸化蛋白质组和动态组的代偿作用评价ULK抑制剂 作为自噬抑制剂(目标2)。我们的Aim 3研究是基于我们对一种2500基因可药物的应用 基因组CRISPR/Cas9基因功能丧失筛查以确定调节CQ抗肿瘤活性的基因。 识别的增强或降低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损伤反应的介体和 细胞周期调节基因作为两类主要的抗药性促进基因。我们将机械地剖析这些 并确定抑制这些途径的成员如何影响自噬通量。在……里面 综上所述，我们的研究将加深我们对癌症自噬调控的理解，并有助于 靶向自噬治疗KRAS突变癌症的新型联合疗法的发展。

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.