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Ferroptosis, a form of regulated cell death that is driven by iron-dependent phospholipid peroxidation, has been implicated in multiple diseases, including cancer 1 , 2 – 3 , degenerative disorders 4 and organ ischaemia–reperfusion injury (IRI) 5 , 6 . Here, using genome-wide CRISPR–Cas9 screening, we identified that the enzymes involved in distal cholesterol biosynthesis have pivotal yet opposing roles in regulating ferroptosis through dictating the level of 7-dehydrocholesterol (7-DHC)—an intermediate metabolite of distal cholesterol biosynthesis that is synthesized by sterol C5-desaturase (SC5D) and metabolized by 7-DHC reductase (DHCR7) for cholesterol synthesis. We found that the pathway components, including MSMO1 , CYP51A1 , EBP and SC5D , function as potential suppressors of ferroptosis, whereas DHCR7 functions as a pro-ferroptotic gene. Mechanistically, 7-DHC dictates ferroptosis surveillance by using the conjugated diene to exert its anti-phospholipid autoxidation function and shields plasma and mitochondria membranes from phospholipid autoxidation. Importantly, blocking the biosynthesis of endogenous 7-DHC by pharmacological targeting of EBP induces ferroptosis and inhibits tumour growth, whereas increasing the 7-DHC level by inhibiting DHCR7 effectively promotes cancer metastasis and attenuates the progression of kidney IRI, supporting a critical function of this axis in vivo. In conclusion, our data reveal a role of 7-DHC as a natural anti-ferroptotic metabolite and suggest that pharmacological manipulation of 7-DHC levels is a promising therapeutic strategy for cancer and IRI. 7-Dehydrocholesterol (7-DHC) is a natural anti-ferroptotic metabolite and pharmacological manipulation of 7-DHC levels shows promise as a therapeutic strategy for cancer and ischaemia–reperfusion injury.

The creation of genome-wide libraries for CRISPR knockout (CRISPRko), interference (CRISPRi), and activation (CRISPRa) has enabled the systematic interrogation of gene function. Here, we show that our recently-described CRISPRko library (Brunello) is more effective than previously published libraries at distinguishing essential and non-essential genes, providing approximately the same perturbation-level performance improvement over GeCKO libraries as GeCKO provided over RNAi. Additionally, we present genome-wide libraries for CRISPRi (Dolcetto) and CRISPRa (Calabrese), and show in negative selection screens that Dolcetto, with fewer sgRNAs per gene, outperforms existing CRISPRi libraries and achieves comparable performance to CRISPRko in detecting essential genes. We also perform positive selection CRISPRa screens and demonstrate that Calabrese outperforms the SAM approach at identifying vemurafenib resistance genes. We further compare CRISPRa to genome-scale libraries of open reading frames (ORFs). Together, these libraries represent a suite of genome-wide tools to efficiently interrogate gene function with multiple modalities. Genome-wide libraries for CRISPR knockout, interference, and activation have allowed the systemic interrogation of gene function. Here, the authors evaluate the Brunello CRISPRko library and introduce Dolcetto and Calabrese for CRISPRi and CRISPRa, respectively.

Adeno-associated virus (AAV) forms the basis for several commercial gene therapy products and for countless gene transfer vectors derived from natural or synthetic viral isolates that are under intense preclinical evaluation. Here, we report a versatile pipeline that enables the direct side-by-side comparison of pre-selected AAV capsids in high-throughput and in the same animal, by combining DNA/RNA barcoding with multiplexed next-generation sequencing. For validation, we create three independent libraries comprising 183 different AAV variants including widely used benchmarks and screened them in all major tissues in adult mice. Thereby, we discover a peptide-displaying AAV9 mutant called AAVMYO that exhibits superior efficiency and specificity in the musculature including skeletal muscle, heart and diaphragm following peripheral delivery, and that holds great potential for muscle gene therapy. Our comprehensive methodology is compatible with any capsids, targets and species, and will thus facilitate and accelerate the stratification of optimal AAV vectors for human gene therapy. Adeno-associated virus is the basis of many gene therapies and gene transfer vectors. Here the authors report a pipeline to enable side-by-side comparison of pre-selected capsids in a high throughput manner.

N6-methyladenosine (m 6 A) serves as the most common and conserved internal transcriptional modification. However, the roles of m 6 A on cervical cancer (CC) tumorigenesis are still unclear. Here, results indicated that METTL3 was significantly upregulated in CC tissue and cells, which was closely correlated with the lymph node metastasis and poor prognosis of CC patients. MeRIP-Seq analysis revealed the m 6 A profiles in CC cells. Functionally, METTL3 promoted the proliferation and Warburg effect (aerobic glycolysis) of CC cells. Mechanistically, METTL3 targeted the 3’-Untranslated Region (3’-UTR) of hexokinase 2 (HK2) mRNA. Moreover, METTL3 recruited YTHDF1, a m 6 A reader, to enhance HK2 stability. These findings demonstrated that METTL3 enhanced the HK2 stability through YTHDF1-mediated m 6 A modification, thereby promoting the Warburg effect of CC, which might promote a novel insight for the CC treatment.

