Drivers of MAPK-redundant resistance to BRAF inhibition in melanoma

项目摘要 皮肤黑色素瘤是一种侵袭性皮肤癌。仅在美国，就有大约9万例新病例 2017年有9,000人死于黑色素瘤。BRAFV 600 E/K的选择性抑制剂有效地起作用 针对由致癌BRAF驱动的黑色素瘤。不幸的是，耐药性无处不在，大多数患者 会在治疗后的两年内恶化大规模的基因组和转录组分析显示， 对MAPK抑制（MAPKi）的依赖性是MAPK依赖性的或冗余的。MAPK依赖性（耐药约50%） 耐药的特征是MAPK的再激活，通常是通过增加MAPK的突变 信号这些MAPKi抗性黑色素瘤在转录组水平上与它们各自的基因组几乎相同。 进一步支持这些癌症的亲代癌症只是恢复了MAPK。相比之下，MAPK-redundant 耐药（剩余50%）的特征是广泛的转录重编程，通常缺乏 复发性突变从机制上讲，MAPK依赖的抗性集中在Raf同源物的形成上， 对当前一代Raf抑制剂有抗性但对下一代Raf抑制剂敏感的异二聚体 具有二聚体活性。然而，MAPK冗余电阻并不统一，没有得到很好的理解， 共同的治疗目标。这一知识很重要，因为目前没有治疗选择。 具有MAPK冗余抗性的黑素瘤。 为了解决这一关键差距，我们进行了正向遗传筛选，以确定新的机制， 对MAPKi的驱动阻力。该筛选使用我们成熟的睡美人转座子进行 系统该筛选的结果是SFKs和PDGFRβ是首选的MAPK-冗余 BRAFV 600 E黑色素瘤A375和SKMEL 28的抗性机制。然而， 哪些SFK和PDGFRβ驱动MAPK-冗余抗性尚不清楚。在目标1中，我将阐明 SFKs和PDGFRβ驱动的MAPKi抗性的机制，通过检验肌动蛋白重塑 是由SFKs或PDGFRβ驱动的MAPKi抗性的核心，整合素是MAPKi抗性的关键上游。 而Akt是PDGFR抗性程序的关键下游调节者。在 目的2：我将检验SFKs和PDGFRβ通过激活MAPKi的表达而促进MAPKi抵抗的假设。 雅普/塔兹/TEAD/AP-1转录复合体。核心假设是肌动蛋白重塑和细胞内的 雅普/TAZ/AP-1复合物是SFKs-和PDGFRβ-抗性程序的关键介质。

PROJECT SUMMARY Cutaneous melanoma is an aggressive form of skin cancer. In the US alone, approximately 90,000 new cases and 9,000 deaths due to melanoma were reported in 2017. Selective inhibitors of BRAFV600E/K work potently against melanomas driven by oncogenic BRAF. Unfortunately, drug resistance is ubiquitous, and most patients will progress within two years of therapy. Large-scale genome- and transcriptome- profiling reveal that resistance to MAPK inhibition (MAPKi) is either MAPK- dependent or redundant. MAPK-dependent (resistance ~50%) resistance is characterized by the reactivation of MAPK typically through mutations that augment MAPK signaling. These MAPKi-resistant melanomas are nearly identical at the transcriptome level to their respective parental cancers, which further support these cancers simply restore MAPK. By contrast, MAPK-redundant resistance (remaining 50%) is characterized by extensive transcriptional reprogramming and is often devoid of recurrent mutations. Mechanistically, MAPK-dependent resistance is centered on the formation of Raf homo- or heter- dimers that are resistant to current generation Raf inhibitors but sensitive to next-generation Raf inhibitors that have dimer activity. However, MAPK-redundant resistance is not uniform, not well-understood, and lacks a common therapeutic target. This knowledge is important as there are no current treatment options for melanomas with MAPK-redundant resistance. To address this critical gap, we performed a forward genetic screen to identify novel mechanisms that drive resistance to MAPKi. This screen was performed using our well-established Sleeping Beauty Transposon system. The outcome of this screen was that SFKs and PDGFRβ were the preferred MAPK-redundant mechanisms of resistance for BRAFV600E melanomas A375 and SKMEL28. However the precise mechanisms by which SFKs and PDGFRβ drive MAPK-redundant resistance are unknown. In Aim 1, I will elucidate the mechanism of SFKs- and PDGFRβ- driven resistance to MAPKi by testing the hypothesis that actin remodeling is central to MAPKi resistance that is driven by both SFKs or PDGFRβ and that integrins are critical upstream of the SFKs resistance program and Akt is a critical downstream meditator of the PDGFR resistance program. In Aim 2 I will test the hypothesis that SFKs and PDGFRβ promotes MAPKi resistance by activating the Yap/Taz/TEAD/AP-1 transcriptional complex. The central hypothesis is that actin remodeling and the YAP/TAZ/AP-1 complex are critical mediators of the SFKs- and PDGFRβ- resistance programs.