Alternative lengthening of telomeres renders cancer cells hypersensitive to ATR inhibitors

Abstract

Cancer cells rely on telomerase or the alternative lengthening of telomeres (ALT) pathway to overcome replicative mortality. ALT is mediated by recombination and is prevalent in a subset of human cancers, yet whether it can be exploited therapeutically remains unknown. Loss of the chromatin remodeling protein ATRX associates with ALT in cancers. Here, we show that ATRX loss compromises cell-cycle regulation of the telomeric non-coding RNA TERRA and leads to persistent association of replication protein A (RPA) with telomeres after DNA replication, creating a recombinogenic nucleoprotein structure. Inhibition of the protein kinase ATR, a critical regulator of recombination recruited by RPA, disrupts ALT and triggers chromosome fragmentation and apoptosis in ALT cells. Importantly, the cell death induced by ATR inhibitors is highly selective for cancer cells that rely on ALT, , suggesting that such inhibitors may be useful for treatment of ALT-positive cancers.Cancer cells overcome replicative senescence by activating telomerase or the alternative lengthening of telomeres (ALT) pathway (1-3). ALT is used in ~5-15% of all human Author ManuscriptAuthor Manuscript Author ManuscriptAuthor Manuscript cancers and is prevalent in specific cancer types, including osteosarcoma and glioblastoma (4). Currently, there are no therapies specifically targeting ALT. ALT relies on recombination to elongate telomeres (3), but how the recombinogenic state of ALT telomeres is established remains elusive. In contrast to cancer cells defective for homologous recombination (HR) and susceptible to Poly(ADP-ribose) polymerase (PARP) inhibition (5, 6), ALT-positive cells are HR-proficient (7). Thus, the reliance of ALT on recombination raises an important question as to whether recombination can be exploited in ALT-positive cancers as a means for targeted therapy.Single-stranded DNA (ssDNA) coated by replication protein A (RPA) is a key intermediate in both DNA replication and HR (8). RPA transiently associates with telomeres during DNA replication, but is released from telomeres after S phase (9, 10). The release of RPA may be an important mechanism to suppress HR at telomeres. The association of RPA with telomeres in S phase is facilitated by TERRA, the telomere repeat-containing RNA, which is also present at telomeres during this period (9,(11)(12)(13). To investigate how ALT is established, we determined whether the association of TERRA with telomeres is altered in ALT cells. TERRA colocalized with the telomere-binding protein TRF2 in telomerasepositive HeLa cervical cancer cells ( fig. S1) (9). However, in both HeLa and telomerasepositive SJSA1 osteosarcoma cells ( fig. S24B), the number of TERRA foci declined from S phase to G2 ( Considering that RPA is released from telomeres in G2/M when TERRA is repressed by ATRX (9), we examined whether ATRX is required for the release of RPA. In HeLa cells, numerous small replication-associated RPA foci (type-A RPA foci) were detected in S phase (Fig. S7). As cells progressed from S to ...

Alternative lengthening of telomeres and loss of ATRX are frequent events in pleomorphic and dedifferentiated liposarcomas

“…Remarkably, inhibition of protein kinase ATR has been shown to selectively cause apoptosis of cells with alternative lengthening of telomeres, a discovery that may endow the current findings with therapeutic implications. 35 In addition, the finding that a mitotic count of 5 in 10 high-power fields effectively dichotomized the dedifferentiated liposarcoma cases into two distinct prognostic groups supports the notion that at least five mitotic figures per 10 high-power fields are required for diagnosing conventional dedifferentiated liposarcoma 30 and indicates that the concept of low-grade dedifferentiated liposarcomas might warrant further clarification factoring in mitosis.…”

Comprehensive screening of alternative lengthening of telomeres phenotype and loss of ATRX expression in sarcomas

“…Recently, ataxia telangiectasia and Rad3-related kinase (ATR) was shown to play crucial roles in homologous recombination and alternative lengthening of telomeres, and inhibition of this kinase caused apoptosis of alternative lengthening of telomeres-positive cancer cells. 38 Identification of alternative lengthening of telomeres and loss of ATRX in sarcomas may provide new opportunities to treat these aggressive neoplasms.…”

Telomere elongation chooses TERRA ALTernatives

“…As mentioned above, TERRA is not down-regulated in ALT cells during S to G2/M phase progression and, similarly, its gradual decline in S to G2/M is prevented in telomerase-positive cells depleted for ATRX. 27,28 These data suggest that the lack of ATRX in ALT cells might be responsible for the loss of regulation of TERRA levels during the cell cycle and re-expressing functional ATRX in ALT cells should uncover whether this hypothesis is correct. Moreover, while RPA is normally released from telomeres in G2/ M, depletion of ATRX in telomerase-positive cells averts this event and, consistently, RPA foci can readily be detected in ALT cells during G2/M.…”

“…[24][25][26] Moreover, contrary to what is observed in telomerase positive cancer cells, TERRA levels do not decline during progression from S-phase to G2/M, possibly due to a lack of ATRX activity. 27,28 Thus increased transcription and loss of its cell cycle regulation account for the overall increase of TERRA cellular levels characteristic of ALT lines.…”