Metabolic changes in T cells can affect the genomic methylation status of key transcription factors and regulate the fate decision between induced regulatory T cells and T helper 17 cells. (DING, Sheng , 23475; Biological Sciences - Letter) Sheng Ding and colleagues address the question of how metabolic changes in T cells can affect the genomic methylation status of key transcription factors and regulate the fate decision between regulatory T and T H 17 T cells. This study demonstrates the ability of a small molecule inhibitor to target a specific metabolic pathway, a finding which may lead to the development of novel therapeutics against T H 17 mediated auto-immune diseases. Metabolism has been shown to integrate with epigenetics and transcription to modulate cell fate and function 1 , 2 , 3 . Beyond meeting the bioenergetic and biosynthetic demands of T-cell differentiation 4 , 5 , 6 , 7 , 8 , whether metabolism might control T-cell fate by an epigenetic mechanism is unclear. Here, through the discovery and mechanistic characterization of a small molecule, (aminooxy)acetic acid, that reprograms the differentiation of T helper 17 (T H 17) cells towards induced regulatory T (iT reg ) cells, we show that increased transamination, mainly catalysed by GOT1, leads to increased levels of 2-hydroxyglutarate in differentiating T H 17 cells. The accumulation of 2-hydroxyglutarate resulted in hypermethylation of the Foxp3 gene locus and inhibited Foxp3 transcription, which is essential for fate determination towards T H 17 cells. Inhibition of the conversion of glutamate to α-ketoglutaric acid prevented the production of 2-hydroxyglutarate, reduced methylation of the Foxp3 gene locus, and increased Foxp3 expression. This consequently blocked the differentiation of T H 17 cells by antagonizing the function of transcription factor RORγt and promoted polarization into iT reg cells. Selective inhibition of GOT1 with (aminooxy)acetic acid ameliorated experimental autoimmune encephalomyelitis in a therapeutic mouse model by regulating the balance between T H 17 and iT reg cells. Targeting a glutamate-dependent metabolic pathway thus represents a new strategy for developing therapeutic agents against T H 17-mediated autoimmune diseases.

Targeting exosome biogenesis and release may have potential clinical implications for cancer therapy. Herein, we have optimized a quantitative high throughput screen (qHTS) assay to identify compounds that modulate exosome biogenesis and/or release by aggressive prostate cancer (PCa) CD63-GFP-expressing C4-2B cells. A total of 4,580 compounds were screened from the LOPAC library (a collection of 1,280 pharmacologically active compounds) and the NPC library (NCGC collection of 3,300 compounds approved for clinical use). Twenty-two compounds were found to be either potent activators or inhibitors of intracellular GFP signal in the CD63-GFP-expressing C4-2B cells. The activity of lead compounds in modulating the secretion of exosomes was validated by a tunable resistive pulse sensing (TRPS) system (qNano-IZON) and flow cytometry. The mechanism of action of the lead compounds in modulating exosome biogenesis and/or secretion were delineated by immunoblot analysis of protein markers of the endosomal sorting complex required for transport (ESCRT)-dependent and ESCRT-independent pathways. The lead compounds tipifarnib, neticonazole, climbazole, ketoconazole, and triademenol were validated as potent inhibitors and sitafloxacin, forskolin, SB218795, fenoterol, nitrefazole and pentetrazol as activators of exosome biogenesis and/or secretion in PC cells. Our findings implicate the potential utility of drug-repurposing as novel adjunct therapeutic strategies in advanced cancer.

Epigenetic dysregulation is a defining feature of tumorigenesis that is implicated in immune escape 1 , 2 . Here, to identify factors that modulate the immune sensitivity of cancer cells, we performed in vivo CRISPR–Cas9 screens targeting 936 chromatin regulators in mouse tumour models treated with immune checkpoint blockade. We identified the H3K9 methyltransferase SETDB1 and other members of the HUSH and KAP1 complexes as mediators of immune escape 3 – 5 . We also found that amplification of SETDB1 (1q21.3) in human tumours is associated with immune exclusion and resistance to immune checkpoint blockade. SETDB1 represses broad domains, primarily within the open genome compartment. These domains are enriched for transposable elements (TEs) and immune clusters associated with segmental duplication events, a central mechanism of genome evolution 6 . SETDB1 loss derepresses latent TE-derived regulatory elements, immunostimulatory genes, and TE-encoded retroviral antigens in these regions, and triggers TE-specific cytotoxic T cell responses in vivo. Our study establishes SETDB1 as an epigenetic checkpoint that suppresses tumour-intrinsic immunogenicity, and thus represents a candidate target for immunotherapy. A CRISPR–Cas9 screen of chromatin regulators in mouse tumour models treated with immune checkpoint blockade identifies SETDB1 as an epigenetic checkpoint protein that suppresses tumour-intrinsic immunogenicity.

