Identification of Novel Therapeutic Targets in Atm-Deficient Lymphomas Using a Whole Genome CRISPR/CAS-9 Screen

ATM is a principal DNA damage response protein that synchronises a complex network of cellular responses to double stranded DNA breaks. ATM gene is recurrently mutated in a wide range of lymphoid malignancies, including B-cell chronic lymphocytic leukemia (CLL), T-prolymphocytic leukaemia (T-PLL), mantle cell lymphoma (MCL) and diffuse B cell lymphoma (DLBCL). ATM pathway is utilized by many DNA damaging agents and consequently inactivation of this pathway can lead to chemoresistance. Furthermore, in the absence of ATM tumour cells exhibit genomic instability that can lead to clonal selection and evolution even under current targeted treatments. Consequently there is clear need to understand dependency pathways in ATM-deficient tumours and apply tailored targeted therapies that will specifically eliminate those tumour cells.

We have previously presented a novel murine model of ATM-deficiency that spontaneously generate lymphoid tumours, mostly DLBCL. These tumours have been successfully propagated both in recipient mice and in vitro, where several cell lines have been generated.

Genome editing methods, such as CRISPR/CAS-9, permit the targeted disruption of specific genes. Protocols for genome wide screens have been developed based on this technology which can be used to identify genes that are essential for cellular survival. As such, these screens can be used to identify dependency pathways for tumours with specific genetic lesions.

Using lentiviral transduction we established two cell lines that stably expressed CAS-9. We then performed a genome wide CRISPR screen using the GeCKO library to identify novel therapeutic targets in these Atm-deficient tumours. This library consists of 130,209 unique single guide RNA (sgRNAs), targetting 20,611 genes including 1176 miRNAs.

A comparative analysis was performed of sgRNA drop-out following 15 cellular doublings. This revealed a number of pathways including those already known to be synthetically lethal with ATM deficiency, such as ATR and PARP. Pathway analysis of the top genes from this drop-out analysis identified oxidative phosphorylation, the spliceosome, ribosome biogenesis, N-glycan biosynthesis, pyrimidine metabolism and purine metabolism as the most significantly affected pathways. Furthermore, the drop-out screen revealed a number of miRNAs, including MiR-3470a, Mir-3971, MiR-669f and MiR-719.

These data provide a unique molecular assessment of the dependency of ATM-deficient lymphomas and provide a number of novel putative therapeutic targets for treating such tumours.