While several tumor driving genes have been identified in targeted studies, deep sequencing data of primary tumours suggests that many more tumour promoting alterations exist. However, deep sequencing analysis cannot delineate driver genes from passenger genes (those which do not contribute to cancer), nor determine the functional consequences of each genetic aberration. Alternatively, CRISPR/Cas9 can be employed to perform genetic screens. The proto-oncogene c-Myc is known to have abnormal expression in up to 70% of cancers including Burkitt Lymphoma (BL), a common aggressive B cell lymphoma. The Eμ-Myc transgenic mouse model mimics BL in mice and is a valuable tool for identifying rare tumour suppressor genes and elucidating their functional role.
To identify novel tumor suppressor genes, we performed a whole genome in vivo CRISPR knockout screen using Em-Myc transgenic mice which develop lymphoma (mean 100 days). Cas9 expressing Eμ-Myc hematopoietic stem and progenitor cells were transduced with a whole genome sgRNA library and transplanted into lethally irradiated wildtype recipient mice. While several known tumor suppressors arose from our screen, a number of candidates were also identified including the Gator1 complex subunits DEPDC5 and NPRL3 involved in metabolism via negative regulation of the mTORC1 pathway. Deletion of DEPDC5 and NPRL3 considerably accelerated Eμ-Myc driven lymphomagenesis (median survival of 34.5 and 30.5 days respectively) compared to negative control recipient mice (median survival 88 days). Interestingly, 30% of the control lymphomas spontaneously acquired aberrations in the Trp53 signalling pathway while the Depdc5 and Nprl3 deficient lymphomas did.
This demonstrates that Depdc5 and Nprl3 are strong tumor suppressor genes in Eμ-Myc driven lymphoma and do not require loss of Trp53. As such, we aimed to investigate whether Trp53 regulates the Gator1 complex in any form and furthermore, to test whether these lymphomas are sensitive to therapies such as mTOR inhibitors.