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

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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.