Targeting Warburg effect: involvement of lactate transporter MCT1 and its chaperone in cancer cell killing by 18β-glycyrrhetinic acid.

SLC5A11 mediates metformin-induced PD-L1 suppression to enhance cancer immunotherapy through AMPK-IRF1 signaling.

Stimulation of the innate immune system by foreign RNA elicits a potent interferon response and can trigger cell death. The mechanisms by which cells balance a robust response with cell-intrinsic lethality are still being uncovered. Here, using genome-wide CRISPR-Cas9 genetic screens with triphosphorylated RNA stimulation, we discover that promyelocytic leukaemia (PML) nuclear body-localized speckled protein 110 (SP110) is a potent inhibitor of type 1 interferon-driven cell death. Death suppression by SP110 counteracts a toxic activity of SP100, a major constituent of PML bodies. Loss of SP110 leads to mitotic retention of SP100 and PML bodies, which associate with and perturb segregating chromosomes, leading to micronucleus formation, DNA damage and genotoxic cell death. A combination of cryo-electron microscopy, AlphaFold modelling and cellular biochemistry reveals that SP110 dissolves toxic SP100 oligomers via necessary and sufficient direct interactions between their caspase activation and recruitment domains. These data reveal the critical roles of SP100 and SP110 in governing the disassembly of PML bodies during mitosis, as well as the repercussions if this process is misregulated.

Acute kidney injury caused by cisplatin (Cis-AKI) is a major limitation in its clinical use, primarily due to the lack of effective therapeutic targets to mitigate nephrotoxicity. Although several molecular pathways are involved in Cis-AKI, identifying reliable and actionable therapeutic targets has been challenging. Through a CRISPR-based genome-wide screening approach, UBE2M was identified as a novel gene involved in cellular survival during cisplatin-induced stress. However, its expression, biological function, and underlying mechanism in Cis-AKI have not been thoroughly investigated. This study aims to identify key therapeutic targets for Cis- AKI and investigate the role of UBE2M in this condition. A CRISPR-Cas9 genome-wide screening approach was employed to identify key genes involved in cisplatin-induced renal tubular epithelial cell injury. UBE2M, identified as a critical survival factor, was further investigated using both gain- and loss-of-function strategies to explore its biological function and underlying regulatory mechanisms in the Cis-AKI model. CRISPR screening identified UBE2M as a key regulator of cellular survival in Cis-AKI, and subsequent validation experiments confirmed its suppression in cisplatin-induced renal injury models. UBE2M overexpression alleviated apoptosis and renal injury by reducing p53 activation. In contrast, UBE2M knockdown exacerbated these effects, leading to increased apoptosis and renal injury. This study reveals that UBE2M is a critical regulator of cisplatin-induced renal tubular epithelial cell injury. By regulating the p53-mediated apoptotic pathway, UBE2M protects against Cis-AKI. UBE2M could serve as a novel therapeutic target for the prevention and treatment of cisplatin-induced nephrotoxicity.

Microscopy offers an indispensable technique for visualizing biological processes and for defining cytological abnormalities characteristic of disease. However, combining microscopy with the power of pooled CRISPR screening presents considerable technical challenges, hindering application of systematic genetic analysis to imaging-defined phenotypes. Here, we establish a fluorescence microscopy-based CRISPR screening platform that combines ease of implementation with flexible analysis of live-cell or antibody-based molecular markers, including post-translational modifications. Applying this methodology, we systematically identify regulators of primary cilium structure and function in human cells through targeted and genome-wide screens. We further show that integration of screens focused on distinct ciliary phenotypes yields multi-dimensional profiles that delineate precise gene functions. Among the identified hits, TZMP1 (SMIM27) encodes a microprotein at the ciliary transition zone that is required for ciliogenesis in human cells and for ciliary function in Xenopus embryos. More broadly, our approach provides a technological and conceptual strategy for microscopy-based functional genomics.

