Identification of novel regulatory dependencies for Giroblastoma
Tumors of central nervous system are some of the most difficult and deadliest of cancers that can affect mankind. Glioblastoma, also known as glioblastoma multiforme (GBM), is the most aggressive cancer among brains tumors. Glioblastoma is rapidly fatal and with the current treatment regimes, the median overall survival is less than 15 months for GBM patients, therefore advances on both fronts, translational and basic scientific understanding of processes in GBM are needed. Our research focus is to identify and characterize novel functional dependencies required for Glioblastoma. Using a combination of genetic and biochemical approaches to study gene regulation in clinically relevant GBM models, we aim to identify and characterize novel molecular mechanisms of gliomagenesis, hopefully leading to novel therapeutic intervention strategies for GBM patients.
The contribution of epigenetic factors and processes, which include DNA methylation and modification of histone residues, is well established in tumorigenesis; the role of epitranscriptome, post-transcriptional RNA modifications together with the RNA binding protein activity for the modified RNA species, in regulation of tumor malignancy is characterized poorly. In order to identify novel insights into tumorigenic processes during GBM, we will characterize the GBM epitranscriptome. We will perform experiments to identify and characterize novel epigenetic and epitranscriptomic regulators required for Glioblastoma.
Depending on the cell type, m6A modification is present in 10-40% of mRNAs. By itself, the presence of m6A modification in mRNA does not affect the translation of the mRNA. The major layor of gene regulation by m6A is carried out by the proteins to which m6A-containing mRNA is bound. YTHDF1, 2, and 3 are the main binding partners of m6A-modified mRNA. It is currently appreciated that the translation of YTHDF1-bound m6A containing mRNA is enhanced, while YTHDF2-bound m6A containing mRNAs are targeted for degradation. In the context of glioma, we have found that YTHDF2 depletion affects the proliferation of mouse and human glioma cells. m6A-containing mRNAs are bound to YTHDF proteins, but currently it is not clear what determined the specificity of the YTHDF-binding to their target mRNAs. We have generated several cellular and mouse model systems to investigate the determinants of YTHDF1-3 target specificity. These include knock-out mice and knock-out embryonic stem cells derived from mice as well as derived using crispr/cas9 technology. Furthermore, we have generated knock-in mice and embryonic stem cells for YTHDF1-3 proteins. Using these reagents, we will investigate:
- Characterize the functional requirement of YTHDF1-3 loss-of-function in stem cells
- Identify the YTHDF1-3 dependent changes in transcriptome and epi-transcriptome (m6A-bound mRNA)
Using the endogenously tagged, YTHDF1-3 ES cells
- Identify the protein-protein interaction partners of YTHDF1-3 by performing pull-down experiments
- Identify the target mRNA bound to YTHDF1-3 by performing CLIP-seq