Loss of tumor cell MHC Class II drives MAPK-inhibitor insensitivity of BRAF-mutant anaplastic thyroid cancers [RNA-seq]

Cancer cells present neoantigens dominantly through MHC class I (MHCI) to drive tumor rejection through cytotoxic CD8+ T-cells. There is growing recognition that a subset of tumors express MHC class II (MHCII), causing recognition of antigens by TCRs of CD4+ T-cells that contribute to the anti-tumor response. We find that mouse BrafV600E-driven anaplastic thyroid cancers (ATC) respond markedly to the RAF + MEK inhibitors dabrafenib and trametinib (dab/tram) and that this is associated with upregulation of MhcII in cancer cells and increased CD4+ T-cell infiltration. A subset of recurrent tumors lose MhcII expression due to silencing of Ciita, the master transcriptional regulator of MhcII, despite preserved interferon gamma signal transduction, which can be rescued by EZH2 inhibition. Orthotopically-implanted Ciita-/- and H2-Ab1-/- ATC cells into immune competent mice become unresponsive to the MAPK inhibitors. Moreover, depletion of CD4+, but not CD8+ T-cells, also abrogates response to dab/tram. These findings implicate MHCII-driven CD4+ T cell activation as a key determinant of the response of Braf-mutant ATCs to MAPK inhibition. Overall design: To probe into the underlying mechanisms of MhcII loss and attenuated Ciita expression we performed bulk RNA-seq and ATAC-seq. We tested mouse cell lines generated from thyroid tumors derived from a genetic engineered anaplastic thyroid cancer mouse model (Tpo-Cre/eYFP/BRaf-CA/Trp53fl/fl; termed Braf-CAV600E/p53). We included one primary (B92) and one recurrent (B36934) cell line after a 96h treatment with DMSO, IFN? (20ng/ml), trametinib (10nM) or the combination of IFN? and trametinib in vitro.

肿瘤细胞主要通过主要组织相容性复合体I类（MHC class I，MHCI）呈递新抗原，以通过细胞毒性CD8+ T细胞介导肿瘤排斥反应。越来越多研究表明，部分肿瘤可表达主要组织相容性复合体II类（MHC class II，MHCII），使CD4+ T细胞的T细胞受体（T cell receptor，TCR）识别抗原，进而参与抗肿瘤免疫应答。本研究发现，由BrafV600E驱动的小鼠间变性甲状腺癌（anaplastic thyroid cancer，ATC）对RAF与MEK双抑制剂达拉非尼（dabrafenib）联合曲美替尼（trametinib，简称dab/tram）治疗应答显著，且该应答与肿瘤细胞中MhcII表达上调及CD4+ T细胞浸润增加密切相关。部分复发肿瘤会因主要组织相容性复合体II类反式激活因子（class II major histocompatibility complex transactivator，CIITA）的基因沉默而丧失MhcII表达，即便其干扰素γ（interferon gamma，IFN-γ）信号转导通路仍保持完整；而zeste同源物2增强子（Enhancer of zeste homolog 2，EZH2）抑制可逆转这一表型。将Ciita基因敲除（Ciita-/-）与H2-Ab1基因敲除（H2-Ab1-/-）的ATC细胞原位移植至免疫健全小鼠体内后，肿瘤对MAPK抑制剂不再产生应答。此外，耗竭CD4+ T细胞（而非CD8+ T细胞）同样会消除肿瘤对dab/tram的治疗应答。上述研究结果表明，MHCII介导的CD4+ T细胞活化是BRAF突变型ATC对MAPK抑制剂治疗应答的关键决定因素。整体实验设计：为探究MhcII表达缺失及Ciita表达减弱的潜在分子机制，本研究开展了批量RNA测序（bulk RNA-seq）与转座酶可及性染色质测序（Assay for Transposase-Accessible Chromatin using sequencing，ATAC-seq）。我们使用源自基因工程间变性甲状腺癌小鼠模型（Tpo-Cre/eYFP/BRaf-CA/Trp53fl/fl，简称Braf-CAV600E/p53）的甲状腺肿瘤细胞系开展实验。实验设置了原代细胞系（B92）与复发细胞系（B36934），分别在体外以二甲基亚砜（Dimethyl sulfoxide，DMSO）、20ng/ml干扰素γ（IFN-γ）、10nM曲美替尼（trametinib）以及干扰素γ联合曲美替尼处理96小时后进行检测。