Integrative plasma and fecal metabolomics identify functional metabolites in adenoma-colorectal cancer progression and as early diagnostic biomarkers.

Changes in plasma and fecal metabolomes in colorectal cancer (CRC) progression (normal-adenoma-CRC) remain unclear. Here, plasma and fecal samples were collected from four independent cohorts of 1,251 individuals (422 CRC, 399 colorectal adenoma [CRA], and 430 normal controls [NC]). By metabolomic profiling, signature plasma and fecal metabolites with consistent shift across NC, CRA, and CRC are identified, including CRC-enriched oleic acid and CRC-depleted allocholic acid. Oleic acid exhibits pro-tumorigenic effects in CRC cells, patient-derived organoids, and two murine CRC models, whereas allocholic acid has opposing effects. By integrative analysis, we found that oleic acid or allocholic acid directly binds to α-enolase or farnesoid X receptor-1 in CRC cells, respectively, to modulate cancer-associated pathways. Clinically, we establish a panel of 17 plasma metabolites that accurately diagnoses CRC in a discovery and three validation cohorts (AUC = 0.848-0.987). Overall, we characterize metabolite signatures, mechanistic significance, and diagnostic potential of plasma and fecal metabolomes in CRC.

Adenosine-to-Inosine RNA editing in cancer: molecular mechanisms and downstream targets.

Adenosine-to-Inosine (A-to-I), one of the most prevalent RNA modifications, has recently garnered significant attention. The A-to-I modification actively contributes to biological and pathological processes by affecting the structure and function of various RNA molecules, including double stranded RNA, transfer RNA, microRNA, and viral RNA. Increasing evidence suggests that A-to-I plays a crucial role in the development of human disease, particularly in cancer, and aberrant A-to-I levels are closely associated with tumorigenesis and progression through regulation of the expression of multiple oncogenes and tumor suppressor genes. Currently, the underlying molecular mechanisms of A-to-I modification in cancer are not comprehensively understood. Here, we review the latest advances regarding the A-to-I editing pathways implicated in cancer, describing their biological functions and their connections to the disease.

FOXP4 is a Direct YAP1 Target that Promotes Gastric Cancer Stemness and Drives Metastasis.

The Hippo-YAP1 pathway is an evolutionally conserved signaling cascade that controls organ size and tissue regeneration. Dysregulation of Hippo-YAP1 signaling promotes initiation and progression of several types of cancer, including gastric cancer (GC). As the Hippo-YAP1 pathway regulates expression of thousands of genes, it is important to establish which target genes contribute to the oncogenic program driven by YAP1 to identify strategies to circumvent it. Here, we identified a vital role of FOXP4 in YAP1-driven gastric carcinogenesis by maintaining stemness and promoting peritoneal metastasis. Loss of FOXP4 impaired GC spheroid formation and reduced stemness marker expression, while FOXP4 upregulation potentiated cancer cell stemness. RNA-seq analysis revealed SOX12 as downstream target of FOXP4, and functional studies established that SOX12 supports stemness in YAP1-induced carcinogenesis. A small molecule screen identified 42-(2-Tetrazolyl)rapamycin as a FOXP4 inhibitor, and targeting FOXP4 suppressed GC tumor growth and enhanced the efficacy of 5-FU chemotherapy in vivo. Collectively, these findings revealed that FOXP4 upregulation by YAP1 in GC regulates stemness and tumorigenesis by upregulating SOX12. Targeting the YAP1-FOXP4-SOX12 axis represents a potential therapeutic strategy for GC.

Peptostreptococcus stomatis promotes colonic tumorigenesis and receptor tyrosine kinase inhibitor resistance by activating ERBB2-MAPK.