Transfer-RNA-derived small RNAs (tsRNAs; also called tRNA-derived fragments) are an abundant class of small non-coding RNAs whose biological roles are not well understood. Here we show that inhibition of a specific tsRNA, LeuCAG3′tsRNA, induces apoptosis in rapidly dividing cells in vitro and in a patient-derived orthotopic hepatocellular carcinoma model in mice. This tsRNA binds at least two ribosomal protein mRNAs ( RPS28 and RPS15 ) to enhance their translation. A decrease in translation of RPS28 mRNA blocks pre-18S ribosomal RNA processing, resulting in a reduction in the number of 40S ribosomal subunits. These data establish a post-transcriptional mechanism that can fine-tune gene expression during different physiological states and provide a potential new target for treating cancer. A 22-nucleotide fragment of a transfer RNA regulates translation by binding to the mRNA of a ribosomal protein and increasing its expression, and downregulation of the fragment in patient-derived liver tumour cells reduces tumour growth in mice. An anticancer tRNA fragment The functional roles of small RNA fragments derived from tRNAs are not well known, but evidence is growing that some play a part in various cellular processes. Mark Kay and colleagues show that a 22-nucleotide fragment from the 3′ end of leucine tRNA can regulate translation. The fragment binds to the mRNA of a ribosomal protein to upregulate its expression. When this interaction is suppressed in human cells in culture, cell death occurs. Decreasing the levels of the tRNA fragment with an antisense oligonucleotide can slow the growth of liver tumours in mice. Technologies aimed at reducing expression of this tRNA fragment might have utility in treating cancer.

Chromatin-binding proteins are critical regulators of cell state in haematopoiesis 1 , 2 . Acute leukaemias driven by rearrangement of the mixed lineage leukaemia 1 gene ( KMT2A r) or mutation of the nucleophosmin gene ( NPM1 ) require the chromatin adapter protein menin, encoded by the MEN1 gene, to sustain aberrant leukaemogenic gene expression programs 3 – 5 . In a phase 1 first-in-human clinical trial, the menin inhibitor revumenib, which is designed to disrupt the menin–MLL1 interaction, induced clinical responses in patients with leukaemia with KMT2A r or mutated NPM1 (ref. 6 ). Here we identified somatic mutations in MEN1 at the revumenib–menin interface in patients with acquired resistance to menin inhibition. Consistent with the genetic data in patients, inhibitor–menin interface mutations represent a conserved mechanism of therapeutic resistance in xenograft models and in an unbiased base-editor screen. These mutants attenuate drug–target binding by generating structural perturbations that impact small-molecule binding but not the interaction with the natural ligand MLL1, and prevent inhibitor-induced eviction of menin and MLL1 from chromatin. To our knowledge, this study is the first to demonstrate that a chromatin-targeting therapeutic drug exerts sufficient selection pressure in patients to drive the evolution of escape mutants that lead to sustained chromatin occupancy, suggesting a common mechanism of therapeutic resistance. Somatic mutations in MEN1 are identified in patients with leukaemia treated with a novel chromatin-targeting therapy, and the mechanism by which these mutations mediate therapeutic resistance is characterized.

The pro-inflammatory state of macrophages, underpinned by their metabolic condition, is essentially affecting their capacity of combating tumor cells. Here we find, via a pooled metabolic gene knockout CRISPR screen that KEAP1 and ACOD1 are strong regulators of the pro-inflammatory state in macrophages. We show that ACOD1 knockout macrophages, generated in our induced pluripotent stem cell-derived CAR-macrophage (CAR-iMAC) platform, are strongly and persistently polarized toward the pro-inflammatory state, which manifests in increased reactive oxygen species (ROS) production, more potent phagocytosis and enhanced cytotoxic functions against cancer cells in vitro. In ovarian or pancreatic cancer mouse models, ACOD1-depleted CAR-iMACs exhibit enhanced capacity in repressing tumors, leading to increased survival. In addition, combining ACOD1-depleted CAR-iMACs with immune checkpoint inhibitors (ICI), such as anti-CD47 or anti-PD1 antibodies, result in even stronger tumor suppressing effect. Mechanistically, the depletion of ACOD1 reduces levels of the immuno-metabolite itaconate, allowing KEAP1 to prevent NRF2 from entering the nucleus to activate an anti-inflammatory program. This study thus lays down the proof of principle for targeting ACOD1 in myeloid cells for cancer immunotherapy and introduces metabolically engineered human iPSC-derived CAR-iMACs cells with enhanced polarization and anti-tumor functions in adoptive cell transfer therapies. The functional-metabolic state of macrophages fundamentally influences the tumour microenvironment, making adoptive cell therapy with pro-inflammatory macrophages an attractive anti-tumour approach. Here authors introduce pluripotent stem cell-derived CAR-macrophage that are depleted of ACOD1, an essential gene in itaconate metabolism, which reprograms them to a pro-inflammatory state enabling enhanced anti-tumour function.