Mechanism of Resistance to Arginine Deiminase Therapy in Advance Melanoma

描述（申请人提供）：皮肤恶性黑色素瘤在晚期发现时可能具有高度致死性，总体治愈率<10%。几乎所有的恶性黑色素瘤都是精氨酸自养型的，因为这些黑色素瘤细胞不表达精氨酸琥珀酸合成酶（AS），这是瓜氨酸生物合成精氨酸的限速酶。因此，黑色素瘤细胞需要来自细胞外来源的精氨酸来存活。当这些黑色素瘤细胞暴露于精氨酸脱亚胺酶（ADI）（其将精氨酸降解为瓜氨酸和氨）时，它们变成精氨酸饥饿，导致细胞死亡;而表达AS的正常细胞能够存活。ADI是一种细菌酶，并且已经配制了聚乙二醇化酶（ADI-PEG 20）。使用ADI-PEG 20治疗晚期黑色素瘤的I/II期临床试验已经产生了有利的结果。我们已经了解到，在ADI不敏感的患者中观察到AS的重新表达。与这些发现相一致，我们发现ADI处理诱导了一些黑素瘤细胞中AS的表达;并且诱导的AS表达与ADI抗性相关。此外，抑制ADI处理的黑色素瘤细胞中AS表达的诱导抑制了耐药性的发展。这些结果表明，AS的再表达是与ADI-PEG 20抗性相关的重要机制，并强调了理解AS诱导机制以克服抗性发展的重要性。我们一直在研究黑色素瘤细胞中AS诱导的机制，发现正调节因子c-Myc和负调节因子HIF-1a参与其中。我们还观察到，在一些黑色素瘤细胞中AS表达诱导失败与c-Myc不能与AS启动子相互作用有关。我们提出了以下四个具体的目标，以进一步阐明AS表达的调控机制：我们的初步结果表明，磷脂酰肌醇3-激酶（PI 3 K）-AKT通路可能参与调节c-Myc介导的AS诱导ADI-PEG 20。我们建议在具体目标1中进一步阐明该途径在诱导中的参与。我们的初步结果表明，ADI-PEG 20处理导致HIF-1a下调。我们建议在特定目标2中阐明下调机制的分子基础。在具体目标3中，我们建议研究c-Myc/HIF-1a在ADI耐药黑色素瘤细胞中维持AS表达的作用。我们还建议使用小分子通过抑制AS表达来克服耐药性。在具体目标4中，我们建议研究黑色素瘤细胞中产生的ADI-PEG-20耐药机制是否可以在来自接受ADI-PEG-20治疗的黑色素瘤患者的组织标本中得到验证。本研究的最终目的是通过充分了解精氨酸可利用性调节AS的分子基础，改善ADI-PEG 20治疗晚期黑色素瘤的治疗效果。 公共卫生相关性：本申请研究了由于在这种人类恶性肿瘤中缺乏氨基琥珀酸合成酶（AS）表达而对使用精氨酸脱亚胺酶（ADI-PEG 20）的人类黑色素瘤的新治疗模式的抗性机制。虽然已发现ADI-PEG 20在体外和I/II期临床试验中具有活性，但由于对药物的抗性，并非所有患者都对ADI-PEG 20治疗的精氨酸剥夺有反应。我们提出了避免耐药性发展的方法，从而提高ADI-PEG 20对人恶性黑色素瘤的治疗效果。

DESCRIPTION (provided by applicant): Malignant melanoma of the skin can be highly lethal when the disease is detected in the advanced stages with overall cure rate of <10%. Almost all of malignant melanoma is autotrophic for arginine because these melanoma cells do not express argininosuccinate synthetase (AS), the rate-limiting enzyme for the biosynthesis of arginine from citrulline. Thus, melanoma cells require arginine from extracellular sources for survival. When these melanoma cells are exposed to arginine deiminase (ADI) which degrades arginine to citrulline and ammonia, they become arginine starvation, leading to cell death; while normal cells which express AS are able to survive. ADI is a bacterial enzyme and a pegylated enzyme (ADI- PEG20) has been formulated. Phase I/II clinical trials using ADI-PEG20 for advanced melanoma have produced favorable results. We have learned that re-expression of AS was observed in the ADI-insensitive patients. Consistent with these findings, we found that ADI treatment induces AS expression in some melanoma cells; and the induced AS expression is associated with ADI resistance. Moreover, suppressing induction of AS expression in ADI-treated melanoma cells inhibited the development of resistance. These results suggest that the re-expression of AS is an important mechanism associated with ADI-PEG20 resistance and underscores the importance of understanding the mechanism(s) of AS induction to overcome the development of resistance. We have been studying the mechanism of AS induction in melanoma cells and discovered that the positive regulator c-Myc and the negative regulator HIF-1a are involved. We have also observed that failure of induction of AS expression in some melanoma cells is associated with the inability of c-Myc to interact with the AS promoter. We propose the following four specific aims to further elucidate the regulatory mechanisms of AS expression: Our preliminary results suggest that phosphatidylinositol 3-kinase (PI3K)-AKT pathway may be involved in the regulation of c-Myc- mediated AS induction by ADI-PEG20. We propose in Specific Aim 1 to further elucidate the involvement of this pathway in the induction. Our preliminary results showed that ADI-PEG20 treatment results in downregulation of HIF-1a. We propose in Specific Aim 2 to elucidate the molecular basis underlying the downregulation mechanism. In Specific Aim 3, we propose to investigate the role of c-Myc/HIF-1a in the maintenance of AS expression in the ADI-resistant melanoma cells. We also propose to use small molecules to overcome the resistance through suppression of AS expression. In Specific Aim 4, we propose to investigate whether ADI-PEG20-resistant mechanisms developed in melanoma cells can be validated in tissue specimens derived from melanoma patients treated with ADI-PEG-20. The ultimate goal of this research is to improve therapeutic outcome in advanced melanoma by ADI-PEG20 treatment by fully understanding the molecular basis of AS regulation by arginine availability. PUBLIC HEALTH RELEVANCE: This application investigates the mechanism of resistance to the new treatment modality of human melanoma with arginine deiminase (ADI-PEG20) due to the lack argininosuccinate synthetase (AS) expression in this human malignancy. While ADI- PEG20 has been found to be active in vitro and in phase I/II clinical trials, not all patients respond to arginine deprivation with ADI-PEG20 therapy due to resistance to the drug. We propose approaches to circumvent the development of resistance, thereby to enhance the treatment efficacy of human malignant melanoma with ADI-PEG20.