Peptostreptococcus stomatis (P. stomatis) is enriched in colorectal cancer (CRC), but its causality and translational implications in CRC are unknown. Here, we show that P. stomatis accelerates colonic tumorigenesis in ApcMin/+ and azoxymethane/dextran sodium sulfate (AOM-DSS) models by inducing cell proliferation, suppressing apoptosis, and impairing gut barrier function. P. stomatis adheres to CRC cells through its surface protein fructose-1,6-bisphosphate aldolase (FBA) that binds to the integrin α6/ß4 receptor on CRC cells, leading to the activation of ERBB2 and the downstream MEK-ERK-p90 cascade. Blockade of the FBA-integrin α6/ß4 abolishes ERBB2-mitogen-activated protein kinase (MAPK) activation and the protumorigenic effect of P. stomatis. P. stomatis-driven ERBB2 activation bypasses receptor tyrosine kinase (RTK) blockade by EGFR inhibitors (cetuximab, erlotinib), leading to drug resistance in xenograft and spontaneous CRC models of KRAS-wild-type CRC. P. stomatis also abrogates BRAF inhibitor (vemurafenib) efficacy in BRAFV600E-mutant CRC xenografts. Thus, we identify P. stomatis as an oncogenic bacterium and a contributory factor for non-responsiveness to RTK inhibitors in CRC.

RUVBL1/2 Blockade Targets YTHDF1 Activity to Suppress m6A-Dependent Oncogenic Translation and Colorectal Tumorigenesis.

The N6-methyladenosine (m6A) RNA binding protein YTHDF1 is frequently overexpressed in colorectal cancer (CRC) and drives chemotherapeutic resistance. To systematically identify druggable targets in CRC with high expression of YTHDF1, we employed a CRISPR/Cas9 screening strategy that revealed RUVBL1 and RUVBL2 as putative targets.RUVBL1/2 were overexpressed in primary CRC samples and represented independent predictors of poor patient prognosis. Functionally, loss of RUVBL1/2 preferentially impaired the growth ofYTHDF1-high CRC cells, patient-derived primary CRC organoids, and subcutaneous xenografts. Mechanistically, YTHFD1 and RUVBL1/2 formed a positive feed-forward circuit to accelerate oncogenic translation. YTHDF1 bound to m6A-modified RUVBL1/2 mRNA to promote translation initiation and protein expression. Co-IP and mass spectrometry identified that RUVBL1/2 reciprocally interacted with YTHDF1 at 40S translation initiation complexes. Consequently, RUVBL1/2 depletion stalled YTHDF1-driven oncogenic translation and nascent protein biosynthesis, leading to proliferative arrest and apoptosis. Ribo-seq revealed that RUVBL1/2 loss impaired the activation of MAPK, RAS and PI3K-AKT signaling induced by YTHDF1. Finally, blockade of RUVBL1/2 by the pharmacological inhibitor CB6644 or vesicle-like nanoparticle-encapsulated siRNAs preferentially arrested the growth of YTHDF1-expressing CRC in vitro and in vivo. Together, this study uncovered that RUVBL1/2 are potential prognostic markers and druggable targets that regulate protein translation in YTHDF1-high CRC.

Peptostreptococcus anaerobius mediates anti-PD1 therapy resistance and exacerbates colorectal cancer via myeloid-derived suppressor cells in mice.

Bacteria such as the oral microbiome member Peptostreptococcus anaerobius can exacerbate colorectal cancer (CRC) development. Little is known regarding whether these immunomodulatory bacteria also affect antitumour immune checkpoint blockade therapy. Here we show that administration of P. anaerobius abolished the efficacy of anti-PD1 therapy in mouse models of CRC. P. anaerobius both induced intratumoral myeloid-derived suppressor cells (MDSCs) and stimulated their immunosuppressive activities to impair effective T cell responses. Mechanistically, P. anaerobius administration activated integrin α2ß1-NF-κB signalling in CRC cells to induce secretion of CXCL1 and recruit CXCR2+ MDSCs into tumours. The bacterium also directly activated immunosuppressive activity of intratumoral MDSCs by secreting lytC_22, a protein that bound to the Slamf4 receptor on MDSCs and promoted ARG1 and iNOS expression. Finally, therapeutic targeting of either integrin α2ß1 or the Slamf4 receptor were revealed as promising strategies to overcome P. anaerobius-mediated resistance to anti-PD1 therapy in CRC.

Targeting squalene epoxidase restores anti-PD-1 efficacy in metabolic dysfunction-associated steatohepatitis-induced hepatocellular carcinoma.

