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Tumourigenesis and cancer progression require enhanced global protein translation1-3. Such enhanced translation is caused by oncogenic and tumour-suppressive events that drive the synthesis and activity of translational machinery4,5. Here we report the surprising observation that leucyl-tRNA synthetase (LARS) becomes repressed during mammary cell transformation and in human breast cancer. Monoallelic genetic deletion of LARS in mouse mammary glands enhanced breast cancer tumour formation and proliferation. LARS repression reduced the abundance of select leucine tRNA isoacceptors, leading to impaired leucine codon-dependent translation of growth suppressive genes, including epithelial membrane protein 3 (EMP3) and gamma-glutamyltransferase 5 (GGT5). Our findings uncover a tumour-suppressive tRNA synthetase and reveal that dynamic repression of a specific tRNA synthetase-along with its downstream cognate tRNAs-elicits a downstream codon-biased translational gene network response that enhances breast tumour formation and growth.

Inborn errors of human IFN-γ-dependent macrophagic immunity underlie mycobacterial diseases, whereas inborn errors of IFN-α/β-dependent intrinsic immunity underlie viral diseases. Both types of IFNs induce the transcription factor IRF1. We describe unrelated children with inherited complete IRF1 deficiency and early-onset, multiple, life-threatening diseases caused by weakly virulent mycobacteria and related intramacrophagic pathogens. These children have no history of severe viral disease, despite exposure to many viruses, including SARS-CoV-2, which is life-threatening in individuals with impaired IFN-α/β immunity. In leukocytes or fibroblasts stimulated in vitro, IRF1-dependent responses to IFN-γ are, both quantitatively and qualitatively, much stronger than those to IFN-α/β. Moreover, IRF1-deficient mononuclear phagocytes do not control mycobacteria and related pathogens normally when stimulated with IFN-γ. By contrast, IFN-α/β-dependent intrinsic immunity to nine viruses, including SARS-CoV-2, is almost normal in IRF1-deficient fibroblasts. Human IRF1 is essential for IFN-γ-dependent macrophagic immunity to mycobacteria, but largely redundant for IFN-α/β-dependent antiviral immunity.

Histone H3 monoaminylations at glutamine(Q) 5 represent an important family of epigenetic markers in neurons that play critical roles in the mediation of permissive gene expression (1, 2). We previously demonstrated that H3Q5 serotonylation(ser) and dopaminylation(dop) are catalyzed by the Transglutaminase 2 (TGM2) enzyme and alter both local and global chromatin states (3, 4). Here, we found that TGM2 additionally functions as an “eraser” of H3 monoaminylations that is capable of “re-writing” these epigenetic marks in cells, including a new class of this modification, H3Q5 histaminylation(his), which displays dynamic diurnal expression in brain and contributes to neural rhythmicity. We found that H3Q5his inhibits binding of the MLL1 complex to the H3 N-terminus and attenuates its methyltransferase activity on H3 lysine(K) 4. We determined that H3Q5 monoaminylation dynamics are dictated by local monoamine concentrations, which are utilized by TGM2. Taken together, we present here a novel mechanism through which a single chromatin regulatory enzyme is capable of sensing chemical microenvironments to affect the epigenetic states of cells.One sentence summaryA single enzyme, TGM2, bidirectionally controls H3 monoaminylation dynamics, which, in turn, facilitate neural rhythmicity.

The Dietary Approach to Stop Hypertension (DASH) diet is a proven intervention to treat hypertension, yet its mechanism is not clearly known. We investigated the change in protein abundance patterns in urine extracellular vesicles (uEV’s) following DASH diet implementation.A pilot study was carried out to compare uEVs isolated using three different methods: a low centrifugation (P20), high centrifugation (P100), and a combination of both (P20 and P100). Uromodulin was removed by size exclusion chromatography and low ionic strength washing. Mass spectrometry analysis identified 1,593 proteins in the combined fraction (P20+P100), 1434 in the P20 fraction and 1229 in the P100 fraction. The combined fraction was chosen for further analysis. Statistical analysis was carried out using R and Limma to identify all proteins that changed before and during 11 days of DASH intervention (p < 0.05) as well as between individual timepoints.Nine hypertensive volunteers were admitted for a 14-day supervised transition from American style diet to DASH diet. First-void urine was collected on days 0, 5, 11 for uEV processing. In total, 1800 proteins were identified across all 27 DASH samples with 22 proteins upregulated and 25 down regulated between day 0 and both days 5 and 11. These included increased abundance of SLC12A3 (NCC) and reduced abundance of Aquaporine 2.These changes could explain the increased urine volume and reduced sodium reabsorption that lead to blood pressure reduction following consumption of the high potassium and low sodium DASH diet. uEVs may serve as a surrogate to a more invasive procedure.Translational StatementMyriad studies have characterized blood pressure reduction following DASH diet implementation, yet its precise mechanism is unclear. Here, we demonstrate for the first time the effect of DASH nutritional changes, on kidney ion channel composition using proteomic analysis of urinary EVs. Using this innovative tool as a substitute for an invasive procedure, we show that the expression of Aquaporine2 increases and NCC decreases in response to DASH which may account for its antihypertensive effect. Our results indicate that urinary EV are a potential biomarker for DASH compliance, and targeting aquaporine2 may be an effective innovative therapeutic strategy for blood pressure reduction.

