BackgroundThe DNA damage response (DDR) safeguards genome integrity, and its disruption contributes to cancer development, therapy response, and resistance. Large-scale sequencing has identified thousands of DDR gene variants in tumors, but the functional consequences of most remain unclear, limiting their clinical interpretation and application.ResultsWe previously developed CRISPR-dependent base editing screens to functionally characterize DDR variants in breast-derived cell lines. Here, we extend this work to triple-negative breast cancer by performing a large-scale base editing screen in MDA-MB-231 cells. We assessed the impact on cellular fitness of ∼11,000 single-guide RNAs (sgRNAs) targeting the coding sequences of 27 DDR genes, primarily involved in homologous recombination (HR) and inter-strand crosslink repair (ICLR). The resulting dataset integrates mutation-associated effects with clinical annotations, enabling functional stratification of variants of uncertain significance.ConclusionCombined with our previous datasets from MCF7 and MCF10A breast-derived cell lines, these results create a standardized, cross-comparable data that uncover both shared and context-specific genetic dependencies. Ultimately, we anticipate this resource will advance the functional interpretation of DDR variants, thereby facilitating the development of precision oncology approaches.

CRISPR-based high-throughput mutagenesis screens enable systematic mapping of mutations to phenotypes, yet deciphering mutation-phenotype links remains challenging. Here, we present ProTiler-Mut, a versatile computational framework that leverages tiling mutagenesis screens, which introduce variants across entire protein sequences, to analyze mutation effects at the levels of residues, substructures, and protein-protein interactions (PPIs). Applying ProTiler-Mut to multi-condition base-editing (BE) screens targeting DNA damage response proteins and T cell regulators, we define a separation-of-function (SoF) category beyond the conventional loss-of-function (LoF) and gain-of-function (GoF) classes, where SoF mutations show the strongest enrichment for ClinVar-annotated pathogenic variants. ProTiler-Mut also identifies candidate substructures that enable functional inference of unscreened pathogenic mutations and prioritizes candidate phenotype-associated PPIs potentially disrupted by functional variants. Using ProTiler-Mut, in cells with elevated programmed cell death 1 (PD-1) expression, we identify pathogenic GoF mutations that constitute a substructure that may disrupt mitogen-activated protein kinase (MAPK)1-RSK1 interactions and lead to MAPK activation. Finally, we show that ProTiler-Mut is applicable across different mutagenesis screening platforms. A record of this paper’s transparent peer review process is included in the supplemental information.

Taxane-based chemotherapy is a main treatment modality for ovarian cancer and other solid tumors, but chemoresistance limits the clinical efficacy. Studies have shown tumor interaction with macrophages in the tumor microenvironment (TME) plays a significant role in taxane resistance, yet the underlying molecular mechanisms are poorly understood. In this study, we employed translatome profiling of paclitaxel-treated cancer cells, live-cell imaging analysis, gene knockdown/knockout, and in vitro cancer-macrophage coculture assays to unravel a novel chemoresistance mechanism mediated by tumor-macrophage interaction via the NOTCH2-JAG1 axis. The in vitro data were further validated by multiple xenograft, syngeneic and patient-derived xenograft mouse tumor models of ovarian cancer as well as ovarian cancer patient samples. We found paclitaxel selectively induced translational upregulation of NOTCH2 via cytoplasmic polyadenylation, and this NOTCH2 upregulation persisted after mitotic exit. Subsequent NOTCH2 activation by JAG1 expressed mainly on the neighboring macrophages promoted tumor cell survival and simulated cytokine release, such as CSF1 and IL-1β, that recruited JAG1-expressing macrophages, thus forming a positive feedback loop that further enhanced the pro-tumor NOTCH2 activity. Genetic depletion or pharmacological inhibition of NOTCH2 with the γ-secretase inhibitor attenuated macrophage infiltration and sensitized tumor response to paclitaxel in multiple preclinical models of ovarian cancer. Moreover, single-cell RNA sequencing analysis identified a JAG1-high macrophage subset that was enriched by paclitaxel treatment and attenuated by NOTCH inhibition. Clinically, high NOTCH2 expression in ovarian tumors was associated with recurrence and shorter progression-free survival of ovarian cancer patients. Paclitaxel-induced translational upregulation of NOTCH2 enables immediate juxtacrine activation by JAG1-positive macrophages, coupling tumor cell survival with immune remodeling in the tumor microenvironment to drive chemoresistance. Our results suggest NOTCH2 is a viable biomarker for paclitaxel resistance and that combining NOTCH2 inhibitor with taxane is an effective therapeutic strategy to selectively disrupt tumor-macrophage interaction and overcome macrophage-mediated taxane resistance in NOTCH2-positive tumors.

