New Strategies for Treatment of NRAS Mutant Melanoma after Progression on Immune Checkpoint Inhibitors

用免疫检查点抑制剂(ICI)治疗转移性黑色素瘤延长了许多黑色素瘤的生命 患者，但绝大多数患者经历了疾病的进展，促使需要替代疗法。 对于约50%的BRAFmut肿瘤患者，BRAF和MEK抑制剂的治疗提供了很好的二线治疗 治疗选项。不幸的是，对于25%-30%的肿瘤患者来说，几乎没有二线治疗的选择 NRAS突变。由于近40%的黑色素瘤患者表现出缺失、突变或表观遗传学沉默 CDK4/6调节因子CDKN2A，我们推测抑制CDK4/6可能诱导NRASmut RBWT肿瘤的反应 伴随着CDKN2A的损失。因为CDKN2A的缺失也会扰乱ARF，ARF是MDM2介导的降解的抑制因子 对于p53WT黑色素瘤，抑制MDM2以恢复细胞周期控制将是至关重要的。我们已经证明了 对ICI获得性耐药的NRASmut黑色素瘤对CDK4/6抑制剂和AN联合治疗的反应 MDM2，肿瘤生长减少，CD8+T细胞向肿瘤内募集增加。这些 肿瘤包含大量表达髓系来源的抑制细胞(MDSCs)的CXCR1，2，这些细胞可以产生 免疫抑制的肿瘤微环境。我们的初步数据显示，当CXCR2在髓系中缺失时 细胞，MDSC对肿瘤的募集减少，肿瘤生长受到抑制。此外，CXCR1，2的系统交付 抑制剂抑制小鼠NRASmut黑色素瘤的生长(p&lt；0.02)，并诱导黑素细胞中CXCR2的缺失 在可诱导的BRAFV600E/PTEN-/-黑色素瘤小鼠模型中阻止黑色素瘤的形成。这些耐人寻味的发现 支持先前的研究表明CXCR2抑制剂在黑色素瘤治疗中的作用。然而，这些机制 对CXCR1，2拮抗反应的共性还有待进一步阐明。前提和假设：CXCR2 通过促进肿瘤形成和诱导免疫抑制肿瘤，在黑色素瘤中发挥关键和促多发性作用 环境。此外，CDK4/6和MDM2的联合抑制显著抑制了小鼠和人的生长 NRASmut黑色素瘤。我们假设CXCR1，2，CDK4/6和MDM2在NRASmut中的共同抑制 对ICI具有获得性耐药的黑色素瘤将抑制肿瘤细胞增殖，诱导肿瘤细胞死亡，刺激抗ICI 肿瘤免疫，并有可能克服对ICI的获得性耐药性。我们提出了三个具体目标。1)检查 CDK4/6i和MDM2i联合CXCR1，2拮抗剂治疗或单独使用CXCR1，2激动剂的能力 增强或恢复NRASmut黑色素瘤的ICI敏感性。2)确定黑素细胞表达的作用 CXCR2在黑色素瘤发生中的作用CXCR2将被删除，以符合诱导小鼠黑色素瘤形成和 该缺失对黑素细胞的凋亡、衰老、分化和增殖的影响将被表征。 3)确定小鼠的研究结果是否转化为人类黑色素瘤，改变了肿瘤的免疫功能 微环境(时间)将随着时间的推移表征CXCR1，2拮抗剂(CXCR1，2i)与或 在人源化NRASmut患者来源的异种移植(PDX)模型中不使用CDK4/6i+MDM2i和ICI治疗 人NRAS-MUT黑色素瘤有机共培养。这些数据将为未来的临床试验提供信息。

Treatment of metastatic melanoma with immune checkpoint inhibitors (ICI) has extended the life of many melanoma patients, but the vast majority of patient experience disease progression, prompting the need for alternate therapies. For ~50% of patients with BRAFmut tumors, treatment with BRAF and MEK inhibitors provide a good second-line treatment option. Unfortunately, there are few second-line options for the 25-30% of patients whose tumors harbor NRAS mutations. Since nearly 40% of all melanoma patients exhibit loss, mutation, or epigenetic silencing of the CDK4/6 regulator CDKN2A, we postulate that inhibition of CDK4/6 may induce response in NRASmut RBWT tumors with loss of CDKN2A. Because loss of CDKN2A also disrupts ARF, a suppressor of MDM2-mediated degradation of p53, it will be essential to also inhibit MDM2 to restore cell cycle control in p53WT melanoma. We have shown that NRASmut melanoma tumors with acquired resistance to ICI respond to co-treatment with a CDK4/6 inhibitor plus an MDM2, demonstrating both with reduced tumor growth and enhanced CD8+T cell recruitment into the tumor. These tumors contain a significant number of CXCR1,2 expressing myeloid-derived suppressor cells (MDSCs) that create an immune suppressive tumor microenvironment. Our preliminary data show that when CXCR2 is deleted in myeloid cells, MDSC recruitment to tumor is reduced and tumor growth is inhibited. Moreover, systemic delivery of a CXCR1,2 inhibitor reduced the growth of NRASmut melanoma in mice (p<0.02), and inducible deletion of CXCR2 in melanocytes blocks melanoma formation in the inducible BRAFV600E/PTEN-/- melanoma mouse model. These intriguing findings support prior studies indicating a role for CXCR2 inhibitors for treatment of melanoma. However, the mechanisms and generality of response to CXCR1,2 antagonism require further elucidation. Premise and Hypothesis: CXCR2 plays critical and pleotropic role in melanoma by promoting tumorigenesis and inducing an immunosuppressive tumor environment. Moreover, combined CDK4/6 and MDM2 inhibition significantly inhibits the growth of mouse and human NRASmut melanoma tumors. We hypothesize that co-inhibition of CXCR1,2, CDK4/6, and MDM2 in NRASmut melanoma with acquired resistance to ICI will inhibit tumor cell proliferation, induce tumor cell death, stimulate anti- tumor immunity, and potentially overcome the acquired resistance to ICI. We propose 3 specific aims. 1)To examine the ability of CDK4/6i plus MDM2i, combined with a CXCR1,2 antagonist treatment, or CXCR1,2 agonist alone, to enhance or restore ICI sensitivity for NRASmut melanoma tumors. 2) To determine the role of melanocyte-expressed CXCR2 in melanoma initiation. CXCR2 will be deleted coincident with induction of melanoma formation in mice and effects of this deletion on melanocyte apoptosis, senescence, differentiation and proliferation will be characterized. 3) To determine whether findings in mouse translate to human melanoma, changes in the tumor immune microenvironment (TIME) will be characterized over time in response to a CXCR1,2 antagonist (CXCR1,2i) with or without CDK4/6i+MDM2i followed by ICI therapy in humanized NRASmut patient-derived xenograft (PDX) models and human NRAS mut melanoma organoid co-cultures. These data will inform future clinical trials.