Epitranscriptomic mechanisms linking tRNA function and the brain proteome to cognition and complex behaviors are not well described. Here, we report bi-directional changes in depression-related behaviors after genetic disruption of neuronal tRNA cytosine methylation, including conditional ablation and transgene-derived overexpression of Nsun2 in the mouse prefrontal cortex (PFC). Neuronal Nsun2-deficiency was associated with a decrease in tRNA m5C levels, resulting in deficits in expression of 70% of tRNAGly isodecoders. Altogether, 1488/5820 proteins changed upon neuronal Nsun2-deficiency, in conjunction with glycine codon-specific defects in translational efficiencies. Loss of Gly-rich proteins critical for glutamatergic neurotransmission was associated with impaired synaptic signaling at PFC pyramidal neurons and defective contextual fear memory. Changes in the neuronal translatome were also associated with a 146% increase in glycine biosynthesis. These findings highlight the methylation sensitivity of glycinergic tRNAs in the adult PFC. Furthermore, they link synaptic plasticity and complex behaviors to epitranscriptomic modifications of cognate tRNAs and the proteomic homeostasis associated with specific amino acids., The link between tRNA modifications, protein translation, and behavior is unclear. Here, the authors show that neuronal Nsun2 deficiency results in codon-specific epitranscriptomic changes of Gly-tRNAs and proteomic changes affecting synaptic signaling and behavior in mice.

Peptidoglycan is a vital component of the bacterial cell wall, and its dynamic remodeling by NlpC/p60 hydrolases is crucial for proper cell division and survival. Beyond these essential functions, we previously discovered that Enterococcus species express and secrete the NlpC/p60 hydrolase-secreted antigen A (SagA), whose catalytic activity can modulate host immune responses in animal models. However, the localization and peptidoglycan hydrolase activity of SagA in Enterococcus was still unclear. In this study, we show that SagA contributes to a triseptal structure in dividing cells of enterococci and localizes to sites of cell division through its N-terminal coiled-coil domain. Using molecular modeling and site-directed mutagenesis, we identify amino acid residues within the SagA-NlpC/p60 domain that are crucial for catalytic activity and potential substrate binding. Notably, these studies revealed that SagA may function via a catalytic Cys-His dyad instead of the predicted Cys-His-His triad, which is conserved in SagA orthologs from other Enterococcus species. Our results provide key additional insight into peptidoglycan remodeling in Enterococcus by SagA NlpC/p60 hydrolases.

Coessentiality mapping has been useful to systematically cluster genes into biological pathways and identify gene functions1-3. Here, using the debiased sparse partial correlation (DSPC) method3, we construct a functional coessentiality map for cellular metabolic processes across human cancer cell lines. This analysis reveals 35 modules associated with known metabolic pathways and further assigns metabolic functions to unknown genes. In particular, we identify C12orf49 as an essential regulator of cholesterol and fatty acid metabolism in mammalian cells. Mechanistically, C12orf49 localizes to the Golgi, binds membrane-bound transcription factor peptidase, site 1 (MBTPS1, site 1 protease) and is necessary for the cleavage of its substrates, including sterol regulatory element binding protein (SREBP) transcription factors. This function depends on the evolutionarily conserved uncharacterized domain (DUF2054) and promotes cell proliferation under cholesterol depletion. Notably, c12orf49 depletion in zebrafish blocks dietary lipid clearance in vivo, mimicking the phenotype of mbtps1 mutants. Finally, in an electronic health record (EHR)-linked DNA biobank, C12orf49 is associated with hyperlipidaemia through phenome analysis. Altogether, our findings reveal a conserved role for C12orf49 in cholesterol and lipid homeostasis and provide a platform to identify unknown components of other metabolic pathways.

More than a normal neurotransmitter The molecular mechanisms underlying the persistence of addiction remain largely unclear. Lepack et al. found that, with cocaine exposure, there is an intracellular accumulation of dopamine in neurons of a brain region called the ventral tegmental area (see the Perspective by Girault). Dopamine associates with chromatin to initiate a previously unknown form of epigenetic regulation called dopaminylation. This modification has an impact on ventral tegmental area function and, consequently, on dopaminergic action potentials. The result is aberrant dopamine signaling in the ventral striatum during periods of drug seeking. Science , this issue p. 197 ; see also p. 134

