Renalase inhibition for treatment of unresectable melanoma

最近治疗转移性黑色素瘤的进展，特别是使用免疫检查点抑制剂，已经导致总生存率的提高。尽管如此，由于发病率增加以及对免疫治疗的原发性和获得性耐药性，黑色素瘤的死亡率继续上升。在之前的研究中，我们发现分泌黄素蛋白Renalase （RNLS）在黑色素瘤细胞中上调，并且在人类肿瘤中的高表达与肿瘤进展和生存率降低有关。在小鼠模型中，RNLS抗体和抑制肽敲除RNLS可抑制肿瘤生长，减少肿瘤细胞中MAPK和PI3K通路的信号传导，并导致CD163+肿瘤相关巨噬细胞（tam）的显著减少。此外，我们发现CD163+ tam通过激活STAT3来增加RNLS的产生，从而促进黑色素瘤的生长，这表明通过直接靶向肿瘤细胞和抑制CD163+ tam来抑制RNLS可能是治疗黑色素瘤的一种有用方法。后一种机制特别有趣，因为CD163+ tam已知有助于抵抗效应T细胞杀死肿瘤细胞。我们拟验证黑素瘤中RNLS表达和信号通路异常调节肿瘤生长和扩散的假说，并进一步研究其机制。此外，我们推测肿瘤细胞和tam中的RNLS调节先天和适应性免疫反应，以促进癌症的生长和扩散。因此，在CD8+ T细胞中，RNLS与PD-1或CTLA4的共抑制比单独使用任何一种方法都能增强抗肿瘤活性。我们将利用我们团队的独特资源，包括新型动物模型，新型RNLS抑制剂的可用性，以及免疫检查点抑制剂治疗患者的黑色素瘤标本，研究将由基础，临床和转化研究人员组成的跨学科团队进行。我们提出三个相互关联但不相互依赖的目标。在Aim 1中，我们将确定RNLS抑制作用影响黑色素瘤细胞内信号通路的机制，以及RNLS在体外和体内调节黑色素瘤先天和适应性免疫反应的机制。在Aim 2中，我们将充分表征RNLS在以BRAF和NRAS突变、tam （CD163+或CD86+）、CD4和CD8+细胞为特征的转移性黑色素瘤肿瘤细胞中的表达，包括接受免疫检查点抑制剂治疗的患者的肿瘤。在Aim 3中，我们将验证抗rnls抗体和抗ctla4或抗pd -1联合治疗在具有与人类肿瘤相关的遗传畸变（BRaf或NRas突变）的免疫能力小鼠黑色素瘤模型中具有协同作用的假设。这些研究的潜在影响是很高的，我们正在开发一种RNLS抑制剂用于临床应用。如果成功，这些研究将导致未来抗rnls联合免疫检查点抑制剂的临床试验。这种方法也可以在其他疾病中进行研究，因此对癌症治疗具有深远的意义。

Recent advances in treating metastatic melanoma, particularly with immune checkpoint inhibitors, have resulted in improved overall survival. Despite this, the mortality from melanoma continues to rise due to the increased incidence and to primary and acquired resistance to immunotherapy. In previous studies we showed that the secreted flavoprotein Renalase (RNLS) is upregulated in melanoma cells and high expression in human tumors is associated with tumor progression and decreased survival. Knock-down of RNLS by RNLS antibodies and an inhibitory peptide in murine models inhibited tumor growth, decreased signaling through the MAPK and PI3K pathways in tumor cells, and caused a marked reduction in CD163+ Tumor Associated Macrophages (TAMs). Furthermore, we showed that increased RNLS production by CD163+ TAMs facilitates melanoma growth by activating STAT3, suggesting that inhibition of RNLS might be a useful approach for treating melanoma by direct tumor cell targeting and by inhibition of CD163+ TAMs. The latter mechanism is particularly interesting as CD163+ TAMs are known to facilitate resistance to tumor cell kill by effector T cells. We propose to test the hypothesis that dysregulated RNLS expression and signaling in melanomas modulates tumor growth and spread and will further study the mechanism thereof. Furthermore, we surmise that RNLS in tumor cells and TAMs modulates both the innate and adaptive immune response to promote cancer growth and spread. Therefore, co-inhibition of RNLS and PD-1 or CTLA4 in CD8+ T cells could result in enhanced anti-tumor activity compared to either approach alone. We will capitalize on unique resources available to our team that include novel animal models, availability of a novel RNLS inhibitor, and access to melanoma specimens from patients treated with immune checkpoint inhibitors, and the studies will be conducted by an interdisciplinary team of basic, clinical and translational researchers. We propose three inter-related but not inter-dependent aims. In Aim 1 we will determine the mechanism by which RNLS inhibition affects signaling pathways within melanoma cells and the mechanism by which RNLS modulates the innate and adaptive immune response to melanoma in vitro and in vivo. In Aim 2 we will fully characterize expression of RNLS in tumor cells characterized for BRAF and NRAS mutations, TAMs (CD163+ or CD86+), CD4 and CD8+ cells in large cohorts of metastatic melanoma tumors, including tumors from patients treated with immune checkpoint inhibitors. In Aim 3 we will test the hypothesis that combination therapy with anti-RNLS antibodies and anti-CTLA4 or anti-PD-1 are synergistic in immune competent murine melanoma models with genetic aberrations relevant to human tumors (mutations in BRaf or NRas). The potential impact of these studies is high – we are developing a RNLS inhibitor for clinical use. If successful, these studies will lead to future clinical trials of the combination anti-RNLS with an immune checkpoint inhibitor. This approach can be studied in other diseases as well and can thus have far-reaching implications for cancer therapy.