Protein arginine methyltransferase 5 (PRMT5) is a synthetic lethal target in methylthioadenosine phosphorylase-deleted (MTAP-null) cancers. Second-generation methylthioadenosine (MTA)-cooperative PRMT5 inhibitors preferentially target MTAP-null cells while largely sparing MTAP-wildtype (MTAP-WT) cells, thereby improving tumor selectivity over first-generation PRMT5 inhibitors. Despite encouraging efficacy and safety signals in early clinical studies, the modest objective response rates (ORRs) observed with these inhibitors suggest that intrinsic or acquired resistance may limit their clinical benefit. Here, we investigated acquired resistance to the MTA-cooperative PRMT5 inhibitor BMS-986504/MRTX1719 in MTAP-null non-small cell lung cancer (NSCLC) cells and sought to identify therapeutic vulnerabilities that emerge upon resistance. Using multiple in vitro-derived resistant models, we found that acquired resistance was accompanied by cross-resistance to mechanistically distinct PRMT5 inhibitors. Notably, this phenotype was not fully explained by altered PRMT5 activity or changes in MTA levels. High-throughput drug screening of paired sensitive and resistant cells revealed increased sensitivity to MEK inhibitors following acquisition of MRTX1719 resistance in KRAS-wildtype NSCLC cells. Consistently, resistant cells exhibited rewired MAPK-related transcriptional programs. Together, these findings identify MEK inhibition as a reproducible collateral vulnerability associated with acquired MRTX1719 resistance in MTAP-null NSCLC models and support further evaluation of MEK inhibition as a potential treatment-switching strategy following resistance.

BackgroundCD47 is a key innate immune checkpoint that enables tumor cells to evade macrophage-mediated clearance.Methods/ResultsTo systematically identify genetic regulators of CD47 surface expression, we performed FACS-based genome-wide CRISPR screens in three murine cancer cell lines B16 (melanoma), MC38 (colon adenocarcinoma), and EMT6 (breast carcinoma). ResultsComparative analysis of cells with high or low CD47 surface expression using DrugZ revealed CD47 itself as the top hit, validating the screens. Notably, DNAJC13 emerged as a consistent and robust regulator of CD47 expression across all three cell lines. Functional validation using DNAJC13-knockout cells confirmed a significant reduction in CD47 surface levels. Furthermore, in co-culture assays with macrophages, DNAJC13-deficient tumor cells exhibited increased susceptibility to phagocytosis, supporting a functional role for DNAJC13 in innate immune evasion. Finally, we verify that DNAJC13-knockout decrease tumor burden when treated with CD47 blockade.ConclusionsOverall, this study highlights a previously unrecognized regulator of CD47 and demonstrates the utility of high-throughput FACS-based CRISPR screening to uncover modulators of key immune checkpoint pathways.