SUMMARYFemale Aedes aegypti mosquitoes impose a severe global public health burden as vectors of multiple viral pathogens. Under optimal environmental conditions, Aedes aegypti females have access to human hosts that provide blood proteins for egg development, conspecific males that provide sperm for fertilization, and freshwater that serves as an egg-laying substrate suitable for offspring survival. As global temperatures rise, Aedes aegypti females are faced with climate challenges like intense droughts and intermittent precipitation, which create unpredictable, suboptimal conditions for egg-laying. Here we show that under drought-like conditions simulated in the laboratory, females retain mature eggs in their ovaries for extended periods, while maintaining the viability of these eggs until they can be laid in freshwater. Using transcriptomic and proteomic profiling of Aedes aegypti ovaries, we identify two previously uncharacterized genes named tweedledee and tweedledum, each encoding a small, secreted protein that both show ovary-enriched, temporally-restricted expression during egg retention. These genes are mosquito-specific, linked within a syntenic locus, and rapidly evolving under positive selection, raising the possibility that they serve an adaptive function. CRISPR-Cas9 deletion of both tweedledee and tweedledum demonstrates that they are specifically required for extended retention of viable eggs. These results highlight an elegant example of taxon-restricted genes at the heart of an important adaptation that equips Aedes aegypti females with “insurance” to flexibly extend their reproductive schedule without losing reproductive capacity, thus allowing this species to exploit unpredictable habitats in a changing world.

In fibrolamellar hepatocellular carcinoma (FLC), hyperammonemic encephalopathy is a common occurrence and occasionally causes death. Using mass spectrometry, we quantitatively analyzed the proteomes of FLC patient’s tumor and adjacent normal, to find pathways that are changed in FLC. These data identified multiple proteins that were altered in the proteome, among these, enzymes involved in metabolism of ammonia. These results were confirmed with immunofluorescence demonstrating that these alterations occur in all tumor cells. These results suggest that FLC cells have defects in the two primary ammonia detoxification pathways in the liver, which are responsible for detoxification of 70% of the ammonia in the body: 1) consumption of ammonia by glutamine synthetase (GLUL), and 2) addition of ammonia by ornithine carbamoyltransferase (OTC) to the urea cycle. Additionally, they also generate extra ammonia because of upregulation of glutaminase (GLS). This was tested with a targeted metabolomics of the reactants and products of these enzymes. The results were consistent with both a loss of the two pathways for consumption of ammonia activation of a pathway for generating ammonia. This production of ammonia is consistent with the observation that surgical resection of fibrolamellar reduces the ammonia in patients. All FLC patients with hyperammonemic encephalopathy and documented urine test results showed increased urinary orotic acid, evidence of blockage of the OTC pathway. This study implies that hyperammonemic encephalopathy in FLC may require alternatives to commonly used therapies. Citation Format: Mahsa Shirani, Solomon Levin, Michael D. Tomasini, James Knox, Bassem Shebl, David Requena, Jackson Clark, Søren Heissel, Hanan Alwaseem, Rodrigo Surjan, Ron Lahasky, Henrik Molina, Barbara Lyons, Rachael D. Migler, Philip Coffino, Sanford M. Simon. Urea cycle metabolism is disturbed in Fibrolamellar carcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6729.

Immune checkpoint blockade therapy has revolutionized cancer care, including the treatment of advanced metastatic disease. However, most patients derive little or no clinical benefit from these therapies and many cancer types are notoriously non-responsive. Motivated by (1) the correlation between tumor neoantigen abundance and anti-tumor immunity and (2) that most cancers are characterized by widespread dysregulation of RNA processing, we reasoned that pharmacologic modulation of RNA splicing might increase cancer cell immunogenicity via the generation of splicing-derived neoantigens. We demonstrated that two compounds which modulate RNA splicing via distinct mechanisms, inhibited tumor growth and enhanced response to immune checkpoint blockade in a manner dependent on host T cells and peptides presented on tumor MHC class I. Critical for their clinical translatability, therapeutic doses of splicing inhibitors were non-toxic, tolerated by the host immune system, and did not affect T cell activation, proliferation, and anti-cancer killing activities. Mechanistically, splicing modulation induced stereotyped, dose-dependent “splicing failure” — dramatic intron retention, alternative exon skipping, etc. — that was consistent across multiple mouse and human tumor types. By combining RNA-seq-based peptide predictions and mass spectrometry of the MHC I-bound immunopeptidome, we identified drug-induced, splicing-derived peptides that promote the expansion of antigen-specific CD8+ T cells and trigger anti-tumor T cell responses in vivo. These data definitively identify splicing modulation as an untapped source of immunogenic peptides and provide a means to enhance response to checkpoint blockade that is readily translatable to the clinic. Citation Format: James D. Thomas, Sydney X. Lu, Emma De Neef, Erich Sabio, Benoit Rousseau, Mathieu Gigoux, David A. Knorr, Benjamin Greenbaum, Yuval Elhanati, Simon J. Hogg, Andrew Chow, Arnab Ghosh, Abigail Xie, Dmitriy Zamarin, Daniel Cui, Caroline Erickson, Michael Singer, Hana Cho, Eric Wang, Bin Lu, Benjamin H. Durham, Harshal Shah, Diego Chowell, Austin M. Gabel, Yudao Shen, Jing Liu, Jian Jin, Matthew C. Rhodes, Richard E. Taylor, Henrik Molina, Jedd D. Wolchok, Taha Merghoub, Luis A. Diaz Jr, Omar Abdel-Wahab, Robert K. Bradley. Pharmacologic modulation of RNA splicing enhances anti-tumor immunity. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5742.

