DePARylation is critical for S phase progression and cell survival.

Poly(ADP-ribose)ylation or PARylation by PAR polymerase 1 (PARP1) and dePARylation by poly(ADP-ribose) glycohydrolase (PARG) are equally important for the dynamic regulation of DNA damage response. PARG, the most active dePARylation enzyme, is recruited to sites of DNA damage via pADPr-dependent and PCNA-dependent mechanisms. Targeting dePARylation is considered an alternative strategy to overcome PARP inhibitor resistance. However, precisely how dePARylation functions in normal unperturbed cells remains elusive. To address this challenge, we conducted multiple CRISPR screens and revealed that dePARylation of S phase pADPr by PARG is essential for cell viability. Loss of dePARylation activity initially induced S-phase-specific pADPr signaling, which resulted from unligated Okazaki fragments and eventually led to uncontrolled pADPr accumulation and PARP1/2-dependent cytotoxicity. Moreover, we demonstrated that proteins involved in Okazaki fragment ligation and/or base excision repair regulate pADPr signaling and cell death induced by PARG inhibition. In addition, we determined that PARG expression is critical for cellular sensitivity to PARG inhibition. Additionally, we revealed that PARG is essential for cell survival by suppressing pADPr. Collectively, our data not only identify an essential role for PARG in normal proliferating cells but also provide a potential biomarker for the further development of PARG inhibitors in cancer therapy.

Genome-wide CRISPR screen reveals the synthetic lethality between BCL2L1 inhibition and radiotherapy.

Radiation therapy (RT) is one of the most commonly used anticancer therapies. However, the landscape of cellular response to irradiation, especially to a single high-dose irradiation, remains largely unknown. In this study, we performed a whole-genome CRISPR loss-of-function screen and revealed temporal inherent and acquired responses to RT. Specifically, we found that loss of the IL1R1 pathway led to cellular resistance to RT. This is in part because of the involvement of radiation-induced IL1R1-dependent transcriptional regulation, which relies on the NF-κB pathway. Moreover, the mitochondrial anti-apoptotic pathway, particularly the BCL2L1 gene, is crucially important for cell survival after radiation. BCL2L1 inhibition combined with RT dramatically impeded tumor growth in several breast cancer cell lines and syngeneic models. Taken together, our results suggest that the combination of an apoptosis inhibitor such as a BCL2L1 inhibitor with RT may represent a promising anticancer strategy for solid cancers including breast cancer.

DePARylation is critical for S phase progression and cell survival.

Poly(ADP-ribose)ylation or PARylation by PAR polymerase 1 (PARP1) and dePARylation by poly(ADP-ribose) glycohydrolase (PARG) are equally important for the dynamic regulation of DNA damage response. PARG, the most active dePARylation enzyme, is recruited to sites of DNA damage via pADPr-dependent and PCNA-dependent mechanisms. Targeting dePARylation is considered an alternative strategy to overcome PARP inhibitor resistance. However, precisely how dePARylation functions in normal unperturbed cells remains elusive. To address this challenge, we conducted multiple CRISPR screens and revealed that dePARylation of S phase pADPr by PARG is essential for cell viability. Loss of dePARylation activity initially induced S phase-specific pADPr signaling, which resulted from unligated Okazaki fragments and eventually led to uncontrolled pADPr accumulation and PARP1/2-dependent cytotoxicity. Moreover, we demonstrated that proteins involved in Okazaki fragment ligation and/or base excision repair regulate pADPr signaling and cell death induced by PARG inhibition. In addition, we determined that PARG expression is critical for cellular sensitivity to PARG inhibition. Additionally, we revealed that PARG is essential for cell survival by suppressing pADPr. Collectively, our data not only identify an essential role for PARG in normal proliferating cells but also provide a potential biomarker for the further development of PARG inhibitors in cancer therapy.

RAD54L2-mediated DNA damage avoidance pathway specifically preserves genome integrity in response to topoisomerase 2 poisons.

