TGF-β/SMAD4信号通路失活促进BRAF(V600)突变型肠癌靶向耐药和免疫逃逸的作用机制及干预研究

BRAF(V600)突变肠癌患者预后极差，BRAF抑制剂联合EGFR抑制剂的双靶治疗有效率低，容易产生耐药且机制不明，临床上缺乏疗效预测标志物。近年来，液体活检技术为靶向治疗耐药监测提供了新思路。我们前期通过对接受双靶治疗的BRAF(V600)突变肠癌患者循环血ctDNA进行动态监测，结果表明：耐药患者的ctDNA中存在TGFBR2/SMAD4基因失活突变。体内外实验沉默TGFBR2/SMAD4基因降低了BRAF(V600)突变肠癌对双靶治疗的敏感性、同时激活MAPK通路、促进肿瘤细胞PD-L1表达、并抑制细胞毒T细胞的杀伤作用。本研究将在此基础上，深入阐明TGF-β/SMAD4信号通路失活如何通过促进MAPK通路的活性和抵抗细胞毒T细胞的杀伤作用介导双靶耐药和免疫逃逸，探索同时靶向TGF-β和PD-1对耐药的逆转作用，为耐药BRAF(V600)突变肠癌患者的诊疗提供新的标志物及干预策略。

BRAF(V600) mutation is found in about 10% of colorectal cancer (CRC) patients and is a poor prognosis factor in standard chemotherapy. Combination of BRAF and EGFR inhibition exerts a better therapeutic effective due to pathway reactivation through EGFR signaling in BRAF inhibitor monotherapy, whereas the resistance develops through undefined mechanisms. Circulating tumor DNA (ctDNA) is a non-invasive approach to assess the genetic evolution of tumors and prognosis in response to therapy, which would help better understanding the treatment response and resistance mechanism in BRAF inhibitor treated colorectal cancer patients..We performed panel next-generation sequencing (NGS) of 425 cancer-related genes in serial plasma samples collect from 19 patients who have BRAF(V600) mutation CRC to track the resistance during the Vemurafenib treatment in combination with Cetuximab (VC) and irinotecan and evaluate the treatment response. .By January 20, 2020, treatment had been discontinued in 8 of the patients due to disease progression, while the other 11 cases were still under treatment. Among them, four patients with innate resistance (n=4) were defined as those with PFS of less than 2 months, while patients (n=15) with acquired resistance were defined as those with PFS of greater than 2 months. The VC regimen demonstrated efficacy in patients with BRAF(V600) mutant mCRC. Changes in levels of ctDNA at 4 weeks predicts therapeutic responses. Acquired TGFBR2 and SMAD4 loss-of-function mutations leads to the inactivation of the classical TGF-β/SMAD4 signaling pathway, were identified as novel resistant mechanism to the combination of BRAF and EGFR inhibition, it is supposed that driven by immune evasion and RAS/RAF/MAPK pathway reactivation. While in vivo studies indicated that combined TGF-β, PD-1, BRAF and EGFR inhibition blockade may overcome the BRAF(V600) mutation CRC with TGFBR2 and SMAD4 loss-of-function mutations to VC regimen resistance. .Our present study are to further explore the molecular mechanisms of TGFBR2 and SMAD4 gene mutations mediated BRAF and EGFR inhibition resistance in BRAF(V600) mutation CRC in vitro and in vivo and looking for the new strategies to overcome the combination of BRAF and EGFR inhibition resistance in BRAF(V600) mutation CRC.