Abstract Natural CRISPR-Cas9 systems provide a rich resource for developing genome editing tools with diverse properties, including genome size, protospacer preference, and PAM specificity. In this study, we screened a panel of 11 Cas9 nucleases orthologous to CjCas9 using a GFP activation assay and identified seven active nucleases. Among these, Cj4Cas9 emerges as particularly noteworthy due to its compact genome size (985 amino acids) and unique PAM preference (5’-NNNGRY-3’). Cj4Cas9 demonstrates efficient disruption of the Tyr gene in mouse zygotes, resulting in an albino phenotype. Furthermore, when delivered via AAV8, Cj4Cas9 achieves efficient genome editing of the Pcsk9 gene in mouse liver, leading to reduced serum cholesterol and LDL-C levels. To enhance its utility, we engineered Cj4Cas9 for higher activity by introducing L58Y/D900K mutations, resulting in a variant termed enCj4Cas9. This variant exhibits a two-fold increase in nuclease activity compared to the wild-type Cj4Cas9 and recognizes a simplified N3GG PAM, considerably expanding its targeting scope. These findings highlight the potential of Cj4Cas9 and its high-activity variants for both fundamental research and therapeutic applications.

Abstract CBFA2T3::GLIS2 fusion positive pediatric acute myeloid leukemia (AML) remains one of the worst prognostic AML subgroups. To uncover innovative targeted therapeutic approaches in this disease subtype we performed genome-scale CRISPR-Cas9 screening that highlighted a strong, selective dependency on JAK2 compared to other types of cancer. Using a doxycycline-inducible JAK2 knockout (KO) system, we validated JAK2 dependency in CBFA2T3::GLIS2 cell lines, observing impaired proliferation in vitro and in vivo and induced apoptosis with JAK2 KO. Both type I (ruxolitinib) and type II (CHZ868) JAK2 inhibitors showed selective in vitro activity in CBFA2T3::GLIS2 positive AML models. To identify resistance and sensitizer mechanisms to JAK2 inhibitors, we used CRISPR-Cas9 ruxolitinib anchor screening in CBFA2T3::GLIS2 AML. sgRNAs targeting negative regulators of the MAPK pathway were enriched in the ruxolitinib-treated cells. Similarly, CBFA2T3::GLIS2 AML sublines grown to resistance under chronic ruxolitinib treatment expressed pathogenic NRAS mutations. Both approaches converged on MAPK pathway activation as a resistance mechanism to ruxolitinib treatment. Combining ruxolitinib with MEK inhibitors showed a synergistic effect in cell lines and patient-derived xenograft (PDX) cells expressing the fusion and in vivo activity in a CBFA2T3::GLIS2 AML PDX, suggesting a potential approach to target this signaling circuitry in this poor outcome AML subtype.

Abstract Background Enhanced metabolic and mitochondrial activity inherent in actively proliferating cancer cells is associated with intracellular redox imbalance that impacts cellular viability. To restore redox homeostasis cancer cells evolve to activate redox protective mechanisms. This differential activation of redox defense pathways compared to normal cells provides a therapeutic window for novel targeted therapies in cancer. Although the heme metabolism emerges as a crucial regulator of redox homeostasis and iron metabolism in cancer cells with frequent alteration in breast cancer, it remains largely unexplored, and no targeted translational approaches have been developed. Heme-regulated redox homeostasis is coordinately maintained through biosynthetic and degradation pathways. As a byproduct of TCA cycle, cytotoxic heme is initially derivatized by heme oxygenases and progressively metabolized to the potent antioxidant bilirubin by two non-redundant biliverdin reductases, BLVRA and BLVRB. BLVRB overexpression has been observed in breast cancers, although its function in breast cancer pathogenesis remains unknown. Methods CRISPR/Cas9 deletion of BLVRB in multiple breast cancer cell lines demonstrated its profound effect on intracellular redox state and cell proliferation in vitro and xenograft models. Integrated proteomic, metabolomic, and lipidomic studies identified and validated BLVRB–mediated adaptive metabolic responses required for breast cancer cell cytoprotection. Results We have established BLVRB as a requisite component of the pro-survival redox defense mechanism in breast cancer cells. Targeted deletion of BLVRB induces reductive stress, leading to alterations in endoplasmic reticulum proteostasis and lipid composition. These defects impact plasma membrane functionality and endosomal recycling of multiple oncogenic receptors, such as HER2 and transferrin receptors. Conclusions These data collectively identify BLVRB as a novel metabolic target in breast cancer, distinct from other redox-regulating pathways. This study, along with our recent progress in developing novel specific BLVRB inhibitors, offers a unique translational opportunity for targeted therapies in personalized breast cancer medicine.

