Instructor Dana-Farber Cancer Institute, United States
Introduction: DNA breaks are one of the most deleterious lesions caused by various genotoxic mechanisms. If DNA breaks are not accurately repaired before mitosis, they can lead to genomic rearrangements and instability. We hypothesized that genes contributing to increased DNA breaks can drive genomic instability and clonal evolution in myeloma (MM). To identify such genes, we developed a genome-wide screen to investigate the impact of gene modulation on expression of 𝛄-H2AX (a marker for DNA breaks).
Methods: We used a whole genome lentiviral expression library from Broad Institute containing 17,255 ORFs representing 12,728 genes. MM cell lines (U266 and JIM3) were transduced (in triplicate), selected in puromycin, cultured, fixed and stained using a fluorescent 𝛄-H2AX antibody. Cells with high levels of DNA breaks were sorted via FACS and DNA sequenced to identify which ORFs induced DNA breaks.
Results: Overexpression of 226 ORFs significantly associated with increased DNA breaks in both MM cell lines. The enriched ORFs were predominantly involved in one of the 6 different functions: 1) Serine/threonine-protein kinases including calcium/calmodulin-dependent protein kinases; 2) Cell cycle cyclin-dependent kinases and phosphatases; 3) Cytoskeleton, chromosome maintenance and mitosis/cytokinesis related proteins; 4) DNA repair and recombination proteins; 5) RNA helicases, and proteins involved in RNA processing, splicing and degradation; 6) Monooxygenases involved in metabolic detoxification of xenobiotics.
In a clinical dataset (GSE24080; n=559), high expression of 20 of these genes correlated with poor OS, 23 with poor EFS and 12 with both poor OS and EFS. In IFM70 (n=170) dataset, high expression of 14 of these genes correlated with poor OS, 8 with poor EFS and 6 with both poor OS and EFS. High expression of 3 genes significantly correlated with both poor OS and EFS in both MM datasets. These included: 1) a kinase (DTYMK; deoxythymidylate kinase), which regulates nucleotide biosynthesis and contributes to DNA replication and repair; 2) a phosphatase (CDC25A; cell division cycle 25A), which is required for G1/S progression and whose degradation in response to DNA damage is essential to prevent cells with chromosomal abnormalities from progressing through cell division; and 3) a nuclease (EXO1; exonuclease 1) with roles in DNA repair and replication. We further validated the role of EXO1 whose expression and activity are elevated in MM. Both gain and loss-of function studies showed that EXO1 contributes to increased DNA replication/proliferation and spontaneous and chemotherapy-induced DNA breaks and genomic instability. We also developed a novel inhibitor of EXO1 that decreases genomic instability and growth of MM cells.
Conclusions: We have identified genes/pathways driving DNA damage in MM. Some of these genes, separately or together, have potential to serve as novel and promising targets to make cancer cells genomically static and are thus currently being further investigated.