Renalase inhibition for treatment of unresectable melanoma

最近在治疗转移性黑色素瘤方面的进展，特别是免疫检查点抑制剂，导致了总存活率的提高。尽管如此，由于黑色素瘤发病率的增加以及对免疫治疗的原始和获得性抵抗，黑色素瘤的死亡率继续上升。在以前的研究中，我们发现分泌型黄素蛋白肾酶(RNLS)在黑色素瘤细胞中表达上调，并且在人类肿瘤中的高表达与肿瘤的进展和生存期的降低有关。用RNLS抗体和抑制肽在小鼠模型中敲除RNLS可抑制肿瘤生长，减少肿瘤细胞中通过MAPK和PI3K通路的信号转导，并导致CD163+肿瘤相关巨噬细胞(TAMs)显著减少。此外，我们发现CD163+TAMS增加RNLS的产生通过激活STAT3促进黑色素瘤的生长，这表明抑制RNLS可能是通过直接靶向肿瘤细胞和抑制CD163+TAMS来治疗黑色素瘤的有用方法。后一种机制特别有趣，因为已知CD163+TAMs有助于通过效应性T细胞抵抗肿瘤细胞的杀伤。我们建议验证黑色素瘤中RNLS表达和信号异常调节肿瘤生长和扩散的假设，并将进一步研究其机制。此外，我们推测肿瘤细胞和TAMs中的RNLS调节先天和获得性免疫反应，以促进肿瘤的生长和扩散。因此，联合抑制CD8+T细胞中的RNLS和PD-1或CTLA4，与单独使用其中一种方法相比，可以增强抗肿瘤活性。我们将利用我们团队可用的独特资源，包括新的动物模型、新型RNLS抑制剂的可用性，以及获得接受免疫检查点抑制剂治疗的患者的黑色素瘤标本，这些研究将由基础、临床和翻译研究人员组成的跨学科团队进行。我们提出了三个相互关联但不相互依赖的目标。在目标1中，我们将确定RNLS抑制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的先天和获得性免疫反应的机制。在目标2中，我们将全面研究RNLS在具有BRAF和NRAS突变特征的肿瘤细胞中的表达，以及大量转移性黑色素瘤肿瘤(包括接受免疫检查点抑制剂治疗的患者的肿瘤)中TAMS(CD163+或CD86+)、CD4和CD8+细胞的表达。在目标3中，我们将测试这样一个假设，即在具有与人类肿瘤相关的遗传突变(BRAF或NRAS突变)的免疫活性小鼠黑色素瘤模型中，抗RNLS抗体和抗CTLA4或抗PD-1的联合治疗是协同的。这些研究的潜在影响很大--我们正在开发一种用于临床的RNLS抑制剂。如果成功，这些研究将导致未来联合抗RNLS和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，因此可以对癌症治疗产生深远的影响。

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.