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.