Nuclear F-actin assembly on damaged chromatin is regulated by DYRK1A and Spir1 phosphorylation.

Nuclear actin-based movements support DNA double-strand break (DSB) repair. However, molecular determinants that promote filamentous actin (F-actin) formation on the damaged chromatin remain undefined. Here we describe the DYRK1A kinase as a nuclear activity that promotes local F-actin assembly to support DSB mobility and repair, accomplished in part by its targeting of actin nucleator spire homolog 1 (Spir1). Indeed, perturbing DYRK1A-dependent phosphorylation of S482 mis-regulated Spir1 accumulation at damaged-modified chromatin, and led to compromised DSB-associated actin polymerization and attenuated DNA repair. Our findings uncover a role of the DYRK1A-Spir1 axis in nuclear actin dynamics during early DSB responses, and highlight the intricate details of nuclear cytoskeletal network in DSB repair and genome stability maintenance.

R-loop resolution by ARIP4 helicase promotes androgen-mediated transcription induction.

Pausing of RNA polymerase II (Pol II) at transcription start sites (TSSs) primes target genes for productive elongation. Coincidentally, DNA double-strand breaks (DSBs) enrich at highly transcribed and Pol II-paused genes, although their interplay remains undefined. Using androgen receptor (AR) signaling as a model, we have uncovered AR-interacting protein 4 (ARIP4) helicase as a driver of androgen-dependent transcription induction. Chromatin immunoprecipitation sequencing analysis revealed that ARIP4 preferentially co-occupies TSSs with paused Pol II. Moreover, we found that ARIP4 complexes with topoisomerase II beta and mediates transient DSB formation upon hormone stimulation. Accordingly, ARIP4 deficiency compromised release of paused Pol II and resulted in R-loop accumulation at a panel of highly transcribed AR target genes. Last, we showed that ARIP4 binds and unwinds R-loops in vitro and that its expression positively correlates with prostate cancer progression. We propose that androgen stimulation triggers ARIP4-mediated unwinding of R-loops at TSSs, enforcing Pol II pause release to effectively drive an androgen-dependent expression program.

Inhibition of aberrantly overexpressed Polo-like kinase 4 is a potential effective treatment for DNA damage repair-deficient uterine leiomyosarcoma.

PURPOSE: Uterine leiomyosarcoma (LMS) is an aggressive sarcoma and a subset of which exhibit DNA repair defects. Polo-like kinase 4 (PLK4) precisely modulates mitosis, and its inhibition causes chromosome missegregation and increased DNA damage. We hypothesize that PLK4 inhibition is an effective LMS treatment. EXPERIMENTAL DESIGN: Genomic profiling of clinical uterine LMS samples was performed, and homologous recombination (HR) deficiency scores were calculated. PLK4 inhibitor (CFI-400945) with and without an ataxia telangiectasia mutated (ATM) inhibitor (AZD0156) were tested in vitro on gynecological sarcoma cell lines SK-UT-1, and SKN, and SK-LMS-1. Findings were validated in vivo using the SK-UT-1 xenograft model in Balb/c nude mouse model. The effects of CFI-400945 were also evaluated in a BRCA2 knockout SK-UT-1 cell line. The mechanisms of DNA repair were analyzed using a DNA damage reporter assay. RESULTS: Uterine LMS had a high HR deficiency score, overexpressed PLK4 mRNA, and displayed mutations in genes responsible for DNA repair. CFI-400945 demonstrated effective antitumor activity in vitro and in vivo. The addition of AZD0156 resulted in drug synergism, largely due to a preference for nonhomologous end-joining (NHEJ) DNA repair. Compared to wild-type cells, BRCA2 knockouts were more sensitive to PLK4 inhibition when both HR and NHEJ repairs were impaired. CONCLUSIONS: Uterine LMS with DNA repair defects is sensitive to PLK4 inhibition because of the effects of chromosome missegregation and increased DNA damage. Loss-of-function BRCA2 alterations or pharmacological inhibition of ATM enhanced the efficacy of PLK4 inhibitor. Genomic profiling of an advanced-stage or recurrent uterine LMS may guide therapy.

Synergistic anticancer effect by targeting CDK2 and EGFR-ERK signaling.

