Project description:MYCN is an oncogene amplified in approximately 20% of all neuroblastomas and 50% of high risk neuroblastoma. It is a transcription factor regulating the expression of genes involved in proliferation and apoptosis.We would like to identify genes potentially regulated by MYCN in a conditional MYCN expression system. The SHEP Tet21N system is a conditional, tetracycline-regulated MYCN expression system established in the MYCN non-amplified SHEP neuroblastoma cell line (Lutz et al., 1996). Expression of MYCN is switched off by the addition of tetracycline to the growth medium. Levels of MYCN protein and RNA in the absence of tetracycline are comparable with those present in MYCN amplified cell lines (Bell et al., 2006). By performing microarray analyses on the Tet21n cell lines in the presence and absence of MYCN we hope to identify genes whose expression had been altered by MYCN either upregulated or downregulated and may be potential MYCN targets. The altered expression of some of these genes will then be validated using quantitative RTPCR and a selection will be further investigated to determine if they are MYCN transcriptional targets and may be potential candidates for anti-MYCN therapies for neuroblastoma Specific objectives 1) To investigate genes which are altered by 2 fold or more in the presence of or absence of MYCN in Tet21N cells. Refs: Bell, E., Premkumar, R., Carr, J., Lu, X., Lovat, P.E., Kees, U.R., Lunec, J. & Tweddle, D.A. (2006). The role of MYCN in the failure of MYCN amplified neuroblastoma cell lines to G1 arrest after DNA damage. Cell Cycle, 5, 2639-47. Lutz, W., Stohr, M., Schurmann, J., Wenzel, A., Lohr, A. & Schwab, M. (1996). Conditional expression of N-myc in human neuroblastoma cells increases expression of alpha-prothymosin and ornithine decarboxylase and accelerates progression into S-phase early after mitogenic stimulation of quiescent cells. Oncogene, 13, 803-12.

Project description:In mammalian cells, gene copy number is tightly controlled to maintain gene expression and genome stability. However, a near-universal molecular feature across cancer types is oncogene amplification, which promotes cancer progression by dramatically increasing the copy number and expression of tumor-promoting genes. For example, in tyrosine kinase inhibitor (TKI)-resistant lung adenocarcinoma (LUAD), oncogene amplification occurs in over 40% of patients’ tumors. Despite the prevalence of oncogene amplification in TKI-resistant tumors, the mechanisms facilitating oncogene amplification are not fully understood. Here, we find that LUAD tumors exhibit a unique chromatin signature demarcated by strong CTCF and cohesin deposition in drug-naïve tumors, which correlates with the boundaries of oncogene amplicons in TKI-resistant LUAD cells. Throughout the acquisition of TKI resistance, we identified a global chromatin priming effect, marked by a dynamic increase of H3K27Ac, cohesin loading, and inter-TAD interactions, which occurs before the onset of oncogene amplification. Furthermore, we have identified that the protein METTL7A, which was previously reported to localize to the ER and inner nuclear membrane, has a novel chromatin regulatory function in binding to amplified oncogenes and regulating cohesin recruitment and inter-TAD interactions. Surprisingly, we discovered that METTL7A remodels the chromatin landscape prior to any noticeable oncogene copy number gains. Furthermore, while METTL7A depletion has little effect on drug-naïve cells, METTL7A depletion prevents the formation of TKI resistant-clones, highlighting the specific role of METTL7A as cells are acquiring resistance. In summary, we discovered an unexpected mechanism required for the acquisition of TKI resistance regulated by a largely uncharacterized factor, METTL7A. This discovery sheds new light into maintenance of oncogene copy number and paves the way to the development of new therapeutics for preventing TKI resistance in LUAD.

			Action	DRS
	DIANN_results.zip	Other
	RawFile_description.xlsx	Xlsx
	Rawfiles.zip	Other
	checksum.txt	Txt
	human2023_contaminants.predicted.speclib	Other

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Extrachromosomal DNA-driven oncogene dosage heterogeneity promotes rapid adaptation to therapy in MYCN-amplified cancers

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