Developing inducible CRISPR to identify molecular targets in melanoma

描述（由申请人提供）：靶向治疗有可能通过提供基于遗传驱动因素的个性化治疗策略来改善癌症护理。BRAF（~50%）和NRAS（~20%）已被确定为黑色素瘤的致癌驱动因素。用特异性抑制剂靶向突变型BRAF已显示出显著的临床影响，导致最近FDA批准了几种药物（vemurafenib和dabrafenib）。由于NRAS已被证明难以靶向NRAS，因此许多努力都是针对破坏明确定义的下游信号传导途径，如MAPK和PI3K信号传导。尽管做出了这些努力，但在突变型NRAS肿瘤中靶向这些途径中的一种或两种在临床上尚未成功，为大量患者留下了很少的治疗选择。因此，需要确定其他治疗靶点。我们最近开发了一种新的黑色素瘤小鼠模型，其利用逆转录病毒递送基因（例如，在Cdkn2a缺失（在黑素瘤中观察到的最常见的肿瘤抑制因子缺失）的情况下，NRAS）特异性地针对黑素细胞的小子集，导致黑素瘤形成。虽然该模型非常适合于解决递送的基因在肿瘤进展中的作用，但它（与所有其他当前模型一样）缺乏以诱导方式消除肿瘤形成后任何基因的通用性。将具有不同传统或诱导型敲除的小鼠杂交将是耗时且成本高昂的，并且仍然主要解决肿瘤起始而不是与人类疾病更相关的肿瘤维持。因此，迫切需要开发具有免疫能力的临床前模型，其允许特异性删除肿瘤形成后不存在药理学试剂的任何靶标。为此，我们建议改进我们的黑色素瘤小鼠模型，以纳入诱导型CRISPR（iCRISPR）技术，从而能够快速体内评估新的分子靶点。我们最近开发了能够在体内诱导NRAS驱动的黑素瘤的病毒载体，其具有通过CRISPR/Cas9的四环素诱导来删除任何特定基因的能力。这种多功能系统能够快速体内筛选任何候选基因在肿瘤维持中的作用。作为原理证明，在目标1中，我们将在我们的NRAS黑色素瘤模型中评估NRAS指导的iCRISPR。目的2和3将评估涉及NRAS肿瘤维持的潜在候选物的作用（Ccnd1和Sos1）。

DESCRIPTION (provided by applicant): Targeted therapies have the potential to improve cancer care by providing personalized treatment strategies based on genetic drivers. BRAF (~50%) and NRAS (~20%) have been identified as oncogeneic drivers in melanoma. Targeting mutant BRAF with specific inhibitors has demonstrated significant clinical impact resulting in recent FDA approval of several agents (vemurafenib and dabrafenib). Since NRAS has proven difficult to target pharmacologically, much effort has been directed at disrupting well defined downstream signaling pathways such as MAPK and PI3K signaling. Despite these efforts, targeting either or both of these pathways in mutant NRAS tumors has not been successful clinically, leaving few treatment options for a significant number of patients. Therefore, additional therapeutic targets need to be identified. We have recently developed a novel mouse model of melanoma that utilizes retroviral delivery of genes (e.g., NRAS) specifically to a small subset of melanocytes resulting in melanoma formation in the context of Cdkn2a loss (most common tumor suppressor loss observed in melanoma). While this model is well suited to address the role of a delivered gene(s) in tumor progression, it (like all other current models) lacks the versatility to eliminate any gene post tumor formation in an inducible manner. Crossing mice with different traditional or inducible knock outs would be time consuming and cost prohibitive and still primarily address tumor initiation rather than tumor maintenance which is more relevant to the human disease. Therefore, there is an urgent need to develop immune-competent pre-clinical models that allow for the specific deletion of any target for which pharmacological agents do not exist post tumor formation. To this end, we propose to advance our melanoma mouse model to incorporate inducible CRISPR (iCRISPR) technology to allow for rapid in vivo assessment of novel molecular targets. We have recently developed viral vectors capable of inducing NRAS driven melanomas in vivo with the capacity to delete any specific gene via tetracycline induction of CRISPR/Cas9. This versatile system enables rapid in vivo screening of any candidate genes' role in tumor maintenance. As proof of principle, in Aim 1 we will evaluate NRAS-directed iCRISPR in our NRAS melanoma model. Aims 2 and 3 will assess the role(s) of potential candidates implicated in NRAS tumor maintenance (Ccnd1 and Sos1).