OBJECTIVE: Squalene epoxidase (SQLE) promotes metabolic dysfunction-associated steatohepatitis-associated hepatocellular carcinoma (MASH-HCC), but its role in modulating the tumour immune microenvironment in MASH-HCC remains unclear. DESIGN: We established hepatocyte-specific Sqle transgenic (tg) and knockout mice, which were subjected to a choline-deficient high-fat diet plus diethylnitrosamine to induce MASH-HCC. SQLE function was also determined in orthotopic and humanised mice. Immune landscape alterations of MASH-HCC mediated by SQLE were profiled by single-cell RNA sequencing and flow cytometry. RESULTS: Hepatocyte-specific Sqle tg mice exhibited a marked increase in MASH-HCC burden compared with wild-type littermates, together with decreased tumour-infiltrating functional IFN-γ+ and Granzyme B+ CD8+ T cells while enriching Arg-1+ myeloid-derived suppressor cells (MDSCs). Conversely, hepatocyte-specific Sqle knockout suppressed tumour growth with increased cytotoxic CD8+ T cells and reduced Arg-1+ MDSCs, inferring that SQLE promotes immunosuppression in MASH-HCC. Mechanistically, SQLE-driven cholesterol accumulation in tumour microenvironment underlies its effect on CD8+ T cells and MDSCs. SQLE and its metabolite, cholesterol, impaired CD8+ T cell activity by inducing mitochondrial dysfunction. Cholesterol depletion in vitro abolished the effect of SQLE-overexpressing MASH-HCC cell supernatant on CD8+ T cell suppression and MDSC activation, whereas cholesterol supplementation had contrasting functions on CD8+ T cells and MDSCs treated with SQLE-knockout supernatant. Targeting SQLE with genetic ablation or pharmacological inhibitor, terbinafine, rescued the efficacy of anti-PD-1 treatment in MASH-HCC models. CONCLUSION: SQLE induces an impaired antitumour response in MASH-HCC via attenuating CD8+ T cell function and augmenting immunosuppressive MDSCs. SQLE is a promising target in boosting anti-PD-1 immunotherapy for MASH-HCC.

Mapping the pancancer metastasis tumor microbiome.

The landscape of the intratumoral microbiome in tumor metastases is largely unchartered. In this issue of Cell, Voest et al. profiled the tumor metastasis-associated microbiome in a pancancer cohort of 4,160 biopsies from 26 cancer types. This dataset offers a useful resource for understanding the role of the microbiome in metastatic cancers.

Defining the biogeographical map and potential bacterial translocation of microbiome in human 'surface organs'.

The microbiome in a specific human organ has been well-studied, but few reports have investigated the multi-organ microbiome as a whole. Here, we aim to analyse the intra-individual inter-organ and intra-organ microbiome in deceased humans. We collected 1608 samples from 53 sites of 7 surface organs (oral cavity, esophagus, stomach, small intestine, appendix, large intestine and skin; n = 33 subjects) and performed microbiome profiling, including 16S full-length sequencing. Microbial diversity varied dramatically among organs, and core microbial species co-existed in different intra-individual organs. We deciphered microbial changes across distinct intra-organ sites, and identified signature microbes, their functional traits, and interactions specific to each site. We revealed significant microbial heterogeneity between paired mucosa-lumen samples of stomach, small intestine, and large intestine. Finally, we established the landscape of inter-organ relationships of microbes along the digestive tract. Therefore, we generate a catalogue of bacterial composition, diversity, interaction, functional traits, and bacterial translocation in human at inter-organ and intra-organ levels.

Streptococcus anginosus promotes gastric inflammation, atrophy, and tumorigenesis in mice.

