Crosstalk of LKB1 and KEAP1 mutations in driving growth of lung adenocarcinoma

 描述(申请人提供)：近25%的肺腺癌(LuAD)存在LKB1基因的缺失或失活突变。作为对代谢压力的反应，野生型LKB1促进分解代谢反应，以产生ATP并保存抗氧化剂(NADPH和GSH)水平。由于LKB1可以抵消代谢应激引起的ROS，该基因在癌症中的失活似乎与预期相反，确实，LKB1缺陷细胞对癌基因转化具有相对抵抗力，对代谢应激敏感。TCGA测序数据显示，约50%的LKB1突变株LuAD也含有Kelch-ECH相关蛋白1(Keap1)突变。LKB1缺陷细胞适应营养和代谢压力的能力受损，能否通过Keap1的平行丢失来克服？我们的初步研究显示，在KrasG12D驱动的小鼠肺癌模型中，Lkb1和Keap1的联合丢失降低了ROS，显著提高了肿瘤生长(组织学上，腺癌)和死亡率。即使在没有致癌Kras信号的情况下，Keap1-/-Lkb1-/-动物也会在肺部形成潜伏期很长的肿瘤。重要的是，在缺乏Nrf2途径激活的情况下，LKB1缺陷的细胞表现出适应代谢应激的能力受损，但这种缺陷可以通过同时失去Keap1信号来修复。Keap1突变导致NRF2功能在NSCLC中获得，从而驱动抗氧化途径和代谢变化。我们假设，LKB1突变的肺腺癌细胞中的Keap1突变导致NRF2的获得，以用于ROS解毒和代谢途径的改变，这对肿瘤细胞的生存至关重要。为了利用这种适应性的脆弱性，我们假设在LKB1和Keap1丢失的肿瘤中抑制NRF2将由于代谢应激而减少肿瘤生长。具体目标1将确定在Lkb1缺乏的肺细胞中选择性地丢失Keap1信号是否维持细胞氧化还原动态平衡并促进肺肿瘤的发生。我们已经建立了选择性缺失Lkb1和Keap1的小鼠模型(有或没有致癌应激-KrasG12D)，表达Nrf2的转基因小鼠结合Lkb1缺失，以及LKB1突变的人肺腺癌细胞株Nrf2获得或Keap1功能丧失。具体目标2将确定Keap1信号丢失与Lkb1信号协同代谢改变以提供生存优势的机制。将进行转录体和稳定同位素分解代谢组学研究。具体目标3将确定在我们的SA1小鼠模型中干扰Nrf2信号是否会减少肿瘤生长并利用遗传和小分子方法提高存活率。除了基因敲除Nrf2，我们的初步研究显示了一种新的小分子(与NCATS合作开发)在体外抑制NRF2的有效性，并将探索其在治疗LKB1突变癌症中靶向Nrf2途径的潜力。这些研究将(A)提供为什么50%的LKB1丢失与LuAD中Keap1突变共存的分子理解(B)创造针对这种合作所必需的临床前知识。

 DESCRIPTION (provided by applicant): Nearly 25% of lung adenocarcinomas (LuAD) have deletions or inactivating mutations of the gene for LKB1. In response to metabolic stress, wildtype LKB1 promotes catabolic reactions for generating ATP and conserving antioxidant (NADPH and GSH) levels. Since LKB1 counteracts ROS resulting from metabolic stress, the inactivation of this gene in cancer seems to be contrary to expectations, and indeed, LKB1-deficient cells are relatively resistant to oncogenic transformation and sensitive to metabolic stress. TCGA sequencing data revealed that ~ 50% of LKB1-mutant LuAD also harbor a Kelch-ECH associated protein 1 (KEAP1) mutation. Can the impaired ability of LKB1-deficient cells to adapt to nutrient and metabolic stress be overcome by parallel loss of KEAP1? Our preliminary studies revealed that combined loss of Lkb1 and Keap1 decrease ROS and dramatically enhances tumor growth (histologically, adenocarcinomas), and mortality in KrasG12D driven mouse model of lung cancer. Even in the absence of oncogenic Kras signaling, Keap1-/-Lkb1-/- animals form tumors in the lungs with long latency. Importantly, LKB1-deficient cells showed impaired ability to adapt to metabolic stress in the absence of Nrf2 pathway activation, but this defect was rescued by simultaneous loss of Keap1 signaling. KEAP1 mutations lead to gain of NRF2 function in NSCLC that drives antioxidant pathways and metabolic alterations. We hypothesize that KEAP1 mutations in LKB1 mutated lung adenocarcinoma cells causes gain of NRF2 for ROS detoxification and metabolic pathway alterations, which are critical for tumor cell survival. To exploit the vulnerability of this adaptation, we hypothesize that inhibiting NRF2 in tumors with loss of LKB1 and KEAP1 will decrease tumor growth due to metabolic stress. Specific aim 1 will determine if selective loss of Keap1 signaling in Lkb1 deficient lung cells maintains cellular redox homeostasis and promotes lung tumorigenesis. We have developed mouse models with selective deletion of Lkb1 and Keap1 (with or without oncogenic stress - KrasG12D), transgenic mice expressing Nrf2 with activating mutation combined with Lkb1 deletion as well as LKB1 mutant human lung adenocarcinoma cell lines with gain of Nrf2 or loss of Keap1 function. Specific aim 2 will determine the mechanisms by which loss of Keap1 signaling cooperates with Lkb1 signaling for metabolic alterations to provide survival advantage. Transcriptomic and stable isotope resolved metabolomics studies will be performed. Specific aim 3 will determine if disruption of Nrf2 signaling in our mouse models of SA 1 will decrease tumor growth and improve survival using genetic as well as small molecule approach. In addition to genetic knockout of Nrf2, our preliminary studies have shown the efficacy of a novel small molecule (developed in collaboration with NCATS) for inhibiting NRF2 in vitro and in which will be explored for its potential for targeting the Nrf2 pathway in treating LKB1-mutant cancers. These studies will (a) provide the molecular understanding of why 50% of LKB1 loss coexist with KEAP1 mutations in LuAD (b) create the preclinical knowledge essential for targeting this cooperation.