The EGFR-RAS-ERK pathway is one of the most important signaling cascades in cell survival, growth, and proliferation. Aberrant activation of this pathway is a common mechanism in various cancers. Here, we report that CDK2 is a novel regulator of the ERK pathway via USP37 deubiquitinase (DUB). Mechanistically, CDK2 phosphorylates USP37, which is required for USP37 DUB activity. Further, USP37 deubiquitinates and stabilizes ERK1/2, thereby enhancing cancer cell proliferation. Thus, CDK2 is able to promote cell proliferation by activating USP37 and, in turn, stabilizing ERK1/2. Importantly, combined CDK1/2 and EGFR inhibitors have a synergetic anticancer effect through the downregulation of ERK1/2 stability and activity. Indeed, our patient-derived xenograft (PDX) results suggest that targeting both ERK1/2 stability and activity kills cancer cells more efficiently even at lower doses of these two inhibitors, which may reduce their associated side effects and indicate a potential new combination strategy for cancer therapy.

Inhibition of PLK4 remodels histone methylation and activates the immune response via the cGAS-STING pathway in TP53-mutated AML.

Acute myeloid leukemia (AML) with TP53 mutation is one of the most lethal cancers and portends an extremely poor prognosis. Based on in silico analyses of druggable genes and differential gene expression in TP53-mutated AML, we identified pololike kinase 4 (PLK4) as a novel therapeutic target and examined its expression, regulation, pathogenetic mechanisms, and therapeutic potential in TP53-mutated AML. PLK4 expression was suppressed by activated p53 signaling in TP53 wild-type AML and was increased in TP53-mutated AML cell lines and primary samples. Short-term PLK4 inhibition induced DNA damage and apoptosis in TP53 wild-type AML. Prolonged PLK4 inhibition suppressed the growth of TP53-mutated AML and was associated with DNA damage, apoptosis, senescence, polyploidy, and defective cytokinesis. A hitherto undescribed PLK4/PRMT5/EZH2/H3K27me3 axis was demonstrated in both TP53 wild-type and mutated AML, resulting in histone modification through PLK4-induced PRMT5 phosphorylation. In TP53-mutated AML, combined effects of histone modification and polyploidy activated the cGAS-STING pathway, leading to secretion of cytokines and chemokines and activation of macrophages and T cells upon coculture with AML cells. In vivo, PLK4 inhibition also induced cytokine and chemokine expression in mouse recipients, and its combination with anti-CD47 antibody, which inhibited the "don't-eat-me" signal in macrophages, synergistically reduced leukemic burden and prolonged animal survival. The study shed important light on the pathogenetic role of PLK4 and might lead to novel therapeutic strategies in TP53-mutated AML.

Ganciclovir-induced mutations are present in a diverse spectrum of post-transplant malignancies.

BACKGROUND: Ganciclovir (GCV) is widely used in solid organ and haematopoietic stem cell transplant patients for prophylaxis and treatment of cytomegalovirus. It has long been considered a mutagen and carcinogen. However, the contribution of GCV to cancer incidence and other factors that influence its mutagenicity remains unknown. METHODS: This retrospective cohort study analysed genomics data for 121,771 patients who had undergone targeted sequencing compiled by the Genomics Evidence Neoplasia Information Exchange (GENIE) or Foundation Medicine (FM). A statistical approach was developed to identify patients with GCV-associated mutational signature (GCVsig) from targeted sequenced data of tumour samples. Cell line exposure models were further used to quantify mutation burden and DNA damage caused by GCV and other antiviral and immunosuppressive drugs. RESULTS: Mutational profiles from 22 of 121,771 patient samples in the GENIE and FM cohorts showed evidence of GCVsig. A diverse range of cancers was represented. All patients with detailed clinical history available had previously undergone solid organ transplantation and received GCV and mycophenolate treatment. RAS hotspot mutations associated with GCVsig were present in 9 of the 22 samples, with all samples harbouring multiple GCV-associated protein-altering mutations in cancer driver genes. In vitro testing in cell lines showed that elevated DNA damage response and GCVsig are uniquely associated with GCV but not acyclovir, a structurally similar antiviral. Combination treatment of GCV with the immunosuppressant, mycophenolate mofetil (MMF), increased the misincorporation of GCV in genomic DNA and mutations attributed to GCVsig in cell lines and organoids. CONCLUSIONS: In summary, GCV can cause a diverse range of cancers. Its mutagenicity may be potentiated by other therapies, such as mycophenolate, commonly co-prescribed with GCV for post-transplant patients. Further investigation of the optimal use of these drugs could help reduce GCV-associated mutagenesis in post-transplant patients.

