Target discovery and combination therapy in KRAS mutant cancer

背景资料。KRAS癌基因是很大一部分肺癌、胰腺癌和结直肠癌的驱动突变。最近，针对KRAS-G12C突变的抑制剂取得突破，这是几十年来第一次针对RAS的靶向治疗。对于其他KRAS突变肿瘤，还没有有效的靶向治疗。癌症患者对KRAS抑制剂的耐药性发展迅速，需要能够预防或延缓耐药的药物组合来延长患者的反应持久性。除了RAS，针对RAS效应通路的激酶抑制剂已经被开发出来，以靶向MAPK和PI3K通路。然而，这些药物还没有在临床试验中证明对KRAS突变肿瘤的疗效。到目前为止，靶向治疗主要集中在阻断OA，而对阻断KRAS突变细胞中的NOA的关注较少。联合靶向NOA和OA可以在KRAS突变肿瘤中产生更好的药物组合，包括正交治疗方案、改善的治疗窗口和更持久的反应。目标。1)使用功能基因组筛选来确定KRAS突变癌细胞中癌基因成瘾(OA)和非癌基因成瘾(NOA)引起的遗传依赖性；2)评估KRAS突变细胞中靶向治疗的机制、选择性，以及导致耐药的机制；以及3)识别正向靶向KRAS突变细胞中的OA和NOA的不同方面的药物组合，以实现更好的治疗窗口。重大活动、重大成果、关键成果。1)基因筛查以识别KRAS突变癌细胞的功能脆弱性。利用共用的CRISPR/Cas9基因KO文库，在不同培养条件下对KRAS突变型癌细胞进行了功能缺失筛选。我们已经确定了一个候选的可药物基因，它对KRAS突变细胞的锚定非依赖性生长至关重要，目前我们正在研究这种遗传依赖性的机制，并探索这一靶点的小分子抑制剂。2)RAS途径抑制剂耐药机制。使用不同的KRAS突变癌细胞系，包括结直肠、胰腺、肺和多发性骨髓瘤细胞，我们评估了导致KRAS抑制剂和MEK抑制剂耐药的遗传和转录后机制。我们目前正在阐明如何逆转这些耐药机制，以提高临床前模型的治疗反应。3)寻找治疗KRAS突变癌的新药物组合。利用高通量药物筛选和假设驱动的方法，我们正在评估药物组合，其中包括靶向RAS OA的抑制剂来阻断致癌的RAS信号，以及靶向NOA通路的抑制剂来加剧KRAS突变癌细胞的致癌压力。我们比较了药物组合在KRAS突变癌细胞和正常上皮细胞中的毒性，以评估治疗窗口，并使用小鼠异种移植瘤和原发肿瘤模型来评估药物组合对KRAS驱动的体内肿瘤的疗效。

BACKGROUND. The KRAS oncogene is a driver mutation in a significant fraction of lung, pancreatic and colorectal cancers. Recently breakthroughs with inhibitors targeting the KRAS-G12C mutant is the first Ras targeted therapies in decades. For other KRAS mutant tumors, there are no effective targeted therapies. Resistance to KRAS inhibitors develop rapidly in cancer patients, and drug combination that can prevent or delay drug resistant are needed to extend the durability of response in patients. In addition to Ras, kinases inhibitors against Ras effector pathways have been developed to target the MAPK and PI3K pathways. However, these agents have not demonstrated efficacy in clinical trials against KRAS mutant tumors. Thus far, targeted therapies have primarily focused on blocking OA, and less attention have been devoted to blocking NOA in KRAS mutant cells. Co-targeting NOA and OA together could lead to better drug combinations with orthogonal therapeutic modalities, improved therapeutic window, and more durable response in KRAS mutant tumors. OBJECTIVES. 1) Using functional genomic screens to identify genetic dependencies resulting from oncogene addiction (OA) and non-oncogene addiction (NOA) in KRAS mutant cancer cells; 2) Evaluating the mechanism, selectivity of targeted therapies in KRAS mutant cells, and the mechanisms leading to drug resistance; and 3) Identify drug combinations that orthogonally target distinct aspects of OA and NOA in KRAS mutant cells to achieve better therapeutic window. MAJOR ACTIVITIES, SIGNIFICANT RESULTS AND KEY OUTCOMES. 1) Genetic screens to identify functional vulnerabilities in KRAS mutant cancer cells. Using pooled CRISPR/Cas9 gene KO libraries, we have carried out loss-of-function screens in KRAS mutant cancer cells under different culture conditions. We have identified a candidate, druggable gene that is critical for the anchorage-independent growth of KRAS mutant cells, and we are currently investigating the mechanism of this genetic dependency and exploring small molecule inhibitors of this target. 2) Mechanisms of drug resistant to Ras pathway inhibitors. Using different KRAS mutant cancer cell lines including colorectal, pancreatic, lung, and multiple myeloma cells, we have evaluated genetic and post-transcriptional mechanisms that contribute to resistance to KRAS inhibitors and MEK inhibitors. We are currently elucidating how these resistance mechanisms can be reversed to improve therapeutic response in pre-clinical models. 3) Identification of novel drug combinations for KRAS mutant cancer. Using high-throughput drug screen and hypothesis-driven approach, we are evaluating drug combinations that involve inhibitors targeting Ras OA to block oncogenic Ras signaling and inhibitors targeting NOA pathways to exacerbate oncogenic stress in KRAS mutant cancer cells. We contrast the toxicity of drug combinations in KRAS mutant cancer cells against that in normal epithelial cells to assess the therapeutic window, and we use mouse xenograft and autochthonous tumor models to evaluate the drug combination's efficacy against KRAS-driven tumors in vivo.