Type II topoisomerases (TOP2) form transient TOP2 cleavage complexes (TOP2ccs) during their catalytic cycle to relieve topological stress. TOP2ccs are covalently linked TOP2-DNA intermediates that are reversible but can be trapped by TOP2 poisons. Trapped TOP2ccs block transactions on DNA and generate genotoxic stress, which are the mechanisms of action of TOP2 poisons. How cells avoid TOP2cc accumulation remains largely unknown. In this study, we uncovered RAD54 like 2 (RAD54L2) as a key factor that mediates a TOP2-specific DNA damage avoidance pathway. RAD54L2 deficiency conferred unique sensitivity to treatment with TOP2 poisons. RAD54L2 interacted with TOP2A/TOP2B and ZATT/ZNF451 and promoted the turnover of TOP2 from DNA with or without TOP2 poisons. Additionally, inhibition of proteasome activity enhanced the chromatin binding of RAD54L2, which in turn led to the removal of TOP2 from chromatin. In conclusion, we propose that RAD54L2-mediated TOP2 turnover is critically important for the avoidance of potential TOP2-linked DNA damage under physiological conditions and in response to TOP2 poisons.

CRISPR/Cas9-Mediated Genome Editing in Cancer Therapy.

The Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system, an RNA-based adaptive immune system found in bacteria and archaea, has catalyzed the development and application of a new generation of gene editing tools. Numerous studies have shown that this system can precisely target a wide range of human genes, including those associated with diseases such as cancer. In cancer research, the intricate genetic mutations in tumors have promoted extensive utilization of the CRISPR/Cas9 system due to its efficient and accurate gene editing capabilities. This includes improvements in Chimeric Antigen Receptor (CAR)-T-cell therapy, the establishment of tumor models, and gene and drug target screening. Such progress has propelled the investigation of cancer molecular mechanisms and the advancement of precision medicine. However, the therapeutic potential of genome editing remains underexplored, and lingering challenges could elevate the risk of additional genetic mutations. Here, we elucidate the fundamental principles of CRISPR/Cas9 gene editing and its practical applications in tumor research. We also briefly discuss the primary challenges faced by CRISPR technology and existing solutions, intending to enhance the efficacy of this gene editing therapy and shed light on the underlying mechanisms of tumors.

FACS-based genome-wide CRISPR screens define key regulators of DNA damage signaling pathways.

DNA damage-activated signaling pathways are critical for coordinating multiple cellular processes, which must be tightly regulated to maintain genome stability. To provide a comprehensive and unbiased perspective of DNA damage response (DDR) signaling pathways, we performed 30 fluorescence-activated cell sorting (FACS)-based genome-wide CRISPR screens in human cell lines with antibodies recognizing distinct endogenous DNA damage signaling proteins to identify critical regulators involved in DDR. We discovered that proteasome-mediated processing is an early and prerequisite event for cells to trigger camptothecin- and etoposide-induced DDR signaling. Furthermore, we identified PRMT1 and PRMT5 as modulators that regulate ATM protein level. Moreover, we discovered that GNB1L is a key regulator of DDR signaling via its role as a co-chaperone specifically regulating PIKK proteins. Collectively, these screens offer a rich resource for further investigation of DDR, which may provide insight into strategies of targeting these DDR pathways to improve therapeutic outcomes.

Omentin-1 ameliorates experimental inflammatory bowel disease via Nrf2 activation and redox regulation.