Abstract The CRISPR/Cas9 based technology has been used for sequential gene editing in E. coli . The plasmids carrying the sgRNA and/or Cas9 genes need to be cured after each round of editing. Curing of these plasmids, particularly the sgRNA plasmid, limits the efficiency of sequential gene editing. In this study, a lethal endotoxin ( ccd B) based counter-selection was established for improving the overall efficiency of sequential gene editing in E. coli . This approach was validated for sequential editing (deletion) of cst A and pps A genes. The experimental results showed that the transformation efficiency sgRNA plasmid (pTargetF- tcr -P L - ccd B-N20) reached to 10 8 -10 9 cfu / µg -DNA , resulting in a 90% of recombination rate for the target gene ( cst A and pps A). Upon completion of cst A gene editing, the sgRNA plasmid (pTargetF- tcr -P L - ccd B-N20( cst A)) were effectively cured through ccd B based counterselection at 42°C, with a 43.75% efficiency. At the end of sequential editing of pps A gene, both Cas9 (25A) and sgRNA (pTargetF- tcr -P L - ccd B-N20( pps A)) plasmids were cured simultaneously through the sac B and ccd B based counterselections by incubating the cells on LB-sucrose (5%) plate at 42°C, achieving a curing rate of 100% for Cas9 plasmid (25A), and 37.5% for sgRNA plasmid (pTargetF- tcr -P L - ccd B-N20( pps A)). These results demonstrated that the endotoxin ( ccd B) based counterselection improved the transformation efficiency of sgRNA plasmid, the recombination rate of the editing target gene, the curing rate of sgRNA plasmid, and the overall efficiency of sequential gene editing.

ABSTRACT Protein translation regulation is critical for cellular responses and development, yet how disruptions during the elongation stage shape these processes remains incompletely understood. Here, we identify and validate a single amino acid substitution (P55Q) in the ribosomal protein RPL-36A of Caenorhabditis elegans that confers complete resistance to high concentrations of the elongation inhibitor cycloheximide (CHX). Heterozygous animals carrying both wild-type RPL-36A and RPL-36A(P55Q) exhibit normal development but intermediate CHX resistance, indicating a partial dominant effect. Leveraging RPL-36A(P55Q) as a single-copy positive selection marker for CRISPR-based genome editing, we introduced targeted modifications into multiple ribosomal protein genes, confirming its broad utility for altering essential loci. In L4-stage heterozygotes, where CHX-sensitive and CHX-resistant ribosomes coexist, ribosome profiling revealed increased start-codon occupancy, suggesting early stalling of CHX sensitive ribosomes. Chronic CHX reduced ribosome collisions, evidenced by fewer disomes and unchanged codon distributions in monosomes. Surprisingly, prolonged elongation inhibition did not activate well characterized stress pathways–including ribosome quality control (RQC), the ribotoxic stress response (RSR), or the integrated stress response (ISR)–as indicated by absence of changes in RPS-10 ubiquitination, eIF2α phosphorylation, PMK-1 phosphorylation, or the transcriptional upregulation of ATF-4 target genes. Instead, RNA-normalized ribosome footprints revealed gene-specific changes in translation efficiency, with nucleolar and P granule components significantly decreased while oocyte development genes were increased. Consistent with these observations, we detected premature oogenesis in L4 animals, suggesting that partial translation elongation inhibition reshapes translation efficiency, to fine-tune developmental timing.

Over the last two decades, new in vivo and in cellulo imaging technologies have uncovered the inherently dynamic nature of transcriptional regulation in embryonic development and, in particular, in the fruit fly D. melanogaster .These technologies have made it possible to characterize the subnuclear and single-molecule dynamics of transcription factors. However, a lack of appropriate fluorescent protein fusions has, until now, limited these studies to only a few of the dozens of important transcription factors in the fruit fly gene regulatory network dictating early development. Here, we report the creation of four new fluorescent protein fusions to Dorsal, a member of the NF-κB/Rel family that initiates dorsal-ventral patterning. We generated and characterized two bright fluorescent protein fusions for Dorsal, meGFP and mNeonGreen, and two photoconvertible fluorescent protein fusions, mEos4a and Dendra2. We show that removal of the DsRed2 cassette commonly used to mark the CRISPR integration restores endogenous Dorsal mRNA and protein levels and enables the fusion allele to rescue a dorsal null allele, meeting the gold standard for endogenous function of a tagged protein in a fruit fly. We then demonstrate that our bright fluorescent protein fusions can be used to dissect the spatiotemporal dynamics of stable Dorsal clusters that traverse the nucleoplasm and uncovered that these clusters preferentially interact with active sites of Dorsal-modulated transcription. We further demonstrate that our photoconvertible fluorescent protein fusions make it possible to detect individual molecules of Dorsal in the nuclei of developing embryos. These new fluorescent protein fusions constitute a valuable resource for the community to elucidate the role of Dorsal activator dynamics in dictating fruit fly early embryonic development.

