GCDH Addiction in Melanoma

项目摘要 在这个修订的R01申请中，我们建议描述一种新的潜在的细胞凋亡控制机制 通过线粒体GCDH酶，并延伸小分子GCDH抑制剂的开发来治疗 黑色素瘤。在我们的初步结果中，我们发现对GCDH活性的依赖是细胞存活的关键 黑色素瘤，但不是肝脏、乳腺或前列腺肿瘤细胞，这一发现已在患者样本中得到证实 在黑色素瘤中，GCDH的表达与生存期呈负相关。我们的研究表明 GCDH抑制后黑色素瘤细胞系的凋亡诱导，这种表型依赖于上游 DHTKD1酶。在黑色素瘤中介导GCDH活性的关键是NRF2，它受到戊二醛的作用 在抑制GCDH时。NRF2戊二酸化促进其稳定性和对凋亡的UPR的反式激活 信号由ATF4、ATF3、CHOP和CHAC1组成，后者是未折叠蛋白的组成部分 回应(普遍定期审议)。抑制GCDH有效地诱导黑色素瘤细胞凋亡UPR信号转导，但对肝脏无明显作用 或乳腺癌细胞，证实了该通路在细胞系和患者肿瘤中的选择性 样本。这些观察结果为我们的假设提供了基础，即GCDH介导的调节 NRF2-UPR信号通路构成了控制黑色素瘤细胞存活的新途径。我们的研究将 (I)绘制黑色素瘤中NRF2戊二酸化途径的图谱，产生针对戊二酸化NRF2的抗体，监测 黑色素瘤患者治疗有效和无效患者标本中NRF2的戊二醛作用(II) 评估GCDH如何影响肿瘤的发生、发展以及对靶向和免疫治疗的反应 遗传小鼠模型。将对YUMM1.7、MARS、B16F10进行修饰，以表达GCDH的可诱导KD 在将它们接种到同基因的WT或GCDH KO小鼠模型之前，将事先和 后续治疗(Iii)发展GCDH抑制剂作为新的治疗方法。印度国家石油公司-0690564 在体外和培养的黑色素瘤细胞中得到证实，在那里它表现出对GCDH的遗传失活并抑制 小鼠的黑色素瘤。我们将表征并进一步开发一类新的靶向GCDH的抑制剂 并用小鼠模型确定它们在培养和体内的有效性。我们的研究将建立新的 当我们定义GCDH控制黑色素瘤肿瘤命运的潜在机制时，GCDH信号的范例。 了解黑色素瘤患者对GCDH的依赖程度为黑色素瘤的开发和评估提供了基础 新的GCDH抑制剂选择性地靶向这些癌症，可能还有其他选定的癌症。

Project Summary In this revised R01 application we propose to characterize a novel mechanism underlying the control of apoptosis by the mitochondrial enzyme GCDH, and to extend the development of small molecule GCDH inhibitors to treat melanoma. In our preliminary results we discovered that addiction to GCDH activity is critical for cell survival in melanoma, but not liver, breast or prostate tumor cells, a discovery that was confirmed in patient specimens where inverse correlation between GCDH expression and survival is seen in melanoma. Our studies reveal induction of apoptosis in melanoma cell lines following GCDH inhibition, a phenotype dependent on the upstream DHTKD1 enzyme. Key in mediating GCDH activities in melanoma is NRF2, which is subjected to glutarylation upon inhibition of GCDH. NRF2 glutarylation promotes its stability and transactivation of an apoptotic UPR signaling consisting of ATF4, ATF3, CHOP and CHAC1, the latter being components of the Unfolded Protein Response (UPR). Inhibition of GCDH effectively induces apoptotic UPR signaling in melanoma but not in liver or breast cancer cells, substantiating the selectivity of the pathway as mapped in cell lines and patient tumor samples. These observations provide the foundation for our hypothesis that GCDH-mediated regulation of NRF2-UPR signaling constitutes a novel pathway controlling melanoma cell survival. Our studies will (i) map the NRF2 glutarylation pathway in melanoma, generate antibodies specific to glutarylated NRF2, monitor NRF2 glutarylation in specimens from melanoma patients both responsive and non-responsive to therapy (ii) assess how GCDH impacts tumor development, progression and response to targeted and immuno therapy in genetic mouse models. YUMM1.7, MaRas, B16F10 lines will be modified to express inducible KD of GCDH before their inoculation into syngeneic WT or GCDH KO mouse models which will be monitored prior and following therapy (iii) advance development GCDH inhibitors as novel therapeutic modalities. SBI-0690564 was confirmed in vitro and in cultured melanoma cells, where it phenocopies genetic inactivation of GCDH and inhibits melanoma tumors in mice. We will characterize and further develop a novel class of inhibitors to target GCDH and determine their effectiveness in culture and in vivo using mouse models. Our studies will establish novel paradigm for GCDH signaling as we define mechanisms underlying GCDH control melanoma tumor fate. Understanding the addiction to GCDH in melanoma provides the foundation for the development and evaluation of novel GCDH inhibitors to selectively target these and possibly other select cancers.