AIMS: Omentin-1 production is decreased in patients with IBD. However, the specific role of Omentin-1 in IBD has not been fully elucidated. This study aimed to investigate the expression and role of Omentin-1 in IBD and the potential mechanisms. MAIN METHODS: We collected human serum and colon biopsy samples at the Wuhan Union Hospital. Omentin-1 recombinant protein was injected intraperitoneally in a DSS-induced experimental IBD mouse model. Omentin-1 levels were measured in IBD patients, colitis mice, and LPS-induced HT-29 cells. Omentin-1 and/or a Nrf2 specific inhibitor (ML385) were administered to DSS mice and LPS-induced HT-29 cells. The effects of Omentin-1 on inflammation, intestinal barrier function, Nrf2 pathway, oxidative stress, and NF-κB signaling were detected in vivo and in vitro. KEY FINDINGS: Serum Omentin-1 levels were significantly reduced in UC and CD patients compared with controls (173.7 (IQR, 120.1-221.2) ng/ml, 80.8 (43.8-151.8) ng/ml, and 270.7 (220.7-306.5) ng/ml, respectively). The levels of Omentin-1 were also significantly lower in colitis mice and LPS-induced HT-29 cells. Omentin-1 treatment effectively ameliorated inflammation and impaired intestinal barrier, decreased ROS and MDA levels, and increased GSH and SOD production in the DSS-induced colitis mice and LPS-induced HT-29 cells. Mechanically, Omentin-1 repaired the intestinal barrier by activating Nrf2, then improving oxidative stress and inhibiting NF-κB signaling. Furthermore, the interaction between Omentin-1 and Nrf2 was identified. SIGNIFICANCE: Omentin-1 activates the Nrf2 pathway to regulate redox balance, ultimately protecting intestinal barrier function and reducing intestinal inflammation. In general, Omentin-1 can be used as a promising therapeutic target for IBD.

Genome-Wide CRISPR Screens Reveal ZATT as a Synthetic Lethal Target of TOP2-Poison Etoposide That Can Act in a TDP2-Independent Pathway.

Etoposide (ETO) is an anticancer drug that targets topoisomerase II (TOP2). It stabilizes a normally transient TOP2-DNA covalent complex (TOP2cc), thus leading to DNA double-strand breaks (DSBs). Tyrosyl-DNA phosphodiesterases two (TDP2) is directly involved in the repair of TOP2cc by removing phosphotyrosyl peptides from 5'-termini of DSBs. Recent studies suggest that additional factors are required for TOP2cc repair, which include the proteasome and the zinc finger protein associated with TDP2 and TOP2, named ZATT. ZATT may alter the conformation of TOP2cc in a way that renders the accessibility of TDP2 for TOP2cc removal. In this study, our genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screens revealed that ZATT also has a TDP2-independent role in promoting cell survival following ETO treatment. ZATT KO cells showed relatively higher ETO sensitivity than TDP2-KO cells, and ZATT/TDP2 DKO cells displayed additive hypersensitivity to ETO treatment. The study using a series of deletion mutants of ZATT determined that the N-terminal 1-168 residues of ZATT are required for interaction with TOP2 and this interaction is critical to ETO sensitivity. Moreover, depletion of ZATT resulted in accelerated TOP2 degradation after ETO or cycloheximide (CHX) treatment, suggesting that ZATT may increase TOP2 stability and likely participate in TOP2 turnover. Taken together, this study suggests that ZATT is a critical determinant that dictates responses to ETO treatment and targeting. ZATT is a promising strategy to increase ETO efficacy for cancer therapy.

TDP1-independent pathways in the process and repair of TOP1-induced DNA damage.

Anticancer drugs, such as camptothecin (CPT), trap topoisomerase I (TOP1) on DNA and form TOP1 cleavage complexes (TOP1cc). Alternative repair pathways have been suggested in the repair of TOP1cc. However, how these pathways work with TDP1, a key repair enzyme that specifically hydrolyze the covalent bond between TOP1 catalytic tyrosine and the 3'-end of DNA and contribute to the repair of TOP1cc is poorly understood. Here, using unbiased whole-genome CRISPR screens and generation of co-deficient cells with TDP1 and other genes, we demonstrate that MUS81 is an important factor that mediates the generation of excess double-strand breaks (DSBs) in TDP1 KO cells. APEX1/2 are synthetic lethal with TDP1. However, deficiency of APEX1/2 does not reduce DSB formation in TDP1 KO cells. Together, our data suggest that TOP1cc can be either resolved directly by TDP1 or be converted into DSBs and repaired further by the Homologous Recombination (HR) pathway.