Streptococcus anginosus (S. anginosus) was enriched in the gastric mucosa of patients with gastric cancer (GC). Here, we show that S. anginosus colonized the mouse stomach and induced acute gastritis. S. anginosus infection spontaneously induced progressive chronic gastritis, parietal cell atrophy, mucinous metaplasia, and dysplasia in conventional mice, and the findings were confirmed in germ-free mice. In addition, S. anginosus accelerated GC progression in carcinogen-induced gastric tumorigenesis and YTN16 GC cell allografts. Consistently, S. anginosus disrupted gastric barrier function, promoted cell proliferation, and inhibited apoptosis. Mechanistically, we identified an S. anginosus surface protein, TMPC, that interacts with Annexin A2 (ANXA2) receptor on gastric epithelial cells. Interaction of TMPC with ANXA2 mediated attachment and colonization of S. anginosus and induced mitogen-activated protein kinase (MAPK) activation. ANXA2 knockout abrogated the induction of MAPK by S. anginosus. Thus, this study reveals S. anginosus as a pathogen that promotes gastric tumorigenesis via direct interactions with gastric epithelial cells in the TMPC-ANXA2-MAPK axis.

Spatial proteomics of immune microenvironment in nonalcoholic steatohepatitis-associated hepatocellular carcinoma.

BACKGROUND AND AIMS: NASH-HCC is inherently resistant to immune checkpoint blockade, but its tumor immune microenvironment is largely unknown. APPROACH AND RESULTS: We applied the imaging mass cytometry to construct a spatially resolved single-cell atlas from the formalin-fixed and paraffin-embedded tissue sections from patients with NASH-HCC, virus-HCC (HBV-HCC and HCV-HCC), and healthy donors. Based on 35 biomarkers, over 750,000 individual cells were categorized into 13 distinct cell types, together with the expression of key immune functional markers. Higher infiltration of T cells, myeloid-derived suppressor cell (MDSCs), and tumor-associated macrophages (TAMs) in HCC compared to controls. The distribution of immune cells in NASH-HCC is spatially heterogeneous, enriched at adjacent normal tissues and declined toward tumors. Cell-cell connections analysis revealed the interplay of MDSCs and TAMs with CD8 + T cells in NASH-HCC. In particular, exhausted programmed cell death 1 (PD-1 + )CD8 + T cells connected with programmed cell death-ligand 1 (PD-L1 + )/inducible T cell costimulator (ICOS + ) MDSCs and TAMs in NASH-HCC, but not in viral HCC. In contrast, CD4 + /CD8 + T cells with granzyme B positivity were reduced in NASH-HCC. Tumor cells expressed low PD-L1 and showed few connections with immune cells. CONCLUSIONS: Our work provides the first detailed spatial map of single-cell phenotypes and multicellular connections in NASH-HCC. We demonstrate that interactions between MDSCs and TAMs with effector T cells underlie immunosuppression in NASH-HCC and are an actionable target.

The molecular classification of cancer-associated fibroblasts on a pan-cancer single-cell transcriptional atlas.

BACKGROUND: Cancer-associated fibroblasts (CAFs), integral to the tumour microenvironment, are pivotal in cancer progression, exhibiting either pro-tumourigenic or anti-tumourigenic functions. Their inherent phenotypic and functional diversity allows for the subdivision of CAFs into various subpopulations. While several classification systems have been suggested for different cancer types, a unified molecular classification of CAFs on a single-cell pan-cancer scale has yet to be established. METHODS: We employed a comprehensive single-cell transcriptomic atlas encompassing 12 solid tumour types. Our objective was to establish a novel molecular classification and to elucidate the evolutionary trajectories of CAFs. We investigated the functional profiles of each CAF subtype using Single-Cell Regulatory Network Inference and Clustering and single-cell gene set enrichment analysis. The clinical relevance of these subtypes was assessed through survival curve analysis. Concurrently, we employed multiplex immunofluorescence staining on tumour tissues to determine the dynamic changes of CAF subtypes across different tumour stages. Additionally, we identified the small molecule procyanidin C1 (PCC1) as a target for matrix-producing CAF (matCAF) using molecular docking techniques and further validated these findings through in vitro and in vivo experiments. RESULTS: In our investigation of solid tumours, we identified four molecular clusters of CAFs: progenitor CAF (proCAF), inflammatory CAF (iCAF), myofibroblastic CAF (myCAF) and matCAF, each characterised by distinct molecular traits. This classification was consistently applicable across all nine studied solid tumour types. These CAF subtypes displayed unique evolutionary pathways, functional roles and clinical relevance in various solid tumours. Notably, the matCAF subtype was associated with poorer prognoses in several cancer types. The targeting of matCAF using the identified small molecule, PCC1, demonstrated promising antitumour activity. CONCLUSIONS: Collectively, the various subtypes of CAFs, particularly matCAF, are crucial in the initiation and progression of cancer. Focusing therapeutic strategies on targeting matCAF in solid tumours holds significant potential for cancer treatment.

