Combating melanoma resistance by targeting ABL1/2

摘要 尽管过去十年治疗取得了进步，但转移性黑色素瘤的五年存活率仍然很低。 (23%)。BRAF突变发生在50%-75%的黑色素瘤中，BRAF和MEK抑制剂(BRAFi， Meki)是有效的治疗方法；然而，对大多数患者来说，获得性耐药性迅速发展。研究结束 在过去的十年中，已经确定了黑色素瘤细胞对BRAF/MEK产生耐药性的机制 抑制剂，但到目前为止，还没有FDA批准的方案来逆转BRAFi/Meki耐药性并增加 5年生存率。在这里，我们证明了ABL家族的非受体酪氨酸的活性 在BRAFi或BRAFi/Meki抗性和ABL1/2驱动抗性之后，激酶被增强。此外， FDA批准的抗白血病药物尼洛替尼靶向ABL1/2显著逆转BRAFi/Meki (达普拉非尼/曲美替尼)耐药，体内，诱导肿瘤延长消退。基于这些令人兴奋的 发现，我们建议的中心假设是ABL1/2是激活ERK依赖或 -驱动BRAFi/Meki耐药的独立途径；因此，靶向ABL1/2可能有效地克服 抵抗并最终延长治疗难治性黑色素瘤患者的生存时间。我们的长期， 翻译的目标是开发一种新的方案来提高转移性患者的5年存活率 黑色素瘤。我们提出了一个全面的假设驱动的实验设计和多学科 接近。目标1将定义ABL1/2驱动电阻的机制。为了实现我们的目标， 使用亲本/耐药黑色素瘤细胞系的生化、分子生物学和细胞生物学方法， 药理抑制剂/RNAi、质谱学、异种移植、GEM、PDX和体内同源基因方法， 以及从临床试验(目标3)收集的人体组织将被用来检验ABL1/2的假设 MAP3K激活MEK/ERK/MYC(ERK依赖的抗性)；诱导ERBB2/ERBB3激活 PI3K/AKT(ERK非依赖性耐药)；改变免疫细胞浸润。在目标2中，条件培养液来自 亲本/耐药细胞系，使用体外和体内模型进行的共培养实验，以及从 动物和患者将被用来测试ABL1/2驱动的分泌体有助于 布拉菲/梅基抵抗运动。最后，Aim 3是一项多点、无缝的I/II期临床试验，旨在测试 尼洛替尼联合达普拉非尼+曲美替尼(BRAFi/Meki)治疗难治性患者。其他内容 终点包括确定推荐的第二阶段剂量、基于第二阶段数据的初始疗效(PFS)、 PK/PD研究，以及初步的探索性生物标志物开发。这些研究的结果不仅会增加 我们对黑色素瘤如何抵抗BRAFi/Meki影响的理解，也将为 大型、多中心、随机临床试验。

SUMMARY Despite treatment advances over the past decade, five-year survival rates for metastatic melanoma remain low (23%). BRAF mutations occur in 50-75% of melanoma, and a combination of BRAF and MEK inhibitors (BRAFi, MEKi) are effective therapies; however, acquired resistance rapidly develops for most patients. Research over the past decade has identified mechanisms by which melanoma cells develop resistance to BRAF/MEK inhibitors, but thus far, there are no FDA-approved regimens that reverse BRAFi/MEKi resistance and increase the 5-year survival rate. Here, we demonstrate that the activities of the ABL family of non-receptor tyrosine kinases are potentiated following BRAFi or BRAFi/MEKi resistance, and ABL1/2 drive resistance. Moreover, targeting ABL1/2 with an FDA-approved anti-leukemia drug, nilotinib, dramatically reverses BRAFi/MEKi (dabrafenib/trametinib) resistance, in vivo, inducing prolonged tumor regression. Based on these exciting findings, the central hypothesis of our proposal is that ABL1/2 are key nodes that activate ERK-dependent or -independent pathways to drive BRAFi/MEKi resistance; thus, targeting ABL1/2 may be effective for overcoming resistance and ultimately extending survival for patients with treatment-refractory melanomas. Our long-term, translational goal is to develop a novel regimen to improve the 5-year survival rate for patients with metastatic melanoma. We propose a comprehensive hypothesis-driven experimental design and multi-disciplinary approach. Aim 1 will define mechanisms by which ABL1/2 drive resistance. To achieve our objective, biochemical, molecular biological and cell biological approaches using parental/resistant melanoma cell lines, pharmacological inhibitors/RNAi, mass spectrometry, xenograft, GEM, PDX, and syngeneic in vivo approaches, as well as human tissue collected from the clinical trial (Aim 3) will be used to test the hypothesis that ABL1/2 drive MAP3K activation of MEK/ERK/MYC (ERK-dependent resistance); induce ERBB2/ERBB3 activation of PI3K/AKT (ERK-independent resistance); and alter immune cell infiltration. In Aim 2, conditioned medium from parental/resistant cell lines, co-culture experiments using in vitro and in vivo models, and serum collected from animals and patients will be used to test the prediction that the ABL1/2-driven secretome contributes to BRAFi/MEKi resistance. Finally, Aim 3 is a multi-site, seamless Phase I/II clinical trial that tests the safety of combining nilotinib with dabrafenib+trametinib (BRAFi/MEKi) for treatment-refractory patients. Additional endpoints include identifying the recommended Phase II dose, initial efficacy (PFS) based on Phase II data, PK/PD studies, and initial exploratory biomarker development. Results from these studies not only will increase our understanding for how melanomas resist the effects of BRAFi/MEKi but also will lay the groundwork for a large, multi-center, randomized clinical trial.