Abstract

Background

The rapid expansion of cancer genome sequencing projects has led to the identification of numerous somatic mutations with undefined function. In the context of colorectal cancer (CRC), mutations commonly occur in a small number of well-studied genes such as APC, KRAS, TP53, which are currently used to guide the treatment selection. However, many mutations occur in genes, which are still yet to be functionally characterized. Although these genes are mutated at moderate/low frequency in CRC, they could be relevant for CRC formation and therefore pose as potential new druggable targets. 

Method

To identify genes that cooperate with loss of Apc to promote intestinal tumorigenesis, we performed a genome-wide CRISPR knockout screen in Apc heterozygous murine intestinal 3D organoids and we used R-spondin withdrawal from the culture medium as selection for tumour initiation. This provides a strong selection assay for wnt independent growth, which is a property of intestinal tumour initiation.

Results

By next generation sequencing, we identified around 700 hits that are potentially involved in CRC initiation. As expected, we found an over representation of a number of well-established wnt regulators genes (Apc or β-catenin) along with genes with poorly characterized role in CRC. We are currently generating a sub-library containing gRNAs targeting the identified hits for further validation. In parallel, we are carrying out characterization of a number of selected candidates such as Fbxw8 and Lzts2. Interestingly, loss of Fbxw8 induces increased clonogenic potential of Apc heterozygous organoids, without altering the expression of stem cell markers, resembling a fetal-like/repair phenotype.

Conclusion

Our genome-wide approach provides a workframe for understanding the relevance of the entire spectrum of mutations in CRC and suggests that moderate/low frequency mutated genes, rather than a “mutational noise”, can contribute to CRC initiation.