Extrachromosomal circular DNA (ecDNA) is frequently generated within the nucleus, contributing to genome dynamics and heterogeneity, thereby promoting cancer cell evolution and adaptation. However, the mechanisms underlying ecDNA biogenesis remain poorly understood. Here, using genome-wide CRISPR screening in human cells, we identified the BRCA1-A and the LIG4 complexes as key drivers of ecDNA production. Following DNA segmentation, the upstream BRCA1-A complex protects DNA ends from excessive resection, promoting end-joining for circularization. Conversely, the MRN complex, which mediates end resection and thus antagonizes the BRCA1-A complex, suppresses ecDNA formation. Downstream, LIG4 conservatively mediates ecDNA production by joining the free ends of the DNA fragments. Furthermore, ecDNA from patient tumors harbors junction sites with a LIG4 signature. Notably, disruption of either LIG4 or the BRCA1-A complex in cancer cells impairs ecDNA-mediated adaptation, hindering the development of resistance to both chemotherapy and targeted therapies. Together, our study reveals the roles of the LIG4 and BRCA1-A complexes in ecDNA biogenesis, and uncovers therapeutic targets to block ecDNA-mediated adaptation for cancer treatment.

Dedifferentiated liposarcoma (DDLPS) is a rare cancer defined by amplification of MDM2 and CDK4. Conventional chemotherapy (doxorubicin) and targeted inhibition of MDM2 and CDK4 show sporadic responses, but most tumors display primary resistance. Here, we used an unbiased approach to identify therapeutic strategies sensitizing to these DDLPS therapies. Three parallel genome-wide CRISPR-Cas9 knockout screens were conducted in DDLPS cells to sensitize to palbociclib (CDK4 inhibitor), nutlin-3a (MDM2 inhibitor) or doxorubicin. Top screen hits were validated and characterized in both in vitro and in vivo models, while clinical data were used to corroborate molecular findings. Inactivation of genes related to G1/S transition (CDK2, CKS1B, E2F3 and CCNE1) and non-homologous end-joining (NHEJ; TDP2, PRKDC and XRCC4), enhanced sensitivity to palbociclib and doxorubicin, respectively. Genetic perturbation of TDP2 or pharmacological inhibition of DNA-PKcs using peposertib synergized with prolonged administration of low-dose doxorubicin to induce cell cycle arrest and senescence, and subsequent senolytic treatment with Bcl2 inhibitor navitoclax triggered senescent cells to undergo apoptosis. Despite the amplification of MDM2, senescence was mediated by p53. Consistently, TCGA and DepMap data suggested p53 activity in DDLPS. These findings provide a rationale for targeting the NHEJ pathway to enhance the efficacy of low-dose doxorubicin in DDLPS, highlighting a potential therapeutic strategy exploiting p53-mediated cell cycle arrest and senescence. Furthermore, this study provides evidence of maintained baseline p53 activity in MDM2-amplified DDLPS.

RECQL4, a RecQ family helicase, is essential for DNA replication and genome stability. Mutations in RECQL4 cause severe human disorders yet we do not fully understand its functions, particularly regarding ATP-dependent helicase activity. To understand RECQL4's functions further, we performed a genome-wide forward genetic screen using a murine model harbouring patient-like RECQL4 mutations. We identify KLHDC3, a substrate-binding subunit of the Cullin-RING ligase E3 complex, loss as the most significant rescue allele. KLHDC3 loss restores proliferation and replication in RECQL4-deficient cells by stabilizing trace levels of a truncated RECQL4 fragment containing the N-terminal 480 amino acids, lacking the helicase and C-terminal regions. This RECQL4 fragment forms after Cre-mediated recombination of the Recql4fl allele and contains a neo-degron sequence specific for KLHDC3. Although this mechanism does not apply to human mutations, it demonstrates that minimal RECQL4 levels, without any ATPase domain/activity, are sufficient to support DNA replication. This demonstrates that RECQL4 is an essential and non-redundant regulator of DNA replication and cell viability and that this activity does not require its ATP-dependent helicase activity.

