Epigenetic and epitranscriptomic determinants of treatment resistance in cancer

Biology textbooks teach that the central dogma is that DNA is transcribed to mRNA and mRNA is translated to protein. This simplification overlooks the complex processes that enable selective exon transcription and translation which are essential for normal cellular function and the deregulation of which are now known to contribute to cancer and other diseases. Therefore this project will investigate the fundamental mechanisms that enables the regulation of selective transcription and translation in normal and malignant cells.

Our recent work has identified novel mechanisms involved in alternative exon utilisation. We have found that epigenetic mechanisms involving histone lysine methylation and epitranscriptomic mechanisms involving RNA methylation appear to converge to (i) select which regions of DNA are actively transcribed and (ii) which exons within mRNAs are translated. Most importantly we have found that these mechanisms are disrupted in cancer cells as compared to non-malignant cells, enabling these cancer cells to selectively change isoform expression to evade current cancer treatments. This leads to the hypothesis that aberrant RNA methylation contributes to the pro-oncogenic alternatively spliced transcriptome found in cancer. As metastatic cancer is incurable, new treatments are urgently required. Our research will therefore establish the fundamental science as to whether new drugs which target RNA-methylation can reverse resistance to existing cancer drugs and thereby in principle extend the life of patients with metastatic disease.

The specific aim of this project is to decipher the function of the YTHDC1 m6A reader in determining exon utilization. To do this we will compare YTHDC1 function in the context of our existing data on m6A modifiers (METTL3, METTL14, CBLL1, FTO and ALKBH5) and histone lysine demethylases (KDM1A, KDM5B, KDM7A) which regulate alternative splicing.

To do this the DTP student will
1. assess expression of the YTHDC1 m6A reader in our human prostate and breast cancer specimens (expression of all other relevant markers have already been determined).
2. use CRISPR-Cas9 and/ or siRNA to selectively target YTHDC1 in breast and prostate cancer cell lines and determine the effect on androgen and estrogen regulated gene expression by qRTPCR
3. use RNAseq and western blotting to determine the relative role of YTHDC1 and the m6A and histone lysine demethylases in alternative splicing and exon utilization of the AR protein
4. will compare the effects of functional depletion of YTHDC1, and other components of the m6A regulating complex, with new pharmaco-inhibitors of RNA methyltransferases on the transcriptome, proliferation and in vitro invasion of cancer cells.
Collectively these aims will advance fundamental understanding of the role of YTHDC1 in gene regulation and will guide the future development of m6A-targeted therapies in cancer.

The DTP student will receive training in the following techniques
1. Cell culture, CRISPR-cas9, in vitro pharmacology, reporter assays
2. Basic molecular biology, cloning, qRTPCR, western blotting
3. Bioinformatics: our group has optimized pipelines and existing datasets already available for comparison
4. Clinical genomics and data interpretation: the student will learn how to complete immunohistochemistry and clinical correlations