Telomerase-Mediated Repair of Induced DNA Breaks Leads to Increased Genomic Instability in Multiple Myeloma Cells: Possible Mechanism and Translational Significance

Telomerase has multiple functions. We here reported that besides telomere maintenance, telomerase is also involved in the repair of DNA breaks in myeloma (MM) cells. By sequence analyses of “TTAGGG” enriched genomic fragments, we also mapped the interstitial telomeric repeat sequences (ITS) and demonstrate that telomerase mediated repair involves the insertion of ITS within cancer genome. More importantly, we show that such insertions which occur at higher frequency in cancer vs. normal cells, are reduced in the presence of telomerase inhibitor (TI).

We hypothesized that if the ITS if inserted in large numbers following massive DNA breaks, induced by a genotoxic agent, could become substrates for recombination (which is elevated in myeloma) leading to increased genomic instability. To investigate if repair of induced DNA breaks by telomerase would contribute to genomic instability, DNA breaks in RPMI cells were induced by UV, cells cultured in the presence of TI or control oligo for four 4 days, drug was then withdrawn and cells continued in culture for 30 days. Cells were harvested at days 10, 20 and 30 and their genomic DNA evaluated for copy number changes using SNP6.0 arrays. Genome of day 10 for each sample was used as baseline to identify genomic changes in corresponding day 20 and day 30 samples. We observed a gradual increase in deletion events in UV treated control cells from 14-fold at day 20 to 147-fold at day 30, relative to corresponding changes in UV-TI samples at respective time points. Likewise, the amplifications were massively increased (by 500-fold) at day 30, relative to changes in UV-TI cells at the respective time points. When we compared the fold changes in copy number in these samples relative to UV unexposed baseline control cells, changes in TI-treated cells remained close to the baseline till day 30. However, there was a sharp rise in copy number changes in UV treated control cells at corresponding time point. These data indicate that a brief telomerase inhibition during induced DNA damage may prevent late genomic instability. We have further evaluated the translational significance of these findings. We investigated if inhibition of telomerase mediated repair could sensitize cancer cells to DNA damaging chemotherapeutic agents. RPMI MM cells, pretreated with TI or control oligo for 60 hours, were incubated with melphalan at various concentrations for 48 hrs and cell viability assessed. Cell death in cells pretreated with TI was significantly higher than control cells at each dose level suggesting that TI pretreatment sensitizes cells to DNA damaging agents. Telomerase mediated repair involving insertion of telomeric repeats at interstitial break sites prevents immediate genomic loss, and inhibition of this repair sensitizes MM calls to DNA damaging agent.

We conclude that presence of a TI during treatment with a DNA damaging agent would not only enhance the efficacy of treatment but may also protect cancer cells against therapy induced late genomic instability and evolution of aggressive malignant clones.