增强KRAS突变细胞免疫原性并调节肠道抗肿瘤微环境的新机制

Mutations in the KRAS oncogene represent one of the most prevalent genetic alterations in colorectal cancer (CRC), the third leading cause of cancer-related death in the world. Besides their well-characterized functions in driving tumor progression, KRAS mutations can induce tumor immunoresistance by increasing the expression of PD-L1 and immunosuppressive cytokines directly or via downstream signal pathways. KRAS mutation has been linked to resistance to the agents utilized in front-line therapy of CRC. KRAS mutation in CRC activates transforming growth factor-beta (TGF-beta)-activated kinase 1 (TAK1) to promote tumor progression. Our preliminary studies showed that TAK1 inhibitor NG25 induced apoptosis of KRAS mutant CRC cells and the percentage of apoptosis were positively correlated with host immunity in orthotopic CRC mouse models. The number of CD3+CD8 + T cells was increased as well as PD-L1 expression was decreased in both CT26 (KRAS mutant CRC cells) and CD3+ cells, which were treated with NG25 in the mixed culture system. Based on above, we hypothesize that inhibition of TAK1 could induce KRAS mutant cells immunogenic death and enhance antitumor immune responses in colorectal cancer. Our project is going to clarify that the apoptosis induced by TAK1 inhibitor is immunogenic; TAK1 can modulate immune related-cytokines to recruit immune cells and/or induce their differentiation, and downregulate PD-L1 expression, then produce a therapeutically effective anti-KRAS mutant CRC response. In summary, inhibition of TAK1 could induce apoptosis in KRAS mutant cells and activate anti-tumor immunity simultaneously in CRC microenvironment. It may serve as a novel biomarker and potential therapeutic target in KRAS mutant CRC.

KRAS基因突变促进肠上皮细胞异常增生并抑制肠道免疫微环境是结直肠癌（Colorectal Cancer, CRC）复发和难治的重要原因。转化生长因子-β激活激酶1（TAK1）是KRAS突变CRC生存依赖的激酶。我们在小鼠原位结肠癌研究中发现，TAK1抑制剂特异性诱导KRAS突变细胞凋亡，并与免疫反应正相关；抑制TAK1可使体外混合培养体系中CD8+T数量增多，肿瘤和CD3+细胞表面PD-L1表达下降，提示TAK1可调控KRAS突变CRC免疫微环境。据此提出假设：TAK1抑制剂诱导KRAS突变细胞免疫原性死亡并激活抗肿瘤免疫反应。本课题将阐明TAK1通过调节KRAS突变肿瘤免疫微环境中细胞因子并下调PD-L1表达，逆转KRAS突变相关免疫逃逸的机制。研究结果将验证TAK1具有抑制KRAS突变细胞异常增殖和激活免疫微环境的双重效应，是靶向和免疫联合治疗KRAS突变CRC的潜在靶点。

结直肠癌的发生是基因突变和肿瘤微环境共同作用的结果。原癌基因KRAS在结直肠癌患者中突变率高达40%，作为结直肠癌早期出现的基因改变，活化的KRAS激活下游生存通路促进细胞异常增殖，参与了肿瘤发生、发展和耐药的过程。同时突变的KRAS可通过调控细胞因子或免疫检控点PD-L1促进免疫抑制微环境形成，导致肿瘤细胞免疫逃逸。因此如何能在杀死KRAS突变肿瘤细胞的同时重新激活肿瘤免疫反应是治疗KRAS突变肿瘤的重要研究方向。. 转化生长因子β (TGF-β)激活性激酶1 (transforming growth factor-beta-activated kinase 1, TAK1) 是丝裂原活化激酶(MAP3K)家族一员，是维持结肠癌KRAS突变细胞生存重要的激酶。过度激活的TAK1不但通过活化KRAS相关信号通路进而调控肿瘤发生发展，而且在肠道炎症和免疫反应中发挥重要作用。.通过临床样本、体内细胞实验、体内动物实验，我们发现：1. KRAS基因突变和TAK1蛋白表达与结直肠癌的临床病理特征存在相关性。2. TAK1抑制剂NG25诱导KRAS突变细胞发生caspase依赖的细胞凋亡。3.在KRAS突变的结直肠癌细胞中，NG25促进免疫原性的细胞死亡。4.小鼠原位结直肠癌模型及体外细胞培养证实NG25通过抑制NF-κB的活性，降低肿瘤细胞表面免疫检查位点配体PD-L1的表达。5. 体内及体外淋巴细胞-肿瘤细胞混合培养证实NG25可以促进CD3+CD8+细胞增多。6.原位结直肠癌小鼠NG25治疗前后，肿瘤局部免疫相关细胞因子表达谱发生明显改变，肿瘤免疫反应被激活。. 上述研究结果说明TAK1与KRAS突变肿瘤的生存和微环境中炎症免疫反应密切相关，抑制TAK1可以诱导KRAS突变细胞发生免疫原性细胞死亡，并通过调节细胞因子以及免疫监控点PD-1/PD-L1，激活肿瘤特异性免疫反应。因此以TAK1为靶点可抑制KRAS突变肿瘤细胞增殖的同时激活肿瘤免疫反应，有望成为KRAS突变结直肠癌患者临床治疗的方向。.