GMPS-GMPR Axis Melanoma Progression and Therapy

恶性黑色素瘤是人类最具侵袭性的癌症之一。它在体内转移的能力 与其对传统化疗药物的众所周知的耐药性相结合，导致黑色素瘤 极难治愈。因此，转移性黑色素瘤患者的中位生存期仅为8.5 月份。获得侵袭能力是一个先决条件，这种能力已经出现在原发性黑色素瘤中。 对于转移，被认为是与不良预后相关的关键事件。 我们最近证实了鸟苷一磷酸还原酶(GMPR)的表达。 参与嘌呤核苷酸从头合成的酶在侵袭阶段被下调 人类黑色素瘤。功能丧失和功能获得实验表明，GMPR抑制 黑色素瘤细胞形成侵袭足，降解细胞外基质，侵袭(体外和体内)，并生长为 肿瘤异种移植。我们进一步证明了鸟苷一磷酸合成酶(GMPS)的耗竭 GMPR的功能性拮抗剂可降低活性(GTP结合)RAC1、RHOA和RHOC。我们假设 GMPS、GMPR，也许还有其他鸟苷生物合成酶，调节上述酶的活性 Rho-GTP酶通过调节这些Rho-GTP酶附近的GTP水平。这一假设将是 在特定目标1中进行测试。 我们证明了GMPR的活性可以通过磷酸化来调节。无偏的体外激活酶 筛选确定了几个潜在的GMPR磷酸化候选蛋白。GTP的调制 水平从未被认为是任何一种激酶侵袭的调节机制。因此，在 具体目标2，使用功能方法，我们将测试候选人调节GTP水平的能力，Rho- GTPase活性与细胞侵袭力呈GMPR依赖关系。 目前，对于患有野生型BRAF和突变NRAS的黑色素瘤患者，还没有有效的化疗方法。 突变型BRAF患者的存活率通过引入其抑制剂维莫拉非尼(VEM)而得到改善。 然而，快速发展的耐药性绕过了VEM的效果。尽管这种抗性的机制 不同的是，几个独立选择的VEM抗性黑色素瘤细胞株具有更高的侵袭力 才能。我们证明了几种GTP耗尽剂可以显著影响黑色素瘤的异种移植。 免疫功能低下和免疫功能正常的小鼠的生长。因此，在具体目标3中，我们将评估 其中一种药物单独或与现有的抗黑色素瘤疗法联合治疗在几种情况下的疗效 临床前黑色素瘤模型。

Malignant melanoma is one of the most aggressive types of human cancers. Its ability to metastasize in combination with its notorious resistance to conventional chemotherapeutical agents makes melanoma extremely difficult to cure. Consequently, the median survival of patients with metastatic melanoma is only 8.5 months. The acquisition of invasion capability, which occurs already in primary melanomas, is a prerequisite for metastasis and is considered a critical event associated with poor prognosis. We have recently demonstrated that expression of guanosine monophosphate reductase (GMPR), an enzyme involved in the de novo biosynthesis of purine nucleotides, was downregulated at the invasive stages of human melanoma. Loss- and gain-of-function experiments revealed that GMPR suppresses the ability of melanoma cells to form invadopodia, degrade extracellular matrix, invade (in vitro and in vivo), and grow as tumor xenografts. We further demonstrated that depletion of guanosine monophosphate synthase (GMPS), a functional antagonist of GMPR, decreases active (GTP-bound) RAC1, RHOA and RHOC. We hypothesized that GMPS, GMPR, and perhaps other guanylate biosynthesis enzymes, regulate the activity of the above RHO-GTPases via modulation of GTP levels in the vicinity of these RHO-GTPases. This hypothesis will be tested in Specific Aim 1. We demonstrated that the activity of GMPR can be regulated by phosphorylation. Unbiased in vitro kinase screening identified several kinases as potential candidates for GMPR phosphorylation. Modulation of GTP levels has never been considered as a mechanism of regulation of invasion by any kinase. Therefore, in Specific Aim 2, using functional approaches, we will test candidates' ability to regulate GTP levels, RHO- GTPase activity and cell invasion in a GMPR-dependent manner. Currently, no efficient chemotherapy exists for melanoma patients with wildtype BRAF and mutant NRAS. Survival of patients with mutant BRAF was improved by the introduction of its inhibitor vemurafenib (VEM), however rapidly developing resistance circumvents VEM efficacy. Although the mechanisms of such resistance vary, several melanoma cell lines independently selected for VEM resistance possessed increased invasion ability. We demonstrated that several GTP-depleting agents can significantly affect melanoma xenograft growth in immunocompromised and immunocompetent mice. Therefore, in Specific Aim 3, we will evaluate the efficacy of one of such agents alone or in combination with existing anti-melanoma therapy in several preclinical melanoma models.