Abstract The prognosis of colorectal cancer (CRC) is often fatal, and the underlying mechanisms are unclear. Dysregulation of enhancers is involved in several tumors, and this area is expected to become a new frontier in tumor therapy. Here, we identified a novel activated enhancer ‘Enhancer X’ from the Gene Expression Omnibus database and experimentally verified its associated RNA (EX-eRNA) in CRC. To date, there are no reports on the function of Enhancer X, and its role in CRC remains unknown. We aimed to investigate the biological functions and underlying mechanism of Enhancer X in CRC. EX-eRNA expression was elevated in CRC cells and tissues and positively correlated with CRC metastasis and shorter survival. Enhancer X knockout (CRISPR/Cas9) or EX-eRNA knockdown (siRNAs) inhibited CRC cell growth in vivo and in vitro. Mechanistically, Enhancer X activation and transcription to EX-eRNA require the binding of CEBPB to a “GTTGTGTCAC” motif within the enhancer region. Subsequently, EX-eRNA, serves as a scaffold for MED25 to stabilize the chromatin loop between Enhancer X region and the KRT80 promoter, thereby promoting KRT80 transcription and driving CRC tumorigenesis. Thus, we conclude that novel enhancer X activated by CEBPB drives colorectal carcinogenesis via EX-eRNA-mediated DNA loop promotion of KRT80 transcription, and that targeting the CEBPB / Enhancer X / EX-eRNA / KRT80 molecular axis constitutes a promising approach to treat CRC.

Pyridoxine-dependent epilepsy (PDE) is a rare neurometabolic disorder of lysine catabolism caused by bi-allelic variants in ALDH7A1 . This enzyme deficiency leads to the accumulation of neurotoxic metabolites, pyridoxal-phosphate inactivation and consequently severe neurological symptoms. Current treatments, including vitamin B6 supplementation and lysine-restricted diets, partially alleviate seizures and intellectual disability but are not curative. To explore underlying mechanisms and potential therapies, we generated patient-derived human induced pluripotent stem lines (hiPSC) that were subsequently differentiated into astrocytes, the primary source of ALDH7A1 in the brain and key regulators of metabolic homeostasis. Metabolomic analyses confirmed elevated PDE biomarkers, and RNA sequencing revealed gene expression changes consistent with increased oxidative stress. Oxidative damage was validated by markers of DNA oxidation and lipid peroxidation. In addition, dysregulated oxygen consumption rates suggested mitochondrial dysfunction in PDE astrocytes. Notably, these pathological phenotypes were alleviated by downregulating AASS , the first enzyme of the lysine catabolism, by using CRISPR/Cas9 editing or antisense oligonucleotides (AON). This demonstrates that lysine catabolism underlies these phenotypes and highlights the therapeutic potential of AON therapy targeting AASS to reduce neurotoxic metabolite accumulation. These findings provide a promising strategy for developing targeted treatments for PDE and other rare neurometabolic disorders.

Abstract Background: Genomic editing technologies provide rich opportunities to approach inherited human diseases, but gene delivery strategies are still challenging. While engineered induced pluripotent stem cells or adeno-associated viral vectors may be associated with severe adverse effects, mesenchymal stromal cells (MSC) have abundantly been used for various cell therapeutic strategies and have been proven to be safe in a large number of clinical trials. MSC can be expanded ex vivo on a large scale, and genetic engineering of these somatic stem cells may therefore offer an alternative mode of gene delivery. Methods: We applied the guide RNA-directed CRISPR/Cas9 synergistic activation mediator (SAM) technology in immortalized (iMSC) and primary human MSC (pMSC) in order to activate gene expression of CEBPA and CIITA . Results: After meticulous protocol optimization, both genes were successfully induced in MSC. For CIITA induction, we confirmed expression of the downstream target HLA-DR on mRNA and protein level. Unlike adipogenic, chondrogenic or osteogenic genes, which are naturally induced in MSC upon in vitro differentiation, HLA class II molecules are not constitutively expressed in MSC. Lentiviral and transposon-based delivery strategies were applied, but only with lentiviral transduction of the SAM sequences, CEBPA, CIITA, HLA-DR gene and HLA-DR protein expression were achieved in pMSC. Conclusion: Lentiviral transduction of the guided SAM system was capable to induce transcriptionally silent genes in iMSC and pMSC. The resulting genetically engineered somatic stem cells may be suitable and promising for cell-based therapeutic strategies.

