Impact of N-Acetylgalactosamine-4-Sulfatase (Arylsulfatase B) on the Progression of Melanoma

恶性黑色素瘤的发病率和死亡率是退伍军人及其家人面临的重大问题， 和普通公众。在早期诊断和治疗方面取得了进展，特别是取得了进展 在检查点抑制疗法中。然而，并不是所有的患者都对这种方法有反应，治疗有很大的进步。 毒性。在黑色素瘤的治疗方面，仍有未得到满足的需求。这个项目展示了一种 新的和潜在的突破性治疗方法，基于酶N- 乙酰氨基半乳糖-4-硫酸酯酶，也称为芳基硫酸酯酶B(ARSB)。这种酶能去除硫酸盐基团 从软骨素4-硫酸盐(C4S)和皮肤素硫酸盐中提取，是降解这些硫酸盐所必需的 糖胺聚糖。我们之前的工作表明，ARSB活性较低与更具攻击性相关 黑色素瘤。此外，ARSB的下降会导致黑色素瘤蛋白多糖软骨素的表达增加 硫酸盐蛋白多糖(CSPG)4和基质金属蛋白酶原-MMP2促进侵袭。其他 实验表明，ARSB沉默后，黑色素瘤细胞中PD-L1的表达增加。 在这个项目中，我们将确定ARSB下降增加的转录机制 PD-L1在正常黑素细胞和黑色素瘤细胞中的表达实验将展示如何减少和减少 ARSB的增加影响黑色素瘤细胞的存活和细胞内信号转导。抗PD1治疗对血管紧张素转换酶活性的影响 在ARSB沉默后，黑色素瘤细胞将在与免疫细胞共培养的活细胞中进行测试。其他 对B16F10和Yumm小鼠黑色素瘤模型以及人源化小鼠异种移植模型的研究将 显示ARSB的调节与检查点抑制剂治疗相关的对进展的影响 原发和转移性黑色素瘤。该项目基于30多种出版物，其中托巴克曼博士和 合作者已经确定了ARSB下降的生物学后果。包含项目的上一份报告 合作者，Arkadiusz Dudek，医学博士，杰出的肿瘤学家和浓厚兴趣的研究员， 免疫肿瘤学的背景和临床经验表明，随着ARSB的增加，ARSB的下降 黑色素瘤细胞系的侵袭性，以及CSPG4和支持MMP2的表达显著增加。 ARSB先天缺陷存在于溶酶体储积症粘多糖症(MPS)VI， 其中突变导致ARSB活性显著降低。来自前列腺、乳腺、结肠的恶性细胞， 以及在黑色素瘤细胞和组织中，我们已经证明ARSB的表达和活性是 与正常黑素细胞和组织相比，黑素细胞减少。ARSB的下降导致硫酸盐的积累 糖胺聚糖软骨素4-硫酸盐和皮肤素硫酸盐，ARSB通常从它们中去除4- 在非还原端的硫酸盐基团，并需要启动它们的降解。随着ARSB、C4S的下降 积聚，它与关键分子的相互作用被破坏。我们已经证明了Galectin-3，一种共同的- 与其他重要分子结合的转录激活剂，包括胰岛素受体，结合较少 C4S的硫酸盐化程度更高，并增加了核转位和与转录因子的相互作用。 相反，SHP2(PTPN11)，普遍存在的非膜Src同源区2(SH2)蛋白 酪氨酸磷酸酶2，更多地与高度硫酸盐化的C4S结合，较少可用于去磷酸化 关键的信号分子，包括磷酸化ERK1/2。因此，转录事件引起的改变 C4S的硫酸盐化对重要的细胞过程有深远的影响。 该项目将为ARSB的变化以及由此导致的软骨变化提供新的见解 4-硫酸化、信号转导和转录事件，对黑色素瘤进展和对 检查点抑制。这一发现可能会导致治疗黑色素瘤的新方法和改进 以应对检查点抑制，从而减少黑色素瘤的痛苦、发病率和死亡率。

The morbidity and mortality of malignant melanoma are significant problems for veterans, their families, and the general public. Progress has been made in early diagnosis and in treatment, in particular with advances in checkpoint inhibition therapy. However, not all patients respond to this approach and the treatment has major toxicity. There is a continuing unmet need for improvement in treatment of melanoma. This project presents a novel and potentially breakthrough treatment approach, based on the impact of the enzyme N- acetylgalactosamine-4-sulfatase, also known as arylsulfatase B (ARSB). This enzyme removes sulfate groups from chondroitin 4-sulfate (C4S) and dermatan sulfate and is required for the degradation of these sulfated glycosaminoglycans. Our previous work has shown that lower ARSB activity is associated with more aggressive melanomas. Also, decline in ARSB leads to increased expression of the melanoma proteoglycan chondroitin sulfate proteoglycan (CSPG)4 and of the matrix metalloproteinase pro-MMP2 which facilitates invasion. Other experiments have shown increase in PD-L1 expression in melanoma cells following ARSB silencing. In this project, we will determine the transcriptional mechanism by which decline in ARSB increases expression of PD-L1 in normal melanocytes and melanoma cells. Experiments will show how decline and increase in ARSB affect melanoma cell survival and intracellular signaling. The impact of anti-PD1 treatment on melanoma cell survival will be tested in live cell co-culture with immune cells following ARSB silencing. Other studies in the B16F10 and YUMM mouse models of melanoma and in a humanized mouse xenograft model will show the impact of modulation of ARSB in association with checkpoint inhibitor treatment on the progression of primary and metastatic melanomas. This project is based on over 30 publications in which Dr. Tobacman and collaborators have identified biological consequences of decline in ARSB. A previous report with project collaborator, Arkadiusz Dudek, M.D., Ph.D., a distinguished oncologist and investigator with strong interest, background, and clinical experience in immuno-oncology, identified decline in ARSB with increasing aggressiveness of melanoma cell lines, as well as significant increases in expression CSPG4 and pro-MMP2. Inborn deficiency of ARSB is present in the lysosomal storage disease Mucopolysaccharidosis (MPS) VI, in which mutations lead to marked reduction of ARSB activity. In malignant cells from prostate, breast, colon, and liver, as well as in melanoma cells and tissue, we have shown that expression and activity of ARSB are reduced, compared to normal melanocytes and tissue. Decline in ARSB leads to accumulation of the sulfated glycosaminoglycans chondroitin 4-sulfate and dermatan sulfate, from which ARSB normally removes the 4- sulfate group at the non-reducing end and is required to initiate their degradation. With decline in ARSB, C4S accumulates and its interactions with critical molecules are disrupted. We have shown that galectin-3, a co- transcriptional activator which binds to other important molecules, including the insulin receptor, binds less to more highly sulfated C4S and has increased nuclear translocation and interaction with transcription factors. Inversely, SHP2 (PTPN11), the ubiquitous non-membrane Src homology region 2 (SH2)-containing protein tyrosine phosphatase 2, binds more with more highly sulfated C4S and is less available for dephosphorylation of critical signaling molecules, including phospho-ERK1/2. Hence, the transcriptional events arising from altered sulfation of C4S have profound impact on vital cellular processes. This project will provide new insight into how changes in ARSB, and the resulting changes in chondroitin 4-sulfation and signaling and transcriptional events, impact on melanoma progression and on response to checkpoint inhibition. The findings may lead to new approaches to treatment of melanoma and to improvement in response to checkpoint inhibition, resulting in reduced suffering, morbidity, and mortality from melanoma.