H. pylori-induced NF-κB-PIEZO1-YAP1-CTGF axis drives gastric cancer progression and cancer-associated fibroblast-mediated tumour microenvironment remodelling.

BACKGROUND: Gastric cancer (GC) is one of the most common tumours in East Asia countries and is associated with Helicobacter pylori infection. H. pylori utilizes virulence factors, CagA and VacA, to up-regulate pro-inflammatory cytokines and activate NF-κB signaling. Meanwhile, the PIEZO1 upregulation and cancer-associated fibroblast (CAF) enrichment were found in GC progression. However, the mechanisms of PIEZO1 upregulation and its involvement in GC progression have not been fully elucidated. METHODS: The CAF enrichment and clinical significance were investigated in animal models and primary samples. The expression of NF-κB and PIEZO1 in GC was confirmed by immunohistochemistry staining, and expression correlation was analysed in multiple GC datasets. GSEA and Western blot analysis revealed the YAP1-CTGF axis regulation by PIEZO1. The stimulatory effects of CTGF on CAFs were validated by the co-culture system and animal studies. Patient-derived organoid and peritoneal dissemination models were employed to confirm the role of the PIEZO1-YAP1-CTGF cascade in GC. RESULTS: Both CAF signature and PIEZO1 were positively correlated with H. pylori infection. PIEZO1, a mechanosensor, was confirmed as a direct downstream of NF-κB to promote the transformation from intestinal metaplasia to GC. Mechanistic studies revealed that PIEZO1 transduced the oncogenic signal from NF-κB into YAP1 signaling, a well-documented oncogenic pathway in GC progression. PIEZO1 expression was positively correlated with the YAP1 signature (CTGF, CYR61, and c-Myc, etc.) in primary samples. The secreted CTGF by cancer cells stimulated the CAF infiltration to form a stiffened collagen-enrichment microenvironment, thus activating PIEZO1 to form a positive feedback loop. Both PIEZO1 depletion by shRNA and CTGF inhibition by Procyanidin C1 enhanced the efficacy of 5-FU in suppressing the GC cell peritoneal metastasis. CONCLUSION: This study elucidates a novel driving PIEZO1-YAP1-CTGF force, which opens a novel therapeutic avenue to block the transformation from precancerous lesions to GC. H. pylori-NF-κB activates the PIEZO1-YAP1-CTGF axis to remodel the GC microenvironment by promoting CAF infiltration. Targeting PIEZO1-YAP1-CTGF plus chemotherapy might serve as a potential therapeutic option to block GC progression and peritoneal metastasis.

TRIP6 disrupts tight junctions to promote metastasis and drug resistance and is a therapeutic target in colorectal cancer.

Metastasis is the primary cause of death in colorectal cancer (CRC). Thyroid hormone receptor interacting protein 6 (TRIP6) is an adaptor protein that regulates cell motility. Here, we aim to elucidate the role of TRIP6 in driving CRC tumorigenesis and metastasis and evaluate its potential as a therapeutic target. TRIP6 mRNA is up-regulated in CRC compared to adjacent normal tissues in three independent cohorts (all P < 0.0001), especially in liver metastases (P < 0.001). High TRIP6 expression predicts poor prognosis of CRC patients in our cohort (P = 0.01) and TCGA cohort (P = 0.02). Colon-specific TRIP6 overexpression (Trip6KIVillin-Cre) in mice accelerated azoxymethane (AOM)-induced CRC (P < 0.05) and submucosal invasion (P < 0.0001). In contrast, TRIP6 knockout (Trip6+/- mice) slowed tumorigenesis (P < 0.05). Consistently, TRIP6 overexpression in CRC cells promoted epithelial-mesenchymal transition (EMT), cell migration/invasion in vitro, and metastases in vivo (all P < 0.05), whereas knockdown of TRIP6 exerted opposite phenotypes. Mechanistically, TRIP6 interacted PDZ domain-containing proteins such as PARD3 to impair tight junctions, evidenced by decreased tight junction markers and gut permeability dysfunction, inhibit PTEN, and activate oncogenic Akt signaling. TRIP6-induced pro-metastatic phenotypes and Akt activation depends on PARD3. Targeting TRIP6 by VNP-encapsulated TRIP6-siRNA synergized with Oxaliplatin and 5-Fluorouracil to suppress CRC liver metastases. In conclusion, TRIP6 promotes CRC metastasis by directly interacting with PARD3 to disrupt tight junctions and activating Akt signaling. Targeting of TRIP6 in combination with chemotherapy is a promising strategy for the treatment of metastatic CRC.

