Systematic investigation of BRCA1-A, -B, and -C complexes and their functions in DNA damage response and DNA repair.

BRCA1, a breast cancer susceptibility gene, has emerged as a central mediator that brings together multiple signaling complexes in response to DNA damage. The A, B, and C complexes of BRCA1, which are formed based on their phosphorylation-dependent interactions with the BRCA1-C-terminal domains, contribute to the roles of BRCA1 in DNA repair and cell cycle checkpoint control. However, their functions in DNA damage response remain to be fully appreciated. Specifically, there has been no systematic investigation of the roles of BRCA1-A, -B, and -C complexes in the regulation of BRCA1 localization and functions, in part because of cellular lethality associated with loss of CtIP protein, which is an essential component in BRCA1-C complex. To systematically investigate the functions of these complexes in DNA damage response, we depleted a key component in each of these complexes. We used the degradation tag system to inducibly deplete endogenous CtIP and obtained a series of RAP80/FANCJ/CtIP single-, double-, and triple-knockout cells. We showed that loss of BRCA1-B/FANCJ and BRCA1-C/CtIP, but not BRCA1-A/RAP80, resulted in reduced cell proliferation and increased sensitivity to DNA damage. BRCA1-C/CtIP and BRCA1-A/RAP80 were involved in BRCA1 recruitment to sites of DNA damage. However, BRCA1-A/RAP80 was not essential for damage-induced BRCA1 localization. Instead, RAP80/H2AX and CtIP have redundant roles in BRCA1 recruitment. Altogether, our systematic analysis uncovers functional differences between BRCA1-A, -B, and -C complexes and provides new insights into the roles of these BRCA1-associated protein complexes in DNA damage response and DNA repair.

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.

Proteomic analysis of ferroptosis pathways reveals a role of CEPT1 in suppressing ferroptosis.

Ferroptosis has been recognized as a unique cell death modality driven by excessive lipid peroxidation and unbalanced cellular metabolism. In this study, we established a protein interaction landscape for ferroptosis pathways through proteomic analyses, and identified choline/ethanolamine phosphotransferase 1 (CEPT1) as a lysophosphatidylcholine acyltransferase 3 (LPCAT3)-interacting protein that regulates LPCAT3 protein stability. In contrast to its known role in promoting phospholipid synthesis, we showed that CEPT1 suppresses ferroptosis potentially by interacting with phospholipases and breaking down certain pro-ferroptotic polyunsaturated fatty acid (PUFA)-containing phospholipids. Together, our study reveals a previously unrecognized role of CEPT1 in suppressing ferroptosis.

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.

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.

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.

Actin cytoskeleton vulnerability to disulfide stress mediates disulfidptosis.

SLC7A11-mediated cystine uptake suppresses ferroptosis yet promotes cell death under glucose starvation; the nature of the latter cell death remains unknown. Here we show that aberrant accumulation of intracellular disulfides in SLC7A11high cells under glucose starvation induces a previously uncharacterized form of cell death distinct from apoptosis and ferroptosis. We term this cell death disulfidptosis. Chemical proteomics and cell biological analyses showed that glucose starvation in SLC7A11high cells induces aberrant disulfide bonds in actin cytoskeleton proteins and F-actin collapse in a SLC7A11-dependent manner. CRISPR screens and functional studies revealed that inactivation of the WAVE regulatory complex (which promotes actin polymerization and lamellipodia formation) suppresses disulfidptosis, whereas constitutive activation of Rac promotes disulfidptosis. We further show that glucose transporter inhibitors induce disulfidptosis in SLC7A11high cancer cells and suppress SLC7A11high tumour growth. Our results reveal that the susceptibility of the actin cytoskeleton to disulfide stress mediates disulfidptosis and suggest a therapeutic strategy to target disulfidptosis in cancer treatment.

Discovery of toxoflavin, a potent IRE1α inhibitor acting through structure-dependent oxidative inhibition.

Inositol-requiring enzyme 1α (IRE1α) is the most conserved endoplasmic reticulum (ER) stress sensor with two catalytic domains, kinase and RNase, in its cytosolic portion. IRE1α inhibitors have been used to improve existing clinical treatments against various cancers. In this study we identified toxoflavin (TXF) as a new-type potent small molecule IRE1α inhibitor. We used luciferase reporter systems to screen compounds that inhibited the IRE1α-XBP1s signaling pathway. As a result, TXF was found to be the most potent IRE1α RNase inhibitor with an IC50 value of 0.226 µM. Its inhibitory potencies on IRE1α kinase and RNase were confirmed in a series of cellular and in vitro biochemical assays. Kinetic analysis showed that TXF caused time- and reducing reagent-dependent irreversible inhibition on IRE1α, implying that ROS might participate in the inhibition process. ROS scavengers decreased the inhibition of IRE1α by TXF, confirming that ROS mediated the inhibition process. Mass spectrometry analysis revealed that the thiol groups of four conserved cysteine residues (CYS-605, CYS-630, CYS-715 and CYS-951) in IRE1α were oxidized to sulfonic groups by ROS. In molecular docking experiments we affirmed the binding of TXF with IRE1α, and predicted its binding site, suggesting that the structure of TXF itself participates in the inhibition of IRE1α. Interestingly, CYS-951 was just near the docked site. In addition, the RNase IC50 and ROS production in vitro induced by TXF and its derivatives were negative correlated (r = -0.872). In conclusion, this study discovers a new type of IRE1α inhibitor that targets a predicted new alternative site located in the junction between RNase domain and kinase domain, and oxidizes conserved cysteine residues of IRE1α active sites to inhibit IRE1α. TXF could be used as a small molecule tool to study IRE1α's role in ER stress.

USP7 substrates identified by proteomics analysis reveal the specificity of USP7.

