Recent technological advances include CRISPR based technologies as well as advances in screening technologies are revolutionising our ability to use functional genomics. Research in the Rosenbluh lab uses state of the art functional genomic tools including pooled CRISPR loss/gain of function screens and apply these technologies towards identifying genes associated with cancer and developing new approaches to treat cancer.
Advances in oligo synthesis technologies make it possible to synthesis up to 100,000 different sequences in just a few days. This enables very rapid generation of pooled sgRNA libraries that target every known human or mouse coding gene. Pooled libraries are then used to generate a pooled lentiviral library that is used to infect cells at low MOI (this insures that every infected cell receives only one virus). Following propagation genomic DNA is extracted and next generation sequencing is used to quantify sgRNA abundance.
Current project in the lab
Targeting of β-catenin driven colon cancers
The WNT/β-catenin signaling pathway plays a major role in normal development and is aberrantly active in cancer. In normal homeostasis, the destruction complex phosphorylates serine residues in β-catenin leading to its degradation by the proteasome. Upon binding of the WNT ligand to the FZD/LRP receptor the destruction complex is inactivated leading to accumulation and nuclear translocation of β-catenin. In the nucleus β-catenin binds to transcription regulators that drive the expression of genes that promote cell proliferation and differentiation.
Genomic alterations leading to aberrant β-catenin activity are found in the majority of colon cancers and suffice to drive tumor growth. Specifically, inactivating mutations in the tumor suppressor APC are found in ~80% of colon cancers, and we have recently reported that the remaining 20% of colon cancers harbor loss of function mutation in RNF43, a negative regulator of the WNT/β-catenin signaling pathway, suggesting that deregulation of β-catenin activity is required in nearly all colon cancers. Furthermore, studies in animal models and cultured cell lines demonstrate that β-catenin activity is required for tumor progression even in advanced cancers. However, despite the indisputable evidence demonstrating the requirement of this pathway for colon cancer pathogenesis we still lack strategies for direct targeting of β-catenin.
Project in the lab include defining how YAP1 regulates β-catenin driven colon cancer and development of approaches for specific targeting of a subclass of β-catenin driven cancers that harbour APC deletions.
Systematic identification and validation of breast cancer risk genes through follow up of genome-wide association studies.
Using genome wide association studies (GWAS) our collaborator Prof. Georgia Chenevix-Trench (QIMR, Brisbane) and her team have identified 179 breast cancer (BC) risk loci (Michailidou et al. Nature, 2017). The functional mechanism behind the associations usually involves perturbed regulation of target gene transcription by risk single nucleotide polymorphisms (SNPs) lying in regulatory elements positioned some distance from the target. The nearest gene to the GWAS ‘hit’ is not necessarily the target of the association, and for some loci there are multiple gene targets. This project uses large scale pooled clustered regularly interspaced short palindromic repeats (CRISPR) knockout and activation screens of all the predicted target coding and non-coding genes at these loci. We will evaluate the effect of over-expressing or suppressing the expression of all candidate BC risk genes on the ability of cells to proliferate in vitro, and in immune-deficient mice, as well as their ability to promote bypass of cellular senescence, in order to identify determinants of BC risk. In addition to enhancing our understanding of the genetic variants associated with BC risk, these experiments will enable new strategies for risk reduction therapies.
Identification of alternative transcripts that drive gastric cancer
Gastric cancer is a leading cause of mortality. Currently only a limited number gastric cancer treatments are available and new treatment options are necessary. Our collaborator Prof. Patrick Tan (NUS-Duke, Singapore) used ChIP-Seq from normal and gastric cancers and has identified a class of genes that in gastric cancers are expressed from an alternative promoter (Qamra et al Cancer Discovery, 2017). Using a verity of CRISPR based approaches we are identifying alternative transcripts that are essential for proliferation of gastric cancer and could be used as targets for development of gastric cancer specific therapies.