SAMHD1 acts at stalled replication forks to prevent interferon induction.

SAMHD1 was previously characterized as a dNTPase that protects cells from viral infections. Mutations in SAMHD1 are implicated in cancer development and in a severe congenital inflammatory disease known as Aicardi-Goutières syndrome. The mechanism by which SAMHD1 protects against cancer and chronic inflammation is unknown. Here we show that SAMHD1 promotes degradation of nascent DNA at stalled replication forks in human cell lines by stimulating the exonuclease activity of MRE11. This function activates the ATR-CHK1 checkpoint and allows the forks to restart replication. In SAMHD1-depleted cells, single-stranded DNA fragments are released from stalled forks and accumulate in the cytosol, where they activate the cGAS-STING pathway to induce expression of pro-inflammatory type I interferons. SAMHD1 is thus an important player in the replication stress response, which prevents chronic inflammation by limiting the release of single-stranded DNA from stalled replication forks.

SAMHD1 and the innate immune response to cytosolic DNA during DNA replication.

Cytosolic DNA of endogenous or exogenous origin is sensed by the cGAS-STING pathway to activate innate immune responses. Besides microbial DNA, this pathway detects self-DNA in the cytoplasm of damaged or abnormal cells and plays a central role in antitumor immunity. The mechanism by which cytosolic DNA accumulates under genotoxic stress conditions is currently unclear, but recent studies on factors mutated in the Aicardi-Goutières syndrome cells, such as SAMHD1, RNase H2 and TREX1, are shedding new light on this key process. In particular, these studies indicate that the rupture of micronuclei and the release of ssDNA fragments during the processing of stalled replication forks and chromosome breaks represent potent inducers of the cGAS-STING pathway.

Inhibition of Ataxia-Telangiectasia Mutated and RAD3-Related (ATR) Overcomes Oxaliplatin Resistance and Promotes Antitumor Immunity in Colorectal Cancer.

Although many patients with colorectal cancer initially respond to the chemotherapeutic agent oxaliplatin, acquired resistance to this treatment remains a major challenge to the long-term management of this disease. To identify molecular targets of oxaliplatin resistance in colorectal cancer, we performed an shRNA-based loss-of-function genetic screen using a kinome library. We found that silencing of ataxia-telangiectasia mutated and RAD3-related (ATR), a serine/threonine protein kinase involved in the response to DNA stress, restored oxaliplatin sensitivity in a cellular model of oxaliplatin resistance. Combined application of the ATR inhibitor VE-822 and oxaliplatin resulted in strong synergistic effects in six different colorectal cancer cell lines and their oxaliplatin-resistant subclones, promoted DNA single- and double-strand break formation, growth arrest, and apoptosis. This treatment also increased replicative stress, cytoplasmic DNA, and signals related to immunogenic cell death such as calreticulin exposure and HMGB1 and ATP release. In a syngeneic colorectal cancer mouse model, combined administration of VE-822 and oxaliplatin significantly increased survival by promoting antitumor T-cell responses. Finally, a DNA repair gene signature discriminated sensitive from drug-resistant patients with colorectal cancer. Overall, our results highlight the potential of ATR inhibition combined with oxaliplatin to sensitize cells to chemotherapy as a therapeutic option for patients with colorectal cancer. SIGNIFICANCE: These findings demonstrate that resistance to oxaliplatin in colorectal cancer cells can be overcome with inhibitors of ATR and that combined treatment with both agents exerts synergistic antitumor effects.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/11/2933/F1.large.jpg.

DDK Has a Primary Role in Processing Stalled Replication Forks to Initiate Downstream Checkpoint Signaling.

CDC7-DBF4 kinase (DDK) initiates DNA replication in eukaryotes by activating the replicative MCM helicase. DDK has diverse and apparently conflicting roles in the replication checkpoint response in various organisms, but the underlying mechanisms are far from settled. We show that human DDK promotes limited resection of newly synthesized DNA at stalled replication forks or sites of DNA damage to initiate replication checkpoint signaling. DDK is also required for efficient fork restart and G2/M cell cycle arrest. DDK exhibits genetic interactions with the ssDNA exonuclease EXO1 and phosphorylates EXO1 in vitro. EXO1 is also required for nascent strand degradation following exposure to HU, so DDK might regulate EXO1 directly. Lastly, sublethal DDK inhibition causes various mitotic abnormalities, which is consistent with a checkpoint deficiency. In summary, DDK has a primary and previously undescribed role in the replication checkpoint to promote ssDNA accumulation at stalled forks, which is required to initiate a robust checkpoint response and cell cycle arrest to maintain genome integrity.