Lisa Hoeg

Approximately 10% of bladder, upper urinary tract, and endometrial cancers harbor deleterious somatic alterations in nucleotide excision repair (NER) pathway genes. Although platinum-based chemotherapy is the standard treatment for NER-deficient tumors, its utility is limited by nephrotoxicity, motivating alternative strategies that avoid direct DNA targeting. Here, we employed a genome-wide CRISPR-Cas9 screen to identify synthetic lethal vulnerabilities in NER-deficient cancer cells. An ERCC4 -null RT112/84 bladder cancer cell line was generated and validated by Sanger sequencing and Western blotting, and functional NER deficiency was confirmed using a host cell reactivation assay. Parallel screens in ERCC4 -deficient and isogenic wild-type cells. nominated SLC25A28 as the top-ranked synthetic lethal candidate in ERCC4 -deficient cells, an interaction further supported by DepMap dependency. Clonogenic assays confirmed that loss of SLC25A28 selectively impaired cell viability in cells deficient for ERCC2 , ERCC3 , ERCC4 , or ERCC5 and inducible knockout selectively suppressed ERCC4 -deficient xenograft growth in-viv o. Mechanistically, RNA sequencing followed by gene set enrichment analysis revealed enrichment of oxidative stress-related pathways, including cellular responses to reactive oxygen species (ROS) and DNA damage signatures, upon SLC25A28 loss. These transcriptional changes are consistent with increased oxidative stress and DNA damage, placing heightened demand on NER-mediated repair. Direct measurement confirmed increased intracellular ROS. Collectively, our nominates SLC25A28 as a previously unrecognized synthetic lethal vulnerability in NER-deficient cancers and implicate mitochondrial iron homeostasis as a candidate metabolic dependency that may be targeted independently of platinum-based chemotherapy. Significance: This study identifies SLC25A28 as a synthetic lethal vulnerability in nucleotide excision repair–deficient cancers, linking mitochondrial iron homeostasis and oxidative stress to selective DNA damage intolerance and therapeutic opportunity.

Summary: Incomplete DNA replication and chromosome breakage during mitosis pose major threats to chromosome segregation. The CIP2A-TOPBP1 complex acts to mitigate this peril, but its exact role is not yet understood. Here, we report that DDIAS acts as a DNA-binding effector of the CIP2A-TOPBP1 complex. DDIAS directly interacts with TOPBP1 and the disruption of this interaction or inactivation of its single-stranded DNA (ssDNA)-binding ability impairs genome integrity and causes synthetic lethality with BRCA1 and BRCA2 deficiency. DDIAS does not promote the putative end-tethering function of CIP2A-TOPBP1 but rather acts to suppress ssDNA during mitosis. This role is emphasized by a pronounced genetic interaction between the genes coding for DDIAS and DNA polymerase (. We conclude that DDIAS defines a mitotic DNA damage response that mitigates the threat of mitotic ssDNA arising from DNA replication stress, which we infer is a liability to accurate chromosome segregation. (144 words).

Tumor suppressor p53 (TP53) is frequently mutated in cancer, often resulting not only in loss of its tumor-suppressive function but also acquisition of dominant-negative and even oncogenic gain-of-function traits. While wild-type p53 levels are tightly regulated, mutants are typically stabilized in tumors, which is crucial for their oncogenic properties. Here, we systematically profiled the factors that regulate protein stability of wild-type and mutant p53 using marker-based genome-wide CRISPR screens. Most regulators of wild-type p53 also regulate p53 mutants, except for p53 R337H regulators, which are largely private to this mutant. Mechanistically, FBXO42 emerged as a positive regulator for a subset of p53 mutants, working with CCDC6 to control USP28-mediated mutant p53 stabilization. Additionally, C16orf72/HAPSTR1 negatively regulates both wild-type p53 and all tested mutants. C16orf72/HAPSTR1 is commonly amplified in breast cancer, and its overexpression reduces p53 levels in mouse mammary epithelium leading to accelerated breast cancer. This study offers a network perspective on p53 stability regulation, potentially guiding strategies to reinforce wild-type p53 or target mutant p53 in cancer.

