Jack Faulkner
Research Mentor(s): John Prensner
Department or Program: Pediatrics and Biological Chemistry
Authors: Jack Faulkner, Anwesha Dasgupta, John Prensner
Session: Session 2: 1:00pm-1:50pm
Poster: 15
Abstract
Medulloblastoma is the most frequent malignant brain tumor in childhood, most commonly occurring in children ages 0-9. There are four different medulloblastoma subgroups, each characterized by molecular differences. Among these, children with MYC or MYCN amplifications, which occur in the Group 3 and Sonic Hedgehog (SHH) subgroups, respectively, have a dismal overall survival and the highest rate of metastases. Biologically, MYC and MYCN have overlapping functions as DNA-binding transcription factors; however, MYC overexpression is observed among a broader range of cancers and tissues, while MYCN has a more specific expression pattern. Extensive previous studies have shown that both of these genes are significant contributors to oncogenesis by initiating and maintaining rapid cell division and other cellular programs. We have hypothesized that MYC(N) upregulation in medulloblastoma might stimulate the production of neoantigens from non-canonical proteins that could be utilized in efforts to develop novel immunotherapy options. Current therapies for treating medulloblastoma are not completely tailored to each subgroup; this project could identify new neoantigens produced by transcription factor activity that can be used to better treat this cancer.
To pursue this hypothesis, UW228 and ONS76 human medulloblastoma cell lines were engineered to express plasmids encoding V5-tagged MYC or MYCN cDNAs, using GFP as a control. Cells were propagated using standard cell culture techniques. To evaluate the level of overexpression, immunoblot analyses of MYC and MYCN protein were performed using SDS-PAGE western blots, along with qPCR to estimate cDNA RNA expression. To characterize the impact of MYC(N) on cell proliferation rate, we used CellTiter-Glo assays to measure cellular ATP abundance.
From the collected data, I confirmed that MYC cDNAs were present in cells and resulted in ectopic expression of the MYC transgenes. I have not fully confirmed MYCN cDNA expression due to optimization needed for western blot antibodies. I am likewise in the process of validating qPCR assays for these genes, which are expected to be finished within the next month. I found that the UW228 MYC and MYCN cell lines were proliferating faster than UW228 parental and GFP lines. This was hypothesized as MYC/MYCN overexpression has been linked to greater cellular metabolism and cellular division. The data also exemplified that all ONS76 cell lines proliferated at similar rates. While these results were surprising, they could be due to a variation among cell lines in the number of cells initially seeded in the 96 well plate.
Overall, these results are broadly supportive of a role for MYC(N) in cell proliferation in medulloblastoma. Moving forward, we have been working to establish and validate cell culture model systems for the analysis of immunopeptides on the cell surface. The next steps in this research will be to assess HLA expression through flow cytometry and confirmation of HLA genotype of the cells. In summary, we have been working to identify novel antigens produced by MYC and MYCN expression in high-risk forms of medulloblastoma, where patient outcomes are poor. Completion of his research will benefit anyone connected to someone affected by medulloblastoma, whether as a friend, family member, or the patient themselves.
