Cancer is one of the major causes of death in Australia. For decades, the origin of cancer was attributed to genetic mutations. However, their involvement in gene regulation and cancer has illuminated the prospect of novel therapies. Epigenetic marks are heritable covalent modifications in the DNA or associated proteins. Epigenetic modifications provide the mechanisms by which a cell “knows” and “remembers” which genetic information to read and which to ignore. Epigenetic modifications include DNA methylation and modifications in the proteins that the DNA is wrapped around. Abnormal epigenetic modifications are frequently observed in cancer. In contrast to genetic mutations, epigenetic modifications are reversible and this can be used to restore the normal state of gene expression in the cancer. In this proposal, we aim to reverse the epigenetic modifications of key breast cancer drivers. We propose the development novel and more selective technologies able to stably suppress the genes that cause breast cancer and breast cancer spread.
The comprehensive genome-wide maps of epigenetic modifications in cancer revealed the deep involvement of epigenetics in cancer; in the majority of cancers, tumor suppressor genes are more frequently inactivated by epigenetic mutations than by genetic mutations1. Importantly, some of the key drivers in cancer cannot be targeted by current therapies. In this regard, we chose key oncogenic drivers often overexpressed in aggressive breast cancers: SOX2, MYC, KRAS, C11ORF67 and FOXM1. We aim to develop epigenomic tools to precisely re-write the specific epigenetic modifications controlling the expression of these oncogenes. We propose the generation of programmable DNA-binding proteins that ferry epigenome-modifers to stably silence these key targets. In our lab, the oncogene SOX2 was successfully methylated and down-regulated by zinc finger proteins (ZFPs) fused to the catalytic domain of DNA methyltransferase 3A (DNMT3A)2. Recently, we also induced DNA methylation on the Estrogen Receptor Receptor-α in cancer using a ZFP linked to a DNA methylatransferase3. In this project, we propose the construction of a sequence specific DNA-binding domain engineered from another state-of-the art technology, the bacterial Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) combined with an epigenetic silencing domain. The advantage of CRISPR system is that it is a protein-RNA complex in which the information to bind the target gene is provided by a guide RNA. The protein component of CRISPRs will be linked to novel combinations of epigenetic modifiers promoting long lasting chromatin condensation and gene silencing to establish both DNA and histone methylation and chromatin condensation. The outcomes of this research are novel proteins able to catalyse local reconfiguration of the chromatin state to permanently suppress oncogenic gene expression. Thus, this work will be highly transformative by providing long lasting strategies to suppress breast cancer growth.
Objective: To develop novel epigenome editing proteins (epiCRISPRs) to selectively inhibit oncogenic drivers of aggressive breast cancers. We hypothesise that the induction of epigenetic silencing in these key oncogenic drivers lead to a long lasting oncogenic silencing.
Aim1. Develop novel epigenome reprogramming tools to edit the epigenetic pattern of the targeted oncogenes (MYC, FOXM1, ZFN703, SOX2, KRAS, C11Orf67). By examining different epigenetic modifiers we will determine the epigenetic marks that “fine tune” and maximize the silencing effect.
Aim2. Determine the capacity of epiCRISPR to promote long lasting phenotypic changes e.g. inhibition of cell growth, suppression cell invasion and increased cell death in combination with chemotherapy agents.
Stable oncogenic silencing in vivo by programmable and targeted de novo DNA methylation in breast cancer. Stolzenburg S, Beltran AS, Swift-Scanlan T, Rivenbark AG, Rashwan R, Blancafort P.
Oncogene. 2015 Oct;34(43):5427-35. doi: 10.1038/onc.2014.470. Epub 2015 Feb 16.
Associate Professor Pilar Blancafort – [email protected]