Abstract Background Alzheimer’s disease (AD) is characterised by progressive cognitive decline and accumulation of pathological markers such as β-amyloid (Aβ) plaques and Tau tangles. Emerging evidence suggests these markers can also be detected in the retina, positioning it as a potential surrogate for investigating AD pathophysiology. The retinal pigment epithelium (RPE) shares features with the brain and is critical for retinal health, yet its role in AD pathology remains underexplored. Methods We generated RPE cells from human induced pluripotent stem cells carrying the PSEN1 H163R pathogenic variant for AD, alongside its CRISPR-corrected isogenic control. AD-associated phenotypes were assessed. Results RPE cell cultures from the two cohorts displayed expression of Aβ and Tau, with notable differences in levels and organisation. Total Aβ 1−42 and Aβ 1−42:1−40 ratio in PSEN1 H163R RPE cell lysates were significantly elevated compared to the CRISPR isogenic controls and volume of Aβ + deposits was significantly larger in PSEN1 H163R RPE cells. Total and phosphorylated Tau proteins were also detected in both cohorts, with altered spatial organisation and localisation of pTau in PSEN1 H163R . Proteomic profiling identified more than 1,800 significantly dysregulated proteins in PSEN1 H163R RPE cells, including key AD-related proteins such as MAPT, APP, APBB1 and NRBF2. Upregulated pathways involved autophagy, intracellular trafficking and neurodegeneration, while downregulated pathways implicated mitochondrial respiration, RNA metabolism, and protein folding. Proteomics analysis of conditioned media further revealed altered secretion of matrix-associated proteins as well as increased APOE and APP in PSEN1 H163R RPE samples. PSEN1 H163R RPE cells demonstrated dysregulation in melanosome biogenesis, marked by decreased expression of core melanogenic proteins (PMEL, TYR, DCT) by proteomics analysis; and altered melanosome morphology and pigmentation by electron microscopy. Conclusion In conclusion, these findings support the RPE as a relevant and accessible in vitro model for AD research, offering insights into the role of PSEN1 in Aβ and Tau dysregulation, disease mechanisms and melanosome biogenesis, providing a promising approach to understand PSEN1 biology in the context of disease and potential biomarker discovery. It is also the first to describe a relationship between PSEN1 H163R and melanosomes in a human cellular model.

The rapid development of CRISPR-Cas gene editing technologies has revolutionized genetic medicine, offering unprecedented precision and potential for treating a wide array of genetic disorders. However, assessing the risks of unintended gene editing effects remains critical, and is complicated by new editing modalities and unclear analytical guidelines. We present UNCOVERseq (Unbiased Nomination of CRISPR Off-target Variants using Enhanced RhPCR), an improved in cellulo off-target nomination workflow designed to sensitively nominate off-target sites (<0.01% editing) with defined input requirements and analytical process controls to provide empirical performance evidence across diverse circumstances. Using this workflow, we nominated off-targets across 192 guide RNAs (gRNAs) and demonstrated superior performance compared to existing methodologies. We identified a subset of six gRNAs with a dynamic range of specificity and confirmed the relevance and high true positive rate of our nomination method, providing relative risk assessments for multiple modalities ( S.p. Cas9 and derived high-fidelity variants / base editors) in a translational system involving hematopoietic stem and progenitor cells (HSPCs). Additionally, we established that double-strand break (DSB) editing retains a strong, positive rank correlation to single-strand break (SSB)-mediated base editing, highlighting the importance of DSB nomination sites as candidate loci for base editing. Overall, UNCOVERseq improves informed risk assessment of gene editing in translational systems by enhancing the quality of off-target nomination.

ABSTRACT We demonstrate here the use of optical genome mapping (OGM) to detect genetic alterations arising from gene editing by various technologies in human induced pluripotent stem cells (iPSCs). OGM enables an unbiased and comprehensive analysis of the entire genome, allowing the detection of genomic structural variants (SVs) of all classes with a quantitative variant allele frequency (VAF) sensitivity of 5%. In this pilot study, we conducted a comparative dual analysis between the parental iPSCs and the derived cells that had undergone gene editing using various techniques, including transposons, lentiviral transduction, and CRISPR-Cas9-mediated safe harbor locus insertion at the adeno-associated virus integration site 1 (AAVS1). These analyses demonstrated that iPSCs that had been edited using transposons or lentiviral transduction resulted in a high number of transgene insertions in the genome. In contrast, CRISPR-Cas9 technology resulted in a more precise and limited transgene insertion, with only a single target sequence observed at the intended locus. These studies demonstrate the value of OGM to detect genetic alterations in engineered cell products and suggests that OGM, together with DNA sequencing, could be a valuable tool when evaluating genetically modified iPSCs for research and therapeutic purposes.