High-throughput mutagenesis screens are powerful tools for mapping mutations to phenotypes. However, deciphering the molecular mechanisms that link mutations to phenotypic outcomes remains a significant challenge. Here, we present ProTiler-Mut, a versatile computational framework that harnesses tiling mutagenesis screens, which introduce variants across entire protein sequences, to facilitate investigation of mutation-to-phenotype associations at multiple levels, including individual residues, protein substructures, and protein-protein interactions (PPIs). As demonstrated through our analyses of base editing (BE) screens targeting DNA Damage Response (DDR) proteins and T cell regulators, ProTiler-Mut provides novel insights into the mutation-phenotype linkages, including: i) refined classification of mutation that reveals separation-of-function (SOF) category beyond the conventional binary classification of loss-of-function (LOF) and gain-of-function (GOF); ii) definition of phenotype-associated hotspot substructures that enable the inference of the function of unscreened pathogenic mutations; and iii) identification of phenotype-associated PPIs disrupted by functional mutations. Through ProTiler-Mut analyses, we identified a substructure harboring pathogenic GOF mutations that disrupt interactions between the kinases MAPK1 and RSK1, leading to MAPK1 activation and elevated expression of the immune checkpoint receptor PD-1. Furthermore, we demonstrate the applicability of ProTiler-Mut to various mutagenesis screening platforms, highlighting its broad utility and generalizability. ### Competing Interest Statement The authors have declared no competing interest. NIH, , R35GM137927, R35GM153387, R01CA197774 John S. Dunn Foundation, https://ror.org/03yhprz62,

Aging is a complex biological process that involves multi-level structural and physiological changes. Aging is a major risk factor for many chronic diseases. The accumulation of senescent cells changes the tissue microenvironment and is closely associated with the occurrence and development of tissue and organ fibrosis. Fibrosis is the result of dysregulated tissue repair response in the development of chronic inflammatory diseases. Recent studies have clearly indicated that SIRT2 is involved in regulating the progression of fibrosis, making it a potential target for anti-fibrotic drugs. SIRT2 is a NAD+ dependent histone deacetylase, shuttling between nucleus and cytoplasm, and is highly expressed in liver, kidney and heart, playing an important role in the occurrence and development of aging and fibrosis. Therefore, we summarized the role of SIRT2 in liver, kidney and cardiac fibrosis during aging.

Taxanes are widely used in chemotherapy, but primary and acquired resistance limit the clinical efficacy. Studies have shown tumor interaction with macrophages in the tumor microenvironment (TME) plays a significant role in taxane resistance, yet therapeutic strategies that directly deplete or repolarize macrophages are challenging and with considerable risk of side effects. Here we uncovered that tumor-macrophage interaction can be selectively targeted by inhibiting post-mitotic NOTCH2-JAG1 juxtacrine signaling in the TME, which strongly sensitizes paclitaxel response. Using translatome profiling, we found significant NOTCH2 upregulation during paclitaxel-induced prolonged mitosis. NOTCH2 was subsequently activated in the post-mitotic G1 phase by JAG1 expressed on the neighboring macrophages and tumor cells, which promoted tumor cell survival and upregulated cytokines that recruited JAG1-expressing macrophages, thus generating a positive feedback loop that further enhanced the pro-tumor NOTCH2 activity. By targeting this NOTCH2-JAG1 axis using NOTCH2 shRNA or a pan-NOTCH inhibitor, macrophage recruitment and paclitaxel resistance were significantly attenuated in multiple mouse tumor models of ovarian cancer. Clinical samples from paired primary and recurrent ovarian cancer patients also showed significant correlation of higher NOTCH2 expression with worse prognosis. Our results thus point to combining NOTCH2 inhibitor with taxane as an effective therapeutic strategy to selectively disrupt tumor-macrophage interaction in the TME and overcome macrophage-mediated taxane resistance in NOTCH2-positive tumors. ### Competing Interest Statement The authors have declared no competing interest.

Glycine- and arginine-rich (GAR) motifs, commonly found in RNA-binding and-processing proteins, can be symmetrically (SDMA) or asymmetrically (ADMA) dimethylated at the arginine residue by protein arginine methyltransferases. Arginine-methylated protein motifs are usually read by Tudor domain-containing proteins. Here, using a GFP-Trap, we identify a non-Tudor domain protein, squamous cell carcinoma antigen recognized by T cells 3 (SART3), as a reader for SDMA-marked GAR motifs. Structural analysis and mutagenesis of SART3 show that aromatic residues lining a groove between two adjacent aromatic-rich half-a-tetratricopeptide (HAT) repeat domains are essential for SART3 to recognize and bind to SDMA-marked GAR motif peptides, as well as for the interaction between SART3 and the GAR-motif-containing proteins fibrillarin and coilin. Further, we show that the loss of this reader ability affects RNA splicing. Overall, our findings broaden the range of potential SDMA readers to include HAT domains.

