Targeting the developmental pathways Notch and ERBB for melanoma therapy

描述（由申请人提供）：黑色素瘤仍然是最致命的皮肤癌，五年生存率仅为15%。特异性靶向突变BRAF的药物的出现代表了黑色素瘤患者护理的重大改进。然而，这些疗法在治疗的6至12个月内受到耐药性发展的阻碍，并且仅适用于包括大约50%的黑色素瘤病例的突变BRAF患者。在这里，我们将研究一种新方法的治疗益处和分子机制，该方法靶向Notch和ERBB作为替代治疗，可以使患者受益于驱动黑色素瘤的突变谱。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发挥重要作用，但在各种癌症中被不适当地激活。我们最近发现了Notch 1和ERBB 3信号在黑色素瘤中的功能性串扰。我们发现，活性Notch 1（Notch 1 NIC）和活性（磷酸化）ERBB 3和ERBB 2在超过70%的黑色素瘤肿瘤中显着相关，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通过调节ERBB 3和4的配体NRG 1（神经调节蛋白1）表达直接促进ERBB 3活化。一旦被激活，ERBB 3特异性地选择ERBB 2以促进黑色素瘤细胞存活。总之，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，它们的协同活性有助于细胞存活和肿瘤扩张。事实上，在阻止 因此，通过结合γ-分泌酶抑制剂（GSI）以阻断Notch活化和酪氨酸激酶抑制剂（TKI）以抑制ERBB 3/2诱导的协同效应，可触发适度的效应，导致黑素瘤细胞活力丧失90%，无论细胞是否携带野生型或突变的BRAF。初步数据还显示，通过使用耐受性良好的给药方案，联合治疗促进黑色素瘤肿瘤消退，而没有明显的体重减轻，这是一种通常与GSI治疗相关的副作用。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IKK-β的泛素化和蛋白酶体依赖性降解，导致NF κ B活性的完全消除。因此，我们认为发育途径Notch和ERBB在黑色素瘤的生存和扩展中是关键的，阻断这些途径可能具有以前未被认识到的治疗意义。我们将：1）确定组合的抗Notch-ERBB靶向疗法在BRAF和RAS依赖性转基因小鼠黑素瘤模型中的功效，所述转基因小鼠黑素瘤模型最好地概括了人类疾病的突变和生物学特征; 2）剖析Notch 1和ERBB 3调节KEAP 1的机制以及Notch和ERBB信号传导下游NFkB的作用;和3）检查活性Notch 1（Notch 1 NIC）和磷酸化ERBB 3和NFkB表达之间的关联以及它们与黑素瘤患者存活率的相关性。我们期望从这项工作中获得的知识为新的治疗方法提供理论基础，这些治疗方法可以使更大的黑色素瘤患者群体受益。

DESCRIPTION (provided by applicant): 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.