Traditional Medicine Pien Tze Huang Suppresses Colorectal Tumorigenesis Through Restoring Gut Microbiota and Metabolites.

BACKGROUND & AIMS: Pien Tze Huang (PZH) is a well-established traditional medicine with beneficial effects against inflammation and cancer. We aimed to explore the chemopreventive effect of PZH in colorectal cancer (CRC) through modulating gut microbiota. METHODS: CRC mouse models were established by azoxymethane plus dextran sulfate sodium treatment or in Apcmin/+ mice treated with or without PZH (270 mg/kg and 540 mg/kg). Gut barrier function was determined by means of intestinal permeability assays and transmission electron microscopy. Fecal microbiota and metabolites were analyzed by means of metagenomic sequencing and liquid chromatography mass spectrometry, respectively. Germ-free mice or antibiotic-treated mice were used as models of microbiota depletion. RESULTS: PZH inhibited colorectal tumorigenesis in azoxymethane plus dextran sulfate sodium-treated mice and in Apcmin/+ mice in a dose-dependent manner. PZH treatment altered the gut microbiota profile, with an increased abundance of probiotics Pseudobutyrivibrio xylanivorans and Eubacterium limosum, while pathogenic bacteria Aeromonas veronii, Campylobacter jejuni, Collinsella aerofaciens, and Peptoniphilus harei were depleted. In addition, PZH increased beneficial metabolites taurine and hypotaurine, bile acids, and unsaturated fatty acids, and significantly restored gut barrier function. Transcriptomic profiling revealed that PZH inhibited PI3K-Akt, interleukin-17, tumor necrosis factor, and cytokine-chemokine signaling. Notably, the chemopreventive effect of PZH involved both microbiota-dependent and -independent mechanisms. Fecal microbiota transplantation from PZH-treated mice to germ-free mice partly recapitulated the chemopreventive effects of PZH. PZH components ginsenoside-F2 and ginsenoside-Re demonstrated inhibitory effects on CRC cells and primary organoids, and PZH also inhibited tumorigenesis in azoxymethane plus dextran sulfate sodium-treated germ-free mice. CONCLUSIONS: PZH manipulated gut microbiota and metabolites toward a more favorable profile, improved gut barrier function, and suppressed oncogenic and pro-inflammatory pathways, thereby suppressing colorectal carcinogenesis.

METTL3 drives NAFLD-related hepatocellular carcinoma and is a therapeutic target for boosting immunotherapy.

Non-alcoholic fatty liver disease (NAFLD) is an emerging risk factor of hepatocellular carcinoma (HCC). However, the mechanism and target therapy of NAFLD-HCC are still unclear. Here, we identify that the N6-methyladenosine (m6A) methyltransferase METTL3 promotes NAFLD-HCC. Hepatocyte-specific Mettl3 knockin exacerbated NAFLD-HCC formation, while Mettl3 knockout exerted the opposite effect in mice. Single-cell RNA sequencing revealed that METTL3 suppressed antitumor immune response by reducing granzyme B (GZMB+) and interferon gamma-positive (IFN-γ+) CD8+ T cell infiltration, thereby facilitating immune escape. Mechanistically, METTL3 mediates sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP) mRNA m6A to promote its translation, leading to the activation of cholesterol biosynthesis. This enhanced secretion of cholesterol and cholesteryl esters that impair CD8+ T cell function in the tumor microenvironment. Targeting METTL3 by single-guide RNA, nanoparticle small interfering RNA (siRNA), or pharmacological inhibitor (STM2457) in combination with anti-programmed cell death protein 1 (PD-1) synergized to reinvigorate cytotoxic CD8+ T cells and mediate tumor regression. Together, METTL3 is a therapeutic target in NAFLD-HCC, especially in conjunction with immune checkpoint blockade (ICB) therapy.

