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Alternative lengthening of telomeres (ALT) is a telomerase-independent telomere maintenance mechanism utilized by about 15% of cancers. Orphan nuclear receptors (NRs), such as COUP-TF1, COUP-TF2, EAR2, TR2, and TR4, associate with telomeres of ALT cells by binding to variant telomeric repeats. However, how these orphan NRs function in the ALT pathway remains to be characterized. Here, we have established an ALT-inducing cell model by tethering orphan NRs to telomeres in non-ALT BJ fibroblast cells. We demonstrate that recruitment of orphan NRs to telomeres is sufficient to initiate formation of ALT-associated promyelocytic leukemia nuclear bodies (APBs) and telomeric DNA synthesis at APBs. We found that the ability of orphan NRs to initiate APB formation and recombination is dependent on the orphan NR AF2 domain, the zinc-finger protein ZNF827, and PML protein. Depletion of orphan NRs in ALT cell lines reduced APB formation and telomeric DNA synthesis, confirming the role of orphan NRs in ALT cells. Furthermore, we found that ATRX/DAXX depletion, together with the telomeric localization of orphan NRs, induces APB formation, telomere clustering, and telomeric DNA synthesis more dramatically in non-ALT cells. Accordingly, we propose that these events in ALT, orphan NR recruitment to telomeres and ATRX/DAXX loss, operate in concert to activate the ALT pathway.

Extrachromosomal telomere repeat (ECTR) DNA is unique to cancer cells that maintain telomeres through the alternative lengthening of telomeres (ALT) pathway, but the role of ECTRs in ALT development remains elusive. We found that induction of ECTRs in normal human fibroblasts activated the cGAS-STING-TBK1-IRF3 signaling axis to trigger IFNβ production and a type I interferon response, resulting in cell-proliferation defects. In contrast, ALT cancer cells are commonly defective in sensing cytosolic DNA. We found that STING expression was inhibited in ALT cancer cell lines and transformed ALT cells. Notably, the ALT suppressors histone H3.3 and the ATRX-Daxx histone chaperone complex were also required to activate the DNA-sensing pathway. Collectively, our data suggest that the loss of the cGAS-STING pathway may be required to evade ECTR-induced anti-proliferation effects and permit ALT development, and this requirement may be exploited for treatments specific to cancers utilizing the ALT pathway.