Targeting the developmental pathways Notch and ERBB for melanoma therapy

黑色素瘤仍然是最致命的皮肤癌，五年生存率仅为15%。药物的出现 特异性靶向突变的BRAF代表了黑素瘤患者护理的重大改进。然而这些 在治疗的六至十二个月内，耐药性的发展阻碍了治疗， 仅适用于BRAF突变患者，包括约50%的黑色素瘤病例。这里我们将 研究一种新的靶向发展中国家的方法的治疗益处和分子机制， Notch和ERBB通路作为替代治疗，可以使所有突变谱的患者受益 导致黑色素瘤Notch和ERBB是进化上保守的信号级联， 在胚胎发生和干细胞更新中，但在各种癌症中被不适当地激活。我们最近 确定了黑色素瘤中Notch 1和ERBB 3信号之间的功能性串扰。我们发现， （Notch 1 NIC）和活性（磷酸化）ERBB 3和ERBB 2在超过70%的黑色素瘤中显著相关 这表明这些受体在黑色素瘤中被共同激活。我们已经证明，Notch 1直接 通过调节ERBB 3和4的配体NRG 1（神经调节蛋白1）表达促进ERBB 3活化。一旦 当ERBB 3被激活时，ERBB 3特异性地选择ERBB 2以促进黑素瘤细胞存活。我们的数据表明 Notch 1和ERBB 3/ERBB 2信号在黑色素瘤中同时重新激活， 有助于细胞存活和肿瘤扩张。事实上，虽然阻断任何一种途径都会产生适度的影响， 将γ-分泌酶抑制剂（GSI）与酪氨酸激酶抑制剂（TKI）组合以阻断Notch活化， 抑制ERBB 3/2增强协同效应，导致90%的黑色素瘤细胞活力丧失，无论是否 细胞携带野生型或突变的BRAF。初步数据还表明，通过使用耐受性良好的分娩方式， 按照时间表，联合治疗促进黑色素瘤肿瘤消退，而没有明显的体重减轻， 通常与GSI治疗有关。从机制上讲，我们首次表明，Notch 1和ERBB 3 抑制导致KEAP 1的失调，KEAP 1是一种（BTB）-Kelch蛋白，通过促进泛素化和 IKK-β的蛋白酶体依赖性降解导致NF κ B活性的完全消除。因此，我们建议 Notch和ERBB的发育途径在黑色素瘤的生存和扩展中起关键作用， 阻断这些途径可能具有以前未认识到的治疗意义。我们将：1）确定 联合抗Notch-ERBB靶向治疗在BRAF和RAS依赖转基因小鼠中的功效 最好地概括人类疾病的突变和生物学特征的黑色素瘤模型; 2）解剖 Notch 1和ERBB 3对KEAP 1的调控机制以及Notch下游NF κ B的作用， ERBB信号传导;和3）检查活性Notch 1（Notch 1 NIC）和磷酸化ERBB 3之间的关联 和NFkB表达以及它们与黑色素瘤患者存活率的相关性。我们希望获得的知识 从这项工作中提供了新的治疗方法，可以受益于更大的黑色素瘤患者群体的基本原理。

Melanoma remains the deadliest form of skin cancer, with a five year survival of only 15%. The advent of drugs specifically targeting mutated BRAF has represented a major improvement in melanoma patient care. Yet, these therapies are hampered by the development of resistance within six to twelve months of treatment, and are applicable exclusively to patients with mutated BRAF that include roughly 50% of melanoma cases. Here we will investigate the therapeutic benefits and molecular mechanisms of a novel approach targeting the developmental pathways Notch and ERBB as alternative treatments that can benefit patients across the spectrum of mutations that drive melanoma. Notch and ERBB are evolutionarily conserved signaling cascades that play essential roles in embryogenesis and stem cell renewal, but are inappropriately activated in various cancers. We have recently identified a functional cross-talk between Notch1 and ERBB3 signaling in melanoma. We find that active Notch1 (Notch1NIC) and active (phosphorylated) ERBB3 and ERBB2 correlate significantly in over 70% of melanoma tumors, suggesting these receptors are co-reactivated in melanoma. We have shown that Notch1 directly promotes ERBB3 activation by regulating NRG1 (neuregulin1) expression, the ligand for ERBB3 and 4. Once activated, ERBB3 specifically co-opts ERBB2 to promote melanoma cell survival. Together, our data suggest Notch1 and ERBB3/ERBB2 signaling are concurrently reactivated in melanoma where their coordinate activity contributes to cell survival and tumor expansion. Indeed, while blocking either pathway triggers modest effects, combining a gamma-secretase inhibitor (GSI) to block Notch activation, and a tyrosine kinase inhibitor (TKI) to inhibit ERBB3/2 elicits synergistic effects, leading to 90% loss of melanoma cell viability regardless of whether cells carry wild type or mutated BRAF. Preliminary data also show that by using a well-tolerated delivery schedule, a combination therapy promotes melanoma tumor regression with no overt weight loss, a side effect often associated with GSI treatments. Mechanistically, we show for the first time that Notch1 and ERBB3 inhibition results in the deregulation of KEAP1, a (BTB)-Kelch protein that by promoting ubiquitination and proteasome-dependent degradation of IKK-beta leads to complete ablation of NFkB activity. Thus, we propose that the developmental pathways Notch and ERBB are pivotal in melanoma survival and expansion, and that blocking these pathways may have previously unappreciated therapeutic implications. We will: 1) Determine the efficacy of a combined anti Notch-ERBB targeted therapy in BRAF and RAS dependent transgenic mouse melanoma models that best recapitulate the mutations and biological features of the human disease; 2) Dissect the mechanisms of regulation of KEAP1 by Notch1 and ERBB3 and the role of NFkB downstream of Notch and ERBB signaling; and 3) examine the association between active Notch1 (Notch1NIC) and phosphorylated ERBB3 and NFkB expression and their correlation with melanoma patient survival. We expect the knowledge gained from this work to provide the rationale for novel treatments that can benefit a larger melanoma patient population.