Deubiquitylating enzymes (DUBs) remove ubiquitin chains from proteins and regulate protein stability and function. USP7 is one of the most extensively studied DUBs, since USP7 has several well-known substrates important for cancer progression, such as MDM2, N-MYC, and PTEN. Thus, USP7 is a promising drug target. However, systematic identification of USP7 substrates has not yet been performed. In this study, we carried out proteome profiling with label-free quantification in control and single/double-KO cells of USP7and its closest homolog, USP47 Our proteome profiling for the first time revealed the proteome changes caused by USP7 and/or USP47 depletion. Combining protein profiling, transcriptome analysis, and tandem affinity purification of USP7-associated proteins, we compiled a list of 20 high-confidence USP7 substrates that includes known and novel USP7 substrates. We experimentally validated MGA and PHIP as new substrates of USP7. We further showed that MGA deletion reduced cell proliferation, similar to what was observed in cells with USP7 deletion. In conclusion, our proteome-wide analysis uncovered potential USP7 substrates, providing a resource for further functional studies.

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.

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.

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.

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.

Non-canonical function of DGCR8 in DNA double-strand break repair signaling and tumor radioresistance.

In response to DNA double-strand breaks (DSBs), repair proteins are recruited to the damaged sites. Ubiquitin signaling plays a critical role in coordinating protein recruitment during the DNA damage response. Here, we find that the microRNA biogenesis factor DGCR8 promotes tumor resistance to X-ray radiation independently of its Drosha-binding ability. Upon radiation, the kinase ATM and the deubiquitinase USP51 mediate the activation and stabilization of DGCR8 through phosphorylation and deubiquitination. Specifically, radiation-induced ATM-dependent phosphorylation of DGCR8 at serine 677 facilitates USP51 to bind, deubiquitinate, and stabilize DGCR8, which leads to the recruitment of DGCR8 and DGCR8's binding partner RNF168 to MDC1 and RNF8 at DSBs. This, in turn, promotes ubiquitination of histone H2A, repair of DSBs, and radioresistance. Altogether, these findings reveal the non-canonical function of DGCR8 in DSB repair and suggest that radiation treatment may result in therapy-induced tumor radioresistance through ATM- and USP51-mediated activation and upregulation of DGCR8.

mTORC1 couples cyst(e)ine availability with GPX4 protein synthesis and ferroptosis regulation.

Glutathione peroxidase 4 (GPX4) utilizes glutathione (GSH) to detoxify lipid peroxidation and plays an essential role in inhibiting ferroptosis. As a selenoprotein, GPX4 protein synthesis is highly inefficient and energetically costly. How cells coordinate GPX4 synthesis with nutrient availability remains unclear. In this study, we perform integrated proteomic and functional analyses to reveal that SLC7A11-mediated cystine uptake promotes not only GSH synthesis, but also GPX4 protein synthesis. Mechanistically, we find that cyst(e)ine activates mechanistic/mammalian target of rapamycin complex 1 (mTORC1) and promotes GPX4 protein synthesis at least partly through the Rag-mTORC1-4EBP signaling axis. We show that pharmacologic inhibition of mTORC1 decreases GPX4 protein levels, sensitizes cancer cells to ferroptosis, and synergizes with ferroptosis inducers to suppress patient-derived xenograft tumor growth in vivo. Together, our results reveal a regulatory mechanism to coordinate GPX4 protein synthesis with cyst(e)ine availability and suggest using combinatorial therapy of mTORC1 inhibitors and ferroptosis inducers in cancer treatment.

Beyond proteins: Ubiquitylation of lipopolysaccharide to fight bacteria.

Mechanisms and functions of protein ubiquitylation and LPS ubiquitylation. LPS ubiquitylation serves as a scaffold to recruit E3 ligases for the ubiquitylation of the membrane of bacteria. Polyubiquitin coat on the bacterial cell surface is one type of "eat-me" signal recognized by the host cells. The abbreviations used in the figure are Pro, Protein; Ub, Ubiquitin; E1, Ubiquitin-activating enzymes; E2, Ubiquitin-conjugating enzymes; E3, Ubiquitin-ligating enzymes; LPS, lipopolysaccharide; RNF213, Ring Finger Protein 213; and LUBAC, linear ubiquitin chain assembly complex.

Selective Labeling and Identification of the Tumor Cell Proteome of Pancreatic Cancer In Vivo.

Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest cancers. Dissecting the tumor cell proteome from that of the non-tumor cells in the PDAC tumor bulk is critical for tumorigenesis studies, biomarker discovery, and development of therapeutics. However, investigating the tumor cell proteome has proven evasive due to the tumor's extremely complex cellular composition. To circumvent this technical barrier, we have combined bioorthogonal noncanonical amino acid tagging (BONCAT) and data-independent acquisition mass spectrometry (DIA-MS) in an orthotopic PDAC model to specifically identify the tumor cell proteome in vivo. Utilizing the tumor cell-specific expression of a mutant tRNA synthetase transgene, this approach provides tumor cells with the exclusive ability to incorporate an azide-bearing methionine analogue into newly synthesized proteins. The azide-tagged tumor cell proteome is subsequently enriched and purified via a bioorthogonal reaction and then identified and quantified using DIA-MS. Applying this workflow to the orthotopic PDAC model, we have identified thousands of proteins expressed by the tumor cells. Furthermore, by comparing the tumor cell and tumor bulk proteomes, we showed that the approach can distinctly differentiate proteins produced by tumor cells from those of non-tumor cells within the tumor microenvironment. Our study, for the first time, reveals the tumor cell proteome of PDAC under physiological conditions, providing broad applications for tumorigenesis, therapeutics, and biomarker studies in various human cancers.