73 p.-5 fig.-1 tab.-11 fig supl.-2 vid. supl., Taxanes are widely used cancer chemotherapeutics. However, intrinsic resistance limits their efficacy without any actionable resistance mechanism. We have discovered a microtubule (MT) plus-end-binding CLIP-170 protein variant, hereafter CLIP-170S, which we found enriched in taxane-resistant cell lines and patient samples. CLIP-170S lacks the first Cap-Gly motif, forms longer comets, and impairs taxane access to its MT luminal binding site. CLIP-170S knockdown reversed taxane resistance in cells and xenografts, whereas its re-expression led to resistance, suggesting causation. Using a computational approach in conjunction with the connectivity map, we unexpectedly discovered that Imatinib was predicted to reverse CLIP-170S-mediated taxane resistance. Indeed, Imatinib treatment selectively depleted CLIP-170S, thus completely reversing taxane resistance. Other RTK inhibitors also depleted CLIP-170S, suggesting a class effect. Herein, we identify CLIP-170S as a clinically prevalent variant that confers taxane resistance, whereas the discovery of Imatinib as a CLIP-170S inhibitor provides novel therapeutic opportunities for future trials., This work was supported in part by the NIH T32 training grant 5T32CA062948 (to K.K.), the NIH T32 training grant 5T32CA062948 (to G.G.), the NIH T32 training grant 5T32CA203702 (to U.D.C.) by Clinical and Translational Science Center at Weill Cornell Medicine NIH/NCATS grant ULTR00457 (to G.G.), the NIH/NCI R01CA228512 (to P.G., M.A.S., and O.E.), NIH/NCI R21 CA216800 (to P.G.), NIH/NCI R01 CA179100 (to P.G.), DoD PC180637 (P.G., A.R.C.), and by the Ministerio de Economía y Competitividad grant BFU2016-75319-R and European Union H2020-MSCA-ITN-ETN/0582 ITN TUBINTRAIN (awarded to J.F.D.). D.S. was supported by the Intramural Research Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development, NIH. Y.L. and M.A.S. were supported, in part, by funds from the Clinical and Translational Science Center (CTSC), National Center for Advancing Translational Sciences (NCATS) of the National Institutes of Health, award # UL1-TR002384-01.

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The development of effective therapies against brain metastasis is currently hindered by limitations in our understanding of the molecular mechanisms driving it. Here we define the contributions of tumour-secreted exosomes to brain metastatic colonization and demonstrate that pre-conditioning the brain microenvironment with exosomes from brain metastatic cells enhances cancer cell outgrowth. Proteomic analysis identified cell migration-inducing and hyaluronan-binding protein (CEMIP) as elevated in exosomes from brain metastatic but not lung or bone metastatic cells. CEMIP depletion in tumour cells impaired brain metastasis, disrupting invasion and tumour cell association with the brain vasculature, phenotypes rescued by pre-conditioning the brain microenvironment with CEMIP+ exosomes. Moreover, uptake of CEMIP+ exosomes by brain endothelial and microglial cells induced endothelial cell branching and inflammation in the perivascular niche by upregulating the pro-inflammatory cytokines encoded by Ptgs2, Tnf and Ccl/Cxcl, known to promote brain vascular remodelling and metastasis. CEMIP was elevated in tumour tissues and exosomes from patients with brain metastasis and predicted brain metastasis progression and patient survival. Collectively, our findings suggest that targeting exosomal CEMIP could constitute a future avenue for the prevention and treatment of brain metastasis.

Heterozygous in-frame mutations in coding regions of human STAT3 underlie the only known autosomal dominant form of hyper IgE syndrome (AD HIES). About 5% of familial cases remain unexplained. The mutant proteins are loss-of-function and dominant-negative when tested following overproduction in recipient cells. However, the production of mutant proteins has not been detected and quantified in the cells of heterozygous patients. We report a deep intronic heterozygous STAT3 mutation, c.1282-89C>T, in 7 relatives with AD HIES. This mutation creates a new exon in the STAT3 complementary DNA, which, when overexpressed, generates a mutant STAT3 protein (D427ins17) that is loss-of-function and dominant-negative in terms of tyrosine phosphorylation, DNA binding, and transcriptional activity. In immortalized B cells from these patients, the D427ins17 protein was 2 kDa larger and 4-fold less abundant than wild-type STAT3, on mass spectrometry. The patients’ primary B and T lymphocytes responded poorly to STAT3-dependent cytokines. These findings are reminiscent of the impaired responses of leukocytes from other patients with AD HIES due to typical STAT3 coding mutations, providing further evidence for the dominance of the mutant intronic allele. These findings highlight the importance of sequencing STAT3 introns in patients with HIES without candidate variants in coding regions and essential splice sites. They also show that AD HIES-causing STAT3 mutant alleles can be dominant-negative even if the encoded protein is produced in significantly smaller amounts than wild-type STAT3.

