The naked mole-rat (NMR, Heterocephalus glaber) is a highly unusual mammal: eusocial, cold-blooded and with remarkable resistance to hypoxia, hypercapnia and acid-induced pain. Furthermore, NMRs are exceptionally long-lived and extremely cancer resistant. Few studies have attempted to explain the reason behind the NMR’s cancer resistance, but most prominently Tian et al. have reported that NMR cells are resistant to tumourigenesis induced by SV40 large T antigen and oncogenic RAS (SV40LT-HRASG12V), a combination of oncogenes sufficient to transform mouse and rat cells. Additionally, Tian et al. have reported that high-molecular weight hyaluronan (HMW-HA) mediates the NMR’s cancer resistance and inhibition of HMW-HA signalling render NMR cells susceptible to transformation by SV40LT-HRASG12V. With the publication of the NMR genome and advances in CRISPR-Cas9 gene editing, I set out to systematically interrogate the NMR genome through a genome-wide CRISPR screen to identify the genes responsible for its cancer resistance. My approach was based on Tian et al.’s finding that the NMR cells are not transformed by SV40LT-HRASG12V unless a further gene is perturbed. Therefore, I aimed to knockout every gene in the NMR genome (a set of ~55,000 predicted genes) in NMR cells expressing SV40LT-HRASG12V and test them for anchorage-independent growth, a hallmark of cancer cells. I, therefore, developed a novel CRISPR gRNA library containing 81116 gRNAs targeting the entire NMR genome. Next, I developed a set of lentiviral vectors to deliver SV40LT-HRASG12V into NMR cells. Using these vectors, I generated 106 different cell lines from 5 different tissues of 11 NMRs and found that, contrary to previous reports, NMR cells are transformed by expression of SV40LT-HRASG12V. Using transcriptomic analysis, I show that both NMR and mouse cells undergo similar gene expression changes in response to introduction of SV40LT-HRASG12V.
Taken together, this thesis challenges previous findings of the field and points to non-cell autonomous processes such as the immune system and cellular microenvironment as key players behind NMR’s cancer resistance. Exploring these new avenues could have profound effect on our understanding of tumour biology.