RNF4 controls the extent of replication fork reversal to preserve genome stability.

Replication fork reversal occurs via a two-step process that entails reversal initiation and reversal extension. DNA topoisomerase IIalpha (TOP2A) facilitates extensive fork reversal, on one hand through resolving the topological stress generated by the initial reversal, on the other hand via its role in recruiting the SUMO-targeted DNA translocase PICH to stalled forks in a manner that is dependent on its SUMOylation by the SUMO E3 ligase ZATT. However, how TOP2A activities at stalled forks are precisely regulated remains poorly understood. Here we show that, upon replication stress, the SUMO-targeted ubiquitin E3 ligase RNF4 accumulates at stalled forks and targets SUMOylated TOP2A for ubiquitination and degradation. Downregulation of RNF4 resulted in aberrant activation of the ZATT-TOP2A-PICH complex at stalled forks, which in turn led to excessive reversal and elevated frequencies of fork collapse. These results uncover a previously unidentified regulatory mechanism that regulates TOP2A activities at stalled forks and thus the extent of fork reversal.

Regulation of Wnt/PCP signaling through p97/VCP-KBTBD7-mediated Vangl ubiquitination and endoplasmic reticulum-associated degradation.

The four-pass transmembrane proteins Vangl1 and Vangl2 are dedicated core components of Wnt/planar cell polarity (Wnt/PCP) signaling that critically regulate polarized cell behaviors in many morphological and physiological processes. Here, we found that the abundance of Vangl proteins is tightly controlled by the ubiquitin-proteasome system through endoplasmic reticulum-associated degradation (ERAD). The key ERAD component p97/VCP directly binds to Vangl at a highly conserved VCP-interacting motif and recruits the E3 ligase KBTBD7 via its UBA-UBX adaptors to promote Vangl ubiquitination and ERAD. We found that Wnt5a/CK1 prevents Vangl ubiquitination and ERAD by inducing Vangl phosphorylation, which facilitates Vangl export from the ER to the plasma membrane. We also provide in vivo evidence that KBTBD7 regulates convergent extension during zebrafish gastrulation and functions as a tumor suppressor in breast cancer by promoting Vangl degradation. Our findings reveal a previously unknown regulatory mechanism of Wnt/PCP signaling through the p97/VCP-KBTBD7-mediated ERAD pathway.

ATM controls the extent of DNA end resection by eliciting sequential posttranslational modifications of CtIP.

DNA end resection is a critical step in the repair of DNA double-strand breaks (DSBs) via homologous recombination (HR). However, the mechanisms governing the extent of resection at DSB sites undergoing homology-directed repair remain unclear. Here, we show that, upon DSB induction, the key resection factor CtIP is modified by the ubiquitin-like protein SUMO at lysine 578 in a PIAS4-dependent manner. CtIP SUMOylation occurs on damaged chromatin and requires prior hyperphosphorylation by the ATM protein kinase. SUMO-modified hyperphosphorylated CtIP is targeted by the SUMO-dependent E3 ubiquitin ligase RNF4 for polyubiquitination and subsequent degradation. Consequently, disruption of CtIP SUMOylation results in aberrant accumulation of CtIP at DSBs, which, in turn, causes uncontrolled excessive resection, defective HR, and increased cellular sensitivity to DSB-inducing agents. These findings reveal a previously unidentified regulatory mechanism that regulates CtIP activity at DSBs and thus the extent of end resection via ATM-dependent sequential posttranslational modification of CtIP.

A DYRK1B-dependent pathway suppresses rDNA transcription in response to DNA damage.

