Combining Immune Therapy with Targeted Therapies to Improve Melanoma Survival

转移性黑色素瘤是美国增长最快的肿瘤类型之一，我们的退伍军人患转移性黑色素瘤的风险更高。 发展成黑素瘤利用免疫检查点抑制剂（ICI）进行免疫治疗的最新治疗进展 彻底改变了晚期/转移性黑色素瘤的治疗方法。靶向PD-1/PD-L1的抗体（纳武单抗， pembrolizumab，atezolizumab）和CTLA-4（ipilimumab）引起强大的抗黑素瘤免疫应答1，2，3。 对于转移性疾病，已经证明用伊匹单抗与纳武单抗组合或单独纳武单抗治疗是有效的。 5-年总生存率（OS）分别为52%和44%，许多患者经历了异常的 反应尽管ICIs的生存结局有所改善，但大多数患者要么没有反应， 对ICI产生抗药性。需要新的治疗策略来改善黑色素瘤患者的预后 用ICI治疗并解决获得性耐药性。开发新的治疗方法， 对ICI治疗表现出原发性或获得性耐药的黑色素瘤患者是我们建议的重点。我们 来自临床前模型的初步数据表明，将Ras模拟物rigosertib（RGS）与ICI组合， 显著降低黑色素瘤肿瘤生长，即使在对ICI反应差的肿瘤中。RGS 治疗导致肿瘤免疫微环境（TIME）富含CD 8 +T细胞、树突状细胞（DC） 和自然杀伤细胞，但不减少肿瘤内髓源性抑制细胞（MDSC）。RGS 还增加黑素瘤肿瘤细胞上的CD 40表达以促进免疫原性肿瘤细胞死亡。在 不表达CD 40的肿瘤细胞，或不表达CD 8 +T细胞的TIME，RGS的抗肿瘤活性 已经消失了我们假设RGS诱导的黑色素瘤细胞中CD 40的表达是黑色素瘤发生的关键因素。 RGS增强的抗肿瘤免疫，并且向RGS +ICI中添加CD 40激动剂将增强CD 40- 介导的免疫原性细胞死亡。我们还建议在RGS+ICI中加入CXCR 1/2拮抗剂， 治疗将阻断MDSC向肿瘤的募集，允许延长的CD 8 +T细胞抗肿瘤活性。中的任一 这些方法将进一步激活抗肿瘤免疫应答以诱导肿瘤消退。 我们将追求三个具体目标来解决这个假设。目标1：确定 RGS诱导肿瘤细胞表面CD 40的表达，并表征肿瘤细胞CD 40在恢复中的作用 免疫活性小鼠黑素瘤模型对ICI的反应。目标二。要评估是否添加了 CD 40激动剂抗体将增强黑色素瘤对RGS + ICI的敏感性并确定机制 使用小鼠模型和人类类器官共培养系统的反应。肿瘤、免疫和 将通过流式细胞术和单细胞（sc）RNAseq评估转录组变化 分析以提供关于该组合疗法如何调节对疗法的反应的机制性见解。目标3： 确定RGS联合CXCR 1/2拮抗剂是否能使黑色素瘤对ICI重新敏感 使用与目标2相似的方法进行治疗。意义：这些研究将产生新的治疗方法 通过增强ICI反应改善退伍军人转移性黑色素瘤治疗的方法 治疗或使ICI抗性肿瘤对ICI治疗重新敏感。

Metastatic melanoma is one of the fastest growing tumor types in the US and our Veterans are at higher risk for developing melanoma. Recent therapeutic advances in immunotherapy utilizing immune checkpoint inhibitors (ICIs) have revolutionized the treatment of advanced/metastatic melanoma. Antibodies targeting PD-1/PD-L1 (nivolumab, pembrolizumab, atezolizumab) and CTLA-4 (ipilimumab) evoke powerful anti-melanoma immune responses1,2,3. For metastatic disease, treatment with ipilimumab combined with nivolumab or nivolumab alone has demonstrated 5-year overall survival (OS) rates of 52% and 44%, respectively, with many patients experiencing exceptional response. Despite improvement in survival outcomes with ICIs, the majority of patients either fail to respond or develop resistance to ICIs. Novel therapeutic strategies are needed to improve outcomes for melanoma patients treated with ICI and to address acquired resistance. Development of new therapeutic approaches for melanoma patients who exhibit primary or acquired resistance to ICI therapy is the focus of our proposal. Our preliminary data from preclinical models indicate that combining the Ras mimetic, rigosertib (RGS) with ICI significantly reduces melanoma tumor growth, even in tumors that are poorly responsive to ICIs. RGS treatment results in a tumor immune microenvironment (TIME) enriched in CD8+T cells, dendritic cells (DCs) and natural killer cells, but does not reduce intratumoral myeloid-derived suppressor cells (MDSCs). RGS also increases CD40 expression on the melanoma tumor cells to facilitate immunogenic tumor cell death. In the absence of CD40 expression by tumor cells, or without CD8+T cells in the TIME, RGS’s anti-tumor activity is lost. We hypothesize that RGS-induced expression of CD40 in melanoma cells is a key component of RGS-enhanced anti-tumor immunity and that addition of a CD40 agonist to the RGS +ICI will enhance CD40- mediated immunogenic cell death. We also propose that addition of a CXCR1/2 antagonist to the RGS+ICI therapy will block MDSC recruitment to tumor, allowing prolonged CD8+T cell anti-tumor activity. Either of these approaches would further activate the anti-tumor immune response to induce tumor regression. We will pursue three specific aims to address this hypothesis. Aim 1: To determine the mechanism by which RGS induces expression of CD40 on tumor cells and to characterize the role of tumor cell CD40 in restoring response to ICI in immunocompetent mouse models of melanoma. Aim 2. To evaluate whether addition of a CD40 agonist antibody will enhance sensitivity of melanoma tumors to RGS + ICI and define mechanisms of response using mouse models and human organoid co-culture systems. Tumoral, immune, and transcriptome changes will be evaluated through multicolor flow cytometry and single cell(sc) RNAseq analysis to provide mechanistic insight on how this combined therapy modulates response to therapy. Aim 3: To determine whether RGS combined with a CXCR1/2 antagonist, can re-sensitize melanoma tumors to ICI therapy using similar approaches as for Aim 2. Significance: These studies will result in new therapeutic approaches to improve treatment of metastatic melanoma in our Veterans by enhancing response to ICI therapies or re-sensitizing ICI-resistant tumors to treatment with ICI.