Profiling and Targeting N-linked Glycosylation in High-Risk Neuroblastomas
Eric J. Rellinger, MD

Basic Science Project Mentors:  Christine Brainson, PhD, Andrew Lane, PhD, and Mark Evers, MD 
Clinical Project Mentor: John D’Orazio, MD, PhD 
External Mentor: K. Sreekumaran Nair, MD, PhD

Neuroblastoma (NB) is the most common extracranial solid tumor in children. MYCN-amplification is present in ~30% of NBs and sufficient for stratifying NBs as high-risk. N-MYC is a proto-oncogene that drives cancer cell growth, angiogenesis, and metabolism. Aberrant glutamine metabolism is a hallmark of MYC-driven cancers. N-MYC enhances expression of the glutamine transporter, ASCT2. ASCT2 has been shown to be critical for MYCN-amplified NB cell survival in vitro and in subcutaneous xenograft formation in vivo.  Our central hypothesis is that aberrant glutamine uptake via ASCT2 is critical for MYCN-amplified NB primary tumor growth and metastasis. We will test a new small molecule inhibitor of ASCT2 (V-9032) in advanced patient-derived orthotopic xenograft models of MYCN-amplified NB. Stable-isotope resolved metabolomics will be used to define aberrant metabolic activated to sustain NB survival in the presence of ASCT2 blockade. We will also seek to define metabolic vulnerabilities uncovered by ASCT2 inhibition utilizing a high-throughput siRNA screen for synthetic lethality in MYCN-amplified NB cell lines.    

TRIM28 fuels prostate cancer growth through SETDB1-mediated epigenetic silencing of androgen metabolic genes UGT2B15 and UGT2B17
Ka-wing Fong, PhD
 

Basic Science Project Mentors: Richard Higashi, PhD, and Xiaoqi Liu, PhD
Clinical Project Mentor: Peng Wang, MD 
External Project Mentor: Douglas R. Spitz, PhD 

Prostate cancer (PCa) is the second-leading cause of cancer-related death in men. The mainstay treatment for metastatic PCa is androgen deprivation therapy (ADT). However, the disease will inevitably return in an incurable form termed castration-resistant prostate cancer (CRPC). Importantly, aberrant androgen receptor (AR) activation in the milieu of low androgen is the main driver of CRPC. Molecular characterization of CRPC paves the way for discovery of novel therapeutic targets, and my previous studies showed that TRIM28 is aberrantly upregulated in advanced PCa. High TRIM28 levels are associated with worse clinical outcomes, which suggests that TRIM28 could serve as a promising target for PCa therapy. It is known that TRIM28 in a complex with trimethyl-histone H3 lysine 9 (H3K9me3)-specific methyltransferase (SETDB1) possesses transcriptional co-repressor activity but its genomic targets remain largely unexplored in PCa. Moreover, the TRIM28 downstream oncogenic pathway has not been comprehensively investigated in PCa. By molecular gene signature analysis, I found that the steroid hormone metabolic pathway is regulated by TRIM28. In particular, I found that the androgen eliminating enzymes UGT2B15 and UGT2B17 are the key TRIM28-repressed genes. It is known that genomic targeting of the TRIM28 complex is facilitated by transcription factors that bind to specific DNA recognition motifs. My data revealed that TRIM28 interacts with AR. In addition, my preliminary results indicated there is co-occupancy of TRIM28, AR and H3K9me3 deposition at UGT2B15 and UGT2B17 gene loci. Based on these findings, I hypothesize that TRIM28 acts together with AR to epigenetically silence the expression of androgen metabolic enzymes through SETDB1-mediated H3K9me3. I propose to test this hypothesis in Aim1. UGT2B15 and UGT2B17 are known to inactivate androgen through glucuronidation using UDP-GlucA as the sugar-donor. Given that the overexpression of UGT2B15 and UGT2B17 correlate to more UDP-GlucA consumption, I hypothesize that levels of UDP-GlucA will decrease in TRIM28 knockdown cells or with treatment of the SETDB1 inhibitor, Mit, in CRPC cells. I propose to test this hypothesis in Aim2. The results from this proposal will not only reveal a novel epigenetic mechanism for regulating androgen glucuronidation, but also have significant implications for the development of SETDB1-targeted therapy for CRPC treatment.
 

Regulation of one-carbon metabolism and purine synthesis by YRDC in hepatocellular carcinoma
Eunus Ali, PhD

Basic Science Project Mentors: Teresa Fan, PhD and Luksana Chaiswing, PhD
Clinical Project Mentor: Zhonglin Hao MD, FACP
External Project Mentor: Martin Hauer-Jensen, MD, PhD, FACS 

Hepatocellular carcinoma (HCC) accounts for >80% of patients with liver cancer and is one of the leading causes of cancer-associated mortalities, both worldwide and in the United States. Recent evidence highlights the involvement of YRDC (YrdC-threonylcarbamoyltransferase domain-containing protein), a tRNA modifying enzyme, in tumor growth via N6-threonylcarbamoylation (t6A) of tRNA in various cancer types, including HCC. Additionally, YRDC has been shown to be overexpressed in different types of cancer and associated with chemotherapeutic drug resistance as well as poor prognosis in HCC patients. However, the precise molecular mechanisms underlying YRDC overexpression in cancers and its involvement in the regulation of tumor metabolism remain unknown. The objective of our study is to define the mechanisms by which YRDC drives cellular growth and investigate the potential of targeting YRDC in cancers, with a specific focus on HCC. Our preliminary data, employing metabolomic and isotope tracing approaches, have shown that YRDC can act as a novel regulator of one-carbon metabolism and purine synthesis in several cancer cells, including HCC cells. We demonstrate that YRDC depletion reduces flux through one-carbon metabolism, thereby restricting the availability of substrates for the synthesis of de novo purine nucleotides. In light of these findings, this proposal focuses on studying YRDC and its impact on tumor metabolism using HCC as a model. We hypothesize that YRDC promotes tumor growth by activating one-carbon metabolism, thereby stimulating de novo purine synthesis. Consequently, targeting YRDC represents a potentially effective strategy against HCC. We will define the mechanisms underlying YRDC upregulation in HCC, we will use isogenic cell settings deleted for the tumor suppressor P53 and expressing mutant KRAS, and examine the role of the MAPK/ERK downstream of KRAS in the regulation of YRDC mRNA and protein levels (Aim1). Then, we will determine the molecular mechanisms by which YRDC stimulates one-carbon metabolism and purine synthesis in HCC cells (Aim2). Finally, we will examine the therapeutic potential of suppressing YRDC to improve HCC, and/or sensitize HCC to chemotherapy (Aim3). Overall, the proposal is significant because it will identify novel regulators of one-carbon metabolism and the de novo purine synthesis pathways, clarify the tumor pro-growth function of YRDC, and identify a novel drug target in HCC treatment. The project is highly innovative because, to date, YRDC has not been linked to the regulation of one-carbon metabolic pathway. The proposal will provide a stepping-stone to aid the applicant to establish an independent research career and the work performed under this award will be used to generate NIH R01 grant proposals.