DNA double-strand breaks (DSBs) at ribosomal gene loci trigger inhibition of ribosomal DNA (rDNA) transcription and extensive nucleolar reorganization, including the formation of nucleolar caps where rDNA DSBs engage with canonical DSB signaling and repair factors. While these nucleolar responses underlie maintenance of rDNA stability, the molecular components that drive each of these events remain to be defined. Here we report that full suppression of rRNA synthesis requires the DYRK1B kinase, a nucleolar DSB response that can be uncoupled from ATM-mediated DSB signaling events at the nucleolar periphery. Indeed, by targeting DSBs onto rDNA arrays, we uncovered that chemical inhibition or genetic inactivation of DYRK1B led to sustained nucleolar transcription. Not only does DYRK1B exhibit robust nucleolar accumulation following laser micro-irradiation across cell nuclei, we further showed that DYRK1B is required for rDNA DSB repair and rDNA copy number maintenance, and that DYRK1B-inactivated cells are hypersensitised to DSBs induced at the rDNA arrays. Together, our findings not only identify DYRK1B as a key signaling intermediate that coordinates DSB repair and rDNA transcriptional activities, but also support the idea of specialised DSB responses that operate within the nucleolus to preserve rDNA integrity.

RECQL5 KIX domain splicing isoforms have distinct functions in transcription repression and DNA damage response.

RecQL5, a mammalian RecQ family protein, is involved in the regulation of transcription elongation, DNA damage response, and DNA replication. Here, we identified and characterized an alternative splicing isoform of RECQL5 (RECQL5ß1), which contains 17 additional amino acid residues within the RECQL5 KIX domain when compared with the canonical isoform (RECQL5ß). RECQL5ß1 had a markedly decreased binding affinity to RNA polymerase II (Pol II) and poorly competed with the transcription elongation factor TFIIS for binding to Pol II. As a result, this isoform has a weaker activity for repression of transcription elongation. In contrast, we discovered that RECQL5ß1 could bind stronger to MRE11, which is a primary sensor of DNA double-strand breaks (DSBs). Furthermore, we found that RECQL5ß1 promoted DNA repair in the RECQL5ß1 rescue cells. These results suggest that RECQL5ß mainly functions as a transcription repressor, while the newly discovered RECQL5ß1 has a specialized role in DNA damage response. Taken together, our data suggest a cellular-functional specialization for each KIX splicing isoform in the cell.

PRMT6 deficiency induces autophagy in hostile microenvironments of hepatocellular carcinoma tumors by regulating BAG5-associated HSC70 stability.

Autophagy is a critical survival factor for cancer cells, whereby it maintains cellular homeostasis by degrading damaged organelles and unwanted proteins and supports cellular biosynthesis in response to stress. Cancer cells, including hepatocellular carcinoma (HCC), are often situated in a hypoxic, nutrient-deprived and stressful microenvironment where tumor cells are yet still able to adapt and survive. However, the mechanism underlying this adaptation and survival is not well-defined. We report deficiency of the post-translational modification enzyme protein arginine N-methyltransferase 6 (PRMT6) in HCC to promote the induction of autophagy under oxygen/nutrient-derived and sorafenib drug-induced stress conditions. Enhanced autophagic flux in HCC cells negatively correlated with PRMT6 expression, with the catalytic domain of PRMT6 critically important in mediating these autophagic activities. Mechanistically, PRMT6 physically interacts and methylates BAG5 to enhance the degradation of its interacting partner HSC70, a well-known autophagy player. The therapeutic potential of targeting BAG5 using genetic approach to reverse tumorigenicity and sorafenib resistance mediated by PRMT6 deficiency in HCC is also demonstrated in an in vivo model. The clinical implications of these findings are highlighted by the inverse correlative expressions of PRMT6 and HSC70 in HCC tissues. Collectively, deficiency of PRMT6 induces autophagy to promote tumorigenicity and cell survival in hostile microenvironments of HCC tumors by regulating BAG5-associated HSC70 stability through post-translational methylation of BAG5. Targeting BAG5 may therefore be an attractive strategy in HCC treatment by suppressing autophagy and inducing HCC cell sensitivity to sorafenib for treatment.

The novel male meiosis recombination regulator coordinates the progression of meiosis prophase I.