Chemical modifications of histones can mediate diverse DNA-templated processes, including gene transcription1–3. Here we provide evidence for a class of histone post-translational modification, serotonylation of glutamine, which occurs at position 5 (Q5ser) on histone H3 in organisms that produce serotonin (also known as 5-hydroxytryptamine (5-HT)). We demonstrate that tissue transglutaminase 2 can serotonylate histone H3 tri-methylated lysine 4 (H3K4me3)-marked nucleosomes, resulting in the presence of combinatorial H3K4me3Q5ser in vivo. H3K4me3Q5ser displays a ubiquitous pattern of tissue expression in mammals, with enrichment observed in brain and gut, two organ systems responsible for the bulk of 5-HT production. Genome-wide analyses of human serotonergic neurons, developing mouse brain and cultured serotonergic cells indicate that H3K4me3Q5ser nucleosomes are enriched in euchromatin, are sensitive to cellular differentiation and correlate with permissive gene expression, phenomena that are linked to the potentiation of TFIID4–6 interactions with H3K4me3. Cells that ectopically express a H3 mutant that cannot be serotonylated display significantly altered expression of H3K4me3Q5ser-target loci, which leads to deficits in differentiation. Taken together, these data identify a direct role for 5-HT, independent from its contributions to neurotransmission and cellular signalling, in the mediation of permissive gene expression. In serotonin-rich tissues, tissue transglutaminase 2 is able to attach serotonin to a glutamine residue in histone H3; this modification mediates permissive gene expression in these tissues.

Thromboembolism (TE) is a common complication in cancer patients and the second leading cause of cancer-related deaths. The incidence of TE varies in different cancer types, with the highest risk in pancreatic ductal adenocarcinoma (PDAC) and in advanced-stage and metastatic cancers. Despite the benefits associated with anti-coagulant therapy for symptomatic TE, the prevention of TE still remains an unmet clinical need due to lack of biomarkers predictive of TE risk and the bleeding risk associated with the routine use of anti-coagulants. Small extracellular vesicles (sEV) mediate cell-to-cell communication. Cancer cells and the tumor microenvironment release large numbers of sEV into the blood circulation and sEVs have displayed a therapeutic and predictive value in systemic diseases. However, the role of sEVs in cancer-associated TE remains to be investigated. Here we show that sEVs from (pre)metastatic lungs of mice with melanoma, breast, lung, and PDAC induce TE in mice and express high levels of integrin beta 2 (ITGB2), while sEVs from tumor cell lines, primary tumors, or other metastasis-bearing organs did not show any pro-thrombotic properties. A specific subtype of interstitial macrophages infiltrating (pre-)metastatic lungs were the main source of ITGB2+ pro-thrombotic sEVs. Blockade of ITGB2 on lung-derived sEVs, or systemically in mice, prevented EV-induced platelet aggregation and TE, and reduced metastasis. Examination of the mechanisms of ITGB2-induced TE showed that EV-associated ITGB2 interacts directly or through fibrin with different binding partners on platelets, and induce their activation and aggregation. Importantly, we found that levels of ITGB2 on sEVs are elevated in the plasma of PDAC patients prior ( Citation Format: Serena Lucotti, Yusuke Ogitani, Candia M. Kenific, Linda Bojmar, Michele Cioffi, Pernille Lauritzen, Henrik Molina, Soeren Heissel, Henry B. Lengel, Xiaohong Jing, Haiying Zhang, Irina Matei, Eileen M. O'Reilly, William R. Jarnagin, David Jones, James B. Bussel, David Kelsen, Jacqueline F. Bromberg, Diane M. Simeone, David Lyden. The lung pro-thrombotic niche drives cancer-associated thromboembolism and metastasis via extracellular vesicle ITGB2 [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C028.

While dysregulation of adipocyte endocrine function plays a central role in obesity and its complications, the vast majority of adipokines remain uncharacterized. We employed bio-orthogonal non-canonical amino acid tagging (BONCAT) and mass spectrometry to comprehensively characterize the secretome of murine visceral and subcutaneous white and interscapular brown adipocytes. Over 600 proteins were identified, the majority of which showed cell type-specific enrichment. We here describe a metabolic role for leucine-rich α-2 glycoprotein 1 (LRG1) as an obesity-regulated adipokine secreted by mature adipocytes. LRG1 overexpression significantly improved glucose homeostasis in diet-induced and genetically obese mice. This was associated with markedly reduced white adipose tissue macrophage accumulation and systemic inflammation. Mechanistically, we found LRG1 binds cytochrome c in circulation to dampen its pro-inflammatory effect. These data support a new role for LRG1 as an insulin sensitizer with therapeutic potential given its immunomodulatory function at the nexus of obesity, inflammation, and associated pathology.