The lysine acetyltransferase KAT5 is a pivotal enzyme responsible for catalyzing histone H4 acetylation in cells. In addition to its indispensable HAT domain, KAT5 also encompasses a conserved Tudor-knot domain at its N-terminus. However, the function of this domain remains elusive, with conflicting findings regarding its role as a histone reader. In our study, we have employed a CRISPR tiling array approach and unveiled the Tudor-knot motif as an essential domain for cell survival. The Tudor-knot domain does not bind to histone tails and is not required for KAT5's chromatin occupancy. However, its absence leads to a global reduction in histone acetylation, accompanied with genome-wide alterations in gene expression that consequently result in diminished cell viability. Mechanistically, we find that the Tudor-knot domain regulates KAT5's HAT activity on nucleosomes by fine-tuning substrate accessibility. In summary, our study uncovers the Tudor-knot motif as an essential domain for cell survival and reveals its critical role in modulating KAT5's catalytic efficiency on nucleosome and KAT5-dependent transcriptional programs critical for cell viability.

High-fidelity Cas9 variants have been developed to reduce the off-target effects of CRISPR systems at a cost of efficiency loss. To systematically evaluate the efficiency and off-target tolerance of Cas9 variants in complex with different single guide RNAs (sgRNAs), we applied high-throughput viability screens and a synthetic paired sgRNA-target system to assess thousands of sgRNAs in combination with two high-fidelity Cas9 variants HiFi and LZ3. Comparing these variants against WT SpCas9, we found that ~20% of sgRNAs are associated with a significant loss of efficiency when complexed with either HiFi or LZ3. The loss of efficiency is dependent on the sequence context in the seed region of sgRNAs, as well as at positions 15-18 in the non-seed region that interacts with the REC3 domain of Cas9, suggesting that the variant-specific mutations in REC3 domain account for the loss of efficiency. We also observed various degrees of sequencedependent off-target reduction when different sgRNAs are used in combination with the variants. Given these observations, we developed GuideVar, a transfer-learning-based computational framework for the prediction of on-target efficiency and off-target effect with high-fidelity variants. GuideVar facilitates the prioritization of sgRNAs in the applications with HiFi and LZ3, as demonstrated by the improvement of signal-to-noise ratios in high-throughput viability screens using these high-fidelity variants.

The histone acetyltransferase p300/CBP is composed of several conserved domains, among which, the TAZ2 domain is known as a protein-protein interaction domain that binds to E1A and various transcription factors. Here we show that TAZ2 has a HAT autoinhibitory function. Truncating p300/CBP at TAZ2 leads to hyperactive HAT and elevated histone H3K27 and H3K18 acetylation in cells. Mechanistically, TAZ2 cooperates with other HAT neighboring domains to maintain the HAT active site in a ‘closed’ state. Truncating TAZ2 or binding of transcription factors to TAZ2 induces a conformational change that ‘opens’ the active site for substrate acetylation. Importantly, genetic mutations that lead to p300/CBP TAZ2 truncations are found in human cancers, and cells with TAZ2 truncations are vulnerable to histone deacetylase inhibitors. Our study reveals a function of the TAZ2 domain in HAT autoinhibitory regulation and provides a potential therapeutic strategy for the treatment of cancers harboring p300/CBP TAZ2 truncations.

