基于BRD4通过c-MYC-MYH9轴调控MYH9与HIF-1α相互作用探讨BET抑制剂防治黑素瘤转移的机制研究

Based on BRD4, the interaction between MYH9 and HIF-1α was studied by using c-myc-myh9 axis to investigate the mechanism of BET inhibitors to prevent and control melanoma metastasis. This study is based on our newly developed BET inhibitor, NHWD-870. Pre-clinical experiments showed that the anti-cancer effect of this inhibitor was 50 fold higher than the traditional BET inhibitor JQ-1. The development and application of this compound will provide a novel opportunity to achieve currently unsatisfied therapeutic effect of JQ-1 on solid tumor, including melanoma. The crucial role of BRD4, a target of NHWD-870 and JQ-1, has been established in melanoma metastasis; however, the mechanism remains unclear. Based on our preliminary results, we hypothesize that “NHWD-870 prohibits invasion, metastasis and tumor-angiogenesis of melanoma through suppressing c-Myc-introduced MYH9 transcription, abolishing MYH9-HIF-1α interaction, and down-regulating HIF targets and signaling pathway”. In the proposed study, we will comprehensively test this hypothesis. We will determine the effects of BRD4 and its inhibition on c-MYC and MYH9 expression, MYH9 and HIF-1α interaction, and melanoma invasion, metastasis and tumor-angiogenesis by conducting NHWD-870 treatment, RNA interference, CRISPR/Cas9-introduced knockout, and gene overexpression, and by applying a hypoxemia-induced mouse model. We will identify key loci and functional domains in down-stream signaling pathway of MYH9 and HIF-1α. This study will discover the working mechanism of BET inhibitors in melanoma and provide novel strategies of combinational treatment to expand its clinical applications.

本项目基于前期开发的新BET抑制剂NHWD-870，临床前数据显示抗肿瘤活性高于JQ-1近50倍。现证实该靶基因BRD4在调控黑素瘤转移中起重要作用，但机制尚不清楚。现结合前期工作基础，提出“BRD4及其抑制剂NHWD-870可能通过c-MYC调节MYH9转录表达，进而影响MYH9与HIF-1α相互作用，调节下游信号通路传导及靶基因表达，调控黑素瘤侵袭、转移和肿瘤血管生成”的科学假说，拟综合利用抑制剂阻断、RNA干扰、CRISPR/Cas9敲除、过表达技术及体内缺氧诱导模型，进一步确证BRD4通过c-MYC-MYH9调节轴影响MYH9与HIF-1α相互作用，解析MYH9与HIF-1α相互作用的关键位点或功能结构域，继而深入阐释NHWD-870干预c-MYC-MYH9轴及其MYH9-HIF-1α相互作用，抑制黑素瘤转移的分子机制，为其临床适应症拓展提供理论依据及多药联合治疗提供新策略。

黑素瘤是最为致命性的恶性皮肤肿瘤，绝大部分患者发现时已经出现转移。转移性黑素瘤患者的五年生存率仅为20%左右。因此，对于转移性黑素瘤的防治一直是研究的热点和难点。本项目基于前期开发的新BET抑制剂NHWD-870，结合前期工作基础，提出了“BRD4及其抑制剂NHWD-870可能通过c-MYC调节MYH9转录表达，进而影响MYH9与HIF-1α相互作用，调节下游信号通路传导及靶基因表达，调控黑素瘤侵袭、转移和肿瘤血管生成”的科学假说。目前本项目已完成全部研究内容，获得如下研究结果：1）BET抑制剂NHWD-870通过BRD4/c-MYC/MYH9/HIF-1α/MIG-7轴抑制肿瘤血管生成，继而阻碍肿瘤的发展和转移。MYH9高表达与肿瘤分化不良、转移和进展显著相关，有望成为预测肿瘤侵袭、转移的新型生物标志物；2）除了下调c-MYC和直接抑制肿瘤细胞增殖的能力外，NHWD-870还可通过抑制BRD4及其靶标HIF1α来抑制CSF1的表达从而阻断肿瘤相关巨噬细胞 (TAM) 的增殖，并最终抑制肿瘤生长，该结果支持进一步开发NHWD-870以治疗实体瘤；3）鉴定SPP1作为BET抑制剂的重要靶标，并提出BET抑制剂通过非经典的NF-ĸB/SPP1途径抑制黑素瘤的进展的新机制；4）报道了用于中国患者和相关动物模型的培养原发性肢端黑素瘤细胞的详细方案，总结了我们的肢端黑素瘤细胞系和现有的肢端黑素瘤细胞系的特点，这将为中国患者的药物反应和个体化治疗研究以及中西方人群黑素瘤的比较研究提供有效的研究工具；5）EEF2K/p-STAT3/SPP1可能是黑素瘤进展中的一种新的致癌途径，这可能是黑素瘤新型联合治疗的靶点；6）eEF2K可能作为预测抗PD-1治疗患者治疗反应和预后的生物标志物，揭示了eEF2K通过控制PD-L1表达来调节TME的重要作用，并提供了抑制eEF2K与ICB治疗的潜在联合治疗策略；7）BRD4抑制剂通过SPINK6/EphA2-EGFR通路抑制黑素瘤转移，SPINK6/EGFR-EphA2轴作为黑素瘤转移的新致癌途径并支持BRD4抑制剂在临床治疗转移性黑素瘤的进一步开发。

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