Pseudomonas aeruginosa, a ubiquitous opportunistic pathogen, coordinates virulence gene expression primarily through three quorum-sensing (QS) systems: Las, Rhl, and Pqs. Despite the functions of QS systems in P. aeruginosa being relatively well-documented, the regulatory mechanisms modulating their activities remain largely unexplored. Here, we isolated and characterized a Las-null strain, PA_HN008, which overproduces pyocyanin (PYO) at the late growth stage due to its active RhlI-RhlR-PqsE pathway. Leveraging PYO production as a phenotypic marker, we conducted a genome-wide screening for upstream QS modulators and successfully identified PA5348 (designated as HupA), the α subunit of the nucleoid-associated protein HU, as a specific activator of the Pqs system and PYO production. RNA-seq and protein-DNA interaction assays further revealed that HupA directly binds to the promoters of pqs and phz genes, thereby inducing PqsE expression and PYO production. This study demonstrates that HupA acts as a novel transcriptional factor to control QS activity and virulence gene expression in P. aeruginosa, which not only reveals the regulatory function of the HU subunit HupA on virulence factor production but also uncovers the sequence-specific DNA-binding capability of HupA for stimulating gene transcription.IMPORTANCEThe nucleoid-associated protein HU is abundant and evolutionarily conserved in bacteria, which typically binds DNA in a sequence-nonspecific manner with high affinity to abnormal DNA structures during DNA damage and then contributes to DNA compaction, replication, recombination, and other physiological functions. However, the potential involvement of HU in regulating quorum sensing (QS), a critical cell-to-cell communication system governing bacterial virulence factor production, is poorly understood. In this study, we demonstrated that the α subunit of the HU protein, namely HupA, is a key regulator that activates the Pqs system and pyocyanin production through genome-wide screening in a clinical isolate PA_HN008. This study reveals the capability of the HU subunit to selectively bind target DNA sequences and stimulate QS activity as well as virulence factor production. Our findings provide novel insights into the biochemical properties of the HU subunit and its regulatory role in bacterial virulence.

Cancer cells reprogramme translation and metabolism to fuel tumorigenesis. Here, we show that hepatocellular carcinoma (HCC) paradoxically maintains low tyrosine levels despite increased uptake and reduced metabolism, redirecting tyrosine to translation via MYC-driven upregulation of tyrosyl-tRNA synthetase 1 (YARS1) and tRNA-TyrGUA. Restricting tyrosine translation availability (RTTA) via dietary limitation, YARS1/tRNA-TyrGUA ablation, tyrosine degradation (TAL), or YARS1 inhibition (tyrosinol) disturbs this adaptation, leading to the mitigation of tumorigenesis and extension of survival. Mechanistically, RTTA reduces tyrosine codon-dependent translation of mitochondrial complex I subunit NDUFB8 and lipid regulator SCD1, causing complex I misassembly, oxidative phosphorylation failure, and lipid peroxidation-induced ferroptosis. Genome-wide CRISPR screening identifies that loss of GPX4 and BCL2 by genetic manipulation or pharmacological treatment enhances the ability of RTTA to inhibit hepatocellular carcinogenesis. Our findings establish RTTA as a therapeutic strategy targeting tyrosine dependency and highlight combinatorial targeting of translation-metabolism crosstalk and ferroptosis pathways in liver cancer.

Construction and initial validation of key gene network for progesterone resistance in endometrial cancer based on genome-wide CRISPR screening.

Patients with malignant peripheral nerve sheath tumors (MPNSTs) have poor outcomes despite multimodal treatment with surgery, radiation, and systemic therapy. The responses to radiotherapy (RT) are mixed, and the biologic mechanisms underlying this heterogeneity in the radiation response of MPNSTs are not understood. Here, we combined bulk and single-cell transcriptomics, genome-wide CRISPR interference screens, and multiplatform molecular analysis across MPNST cells, mouse allograft models, and patients' samples to understand the mediators of the radiation response. Our data revealed that MPNSTs, but not benign plexiform neurofibromas, induced a type I IFN signature that functionally mediated the radiation response. Moreover, irradiation of immunocompetent mouse MPNST allografts led to IFN-mediated T cell recruitment and activation. Both host mouse T cells and intact tumor IFN receptor signaling were required for RT's efficacy in mouse MPNST allografts. Analysis of human MPNST resection specimens demonstrated that increased microenvironmental and CD8+ T cell infiltration were associated with improved local control following RT. These results provide a preclinical rationale for combining immunomodulatory agents targeting IFN signaling to improve radiation responses in MPNSTs and potentially other soft tissue sarcomas.