Postoperative cognitive dysfunction (POCD) is a serious central nervous system complication characterized by impaired memory, reduced information processing ability, and anxiety. Recently, the role of FGF19 in neurological diseases has been reported. However, the effect and mechanisms of FGF19 in improving symptoms of POCD remain unknown. This study aimed to identify the role and exploring the underlying mechanisms of FGF19 in POCD. Here, rats were separated into four different groups, including control, sevoflurane (sev), sev + AAV-empty, and sev + AAV-FGF19 group. Then, the Morris water maze (MWM) test was applied to identify the effect of FGF19 on POCD rats. The result proved that FGF19 improved sevoflurane induced cognitive dysfunction in rats. Subsequently, the expressions of TNF-α, IL-6, IL-1β, and IL-10 were detected to verify the anti-neuroinflammatory effects of FGF19 in POCD rats. Furthermore, DHE fluorescent staining assay showed that FGF19 could inhibit sevoflurane-induced oxidative stress in POCD rats. Besides, NISSL staining and TUNEL assay were applied to reveal that FGF19 could alleviate hippocampal neuron injury induced by sevoflurane in rats. Moreover, mechanistic studies confirmed that FGF19 improved symptoms of POCD by mediated PGC-1α/BDNF/FNDC5 pathway. Together, these results suggested that FGF19 improves sevoflurane-induced POCD in rats through the PGC-1α/BDNF/FNDC5 pathway.

Abstract High-fidelity clustered regularly interspaced palindromic repeats (CRISPR)-associated protein 9 (Cas9) variants have been developed to reduce the off-target effects of CRISPR systems at a cost of efficiency loss. To systematically evaluate the efficiency and off-target tolerance of Cas9 variants in complex with different single guide RNAs (sgRNAs), we applied high-throughput viability screens and a synthetic paired sgRNA–target system to assess thousands of sgRNAs in combination with two high-fidelity Cas9 variants HiFi and LZ3. Comparing these variants against wild-type SpCas9, we found that ∼20% of sgRNAs are associated with a significant loss of efficiency when complexed with either HiFi or LZ3. The loss of efficiency is dependent on the sequence context in the seed region of sgRNAs, as well as at positions 15–18 in the non-seed region that interacts with the REC3 domain of Cas9, suggesting that the variant-specific mutations in the REC3 domain account for the loss of efficiency. We also observed various degrees of sequence-dependent off-target reduction when different sgRNAs are used in combination with the variants. Given these observations, we developed GuideVar, a transfer learning-based computational framework for the prediction of on-target efficiency and off-target effects with high-fidelity variants. GuideVar facilitates the prioritization of sgRNAs in the applications with HiFi and LZ3, as demonstrated by the improvement of signal-to-noise ratios in high-throughput viability screens using these high-fidelity variants.

Emerging evidence suggests that cryptic translation beyond the annotated translatome produces proteins with developmental or physiological functions. However, functions of cryptic non-canonical open reading frames (ORFs) in cancer remain largely unknown. To fill this gap and systematically identify colorectal cancer (CRC) dependency on non-canonical ORFs, we apply an integrative multiomic strategy, combining ribosome profiling and a CRISPR–Cas9 knockout screen with large-scale analysis of molecular and clinical data. Many such ORFs are upregulated in CRC compared to normal tissues and are associated with clinically relevant molecular subtypes. We confirm the in vivo tumor-promoting function of the microprotein SMIMP, encoded by a primate-specific, long noncoding RNA, the expression of which is associated with poor prognosis in CRC, is low in normal tissues and is specifically elevated in CRC and several other cancer types. Mechanistically, SMIMP interacts with the ATPase-forming domains of SMC1A, the core subunit of the cohesin complex, and facilitates SMC1A binding to cis -regulatory elements to promote epigenetic repression of the tumor-suppressive cell cycle regulators encoded by CDKN1A and CDKN2B . Thus, our study reveals a cryptic microprotein as an important component of cohesin-mediated gene regulation and suggests that the ‘dark’ proteome, encoded by cryptic non-canonical ORFs, may contain potential therapeutic or diagnostic targets.

Uncovering conserved 3D protein-ligand binding patterns on the basis of functional groups (FGs) shared by a variety of small molecules can greatly expand our knowledge of protein-ligand interactions. Despite that conserved binding patterns for a few commonly used FGs have been reported in the literature, large-scale identification and evaluation of FG-based 3D binding motifs are still lacking. Here, we propose a computational method, Automatic FG-based Three-dimensional Motif Extractor (AFTME), for automatic mapping of 3D motifs to different FGs of a specific ligand. Applying our method to 233 naturally-occurring ligands, we define 481 FG-binding motifs that are highly conserved across different ligand-binding pockets. Systematic analysis further reveals four main classes of binding motifs corresponding to distinct sets of FGs. Combinations of FG-binding motifs facilitate the binding of proteins to a wide spectrum of ligands with various binding affinities. Finally, we show that our FG-motif map can be used to nominate FGs that potentially bind to specific drug targets, thus providing useful insights and guidance for rational design of small-molecule drugs.