Gene editing technologies have opened the possibility of directly targeting viral DNA in therapeutic applications. In chronically infected hepatocytes with hepatitis B virus (HBV), covalently closed circular DNA (cccDNA) serves as the master template for viral transcripts and gene products. In the present study, we evaluated the outcomes of anti-HBV multiplex gene editing with the CRISPR-Cas9 endonuclease from Staphylococcus aureus (SaCas9) using primary human hepatocytes (PHHs) and HBV mouse models. Nonviral delivery of SaCas9-encoding mRNA and a pair of HBV-targeting guide RNAs (gRNAs) substantially reduced viral biomarkers and intrahepatic HBV DNA copies in vitro and in vivo, suggesting that fragmentation of HBV DNA primarily leads to its degradation. Hybridization capture sequencing analyses indicated that small insertions and deletions (indels) and structural variants including excisions and inversions of the viral sequences were accumulated in the residual HBV DNA. These assays also demonstrated that transient expression of the HBV-targeting SaCas9 significantly suppressed random integration of HBV DNA, while this therapeutic approach was unlikely to affect chromosomal translocations involving viral copies. Taken together, our results suggest that anti-HBV multiplex gene editing eliminates viral DNA from chronically infected hepatocytes, potentially reducing the risk of hepatocarcinogenesis associated with HBV DNA integration.

CRISPR-Cas is a defense system of bacteria and archaea against phages. Parts of the foreign DNA, called spacers, are incorporated into the CRISPR array which constitutes the immune memory. The orientation of CRISPR arrays is crucial for analyzing and understanding the functionality of CRISPR systems and their targets. Several methods have been developed to identify the orientation of a CRISPR array. To predict the orientation, different methods use different features such as the repeat sequences between the spacers, the location of the leader sequence, the Cas genes, or PAMs. However, those features are often not sufficient to predict the orientation with certainty, or different methods disagree. Remarkably, almost all CRISPR systems have been found to insert spacers in a polarized manner at the leader end of the array. We introduce CRISPR-evOr , a method that leverages the resulting patterns to predict the acquisition orientation for (a group of) CRISPR arrays by reconstructing and comparing the likelihood of their evolutionary history with respect to both possible acquisition orientations. The new method is independent of Cas type, leader existence and location, and transcription orientation. CRISPR-evOr is thus particularly useful for arrays that other CRISPR orientation tools cannot predict confidently and to verify or resolve conflicting predictions from existing tools. CRISPR-evOr currently confidently predicts the orientation of 28.3% of the arrays in the considered subset of CRISPRCasdb, which other tools like CRISPRDirection and CRISPRstrand cannot reliably orient. As our tool leverages evolutionary information we expect this percentage to grow in the future when more closely related arrays will be available. Additionally, CRISPR-evOr provides confident decisions for rare subtypes of CRISPR arrays, where knowledge about repeats and leaders and their orientation is limited.

Root cell elongation, the main driver of root growth, is tightly associated with cell wall remodeling, particularly through pectin modifications, which facilitate cell wall loosening and strengthening while maintaining structural integrity. Root cell elongation is precisely regulated by the phytohormone auxin, which has long been known to inhibit this process. The molecular pathways through which auxin influences cell wall modifications remain poorly understood. In this study, we explore the transcriptional regulation of cell wall-related genes by auxin in Arabidopsis thaliana roots. The nuclear auxin pathway altered the expression of numerous cell-wall related genes, suggesting dynamic modification of the cell wall during root cell elongation. We identified novel root-specific polygalacturonases (PGs), enzymes involved in pectin degradation, which we termed POLYGALACTURONASES REGULATED BY AUXIN (PGRAs). PGRAs are expressed specifically in the root epidermis, beginning at the elongation zone. Our results demonstrate that induction of PGRA1 expression initially promotes root cell elongation, while long term overexpression inhibits root growth. Auxin downregulates PGRA1 in the elongation zone, and plants lacking PGRAs fail to increase root growth rate in response to reduced auxin levels. This suggests that auxin downregulates PGRA expression to prevent PGRA-mediated pectin remodeling, thereby contributing to inhibition of root cell elongation. We established a novel link between auxin signaling and pectin modifications in the control of cell growth. These findings provide new insights into the molecular mechanisms through which auxin regulates root cell elongation, highlighting the role of pectin matrix modifications in this process.