Genome-wide screening of potential shell matrix proteins with low complexity regions in molluscs.

Gliomas are the most common primary brain tumors, yet the molecular circuits that drive their malignancy remain incompletely defined. Here, using an integrative, multi-dimensional approach, we aimed to pinpoint key molecular drivers having both functional and clinical relevance to disease progression and tumor aggressiveness in gliomas. Genome-wide CRISPR-Cas9 dependency screen across 70 glioma cell lines was paired with tumor aggressiveness-targeted transcriptomic differential expression and survival analyses to pinpoint critical drivers of disease progression in gliomas. Functional and gene set enrichments as well as protein-protein interaction network analyses were used to identify dominant pathways and key hub genes, followed by independent validation across external transcriptomic and proteomic datasets. Upstream regulator analyses and alternative splicing profiling were performed to nominate regulatory drivers and derive a small nuclear ribonucleoprotein D2 polypeptide (SNRPD2)-associated splicing signature. Initial screening uncovered 222 essential genes (Chronos<-1) in gliomas, 87 of which were overexpressed in tumors displaying proliferative, epithelial-mesenchymal transition, glycolytic, hypoxic, and inflammatory signatures, and were associated with poor overall survival, consistent with aggressive disease biology. These genes converged on alternative splicing regulation, proteasome function, and cell cycle, with spliceosome core component, SNRPD2 emerging as the top hub gene. High SNRPD2 expression was associated with disease aggressiveness, tumor progression, and adverse clinical outcomes. MYC was identified as a putative transcriptional driver of SNRPD2. High SNRPD2 expression was also linked to differential (oncogenic) alternative splicing of multiple cancer-associated genes, correlating with disease aggressiveness and poor clinical outcomes. These data establish SNRPD2 and its associated alternatively spliced repertoire as a central adaptive node linked to disease aggressiveness in gliomas, highlighting it as a potential therapeutic target in glioma patients.

Overcoming lentiviral delivery limitations in hard-to-transduce suspension cells for genome-wide CRISPR screening

Finding Significant Hits in Networks (FISHNET) uses prior biological knowledge, represented as gene interaction networks and gene function annotations, to identify genes that do not meet the genome-wide significance threshold but replicate, nonetheless. Its input is gene-level P-values from any source, including omicsWAS, aggregation of genome-wide association studies P-values, CRISPR screens, or differential expression analysis. It is based on the idea that genes whose P-values are low purely by chance are distributed randomly across networks and functions, so genes with suggestive P-values that cluster in densely connected subnetworks and share common functions are less likely to reflect chance and more likely to replicate. FISHNET combines network and function analysis with permutation-based P-value thresholds to identify a small set of exceptional genes that we call FISHNET genes. Applied to 11 cardiovascular risk traits, FISHNET identified 19 gene-trait relationships that missed genome-wide significance thresholds but, nonetheless, replicated in an independent cohort. The replication rate of FISHNET genes matched that of genes with lower P-values. FISHNET identified a novel association between RUNX1 expression and HDL that is supported by experimental evidence that RUNX1 promotes white fat browning, which increases HDL cholesterol levels. FISHNET also identified an association between LTB expression and BMI that is supported by experimental evidence that higher LTB expression increases BMI via activation of the LTβR pathway. Both associations failed genome-wide significance thresholds, highlighting FISHNET's ability to uncover meaningful relationships missed by traditional methods. FISHNET software is freely available at https://brentlab.github.io/fishnet/.