Goldfish (Carassius auratus auratus) are important ornamental fish that have experienced extreme anthropogenic selection and exhibit diverse phenotypes. Loss of the dorsal fin is a key characteristic that distinguishes the Egg-goldfish from the Wen-goldfish. However, the mechanisms underlying the divergence in dorsal fin development are still unknown between the Egg-goldfish and the Wen-goldfish. In this study, we sought to unravel the postembryonic developmental processes of two goldfish strains, Red Cap Oranda, a representative of the Wen-goldfish, and Ranchu, a representative of the Egg-goldfish. We examined the developmental morphology of the dorsal fin in five-month-old goldfish larvae using both Alcian blue-alizarin red staining and X-ray photography. We showed that the developmental processes of the dorsal fin fold were different in Ranchu compared to Red Cap Oranda, despite similarity in their general developmental processes. We categorized the postembryonic development of Ranchu larvae into four types based on the number of the initial residual dorsal fin fold appearing at protruding-mouth stage. The number, size and position of the initial residual dorsal fin fold appeared at hatching affected and eventually determined the phenotypic variations in dorsal fin defects in Ranchu. In addition, we genotyped the eomesa gene to determine whether it would be a candidate gene responsible for the loss of the dorsal fin in Ranchu. The eomesa CDS sequence exhibited no nonsense mutation in Ranchu. In summary, the absence of the dorsal fin fold during postembryonic development eventually resulted in the loss of the dorsal fin in Ranchu. Further investigation is required to determine whether other genes and developmental pathways play important roles in the loss of the dorsal fin in Ranchu.

With the aging of global population, the incidence of nonalcoholic fatty liver disease (NAFLD) has surged in recent decades. NAFLD is a multifactorial disease that follows a progressive course, ranging from simple fatty liver, nonalcoholic steatohepatitis (NASH) to liver cirrhosis and hepatocellular carcinoma (HCC). It is well established that aging induces pathological changes in liver and potentiates the occurrence and progression of NAFLD, HCC and other age-related liver diseases. Studies of senescent cells also indicate a pivotal engagement in the development of NAFLD via diverse mechanisms. Moreover, nicotinamide adenine dinucleotide (NAD+), silence information regulator protein family (sirtuins), and mechanistic target of rapamycin (mTOR) are three vital and broadly studied targets involved in aging process and NAFLD. Nevertheless, the crucial role of these aging-associated factors in aging-related NAFLD remains underestimated. Here, we reviewed the current research on the roles of aging, cellular senescence and three aging-related factors in the evolution of NAFLD to HCC, aiming at inspiring promising therapeutic targets for aging-related NAFLD and its progression.

The specificity of CRISPR/Cas9 genome editing is largely determined by the sequences of guide RNA (gRNA) and the targeted DNA, yet the sequence-dependent rules underlying off-target effects are not fully understood. To systematically explore the sequence determinants governing CRISPR/Cas9 specificity, here we describe a dual-target system to measure the relative cleavage rate between off- and on-target sequences (off-on ratios) of 1902 gRNAs on 13,314 synthetic target sequences, and reveal a set of sequence rules involving 2 factors in off-targeting: 1) a guide-intrinsic mismatch tolerance (GMT) independent of the mismatch context; 2) an “epistasis-like” combinatorial effect of multiple mismatches, which are associated with the free-energy landscape in R-loop formation and are explainable by a multi-state kinetic model. These sequence rules lead to the development of MOFF, a model-based predictor of Cas9-mediated off-target effects. Moreover, the “epistasis-like” combinatorial effect suggests a strategy of allele-specific genome editing using mismatched guides. With the aid of MOFF prediction, this strategy significantly improves the selectivity and expands the application domain of Cas9-based allele-specific editing, as tested in a high-throughput allele-editing screen on 18 cancer hotspot mutations.