Telomerase Contributes To Repair Of DNA Breaks In Myeloma Cells By Incorporating “TTAGGG” Sequences Within Genome: Biological and Translational Significance

We previously reported that telomerase activity is elevated in multiple myeloma (MM), and its inhibition induces telomere shortening and growth arrest in cancer cells. We have now gone on to study the role of telomerase in DNA break repair and genome maintenance in MM cells. To demonstrate the role of telomerase in DNA break repair: 1) We used g-H2AX staining (marker for DNA breaks) and comet assay, a gel-based technique for detection of DNA breaks in individual cells, and observed that telomerase inhibition leads to significantly increased DNA breaks in MM cells; 2) We have confirmed the repair and re-circularization of a linearized plasmid by telomerase in MM cell extracts; and 3) Demonstrated increased genomic instability, especially deletions, upon telomerase inhibition in MM cells. This does not necessarily suggest role of telomerase in DNA repair as telomerase inhibition with attrition of telomeres can also lead to increased instability. To confirm the direct role of telomerase in DNA repair in MM, we now present the evidence and mechanism of DNA break repair by telomerase by demonstrating: 1) The presence of “TTAGGG” repeats at non-telomeric sites at higher frequency in cancer vs normal cells; and 2) Decline in “TTAGGG” insertions at non-telomeric sites in MM cells following suppression of telomerase. To evaluate rare telomeric insertions in the cancer genome, we created libraries of genomic DNA fragments enriched for “TTAGGG” sequences from primary MM and matching normal PBMCs derived from the same patient. The libraries were sequenced using Illumina platform and reads containing 4 or more telomeric repeats were filtered for further analysis. Telomeric insertion sites were located from unique genomic sequences immediately following TTAGGG at one end of each read. By subtracting telomeric insertions detected in normal cells, from MM cells of same patent, we identified 94 unique loci with telomeric insertion in the primary MM cells. To investigate if telomerase inserts new “TTAGGG” repeats within cancer genome following DNA breaks, UV-treated RPMI cells were incubated with and without telomerase inhibitor for 4 days, cultured without telomerase inhibition for another 6 days, harvested and DNA libraries prepared and enriched for telomeric fragments and subjected to sequencing. DNA from cells preserved before UV treatment (day 0) was used as baseline control and their telomeric insertions were subtracted from UV-treated control and telomerase-inhibited cells. Following induction of DNA breaks by UV, 21 and 3 new telomeric insertions were detected in control and telomerase-inhibited MM cells, respectively, indicating 86% reduction of telomeric insertions within MM cell genome upon telomerase inhibition. Analyses of flanking sequences indicated that 71% of the new telomeric insertions in the UV-treated control cells occurred at sites which did not have any pre-existing “TTAGGG” repeats. Similarly in primary MM cells, 67%, 29% and 4% of the new insertions were observed at positions containing 0, 1 and 2 copies of “TTAGGG” repeats, respectively, indicating that telomerase could use both telomeric as well as non-telomeric DNA as substrate for interstitial telomeric sequence insertions. Evaluation of a few telomeric insertions by Q-PCR confirmed the sequencing data. For an insertion on chr16 (q24.1), a 9.2-fold increase in telomeric signal in UV-treated control relative to background (day 0) cells was observed, whereas the same locus in telomerase-inhibited sample showed near background amplification. We also looked for somatic telomere insertions in 55 largely untreated patients with Waldenström’s macroglobulinemia for whom whole genome sequencing data was available. The absolute number of telomere insertions correlated with the number of somatic structural variants (translocation, inversions, and large deletions) per genome (tau = 0.3 p=0.001) indicating a possible role in DNA double stranded break repair. Thus telomerase contributes to survival of MM and other cancer cells, not only by preventing telomere attrition, but also the repair of DNA breaks which involves the insertion of telomeric repeats within genome. Inhibition of telomerase therefore, may increase the efficacy of chemotherapeutic agents targeting DNA repair. Evaluating interstitial telomeric insertion pattern in cancer could also be a potentially useful tool to study tumor progression or evolution upon treatment.