5-hydroxymethylcytosine and TET Enzymes in Neuroblastoma
Session Number
Project ID: MEDH 29
Advisor(s)
Kelley Moore
Mark Applebaum, The University of Chicago, Department of Pediatrics
Discipline
Medical and Health Sciences
Start Date
17-4-2024 8:35 AM
End Date
17-4-2024 8:50 AM
Abstract
One of the hallmarks of aggressive neuroblastoma is amplification of the MYCN oncogene which defines groups of patients with poor prognosis. The Applebaum Lab has shown that in MYCN-amplified neuroblastoma, 5-hmC (a DNA modification of open chromatin placed by the Ten-Eleven Translocation (TET) proteins) and H3K27me3, a marker of closed chromatin, co-localize. This co-localization results in the repression of genes that would lead to non-malignant differentiation if expressed. We hypothesized that an improved understanding of these mechanisms would allow us to target pathways that promote aggressive neuroblastoma. To test this, we knocked out TET2 and TET3 proteins in SK-N-BE2 neuroblastoma cell lines using a lentivirus CRISPR vector system to deliver Cas9 and sgRNAs. After confirming knocking out the TET 2/3 proteins, we found a weaker presence of 5-hmC among the cells. We then measured cellular proliferation of three unique clones using the MTT assay and found that the SK-N- BE2 cell lines were extremely confluent. This interfered with the results of our MTT assay and more testing is needed to form a conclusion. Thus, knockout of TET2 and TET3 proteins in MYCN-amplified neuroblastoma cells reduced 5-hmC levels and potential impaired cellular proliferation, suggesting that targeting these proteins may be a promising therapeutic strategy.
5-hydroxymethylcytosine and TET Enzymes in Neuroblastoma
One of the hallmarks of aggressive neuroblastoma is amplification of the MYCN oncogene which defines groups of patients with poor prognosis. The Applebaum Lab has shown that in MYCN-amplified neuroblastoma, 5-hmC (a DNA modification of open chromatin placed by the Ten-Eleven Translocation (TET) proteins) and H3K27me3, a marker of closed chromatin, co-localize. This co-localization results in the repression of genes that would lead to non-malignant differentiation if expressed. We hypothesized that an improved understanding of these mechanisms would allow us to target pathways that promote aggressive neuroblastoma. To test this, we knocked out TET2 and TET3 proteins in SK-N-BE2 neuroblastoma cell lines using a lentivirus CRISPR vector system to deliver Cas9 and sgRNAs. After confirming knocking out the TET 2/3 proteins, we found a weaker presence of 5-hmC among the cells. We then measured cellular proliferation of three unique clones using the MTT assay and found that the SK-N- BE2 cell lines were extremely confluent. This interfered with the results of our MTT assay and more testing is needed to form a conclusion. Thus, knockout of TET2 and TET3 proteins in MYCN-amplified neuroblastoma cells reduced 5-hmC levels and potential impaired cellular proliferation, suggesting that targeting these proteins may be a promising therapeutic strategy.