Integrated screens uncover a cell surface tumor suppressor gene KIRREL involved in Hippo pathway.

Cell surface proteins play essential roles in various biological processes and are highly related to cancer development. They also serve as important markers for cell identity and targets for pharmacological intervention. Despite their great potentials in biomedical research, comprehensive functional analysis of cell surface proteins remains scarce. Here, with a de novo designed library targeting cell surface proteins, we performed in vivo CRISPR screens to evaluate the effects of cell surface proteins on tumor survival and proliferation. We found that Kirrel1 loss markedly promoted tumor growth in vivo. Moreover, KIRREL was significantly enriched in a separate CRISPR screen based on a specific Hippo pathway reporter. Further studies revealed that KIRREL binds directly to SAV1 to activate the Hippo tumor suppressor pathway. Together, our integrated screens reveal a cell surface tumor suppressor involved in the Hippo pathway and highlight the potential of these approaches in biomedical research.

Genome-wide CRISPR screens using isogenic cells reveal vulnerabilities conferred by loss of tumor suppressors.

Exploiting cancer vulnerabilities is critical for the discovery of anticancer drugs. However, tumor suppressors cannot be directly targeted because of their loss of function. To uncover specific vulnerabilities for cells with deficiency in any given tumor suppressor(s), we performed genome-scale CRISPR loss-of-function screens using a panel of isogenic knockout cells we generated for 12 common tumor suppressors. Here, we provide a comprehensive and comparative dataset for genetic interactions between the whole-genome protein-coding genes and a panel of tumor suppressor genes, which allows us to uncover known and new high-confidence synthetic lethal interactions. Mining this dataset, we uncover essential paralog gene pairs, which could be a common mechanism for interpreting synthetic lethality. Moreover, we propose that some tumor suppressors could be targeted to suppress proliferation of cells with deficiency in other tumor suppressors. This dataset provides valuable information that can be further exploited for targeted cancer therapy.

Histone chaperone ASF1 acts with RIF1 to promote DNA end joining in BRCA1-deficient cells.

Replication timing regulatory factor 1 (RIF1) acts downstream of p53-binding protein 53BP1 to inhibit the resection of DNA broken ends, which plays critical roles in determining the DNA double-strand break repair pathway choice between nonhomologous end joining and homologous recombination (HR). However, the mechanism by which this choice is made is not yet clear. In this study, we identified that histone chaperone protein ASF1 associates with RIF1 and regulates RIF1-dependent functions in the DNA damage response. Similar to loss of RIF1, we found that loss of ASF1 resulted in resistance to poly (ADP-ribose) polymerase (PARP) inhibition in BRCA1-deficient cells with restored HR and decreased telomere fusion in telomeric repeat-binding protein 2 (TRF2)-depleted cells. Moreover, we showed that these functions of ASF1 are dependent on its interaction with RIF1 but not on its histone chaperone activity. Thus, our study supports a new role for ASF1 in dictating double-strand break repair choice. Considering that the status of 53BP1-RIF1 axis is important in determining the outcome of PARP inhibitor-based therapy in BRCA1- or HR-deficient cancers, the identification of ASF1 function in this critical pathway uncovers an interesting connection between these S-phase events, which may reveal new strategies to overcome PARP inhibitor resistance.

Low-density-lipoprotein-receptor-related protein 1 mediates Notch pathway activation.

The Notch signaling pathway controls cell growth, differentiation, and fate decisions, and its dysregulation has been linked to various human genetic disorders and cancers. To comprehensively understand the global organization of the Notch pathway and identify potential drug targets for Notch-related diseases, we established a protein interaction landscape for the human Notch pathway. By combining and analyzing genetic and phenotypic data with bioinformatics analysis, we greatly expanded this pathway and identified many key regulators, including low-density-lipoprotein-receptor-related protein 1 (LRP1). We demonstrated that LRP1 mediates the ubiquitination chain linkage switching of Delta ligands, which further affects ligand recycling, membrane localization, and stability. LRP1 inhibition led to Notch signaling inhibition and decreased tumorigenesis in leukemia models. Our study provides a glimpse into the Notch pathway interaction network and uncovers LRP1 as one critical regulator of the Notch pathway, as well as a possible therapeutic target for Notch-related cancers.