ABSTRACT Long non-coding RNAs (lncRNAs) are emerging as key regulatory players of coding gene expression in eukaryotes. Here, we investigate the roles of the lncRNAs SVALKA (SVK) and SVALNA (SVN) in regulating CBF1 and CBF3 gene expression in Arabidopsis under cold stress conditions. We used Native Elongation Transcript Sequencing, CRISPR-Cas9 deletion strategies, and RT-qPCR to analyze the transcriptional dynamics and regulatory mechanisms of SVK and SVN . Our results demonstrate that SVK functions as a cis - and trans -acting lncRNA, regulating both CBF1 and CBF3 through RNAPII collision and chromatin remodeling, while SVN serves a cis role by negatively regulating CBF3 via a RNAPII collision mechanism. We identified isoforms of SVK , originating from distinct transcription start sites and undergo alternative splicing to adapt structural stability, crucial for their regulatory functions. This study elucidates the complex interplay of lncRNAs in gene regulation, highlighting their essential roles in modulating responses to environmental stresses. Our findings contribute to a deeper understanding of the mechanisms underlying lncRNA functionality and their significance in gene regulatory networks in eukaryotes.

Freezing behavior, characterised by attentive immobility as a reaction to a perceived threat, is widely studied in the context of fear, anxiety and stress. To uncover the genetic factors underlying this behavior, we conducted a genome-wide association study (GWAS) in kennel-housed beagle dogs. Our analysis identified a single-nucleotide polymorphism (SNP) in intron 5 of the KCNQ3 gene on chromosome 13 associated with freezing behavior in response to unfamiliar environments and people. To validate this finding, we used a zebrafish model, where CRISPR/Cas9-induced kcnq3 deficiencies led to heightened fear and arousal in two behavioral tests. KCNQ3 is implicated in several neurodevelopmental and psychiatric disorders, and our results highlight its evolutionarily conserved role in modulating fear responses. In dogs, an enriched environment can mitigate the adverse effects of KCNQ3 deficiency by reducing threat perception, highlighting the role of gene-environment interactions in shaping behavioral responses. Teaser KCNQ3 influences fear responses across species, with gene-environment interactions shaping freezing behavior in dogs.

ABSTRACT Temozolomide (TMZ) remains the standard of care for glioblastoma; however, its efficacy is frequently influenced by epigenetic mechanisms, notably the methylation status of the O6-methylguanine-DNA methyltransferase (MGMT) promoter. While MGMT promoter hypermethylation is associated with enhanced responsiveness to TMZ, additional epigenetic determinants of TMZ resistance remain largely undefined. In this study, we established TMZ-resistant glioblastoma cell lines that consistently maintained their resistant phenotype both in vitro and in vivo . Transcriptomic analyses revealed a marked upregulation of MGMT expression in these models. To systematically investigate the epigenetic regulators governing TMZ resistance and cell survival, we conducted CRISPR/Cas9-based functional genomic screens using our focused Epigenetic Knock-Out Library (EPIKOL), which targets 800 chromatin regulators alongside selected positive and negative controls. These unbiased screens validated MGMT as a primary mediator of TMZ resistance, confirming the robustness of our approach. Moreover, dropout screens across multiple resistant cell line models identified Retinoblastoma Binding Protein 4 (RBBP4) as a critical vulnerability. Notably, RBBP4 knockout significantly impaired cell proliferation without affecting MGMT expression, suggesting a distinct mechanism supporting the survival of TMZ-resistant glioblastoma cells. Subsequent transcriptomic profiling following RBBP4 loss demonstrated significant downregulation of cell cycle pathways, particularly the G2/M checkpoint. Live-cell imaging and immunofluorescence analyses further revealed increased cell size and multinucleation in RBBP4-deficient cells, indicative of disrupted mitotic progression. Collectively, our results identify RBBP4 as a key regulator of cell cycle progression and survival in TMZ-resistant glioblastoma and highlight its potential as a novel epigenetic target for therapeutic intervention in recurrent disease.

Abstract Endometrial carcinoma (EC), the most common gynecologic cancer type, encompasses multiple molecular subtypes that have consistent prognostic values and are being adopted in clinical practice to guide treatment decisions. However, it remains unclear whether each of these molecular subtypes have unique therapeutic vulnerabilities that can be exploited for advancing the management of ECs. Through analyzing the genomic features of a panel of 39 EC cell lines, we identified multiple tumor cell lines representing each molecular subtype. Histologic and immunochemical analyses of xenografted tumors from these cell lines confirmed their resemblance of cognate primary EC molecular subtypes, both by histology and the protein expression status of mismatch repair genes, p53 and SWI/SNF members in corresponding subtypes. Further investigation of the publicly available genome-wide CRISPR data for EC cell lines identified multiple specific genetic vulnerabilities in mismatch repair-deficient, p53-abnormal and ARID1A and ARID1B-dual deficient EC cell lines, respectively. Particularly, ARID1A and ARID1B-dual deficient EC cells selectively rely on mitochondria oxidative phosphorylation in vitro and in vivo . Therefore, our study demonstrates the utility of EC cell line models for uncovering and validating therapeutic vulnerabilities of each EC molecular subtype.

