DISSECTING AND TARGETING THE ROLE OF GALNT14 IN HIGH-RISK OSTEOSARCOMA

骨肉瘤(OS)是儿童和青少年最常见的骨肿瘤。新辅助化疗方案 自20世纪70年代以来，阿霉素、顺铂和大剂量甲氨蝶呤一直是治疗的标准，但没有 额外的化疗或靶向治疗增加了显著的生存益处，留下了最少的选择 复发和抵抗疾病。这些患者的预后极差，长期预后为 不到20%。因此，更好地了解化疗耐药机制对于制定 我们目前的治疗方法更有效。通过对机构的全面转录分析 化疗耐药患者来源的异种移植(PDX)模型和公开可用的治疗应用 研究生成有效的治疗(目标)OS数据库，我们发现显著提升了 化疗反应差患者的糖基化基因。糖基化是蛋白质后代谢的一种形式 翻译修饰在蛋白质运输、功能和稳定性中起作用的翻译修饰。它还提供了衬底 对于细胞间的相互作用，抗原用于免疫系统的监测，并经常在细胞信号传递中发挥作用。O型 糖基化，即将糖部分加到蛋白质的丝氨酸或苏氨酸残基上，始于 N-乙酰半乳糖基转移酶(GALNTs)。我们的分析发现GALNT14在穷人中过表达 反应性(&lt；90%坏死)OS肿瘤，我们提供了GALNT14表达显著 与OS的总体生存率和无事件生存率呈负相关。GALNT14已被证明可以促进 成人癌症的化疗耐药和转移，如乳腺癌、前列腺癌和肺癌。我们的 假设GALNT14通过促进对儿童OS的疾病进展有显著影响 化疗耐药，是一种新的、可行的小分子抑制治疗靶点。我们建议两个 互补而又独立的目标是解决这一假设。这项提议将协同 尤斯坦博士在肉瘤生物学和建模方面，科勒博士在糖生物学方面成功地完成了 目标。AIM1：GALNT14在骨肉瘤模型中促进化疗耐药的作用 活着。使用商业上可用的和PDX衍生的化疗耐药细胞系，我们正在开发这两种GAIN 以及通过过度表达和CRISPR/Cas9基因敲除方法建立功能丧失的GALNT14模型。vbl.使用 这些模型，我们将在体外和体内评估OS对化疗的敏感性和对肿瘤的影响 成长和进步。AIM2.GALNT14小分子抑制剂的鉴定与评价。在……里面 为此，我们将使用高通量筛选来鉴定抑制纯化的活性的小分子， 重组GALNT14.此外，我们将确定这些分子中的哪些可以在细胞培养中抑制GALNT14。 在这一目标中确定的GALNT14抑制剂有可能成为临床开发的主要候选药物 也可用于研究GALNT14在OS中的机制参与。总体而言，我们的研究将 导致确定治疗高危OS的关键候选治疗方式。

Osteosarcoma (OS) is the most common bone tumor in children and adolescents. Regimens of neoadjuvant doxorubicin, cisplatin, and high dose methotrexate have been the standard of care since the 1970s, but no additional chemo or targeted therapies have added significant survival benefits, leaving minimal options for relapsed and resistant disease. These patients have an extremely poor prognosis, with long term outcomes of less than 20%. Therefore, a better understanding of chemoresistance mechanisms is essential towards making our current therapies more effective. Through comprehensive transcriptomic analysis of institutional chemoresistant patient-derived xenograft (PDX) models and the publicly available Therapeutically Applicable Research to Generate Effective Treatments (TARGET) OS database, we identified significant elevation of glycosylation genes in patients with poor response to chemotherapy. Glycosylation is a form of protein post- translational modification that plays a role in protein trafficking, function, and stability. It also provides substrates for cell-cell interactions, antigens for immune system surveillance, and often plays a role in cell signaling. O-type glycosylation, the addition of sugar moieties to serine or threonine residues to proteins, begins with the family of N-acetylgalactosyltransferase enzymes (GALNTs). Our analysis identified overexpression of GALNT14 in poor responding (<90% necrosis) OS tumors and we provide evidence that expression of GALNT14 has significant inverse correlation with overall survival and event-free survival in OS. GALNT14 has been shown to promote chemotherapy resistance and metastasis in adult cancers, such as of the breast, prostate, and lung. Our hypothesis is GALNT14 has a significant impact on disease progression in pediatric OS by promoting chemotherapy resistance and is a novel, viable therapeutic target for small molecule inhibition. We propose two complementary, yet independent aims to address this hypothesis. The proposal will synergize the expertise of Dr. Yustein, in sarcoma biology and modeling, and Dr. Kohler in glycobiology to successfully accomplish the goals. AIM1: Role of GALNT14 in promoting chemoresistance in osteosarcoma models in vitro and in vivo. Using both commercially available and PDX-derived chemoresistant cell lines we are developing both gain and loss-of-function GALNT14 models through overexpression and CRISPR/Cas9 knockout approaches. Using these models, we will evaluate in vitro and in vivo effects on OS sensitivity to chemotherapy and effects on tumor growth and progression. AIM2. Identification and evaluation of small molecule inhibitors of GALNT14. In this aim, we will use high-throughput screening to identify small molecules that inhibit the activity of purified, recombinant GALNT14. Further, we will determine which of these molecules can inhibit GALNT14 in cell culture. GALNT14 inhibitors identified in this aim have the potential to serve as lead candidates for clinical development and may also be used to investigate the mechanistic involvement of GALNT14 in OS. Overall, our studies will lead to the identification of critical candidate therapeutic modalities for the treatment of high-risk OS.