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 ...

SUMMARYCancer cells overcome replicative senescence by exploiting mechanisms of telomere elongation, a process often accomplished by reactivation of the enzyme telomerase. However, a subset of cancer cells lack telomerase activity and rely on the alternative lengthening of telomeres (ALT) pathway, a recombination-based mechanism of telomere elongation. Although the mechanisms regulating ALT are not fully defined, chronic replication stress at telomeres might prime these fragile regions for recombination. Here, we demonstrate that the replication stress response protein SMARCAL1 is a critical regulator of ALT activity. SMARCAL1 associates with ALT telomeres to resolve replication stress and ensure telomere stability. In the absence of SMARCAL1, persistently stalled replication forks at ALT telomeres deteriorate into DNA double-strand breaks promoting the formation of chromosome fusions. Our studies not only define a role for SMARCAL1 in ALT telomere maintenance, but also demonstrate that resolution of replication stress is a crucial step in the ALT mechanism.

In the originally published Supplemental Information file associated with this article, the Supplemental Experimental Procedures were inadvertently omitted. The Supplemental Information has now been updated online to include these additional experimental reagents and procedures. The authors apologize for the error and for any inconvenience that may have resulted.

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