Meiosis is a specialized cell division for producing haploid gametes in sexually reproducing organisms. In this study, we have independently identified a novel meiosis protein male meiosis recombination regulator (MAMERR)/4930432K21Rik and showed that it is indispensable for meiosis prophase I progression in male mice. Using super-resolution structured illumination microscopy, we found that MAMERR functions at the same double-strand breaks as the replication protein A and meiosis-specific with OB domains/spermatogenesis associated 22 complex. We generated a Mamerr-deficient mouse model by deleting exons 3-6 and found that most of Mamerr-/- spermatocytes were arrested at pachynema and failed to progress to diplonema, although they exhibited almost intact synapsis and progression to the pachytene stage along with XY body formation. Further mechanistic studies revealed that the recruitment of DMC1/RAD51 and heat shock factor 2-binding protein in Mamerr-/- spermatocytes was only mildly impaired with a partial reduction in double-strand break repair, whereas a substantial reduction in ubiquitination on the autosomal axes and on the XY body appeared as a major phenotype in Mamerr-/- spermatocytes. We propose that MAMERR may participate in meiotic prophase I progression by regulating the ubiquitination of key meiotic proteins on autosomes and XY chromosomes, and in the absence of MAMERR, the repressed ubiquitination of key meiotic proteins leads to pachytene arrest and cell death.

Screen identifies DYRK1B network as mediator of transcription repression on damaged chromatin.

DNA double-strand breaks (DSBs) trigger transient pausing of nearby transcription, an emerging ATM-dependent response that suppresses chromosomal instability. We screened a chemical library designed to target the human kinome for new activities that mediate gene silencing on DSB-flanking chromatin, and have uncovered the DYRK1B kinase as an early respondent to DNA damage. We showed that DYRK1B is swiftly and transiently recruited to laser-microirradiated sites, and that genetic inactivation of DYRK1B or its kinase activity attenuated DSB-induced gene silencing and led to compromised DNA repair. Notably, global transcription shutdown alleviated DNA repair defects associated with DYRK1B loss, suggesting that DYRK1B is strictly required for DSB repair on active chromatin. We also found that DYRK1B mediates transcription silencing in part via phosphorylating and enforcing DSB accumulation of the histone methyltransferase EHMT2. Together, our findings unveil the DYRK1B signaling network as a key branch of mammalian DNA damage response circuitries, and establish the DYRK1B-EHMT2 axis as an effector that coordinates DSB repair on transcribed chromatin.

Preemptive Homology-Directed DNA Repair Fosters Complex Genomic Rearrangements in Hepatocellular Carcinoma.

Degree of genomic instability closely correlates with poor prognosis, drug resistance as well as poor survival across human cancer of different origins. This study assessed the relationship between DNA damage response (DDR) and chromosome instability in hepatocellular carcinoma (HCC). We investigated DDR signaling in HCC cells by analyzing DNA damage-dependent redistribution of major DDR proteins to damaged chromatin using immunofluorescence microscopy and Western blotting experimentations. We also performed gene conversion and metaphase analyses to address whether dysregulated DDR may bear any biological significance during hepatocarcinogenesis. Accordingly, we found that HCC cell lines suffered from elevated spontaneous DNA double-strand breaks (DSBs). In addition, analyses of HCC metaphases revealed marked aneuploidy and frequent sister chromatid exchanges when compared to immortalized hepatocytes, the latter of which were further induced following camptothecin-induced DSBs. We propose that genomic instability in HCC may be caused by erroneous DNA repair in a desperate attempt to mend DSBs for cell survival and that such preemptive measures inadvertently foster chromosome instability and thus complex genomic rearrangements.

Perfecting DNA double-strand break repair on transcribed chromatin.

Timely repair of DNA double-strand break (DSB) entails coordination with the local higher order chromatin structure and its transaction activities, including transcription. Recent studies are uncovering how DSBs trigger transient suppression of nearby transcription to permit faithful DNA repair, failing of which leads to elevated chromosomal aberrations and cell hypersensitivity to DNA damage. Here, we summarize the molecular bases for transcriptional control during DSB metabolism, and discuss how the exquisite coordination between the two DNA-templated processes may underlie maintenance of genome stability and cell homeostasis.