The post-translational modification of proteins by SUMO is crucial for cellular viability and mammalian development in part due to the contribution of SUMOylation to genome duplication and repair. To investigate the mechanisms underpinning the essential function of SUMO, we undertook a genome-scale CRISPR/Cas9 screen probing the response to SUMOylation inhibition. This effort identified 130 genes whose disruption reduces or enhances the toxicity of TAK-981, a clinical-stage inhibitor of the SUMO E1-activating enzyme. Among the strongest hits, we validated and characterized NFATC2IP, an evolutionarily conserved protein related to the fungal Esc2 and Rad60 proteins that harbors tandem SUMO-like domains. Cells lacking NFATC2IP are viable but are hypersensitive to SUMO E1 inhibition, likely due to the accumulation of mitotic chromosome bridges and micronuclei. NFATC2IP primarily acts in interphase and associates with nascent DNA, suggesting a role in the postreplicative resolution of replication or recombination intermediates. Mechanistically, NFATC2IP interacts with the SMC5/6 complex and UBC9, the SUMO E2, via its first and second SUMO-like domains, respectively. AlphaFold-Multimer modeling suggests that NFATC2IP positions and activates the UBC9–NSMCE2 complex, the SUMO E3 ligase associated with SMC5/SMC6. We conclude that NFATC2IP is a key mediator of SUMO-dependent genomic integrity that collaborates with the SMC5/6 complex.

DNA replication stress is a threat to genome integrity. The large SNF2-family of ATPases participates in preventing and mitigating DNA replication stress by employing their ATP-driven motor to remodel DNA or DNA-bound proteins. To understand the contribution of these ATPases in genome maintenance, we undertook CRISPR-based synthetic lethality screens with three SNF2-type ATPases: SMARCAL1, ZRANB3 and HLTF. Here we show that SMARCAL1 displays a profound synthetic lethal interaction with FANCM , another ATP-dependent translocase involved in DNA replication and genome stability. Their combined loss causes severe genome instability that we link to chromosome breakage at loci enriched in simple repeats, which are known to challenge replication fork progression. Our findings illuminate a critical genetic buffering mechanism that provides an essential function for maintaining genome integrity.

The post-translational modification of proteins by ubiquitin and ubiquitin-like polypeptides controls multiple cellular processes including the abundance of a large fraction of the proteome. We applied genome-scale CRISPR/Cas9 screens to elucidate the genetic architecture of the response to inhibition of ubiquitin, NEDD8 and SUMO conjugation pathways as well as inhibition of the p97/VCP segregase. This effort identified 395 genes whose disruption alters the fitness of human cells when faced with perturbations in these pathways. We validated that the TMED2 and TMED10 proteins, which are localized to the secretory pathway, promote resistance to p97/VCP inhibition and also characterized NFATC2IP, an evolutionarily conserved protein harboring SUMO-like domains as a major player in promoting genomic integrity when SUMOylation is inhibited. We propose that NFATC2IP acts in interphase cells to promote the SUMO-dependent E3 ligase activity of the SMC5/SMC6 complex, which is critical for SUMO-dependent genome integrity.

53BP1 is a chromatin-binding protein that promotes DNA double-strand break repair through the recruitment of downstream effectors including RIF1, shieldin, and CST. The structural basis of the protein-protein interactions within the 53BP1-RIF1-shieldin-CST pathway that are essential for its DNA repair activity is largely unknown. Here, we used AlphaFold2-Multimer (AF2) to predict all possible pairwise combinations of proteins within this pathway and provide structural models of seven previously characterized interactions. This analysis also predicted an entirely novel binding interface between the HEAT-repeat domain of RIF1 and the eIF4E-like domain of SHLD3. Extensive interrogation of this interface through both in vitro pulldown analysis and cellular assays supports the AF2-predicted model and demonstrates that RIF1-SHLD3 binding is essential for shieldin recruitment to sites of DNA damage, and for its role in antibody class switch recombination and PARP inhibitor sensitivity. Direct physical interaction between RIF1 and SHLD3 is therefore essential for 53BP1-RIF1-shieldin-CST pathway activity.

53BP1 is a chromatin-binding DNA repair protein that promotes DNA double-strand break repair through recruitment of downstream effectors including RIF1, shieldin, and CST. The structural basis of the protein-protein interactions within the 53BP1-RIF1-shieldin-CST pathway that are essential for its DNA repair activity are largely unknown. Here we used AlphaFold2-Multimer (AF2) to predict all possible pairwise combinations of proteins within this pathway and provide structural models of seven previously characterized interactions. This analysis also predicted an entirely novel binding interface between the HEAT-repeat domain of RIF1 and the eIF4E-like domain of SHLD3. Extensive interrogation of this interface through both in vitro pulldown analysis and cellular assays supports the AF2-predicted model and demonstrates that RIF1-SHLD3 binding is essential for shieldin recruitment to sites of DNA damage, and for its role in antibody class switch recombination. Direct physical interaction between RIF1 and SHLD3 is therefore essential for 53BP1-RIF1-shieldin-CST pathway activity.