A genome-wide screen identifies SCAI as a modulator of the UV-induced replicative stress response.

Helix-destabilizing DNA lesions induced by environmental mutagens such as UV light cause genomic instability by strongly blocking the progression of DNA replication forks (RFs). At blocked RF, single-stranded DNA (ssDNA) accumulates and is rapidly bound by Replication Protein A (RPA) complexes. Such stretches of RPA-ssDNA constitute platforms for recruitment/activation of critical factors that promote DNA synthesis restart. However, during periods of severe replicative stress, RPA availability may become limiting due to inordinate sequestration of this multifunctional complex on ssDNA, thereby negatively impacting multiple vital RPA-dependent processes. Here, we performed a genome-wide screen to identify factors that restrict the accumulation of RPA-ssDNA during UV-induced replicative stress. While this approach revealed some expected "hits" acting in pathways such as nucleotide excision repair, translesion DNA synthesis, and the intra-S phase checkpoint, it also identified SCAI, whose role in the replicative stress response was previously unappreciated. Upon UV exposure, SCAI knock-down caused elevated accumulation of RPA-ssDNA during S phase, accompanied by reduced cell survival and compromised RF progression. These effects were independent of the previously reported role of SCAI in 53BP1-dependent DNA double-strand break repair. We also found that SCAI is recruited to UV-damaged chromatin and that its depletion promotes nascent DNA degradation at stalled RF. Finally, we (i) provide evidence that EXO1 is the major nuclease underlying ssDNA formation and DNA replication defects in SCAI knockout cells and, consistent with this, (ii) demonstrate that SCAI inhibits EXO1 activity on a ssDNA gap in vitro. Taken together, our data establish SCAI as a novel regulator of the UV-induced replicative stress response in human cells.

A genome-wide screen reveals that Dyrk1A kinase promotes nucleotide excision repair by preventing aberrant overexpression of cyclin D1 and p21.

Nucleotide excision repair (NER) eliminates highly genotoxic solar UV-induced DNA photoproducts that otherwise stimulate malignant melanoma development. Here, a genome-wide loss-of-function screen, coupling CRISPR/Cas9 technology with a flow cytometry-based DNA repair assay, was used to identify novel genes required for efficient NER in primary human fibroblasts. Interestingly, the screen revealed multiple genes encoding proteins, with no previously known involvement in UV damage repair, that significantly modulate NER uniquely during S phase of the cell cycle. Among these, we further characterized Dyrk1A, a dual specificity kinase that phosphorylates the proto-oncoprotein cyclin D1 on threonine 286 (T286), thereby stimulating its timely cytoplasmic relocalization and proteasomal degradation, which is required for proper regulation of the G1-S phase transition and control of cellular proliferation. We demonstrate that in UV-irradiated HeLa cells, depletion of Dyrk1A leading to overexpression of cyclin D1 causes inhibition of NER uniquely during S phase and reduced cell survival. Consistently, expression/nuclear accumulation of nonphosphorylatable cyclin D1 (T286A) in melanoma cells strongly interferes with S phase NER and enhances cytotoxicity post-UV. Moreover, the negative impact of cyclin D1 (T286A) overexpression on repair is independent of cyclin-dependent kinase activity but requires cyclin D1-dependent upregulation of p21 expression. Our data indicate that inhibition of NER during S phase might represent a previously unappreciated noncanonical mechanism by which oncogenic cyclin D1 fosters melanomagenesis.

HIF1α-dependent hypoxia response in myeloid cells requires IRE1α.

Cellular adaptation to low oxygen tension triggers primitive pathways that ensure proper cell function. Conditions of hypoxia and low glucose are characteristic of injured tissues and hence successive waves of inflammatory cells must be suited to function under low oxygen tension and metabolic stress. While Hypoxia-Inducible Factor (HIF)-1α has been shown to be essential for the inflammatory response of myeloid cells by regulating the metabolic switch to glycolysis, less is known about how HIF1α is triggered in inflammation. Here, we demonstrate that cells of the innate immune system require activity of the inositol-requiring enzyme 1α (IRE1α/XBP1) axis in order to initiate HIF1α-dependent production of cytokines such as IL1ß, IL6 and VEGF-A. Knockout of either HIF1α or IRE1α in myeloid cells ameliorates vascular phenotypes in a model of retinal pathological angiogenesis driven by sterile inflammation. Thus, pathways associated with ER stress, in partnership with HIF1α, may co-regulate immune adaptation to low oxygen.

Non-canonical ATM/MRN activities temporally define the senescence secretory program.

Senescent cells display senescence-associated (SA) phenotypic programs such as stable proliferation arrest (SAPA) and a secretory phenotype (SASP). Senescence-inducing persistent DNA double-strand breaks (pDSBs) cause an immediate DNA damage response (DDR) and SAPA, but the SASP requires days to develop. Here, we show that following the immediate canonical DDR, a delayed chromatin accumulation of the ATM and MRN complexes coincides with the expression of SASP factors. Importantly, histone deacetylase inhibitors (HDACi) trigger SAPA and SASP in the absence of DNA damage. However, HDACi-induced SASP also requires ATM/MRN activities and causes their accumulation on chromatin, revealing a DNA damage-independent, non-canonical DDR activity that underlies SASP maturation. This non-canonical DDR is required for the recruitment of the transcription factor NF-κB on chromatin but not for its nuclear translocation. Non-canonical DDR further does not require ATM kinase activity, suggesting structural ATM functions. We propose that delayed chromatin recruitment of SASP modulators is the result of non-canonical DDR signaling that ensures SASP activation only in the context of senescence and not in response to transient DNA damage-induced proliferation arrest.

CBFA2T3-GLIS2-dependent pediatric acute megakaryoblastic leukemia is driven by GLIS2 and sensitive to navitoclax.

Pediatric acute megakaryoblastic leukemia (AMKL) is an aggressive blood cancer associated with poor therapeutic response and high mortality. Here we describe the development of CBFA2T3-GLIS2-driven mouse models of AMKL that recapitulate the phenotypic and transcriptional signatures of the human disease. We show that an activating Ras mutation that occurs in human AMKL increases the penetrance and decreases the latency of CBF2AT3-GLIS2-driven AMKL. CBFA2T3-GLIS2 and GLIS2 modulate similar transcriptional networks. We identify the dominant oncogenic properties of GLIS2 that trigger AMKL in cooperation with oncogenic Ras. We find that both CBFA2T3-GLIS2 and GLIS2 alter the expression of a number of BH3-only proteins, causing AMKL cell sensitivity to the BCL2 inhibitor navitoclax both in vitro and in vivo, suggesting a potential therapeutic option for pediatric patients suffering from CBFA2T3-GLIS2-driven AMKL.

Neutrophil extracellular traps target senescent vasculature for tissue remodeling in retinopathy.

In developed countries, the leading causes of blindness such as diabetic retinopathy are characterized by disorganized vasculature that can become fibrotic. Although many such pathological vessels often naturally regress and spare sight-threatening complications, the underlying mechanisms remain unknown. Here, we used orthogonal approaches in human patients with proliferative diabetic retinopathy and a mouse model of ischemic retinopathies to identify an unconventional role for neutrophils in vascular remodeling during late-stage sterile inflammation. Senescent vasculature released a secretome that attracted neutrophils and triggered the production of neutrophil extracellular traps (NETs). NETs ultimately cleared diseased endothelial cells and remodeled unhealthy vessels. Genetic or pharmacological inhibition of NETosis prevented the regression of senescent vessels and prolonged disease. Thus, clearance of senescent retinal blood vessels leads to reparative vascular remodeling.