53BP1 loss rescues embryonic lethality but not genomic instability of BRCA1 total knockout mice.

BRCA1 is critical for DNA double-strand break (DSB) repair by homologous recombination (HR). BRCA1 deficient mice are embryonic lethal. Previous studies have shown that 53BP1 knockout (KO) rescues embryonic lethality of BRCA1 hypomorphic mutant mice by restoring HR. Here, we show that 53BP1 KO can partially rescue embryonic lethality of BRCA1 total KO mice, but HR is not restored in BRCA1-53BP1 double knockout (DKO) mice. As a result, BRCA1-53BP1 DKO cells are extremely sensitive to PARP inhibitors (PARPi). In addition to HR deficiency, BRCA1-53BP1 DKO cells have elevated microhomology-mediated end joining (MMEJ) activity and G2/M cell cycle checkpoint defects, causing severe genomic instability in these cells. Interestingly, BRCA1-53BP1 DKO mice rapidly develop thymic lymphoma that is 100% penetrant, which is not observed in any BRCA1 mutant mice rescued by 53BP1 KO. Taken together, our study reveals that 53BP1 KO can partially rescue embryonic lethality caused by complete BRCA1 loss without rescuing HR-related defects. This finding suggests that loss of 53BP1 can support the development of cancers with silenced BRCA1 expression without causing PARPi resistance.

A comprehensive proteomics-based interaction screen that links DYRK1A to RNF169 and to the DNA damage response.

Dysregulation of the DYRK1A protein kinase has been associated with human disease. On the one hand, its overexpression in trisomy 21 has been linked to certain pathological traits of Down syndrome, while on the other, inactivating mutations in just one allele are responsible for a distinct yet rare clinical syndrome, DYRK1A haploinsufficiency. Moreover, altered expression of this kinase may also provoke other human pathologies, including cancer and diabetes. Although a few DYRK1A substrates have been described, its upstream regulators and downstream targets are still poorly understood, an information that could shed light on the functions of DYRK1A in the cell. Here, we carried out a proteomic screen using antibody-based affinity purification coupled to mass spectrometry to identify proteins that directly or indirectly bind to endogenous DYRK1A. We show that the use of a cell line not expressing DYRK1A, generated by CRISPR/Cas9 technology, was needed in order to discriminate between true positives and non-specific interactions. Most of the proteins identified in the screen are novel candidate DYRK1A interactors linked to a variety of activities in the cell. The in-depth characterization of DYRK1A's functional interaction with one of them, the E3 ubiquitin ligase RNF169, revealed a role for this kinase in the DNA damage response. We found that RNF169 is a DYRK1A substrate and we identified several of its phosphorylation sites. In particular, one of these sites appears to modify the ability of RNF169 to displace 53BP1 from sites of DNA damage. Indeed, DYRK1A depletion increases cell sensitivity to ionizing irradiation. Therefore, our unbiased proteomic screen has revealed a novel activity of DYRK1A, expanding the complex role of this kinase in controlling cell homeostasis.

LC8/DYNLL1 is a 53BP1 effector and regulates checkpoint activation.

The tumor suppressor protein 53BP1 plays key roles in response to DNA double-strand breaks (DSBs) by serving as a master scaffold at the damaged chromatin. Current evidence indicates that 53BP1 assembles a cohort of DNA damage response (DDR) factors to distinctly execute its repertoire of DSB responses, including checkpoint activation and non-homologous end joining (NHEJ) repair. Here, we have uncovered LC8 (a.k.a. DYNLL1) as an important 53BP1 effector. We found that LC8 accumulates at laser-induced DNA damage tracks in a 53BP1-dependent manner and requires the canonical H2AX-MDC1-RNF8-RNF168 signal transduction cascade. Accordingly, genetic inactivation of LC8 or its interaction with 53BP1 resulted in checkpoint defects. Importantly, loss of LC8 alleviated the hypersensitivity of BRCA1-depleted cells to ionizing radiation and PARP inhibition, highlighting the 53BP1-LC8 module in counteracting BRCA1-dependent functions in the DDR. Together, these data establish LC8 as an important mediator of a subset of 53BP1-dependent DSB responses.