GMPS-GMPR Axis Melanoma Progression and Therapy

恶性黑色素瘤是最具侵袭性的人类癌症类型之一。它的转移能力 再加上它对常规化疗药物臭名昭著的耐药性， 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 个月原发性黑色素瘤已经具有侵袭能力， 对于转移，并且被认为是与不良预后相关的关键事件。 我们最近证明鸟苷一磷酸还原酶（GMPR）的表达， 一种参与嘌呤核苷酸从头生物合成的酶，在侵袭阶段下调 人类黑色素瘤功能丧失和获得实验表明，GMPR抑制了 黑色素瘤细胞形成侵袭伪足，降解细胞外基质，侵入（体外和体内），并生长为 肿瘤异种移植物我们进一步证明了鸟苷一磷酸合酶（GMPS）的缺失， GMPR的功能性拮抗剂，降低活性（GTP结合）RAC 1、RHOA和RHOC。我们假设 GMPS、GMPR和可能的其他鸟苷酸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 在具体目标1中测试。 我们证明了GMPR的活性可以通过磷酸化来调节。无偏体外激酶 筛选鉴定了几种激酶作为GMPR磷酸化的潜在候选物。GTP的调节 水平从未被认为是任何激酶调节侵袭的机制。因此在 具体目标2，使用功能性方法，我们将测试候选人调节GTP水平，RHO- 以GMPR依赖性方式的GT β活性和细胞侵袭。 目前，对于具有野生型BRAF和突变型NRAS的黑色素瘤患者不存在有效的化疗。 通过引入其抑制剂维罗非尼（VEM）， 然而，迅速发展的耐药性会阻碍VEM的功效。尽管这种抵抗的机制 不同的是，几个独立选择的VEM抗性黑素瘤细胞系具有增加的侵袭性 能力我们证明了几种GTP消耗剂可以显著影响黑色素瘤异种移植物， 在免疫受损和免疫活性小鼠中生长。因此，在具体目标3中，我们将评估 在几种治疗中，单独使用一种这样的药物或与现有的抗黑素瘤疗法联合使用的疗效 临床前黑素瘤模型。

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.