Recurrent breast cancer accounts for most disease-associated mortality and can develop decades after primary tumor therapy. Recurrences arise from residual tumor cells (RTCs) that can evade therapy in a dormant state, however the mechanisms are poorly understood. CRISPR-Cas9 screening identified the transcription factors SOX5/6 as functional regulators of tumor recurrence. Loss of SOX5 accelerated recurrence and promoted escape from dormancy. Remarkably, SOX5 drove dormant RTCs to adopt a cartilage-dependent bone development program, termed endochondral ossification, that was confirmed by [ 18 F]NaF-PET imaging and reversed in recurrent tumors escaping dormancy. In patients, osteochondrogenic gene expression in primary breast cancers or residual disease post-neoadjuvant therapy predicted improved recurrence-free survival. These findings suggest that SOX5-dependent mesodermal transdifferentiation constitutes an adaptive mechanism that prevents recurrence by reinforcing tumor cell dormancy.

The success of cancer immunotherapies is currently limited to a subset of patients, which underscores the urgent need to identify the processes by which tumours evade immunity. Through screening a kinome-wide CRISPR/Cas9 sgRNA library we identified MAP3K7 (TAK1) as a suppressor of CD8+ T cell mediated killing. We demonstrate that TAK1 acts as a cancer-intrinsic checkpoint by integrating signals from T cell-secreted TNF and IFNy effector cytokines to elicit a cytoprotective response. This cytoprotective response profoundly limits the anti-cancer activity these key effector molecules and completely abrogates bystander killing by perforin deficient T cells. Inhibition of the TAK1 checkpoint effectively redirects the combined TNF/IFNy pathway activation to promote inflammatory cell death via RIPK1 and Caspase-8 and simultaneously amplifies the output of the IFNy pathway, thereby priming cells for cytokine-induced cell death. Mechanistically, TAK1 deficiency led to proteasomal degradation of cFLIP, enhancing the formation of Complex II and subversion of other cytoprotective responses. Targeting the TAK1 checkpoint led to profound attenuation of tumour growth in immune competent mice, with minimal impact in immune deficient counterparts. Adoptive cell therapy led to preferential elimination of TAK1 deficient clones. Collectively, our study uncovers a cancer-intrinsic checkpoint controlled by TAK1 activity that switches TNF and IFNy responses from cytoprotective to apoptosis. Cancer cells exploit this to limit cell death in the presence of the cytotoxic lymphoctye cytokines TNF and IFNγ and therapeutic intervention can fully unleash the impact of these effector molecules both on the direct target and bystander cells. These findings highlight the clinical development of TAK1 biologics as a potential strategy to improve cancer immunotherapies through harnessing and enhancing the cytotoxic potential of CTL-derived cytokines.

ABSTRACT Metastatic breast cancer (MBC) is a life-threatening disease with limited therapeutic options. The immune suppressive tumor microenvironment (TME) limits the potency of the antitumor immune response and facilitates disease progression and metastasis. Our current study demonstrates that p38α is a druggable target in the TME that regulates the outcome of the immune-tumor interaction. The study revealed that systemic blockade of p38α reduces metastasis, and this anti-metastatic response is negated by depletion of CD8 + T cells. Single-cell transcriptomic analysis of the immune-TME showed that pharmacological p38 inhibition (p38i) or tumor-specific inactivation of p38α by CRISPR/Cas9 (p38KO) resulted in a less exhausted and more activated CD8 + T cell phenotype. Immunophenotyping analyses demonstrated that p38 blockade reduced the expression of multiple inhibitory receptors on CD8 + T cells (i.e., PD-1, LAG-3, CTLA-4), indicating a reversal of immune exhaustion and enhanced immune activation systemically and in the TME. In contrast, p38 blockade did not exhibit inhibitory effects on T cells in proliferation assays in vitro and did not affect the proportion of regulatory T cells in vivo . The major negative impact of p38 blockade in vivo was on the myeloid populations, such as myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs). Further, tumor p38α activity was required for the expression of cytokines/chemokines and tumor-derived exosomes with high chemotactic capacity for myeloid cells. Altogether, this study highlights a previously unrecognized p38α-driven pathway that promotes an immune suppressive TME and metastasis, and that therapeutic blockade of p38α has important implications for improving antitumor immunity and patient outcomes. STATEMENT OF SIGNIFICANCE This study highlights a previously unrecognized p38α-driven tumor pathway that promotes an immune suppressive microenvironment and metastasis, and that therapeutic blockade of p38α has important implications for improving antitumor immunity and patient outcomes.