Characterizing a cancer spliceosome Cells undergo many genomic changes as they progress toward metastatic cancer. One aspect of this change is to RNA expression and splicing isoforms, but how these differences affect tumor progression is not well characterized. Fish et al. developed a computational framework called pyTEISER that identifies structural cis-regulatory elements that control diverse types of RNA regulation. Applying pyTEISER to models of breast cancer metastasis, they discovered an RNA short-stem-loop element that forms a “structural splicing enhancer” that acts in cis to regulate alternative splicing of RNA transcripts. One of these interactions encompasses the RNA-binding protein SNRPA1 and results in alternative exon inclusion that affects metastatic capacity in xenograft models. Thus, RNA element binding may play a role in splicing regulation and is potentially an important component of the cis-splicing code. Science , this issue p. eabc7531

SUMMARYStress-induced cleavage of transfer RNAs (tRNAs) into tRNA-derived fragments (tRFs) occurs across organisms from yeast to human, yet its mechanistic bases and pathological consequences remain poorly defined. By performing genome-wide small RNA profiling, we detected increased abundance of a Cysteine tRNA fragment (5’-tRFCys) during breast cancer metastatic progression. 5’’-tRFCys is required for efficient breast cancer metastatic lung colonization and metastatic cell survival. We identified Nucleolin as the direct binding partner of 5’-tRFCys. 5’-tRFCys binding enhanced the stability of Nucleolin’s associated pro-metastatic transcripts encoding metabolic enzymes Mthfd1l and Pafah1b1. 5’-tRFCys stabilized these transcripts by promoting Nucleolin oligomerization and the assembly of Nucleolin and its bound transcripts into a higher-order ribonucleoprotein complex. Our findings reveal that a tRF can promote oligomerization of an RNA binding protein into a stabilizing ribonucleoprotein complex containing specific target transcripts, thereby driving specific metabolic pathways underlying cancer progression.

Stress-induced cleavage of transfer RNAs (tRNAs) into tRNA-derived fragments (tRFs) occurs across organisms from yeast to humans; yet, its mechanistic underpinnings and pathological consequences remain poorly defined. Small RNA profiling revealed increased abundance of a cysteine tRNA fragment (5'-tRF

Thromboembolism (TE) is a common complication in cancer patients and the second leading cause of cancer-related deaths. The incidence of TE varies in different cancer types, with the highest risk in lung cancer and pancreatic ductal adenocarcinoma (PDAC), and in advanced-stage and metastatic cancers. Despite the benefits associated with thromboprophylaxis for symptomatic TE, the prevention of TE still remains an unmet clinical need due to lack of biomarkers predictive of TE risk and the bleeding risk associated with the routine use of anti-coagulants. Exosomes are small circulating extracellular vesicles that mediate cell-to-cell communication. Cancer cells and the tumor microenvironment release large numbers of exosomes into the blood circulation and have displayed a therapeutic and predictive value in systemic diseases. Integrins expressed on the surface of exosomes drive their selective organotropism and prepare distant sites for metastatic seeding by establishing favorable pre-metastatic niches. Here we show that exosomes from metastasis-bearing lungs or pre-metastatic lungs of mice with melanoma, breast, lung and pancreatic cancer induce TE in mice and express high levels of integrin beta 2 (ITGB2). Instead, exosomes from tumor cell lines, primary tumors or other metastasis-bearing organs did not show any pro-thrombotic properties. Myeloid cells including monocytes/macrophages and neutrophils infiltrating pre- and post-metastatic lungs were the main source of ITGB2+ pro-thrombotic exosomes. Blockade of ITGB2 on lung-derived exosomes, or systemically in mice, prevented exosome-induced platelet aggregation and TE, and reduced metastasis. Examination of the mechanisms of ITGB2-induced TE showed that exosomal ITGB2 interact directly or through fibrin with different binding partners on platelets, and induce their activation and aggregation. Importantly, we found that exosomal ITGB2 levels are elevated in the plasma of PDAC patients prior to TE events in comparison to PDAC patients with no history of TE, and thus might serve as prognostic biomarker of TE. Together, our results provide the first evidence of the establishment of a pro-thrombotic lung niche in different cancer types. Moreover, we identify exosomal ITGB2 as a new target for the prevention and/or treatment of TE, as well as a potential “liquid biopsy” analyte for the early stratification of patients at high risk of TE. Citation Format: Serena Lucotti, Yusuke Ogitani, Candia M. Kenific, Linda Bojmar, Michele Cioffi, Pernille Lauritzen, Henrik Molina, Soren Heissel, Harry B. Lengel, Xiaohong Jing, Haiying Zhang, Irina Matei, Eileen M. O'Reilly, William R. Jarnagin, David R. Jones, James B. Bussel, David Kelsen, Jacqueline F. Bromberg, Diane M. Simeone, David Lyden. The lung pro-thrombotic niche drives cancer-associated thromboembolism via exosomal ITGB2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3138.