SPRR3 is a small, proline-rich protein that promotes cell proliferation. Overexpressed SPRR3 is associated with cancer and regulates AKT phosphorylation at serine 473. However, the specific cellular mechanisms by which SPRR3 drives proliferation are not fully understood. Using a genome-wide siRNA screen in MCF10A breast epithelial cells for decreased nucleolar number, we identified SPRR3 as a novel regulator of ribosome biogenesis. We used siRNA to deplete SPRR3 and found that it is required for transcription of the pre-ribosomal RNA (pre-rRNA), the earliest step in ribosome biogenesis. Furthermore, this reduction in pre-rRNA transcription triggers the nucleolar stress response (increased TP53 protein and CDKN1A mRNA levels) in both MCF10A cells and A549 lung carcinoma cells. Finally, SPRR3 depletion reduces AKT phosphorylation in both cell lines and correlates with lower levels of the RNAPI catalytic subunit POLR1A. In sum, we establish a new role for the non-nucleolar protein SPRR3 in ribosome biogenesis, specifically pre-rRNA transcription, via its ability to facilitate phosphorylation of AKT.

Genome-wide screen identifies genes conferring sensitivity to the pollutant bisphenol A in Saccharomyces cerevisiae.

A genome-wide knockout screen identified members of the SLC25 family of mitochondrial carrier proteins as important regulators of the rate of de novo mitochondrial protein synthesis. To elucidate this relationship, we generated human cell knockouts for SLC25A25, SLC25A44, SLC25A45, and SLC25A48, which have been shown to exchange adenosine triphosphate-magnesium (ATP-Mg) and phosphate, branched-chain amino acids, methylated basic amino acids, and choline, respectively. Multiomic and functional analyses identified that these four carriers are crucial for mitochondrial translation, biogenesis and function of the oxidative phosphorylation system, as well as mitochondrial morphology. Thermostability screens showed that SLC25A48 is specifically stabilized by choline, and changes in the mitochondrial metabolome and lipidome indicated defects in choline biosynthetic pathways and remodeling of mitochondrial membranes, both consistent with SLC25A48 being a choline transporter. These results highlight the essential roles of specific SLC25 transporters in maintaining mitochondrial structure and function and show that impaired transport of branched-chain amino acids, methylated basic amino acids, ATP-Mg, and choline affects mitochondrial translation.

Phosphatidylinositol-4-phosphate 5-kinase (PI4P5K/PIP5K), a core regulator of phosphatidylinositol signaling pathways, exerts critical regulatory functions in plant cellular signaling networks and developmental processes, and stress response through its kinase activity. However, its functions in cotton are little reported. To comprehensively analyze the PI4P5K gene family in cotton, Genome-wide identification was performed to identify cotton PI4P5K family members and analyzed their gene structure, chromosome distribution, systematic evolution and collinearity, and transcript profiles under salt stress. Moreover, we studied function of GhPI4P5K-D04-2 by transforming it into Arabidopsis and using virus-induced gene silencing (VIGS) system. In this study, we identified 146 PI4P5K family members from four cotton species (G. arboreum, G. raimondii, G. barbadense and G. hirsutum) via genome-wide screening, which were phylogenetically divided into three distinct subgroups. Structural domain analysis revealed conserved PIPKc superfamily domain in all proteins, while chromosomal mapping demonstrated syntenic distribution patterns between subgenomes A and D. Integrated transcriptomic and qRT-PCR analyses uncovered GhPI4P5K-D04-2 as a salt stress-responsive gene. Functional characterization assays demonstrated that overexpressing the GhPI4P5K-D04-2 gene exhibited enhanced tolerance to salt stress in Arabidopsis, whereas cotton plants with GhPI4P5K-D04-2 knockdown via VIGS showed increased sensitivity to salt stress. In conclusion, the findings in this study about PI4P5K gene family and GhPI4P5K-D04-2 gene could lay a foundation for future studies of the biological functions of the cotton PI4P5K genes, and provide a theoretical basis for targeting improvement of cotton salt resistance through genetic manipulation of PIPK pathway.