Genome-wide CRISPR screens reveal cyclin C as synthetic survival target of BRCA2.

Poly (ADP-ribose) polymerase inhibitor (PARPi)-based therapies initially reduce tumor burden but eventually lead to acquired resistance in cancer patients with BRCA1 or BRCA2 mutation. To understand the potential PARPi resistance mechanisms, we performed whole-genome CRISPR screens to discover genetic alterations that change the gene essentiality in cells with inducible depletion of BRCA2. We identified that several RNA Polymerase II transcription Mediator complex components, especially Cyclin C (CCNC) as synthetic survival targets upon BRCA2 loss. Total mRNA sequencing demonstrated that loss of CCNC could activate the transforming growth factor (TGF)-beta signaling pathway and extracellular matrix (ECM)-receptor interaction pathway, however the inhibition of these pathways could not reverse cell survival in BRCA2 depleted CCNC-knockout cells, indicating that the activation of these pathways is not required for the resistance. Moreover, we showed that the improved survival is not due to restoration of homologous recombination repair although decreased DNA damage signaling was observed. Interestingly, loss of CCNC could restore replication fork stability in BRCA2 deficient cells, which may contribute to PARPi resistance. Taken together, our data reveal CCNC as a critical genetic determinant upon BRCA2 loss of function, which may help the development of novel therapeutic strategies that overcome PARPi resistance.

Interactomes of SARS-CoV-2 and human coronaviruses reveal host factors potentially affecting pathogenesis.

Host-virus protein-protein interactions play key roles in the life cycle of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We conducted a comprehensive interactome study between the virus and host cells using tandem affinity purification and proximity-labeling strategies and identified 437 human proteins as the high-confidence interacting proteins. Further characterization of these interactions and comparison to other large-scale study of cellular responses to SARS-CoV-2 infection elucidated how distinct SARS-CoV-2 viral proteins participate in its life cycle. With these data mining, we discovered potential drug targets for the treatment of COVID-19. The interactomes of two key SARS-CoV-2-encoded viral proteins, NSP1 and N, were compared with the interactomes of their counterparts in other human coronaviruses. These comparisons not only revealed common host pathways these viruses manipulate for their survival, but also showed divergent protein-protein interactions that may explain differences in disease pathology. This comprehensive interactome of SARS-CoV-2 provides valuable resources for the understanding and treating of this disease.

Genetic vulnerabilities upon inhibition of DNA damage response.

Because of essential roles of DNA damage response (DDR) in the maintenance of genomic integrity, cellular homeostasis, and tumor suppression, targeting DDR has become a promising therapeutic strategy for cancer treatment. However, the benefits of cancer therapy targeting DDR are limited mainly due to the lack of predictive biomarkers. To address this challenge, we performed CRISPR screens to search for genetic vulnerabilities that affect cells' response to DDR inhibition. By undertaking CRISPR screens with inhibitors targeting key DDR mediators, i.e. ATR, ATM, DNAPK and CHK1, we obtained a global and unbiased view of genetic interactions with DDR inhibition. Specifically, we identified YWHAE loss as a key determinant of sensitivity to CHK1 inhibition. We showed that KLHL15 loss protects cells from DNA damage induced by ATM inhibition. Moreover, we validated that APEX1 loss sensitizes cells to DNAPK inhibition. Additionally, we compared the synergistic effects of combining different DDR inhibitors and found that an ATM inhibitor plus a PARP inhibitor induced dramatic levels of cell death, probably through promoting apoptosis. Our results enhance the understanding of DDR pathways and will facilitate the use of DDR-targeting agents in cancer therapy.

Ubiquitylation in DNA double-strand break repair.