Chromosomal instability (CIN), a characteristic feature of esophageal adenocarcinoma (EAC), drives tumor aggressiveness and therapy resistance, presenting an intractable problem in cancer treatment. CIN leads to constitutive stimulation of the innate immune cGAS–STING pathway, which has been typically linked to anti-tumor immunity. However, despite the high CIN burden in EAC, the cGAS– STING pathway remains largely intact. To address this paradox, we developed novel esophageal cancer models, including a CIN-isogenic model, discovering myeloid-attracting chemokines – with the chemokine CXCL8 (IL-8) as a prominent hit – as conserved CIN-driven targets in EAC. Using high-resolution multiplexed immunofluorescence microscopy, we quantified the extent of ongoing cGAS-activating CIN in human EAC tumors by measuring cGAS-positive micronuclei in tumor cells, validated by orthogonal whole-genome sequencing-based CIN metrics. By coupling in situ CIN assessment with single-nucleus RNA sequencing and multiplex immunophenotypic profiling, we found tumor cell-intrinsic innate immune activation and intratumoral myeloid cell inflammation as phenotypic consequences of CIN in EAC. Additionally, we identified increased tumor cell-intrinsic CXCL8 expression in CIN high EAC, accounting for the inflammatory tumor microenvironment. Using a novel signature of CIN, termed CIN MN , which captures ongoing CIN-associated gene expression, we confirm poor patient outcomes in CIN high tumors with signs of aberrantly rewired cGAS–STING pathway signaling. Together, our findings help explain the counterintuitive maintenance and expression of cGAS–STING pathway components in aggressive, CIN high tumors and emphasize the need to understand the contribution of CIN to the shaping of a pro-tumor immune landscape. Therapeutic strategies aimed at disrupting the cGAS-driven inflammation axis may be instrumental in improving patient outcomes in this aggressive cancer.

A recent study has shown that SKP2 inactivation can prevent cancer initiation by extension of total cell cycle duration without perturbing normal division, which suggests a new strategy for cancer prevention. However, direct in vivo evidence for human SKP2 on cancer initiation and prostatic microenvironment is still lacking and a prostate-specific SKP2 humanized mouse model is critical for developing prostate cancer immunoprevention approaches through targeting human SKP2. We therefore have established a prostate-specific human SKP2 (h SKP2 ) knock-in mouse model by a CRISPR knock-in approach. Overexpression of h SKP2, which is driven by an endogenous mouse probasin promoter, induces prostatic lesions including hyperplasia, mouse prostate intraepithelial neoplasia (mPIN), and low-grade carcinoma and increases prostate weights. Transcriptional profiling by RNA-sequencing analysis revealed significant gene expression alterations in epithelial to mesenchymal transition (EMT), extracellular matrix, and interferon signaling in the prostate of h SKP2 knock-in mice compared to wild-type mice. Single cell deconvolution showed an increase of fibroblasts population and a decrease of CD8 + T cell and B cell populations in the prostate of hSKP2 -knock-in mice. Consistently with these results from the SKP2 humanized mouse, overexpression of hSKP2 in human prostate cancer PC3 cells markedly increased cell migration and invasion and induced the gene expression of EMT and interferon pathways, including FMOD, THY1, PFKP, USP18, IL15, etc. In addition, paired prostate organoids were derived from SKP2 humanized and wild-type mice for drug screening and validated by known SKP2 inhibitors, Flavokawain A and C1. Both of which selectively decrease the viability and alter the morphologies of organoids of h SKP2 knock-in rather than wild-type mice. Our studies provide a well-characterized prostate-specific h SKP2 knock-in mouse model and offer new mechanistic insights for understanding the oncogenic role of SKP2 in shaping the prostatic microenvironment during early carcinogenesis.

1 CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) systems are a fundamental defense mechanism in prokaryotes, where short sequences called spacers are stored in the host genome to recognize and target exogenous genetic elements. Viromics, the study of viral communities in environmental samples, relies heavily on identifying these spacer-target interactions to understand host-virus relationships. However, the choice of sequence search tool to identify putative spacer targets is often overlooked, leading to an unknown impact of downstream inferences in virus-host analysis. Here, we utilize simulated and real datasets to compare popular sequence alignment and search tools, revealing critical differences in their ability to detect multiple matches and handle varying degrees of sequence identity between spacers and potential targets. Finally, we provide general guidelines that may inform future research regarding matching, which is a common practice in studying the complex nature of host-MGE interactions.