Despite its widespread use, the majority of patients with gastric cancer (GC) will not respond to taxane chemotherapy due to resistance mechanisms. Recently, we reported the discovery of a novel truncated variant of the microtubule plus-end binding protein CLIP-170, hereafter CLIP-170S, whose expression is enriched in taxane resistant cell lines and patients with GC. Mass-spec proteomics and 5’-RACE further showed that CLIP-170S lacked the first 155 amino acids, including the Cap-Gly motif required for microtubule plus-end localization. CLIP-170S knockdown reversed taxane resistance in cells and xenografts, whereas its re-expression led to resistance, suggesting causation. Unlike canonical CLIP-170, we showed that CLIP-170S was mislocalized from the MT plus-end to the MT lattice. Computational analysis of RNA-seq data in conjunction with the connectivity map from taxane-sensitive and resistant GC cell lines, predicted imatinib as the top candidate drug to overcome drug resistance. Imatinib treatment completely reversed taxane resistance, as predicted, and did so unexpectedly by selective depletion of CLIP-170S. Other RTK inhibitors also depleted CLIP-170S, suggesting a class effect. We are currently unraveling the molecular mechanisms by which a) CLIP-170S impairs taxane association to its microtubule binding site and b) Imatinib and other RTK inhibitors selectively deplete CLIP-170S. Our data show that CLIP-170S is a clinically prevalent variant that confers taxane resistance in tumors, whereas the discovery of Imatinib as a CLIP-170S inhibitor provides novel therapeutic opportunities for future trials. Citation Format: Prashant V. Thakkar, Katsuhiro Kita, Urko del Castillo, William G. Stone, Giuseppe Galletti, Neel Madhukar, Elena Vila Navarro, Elena Barasoain, Holly V. Goodson, Dan Sackett, José Fernando Díaz, Yao Lu, Arindam RoyChoudhury, Henrik Molina, Olivier Elemento, Manish A. Shah, Paraskevi Giannakakou. Variants of CLIP-170 associated with taxane resistance in tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2623.

Eukaryotic transfer RNAs can become selectively fragmented upon various stresses, generating tRNA‐derived small RNA fragments. Such fragmentation has been reported to impact a small fraction of the tRNA pool and thus presumed to not directly impact translation. We report that oxidative stress can rapidly generate tyrosine‐tRNA(GUA) fragments in human cells—causing significant depletion of the precursor tRNA. Tyrosine‐tRNA(GUA) depletion impaired translation of growth and metabolic genes enriched in cognate tyrosine codons. Depletion of tyrosine tRNA(GUA) or its translationally regulated targets USP3 and SCD repressed proliferation—revealing a dedicated tRNA‐regulated growth‐suppressive pathway for oxidative stress response. Tyrosine fragments are generated in a DIS3L2 exoribonuclease‐dependent manner and inhibit hnRNPA1‐mediated transcript destabilization. Moreover, tyrosine fragmentation is conserved in C. elegans. Thus, tRNA fragmentation can coordinately generate trans‐acting small RNAs and functionally deplete a tRNA. Our findings reveal the existence of an underlying adaptive codon‐based regulatory response inherent to the genetic code.

Summary Although mutations in DNA are the best-studied source of neoantigens that determine response to immune checkpoint blockade, alterations in RNA splicing within cancer cells could similarly result in neoepitope production. However, the endogenous antigenicity and clinical potential of such splicing-derived epitopes have not been tested. Here, we demonstrate that pharmacologic modulation of splicing via specific drug classes generates bona fide neoantigens and elicits anti-tumor immunity, augmenting checkpoint immunotherapy. Splicing modulation inhibited tumor growth and enhanced checkpoint blockade in a manner dependent on host T cells and peptides presented on tumor MHC class I. Splicing modulation induced stereotyped splicing changes across tumor types, altering the MHC I-bound immunopeptidome to yield splicing-derived neoepitopes that trigger an anti-tumor T cell response in vivo. These data definitively identify splicing modulation as an untapped source of immunogenic peptides and provide a means to enhance response to checkpoint blockade that is readily translatable to the clinic.

Plasma thiamine concentrations affect l-asparaginase sensitivity of leukemia cells deficient for thiamine uptake., Tumor environment influences anticancer therapy response but which extracellular nutrients affect drug sensitivity is largely unknown. Using functional genomics, we determine modifiers of l-asparaginase (ASNase) response and identify thiamine pyrophosphate kinase 1 as a metabolic dependency under ASNase treatment. While thiamine is generally not limiting for cell proliferation, a DNA-barcode competition assay identifies leukemia cell lines that grow suboptimally under low thiamine and are characterized by low expression of solute carrier family 19 member 2 (SLC19A2), a thiamine transporter. SLC19A2 is necessary for optimal growth and ASNase resistance, when standard medium thiamine is lowered ~100-fold to human plasma concentrations. In addition, humanizing blood thiamine content of mice through diet sensitizes SLC19A2-low leukemia cells to ASNase in vivo. Together, our work reveals that thiamine utilization is a determinant of ASNase response for some cancer cells and that oversupplying vitamins may affect therapeutic response in leukemia.

