Non-oncogene addiction in cancer cells

背景。我们将NOA定义为癌细胞对生存的应激反应途径的依赖性增加。癌细胞的恶性状态与一系列独特的致癌应激表型相关，包括DNA损伤、代谢应激、蛋白质毒性应激、炎症微环境和免疫监视。致癌应激使癌细胞更加依赖于应激反应途径生存，因此，对这些应激反应途径的破坏更加敏感。相比之下，体内的正常细胞不会经历致癌压力，因此正常细胞对压力反应途径的扰动不那么敏感。癌症与正常细胞依赖应激反应途径的根本差异形成了NOA的理论基础。NOA不同于癌基因依赖，因为应激反应途径基因在癌症基因组中很少发生突变。NOA代表了肿瘤细胞中广泛的合成致死和副依赖机制，靶向NOA可以为目前的肿瘤靶向治疗提供正交治疗方法。目标：这个项目的目标是1.)使用遗传筛选和基于假设的方法确定癌细胞中的NOA基因和遗传途径；2)研究NOA现象的分子机制，了解其在肿瘤发生状态中的重要性；3)探讨NOA作为潜在抗癌药物靶点的治疗意义。我们将主要使用携带KRAS癌基因的癌细胞作为我们的模型系统来实现这些目标，因为KRAS突变肿瘤通常缺乏有效的治疗方法。主要活动、重要成果和关键成果。1)。KRAS突变细胞对RNA剪接因子ERH的NOA。通过KRAS突变细胞中的合成致死性RNAi筛选，我们发现ERH基因是KRAS突变细胞中的合成致死性伴侣。ERH是一种进化上保守的蛋白，其功能尚不清楚。通过质谱分析，我们发现ERH与剪接体蛋白SNRPD3相关，因此是RNA剪接机制的一个组成部分。我们发现ERH对于包括CENPE在内的一组有丝分裂基因的正确剪接和表达是必需的，这对于维持KRAS突变细胞的染色体稳定性至关重要。这项工作定义了ERH蛋白的新功能，揭示了KRAS突变细胞中先前未知的RNA剪接机制的NOA。这项工作表明，选择性地干扰RNA剪接以破坏有丝分裂蛋白的平衡可能是靶向KRAS突变癌细胞的潜在方法。这项工作已经完成并出版了。2)。KRAS突变细胞的NOA向SUMO途径转化生长。通过KRAS突变细胞的合成致死RNAi筛选，我们发现了SUMO通路，特别是SUMO E2连接酶UBE2，在支持KRAS突变癌细胞的生存和转化生长中发挥重要作用。使用质谱法，我们确定了多种蛋白质，其SUMOylation以kras依赖的方式被破坏。通过基因拯救方法，我们发现了几种SUMO靶蛋白，包括KAP1、CHD1和EIF3L，在非锚定条件下对KRAS突变细胞的生存能力至关重要。这项工作确定了SUMO通路在KRAS驱动的肿瘤发生中的新作用，并表明SUMO通路是KRAS突变肿瘤的潜在药物靶点。这项工作已经发表。我们目前正在探索小分子SUMO抑制剂在KRAS突变癌临床前模型中的作用。3) KRAS突变细胞对自噬的NOA。通过对KRAS突变细胞基因依赖性的组合RNAi分析，我们分别确定了Ras信号网络和应激反应通路中关键的OA和NOA组分。我们发现CRAF/RAF1是突变型KRAS的主要促癌因子，自噬E1连接酶ATG7是主要的NOA。这项工作确定了自噬途径与CRAF作为KRAS突变细胞的合理组合的共同靶标。这项工作已经发表。我们目前正在研究自噬促进KRAS突变肿瘤生长和存活的机制。

BACKGROUND. We define NOA as increased dependency of cancer cells on stress-response pathways for survival. The malignant state of the cancer cell is associated with a unique set of oncogenic stress phenotypes involving DNA damage, metabolic stress, proteotoxic stress, inflammatory microenvironment, and immune surveillance. Oncogenic stress renders cancer cells more dependent on stress-response pathways for survival and consequently, more sensitive to the disruption of these stress-response pathways. In contrast, normal cells in the body do no experience oncogenic stress, therefore normal cells are much less sensitive to the perturbation of stress-response pathways. This fundamental difference in cancer vs. normal cell's dependency on stress-response pathways forms the theoretical basis of NOA. NOA is distinct from oncogene dependency because stress response pathway genes are rarely mutated in the cancer genome. NOA represents a broad category of synthetic lethal and collateral dependency mechanisms in cancer cells, and targeting NOA could offer orthogonal therapeutic approaches to current targeted treatment of tumors. OBJECTIVES: The objectives of this project are 1.) Identify NOA genes and genetic pathways in cancer cells using genetic screen and hypothesis-based approaches; 2) Investigate the molecular mechanism that underly a NOA phenomenon and understand its essentiality to the oncogenic state; and 3) Explore the therapeutic implication of NOA as potential cancer drug targets. We will carry out these objectives primarily using cancer cells harboring the KRAS oncogene as our model system as KRAS mutant tumors have generally lacked effective therapies. MAJOR ACTIVITIES, SIGNIFICANT RESULTS AND KEY OUTCOMES. 1.) NOA of KRAS mutant cells to the RNA splicing factor ERH. Through a synthetic lethal RNAi screen in KRAS mutant cells, we have identified the ERH gene as a synthetic lethal partner in KRAS mutant cells. ERH is an evolutionarily conserved protein with poorly understood function. Using mass-spectrometry, we identified ERH associates with the spliceosome protein SNRPD3 and is therefore a component of the RNA splicing machinery. We showed that ERH is required for the proper splicing and expression of a subset of mitotic genes including CENPE that are critical for maintaining chromosomal stability in KRAS mutant cells. This work defined a new function for the ERH protein uncovered a previously unknown NOA in KRAS mutant cells to the RNA splicing machinery. This work suggests that selective perturbation of RNA splicing to disrupt the balance of mitotic proteins could be a potential approach to target KRAS mutant cancer cells. This work has been completed and published. 2.) NOA of KRAS mutant cells to the SUMO pathway for transformed growth. Through a synthetic lethal RNAi screen in KRAS mutant cells, we have identified the SUMO pathway, particularly the SUMO E2 ligase UBE2, to play an important role in supporting the viability and transformation growth of KRAS mutant cancer cells. Using mass-spectrometry, we identified multiple proteins whose SUMOylation are disrupted in a KRAS-dependent manner. Using gene rescue approaches, we showed that several SUMO target proteins, including KAP1, CHD1 and EIF3L, are critical for the viability of KRAS mutant cells under anchorage independent conditions. This work identifies a new role of the SUMO pathway in KRAS-driven oncogenesis and suggests the SUMO pathway as a potential drug target for KRAS mutant tumors. This work has been published. We are currently exploring the effect of small-molecule SUMO inhibitors in preclinical models of KRAS mutant cancer. 3) NOA of KRAS mutant cells to autophagy. Through a combinatorial RNAi analysis of the gene dependency landscape in KRAS mutant cells, we identified critical OA and NOA that components in the Ras signaling network and in stress-response pathways, respectively. We found CRAF/RAF1 as the major onco-effector of mutant KRAS and the autophagy E1 ligase ATG7 as a major NOA. This work identifies the autophagy pathway as a co-target with CRAF as a rational combination for KRAS mutant cells. This work has been published. We are currently investigating the mechanism by which autophagy contributes to the growth and survival of KRAS mutant tumors.