BETTING ON ALTERNATIVES: TARGETING TELOMERIC DILNCRNA AND BET PROTEINS IN ALT CANCER

The Alternative Lengthening of Telomeres (ALT) pathway is a homology-driven, damage-induced mechanism of telomere maintenance (TMM) that sustains the proliferation of 10-15% of human cancers. We have recently discovered that damaged DNA, including deprotected telomeres, triggers de novo transcription of damage-induced long non-coding RNA (dilncRNA), which are essential for the establishment of a full DNA damage response (DDR) and repair. In fact, inhibition of dilncRNA functions using antisense oligonucleotides (ASOs) dampens DDR in a sequence-specific matter, impeding repair. Consistent with the persistence of DDR activation at ALT telomeres, we observed increased transcription of telomeric dilncRNA in ALT cells compared to non-ALT. Since DDR pathways are instrumental to ALT-mediated telomere maintenance and cell survival, we used telomeric ASOs to inhibit dilncRNA in ALT cells and study its consequences. Interestingly, we observed that inhibition of C-rich telomeric dilncRNA by ASO antiC led to an ALT-specific decrease in proliferation, with non-ALT cells remaining unaffected upon the same treatment. ASO antiC in ALT cells impaired S-phase progression, increased features of genomic instability and led to induction of apoptosis. At the molecular level, we observed that inhibition of C-rich telomeric dilncRNA induced accumulation of H2AX at telomeres and reduced the recruitment of RAD51 and 53BP1. Instead, features of break-induced replication (BIR), such as accumulation of RAD52 and POLD3, and C-circles, were upregulated. Overall, these results indicate that C-rich telomeric dilncRNA is necessary for the controlled engagement of selected DDR factors at ALT telomeres and is essential for survival of ALT cancer cells. In addition, in an effort to identify potential therapeutic opportunities synergizing with ASO antiC, we tested a library of bioactive compounds in a high throughput combinatorial screening. From this experiment, it emerged that inhibition of BET (bromodomain-extra-terminal domain) proteins robustly synergized with ASO antiC in reducing ALT cells proliferation. Moreover, the inhibition of these epigenetic readers led to apoptosis activation only in ALT cells, suggesting a TMM-specific role for BET proteins at telomeres. Our results identify C-rich telomeric dilncRNA as a key player in maintaining ALT cells viability, and suggest a previously unknown role of BET proteins at ALT telomeres. The synergy between ASO antiC and BET inhibitors is a promising combination approach against ALT cancers, for which, currently, there are no targeted therapies.