Viruses cleave cellular proteins to remodel the host proteome. The study of these cleavages has revealed mechanisms of immune evasion, resource exploitation, and pathogenesis. However, the full extent of virus-induced proteolysis in infected cells is unknown, mainly because until recently the technology for a global view of proteolysis within cells was lacking. Here, we report the first comprehensive catalog of proteins cleaved upon enterovirus infection and identify the sites within proteins where the cleavages occur. We employed multiple strategies to confirm protein cleavages and assigned them to one of the two enteroviral proteases. Detailed characterization of one substrate, LSM14A, a p body protein with a role in antiviral immunity, showed that cleavage of this protein disrupts its antiviral function. This study yields a new depth of information about the host interface with a group of viruses that are both important biological tools and significant agents of disease., Author summary Enteroviruses are associated with a variety of human diseases, including gastroenteritis, the common cold, hand-foot-and-mouth disease, acute hemorrhagic conjunctivitis, and skin rash. In some cases, the infection can lead to myocarditis, encephalitis, progressive muscle weakness, and paralysis. Exactly how enteroviruses invade human tissues, defeat the host immune system, and alter normal cell biology is unknown. Understanding these cellular and molecular mechanisms will blaze the trail for the development of novel vaccine and therapeutic strategies. Here, we have applied a global N-terminomics approach to investigate how various enteroviruses recruit their proteases to remodel an infected cell, disarm host immunity, and create a favorable environment for their replication. This effort identified several new protease substrates, which we then confirmed by other experimental approaches. To our knowledge, this is the first systematic analysis of host proteins targeted for cleavage during enterovirus infection. The data generated in this study will serve as a valuable resource for the research community in the quest to uncover the molecular details of enterovirus cell biology and disease pathogenesis.

The mimicry of host proteins by viruses contributes to their ability to suppress antiviral immunity and hijack host biosynthetic machinery1. Host adaptation to evade this exploitation depends on host protein functional redundancy2. Non-redundant, essential host proteins have limited potential to adapt without severe consequences3. Histones, which are essential for genome architecture and control of gene expression, are among the most evolutionary conserved proteins4. Here we show that the capsid protein of the flavivirus yellow fever virus (YFV), mimics histone H4 and interferes with chromatin gene regulation by BRD4, a bromodomain and extraterminal domain (BET) protein. Two acetyl-lysine residues of YFV capsid are embedded in a histone-like motif that interacts with the BRD4 bromodomain, affecting gene expression and influencing YFV replication. These findings reveal histone mimicry as a strategy employed by an RNA virus that replicates in the cytosol5 and define convergent and distinct molecular determinants for motif recognition of the viral mimic versus histone H4.

Summary Pancreatic ductal adenocarcinoma (PDAC) cells require substantial metabolic rewiring to overcome nutrient limitations and immune surveillance. However, the metabolic pathways necessary for pancreatic tumor growth in vivo are poorly understood. To address this, we performed metabolism-focused CRISPR screens in PDAC cells grown in culture or engrafted in immunocompetent mice. While most metabolic gene essentialities are unexpectedly similar under these conditions, a small fraction of metabolic genes are differentially required for tumor progression. Among these, loss of heme synthesis reduces tumor growth due to a limiting role of heme in vivo, an effect independent of tissue origin or immune system. Our screens also identify autophagy as a metabolic requirement for pancreatic tumor immune evasion. Mechanistically, autophagy protects cancer cells from CD8+ T cell killing through TNFα-induced cell death in vitro. Altogether, this resource provides metabolic dependencies arising from microenvironmental limitations and the immune system, nominating potential anti-cancer targets.

3-Bromopyruvate (3BP) is a potential anticancer agent viewed as a glycolytic inhibitor that preferentially kills cancer cells through inhibition of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), resulting in severe energy depletion. We previously identified four cysteine residues in GAPDH that are alkylated by 3BP, resulting in its inactivation. However, we also showed that addition of excess pyruvate, the final product of glycolysis, was unable to rescue cells from 3BP treatment. This result indicates that GAPDH may not be the only relevant target and is consistent with the chemical reactivity of 3BP that should result in the modification of cysteine residues in many different proteins. To directly test this hypothesis, we first synthesized a probe of 3BP activity bearing an alkyne functionality, termed AO3BP, and then demonstrated that this probe could modify a variety of proteins in living cells. Subsequent competition of AO3BP labeling with pretreatment by 3BP identified 62 statistically significant proteins of various functions as targets of 3BP, confirming that 3BP labeling is indeed widespread. We conclude that 3BP's cytotoxic impact on cancer cells does not only result from selective inhibition of glycolysis but rather from a more widespread effect on cellular proteins that could be driven by the pharmacokinetics of the 3BP. These pleiotropic consequences should be considered when thinking about the potential toxicity of this highly reactive compound.