Genome integrity is constantly challenged by various DNA lesions with DNA double-strand breaks (DSBs) as the most cytotoxic lesions. In order to faithfully repair DSBs, DNA damage response (DDR) signaling networks have evolved, which organize many multi-protein complexes to deal with the encountered DNA damage. Spatiotemporal dynamics of these protein complexes at DSBs are mainly modulated by post-translational modifications (PTMs). One of the most well-studied PTMs in DDR is ubiquitylation which can orchestrate cellular responses to DSBs, promote accurate DNA repair, and maintain genome integrity. Here, we summarize the recent advances of ubiquitin-dependent signaling in DDR and discuss how ubiquitylation crosstalks with other PTMs to control fundamental biological processes in DSB repair.

Extracellular signal-regulated kinases associate with and phosphorylate DHPS to promote cell proliferation.

The ERK1/2 pathway is one of the most commonly dysregulated pathways in human cancers and controls many vital cellular processes. Although many ERK1/2 kinase substrates have been identified, the diversity of ERK1/2 mediated processes suggests the existence of additional targets. Here, we identified Deoxyhypusine synthase (DHPS), an essential hypusination enzyme regulating protein translation, as a major and direct-binding protein of ERK1/2. Further experiments showed that ERK1/2 phosphorylate DHPS at Ser-233 site. The Ser-233 phosphorylation of DHPS by ERK1/2 is important for its function in cell proliferation. Moreover, we found that higher DHPS expression correlated with poor prognosis in lung adenocarcinoma and increased resistance to inhibitors of the ERK1/2 pathway. In summary, our results suggest that ERK1/2-mediated DHPS phosphorylation is an important mechanism that underlies protein translation and that DHPS expression is a potent biomarker of response to therapies targeting ERK1/2-pathway.

Proteome-wide Analysis Reveals Substrates of E3 Ligase RNF146 Targeted for Degradation.

Specific E3 ligases target tumor suppressors for degradation. Inhibition of such E3 ligases may be an important approach to cancer treatment. RNF146 is a RING domain and PARylation-dependent E3 ligase that functions as an activator of the ß-catenin/Wnt and YAP/Hippo pathways by targeting the degradation of several tumor suppressors. Tankyrases 1 and 2 (TNKS1/2) are the only known poly-ADP-ribosyltransferases that require RNF146 to degrade their substrates. However, systematic identification of RNF146 substrates have not yet been performed. To uncover substrates of RNF146 that are targeted for degradation, we generated RNF146 knockout cells and TNKS1/2-double knockout cells and performed proteome profiling with label-free quantification as well as transcriptome analysis. We identified 160 potential substrates of RNF146, which included many known substrates of RNF146 and TNKS1/2 and 122 potential TNKS-independent substrates of RNF146. In addition, we validated OTU domain-containing protein 5 and Protein mono-ADP-ribosyltransferase PARP10 as TNKS1/2-independent substrates of RNF146 and SARDH as a novel substrate of TNKS1/2 and RNF146. Our study is the first proteome-wide analysis of potential RNF146 substrates. Together, these findings not only demonstrate that proteome profiling can be a useful general approach for the systemic identification of substrates of E3 ligases but also reveal new substrates of RNF146, which provides a resource for further functional studies.

C17orf53 is identified as a novel gene involved in inter-strand crosslink repair.

Ataxia Telangiectasia and Rad3-Related kinase (ATR) is a master regulator of genome maintenance, and participates in DNA replication and various DNA repair pathways. In a genome-wide screen for ATR-dependent fitness genes, we identified a previously uncharacterized gene, C17orf53, whose loss led to hypersensitivity to ATR inhibition. C17orf53 is conserved in vertebrates and is required for efficient cell proliferation. Loss of C17orf53 slowed down DNA replication and led to pronounced interstrand crosslink (ICL) repair defect. We showed that C17orf53 is a ssDNA- and RPA-binding protein and both characteristics are important for its functions in the cell. In addition, using multiple omics methods, we found that C17orf53 works with MCM8/9 to promote cell survival in response to ICL lesions. Taken together, our data suggest that C17orf53 is a novel component involved in ICL repair pathway.