Targeting of SLC25A22 boosts the immunotherapeutic response in KRAS-mutant colorectal cancer.

KRAS is an important tumor intrinsic factor driving immune suppression in colorectal cancer (CRC). In this study, we demonstrate that SLC25A22 underlies mutant KRAS-induced immune suppression in CRC. In immunocompetent male mice and humanized male mice models, SLC25A22 knockout inhibits KRAS-mutant CRC tumor growth with reduced myeloid derived suppressor cells (MDSC) but increased CD8+ T-cells, implying the reversion of mutant KRAS-driven immunosuppression. Mechanistically, we find that SLC25A22 plays a central role in promoting asparagine, which binds and activates SRC phosphorylation. Asparagine-mediated SRC promotes ERK/ETS2 signaling, which drives CXCL1 transcription. Secreted CXCL1 functions as a chemoattractant for MDSC via CXCR2, leading to an immunosuppressive microenvironment. Targeting SLC25A22 or asparagine impairs KRAS-induced MDSC infiltration in CRC. Finally, we demonstrate that the targeting of SLC25A22 in combination with anti-PD1 therapy synergizes to inhibit MDSC and activate CD8+ T cells to suppress KRAS-mutant CRC growth in vivo. We thus identify a metabolic pathway that drives immunosuppression in KRAS-mutant CRC.

Targeting N6-methyladenosine reader YTHDF1 with siRNA boosts antitumor immunity in NASH-HCC by inhibiting EZH2-IL-6 axis.

BACKGROUND & AIMS: RNA N6-methyladenosine (m6A) reader protein YTHDF1 has been implicated in cancer; however, its role in hepatocellular carcinoma (HCC), especially in non-alcoholic steatohepatitis-associated HCC (NASH-HCC), remains unknown. Here, we investigated the functional role of YTHDF1 in NASH-HCC and its interplay with the tumor immune microenvironment. METHODS: Hepatocyte-specific Ythdf1-overexpressing mice were subjected to a NASH-HCC-inducing diet. Tumor-infiltrating immune cells were profiled with single-cell RNA-sequencing, flow cytometry, and immunostaining. The molecular target of YTHDF1 was elucidated with RNA-sequencing, m6A-sequencing, YTHDF1 RNA immunoprecipitation-sequencing, proteomics, and ribosome-profiling. Ythdf1 in NASH-HCC models was targeted by lipid nanoparticle (LNP)-encapsulated small-interfering Ythdf1. RESULTS: YTHDF1 is overexpressed in tumor tissues compared to adjacent peri-tumor tissues from patients with NASH-HCC. Liver-specific Ythdf1 overexpression drives tumorigenesis in dietary models of spontaneous NASH-HCC. Single-cell RNA-sequencing and flow cytometry revealed that Ythdf1 induced accumulation of myeloid-derived suppressor cells (MDSCs) and suppressed cytotoxic CD8+ T-cell function. Mechanistically, Ythdf1 expression in NASH-HCC cells induced the secretion of IL-6, which mediated MDSC recruitment and activation, leading to CD8+ T-cell dysfunction. EZH2 mRNA was identified as a key YTHDF1 target. YTHDF1 binds to m6A-modified EZH2 mRNA and promotes EZH2 translation. EZH2 in turn increased expression and secretion of IL-6. Ythdf1 knockout synergized with anti-PD-1 treatment to suppress tumor growth in NASH-HCC allografts. Furthermore, therapeutic targeting of Ythdf1 using LNP-encapsulated small-interfering RNA significantly increased the efficacy of anti-PD-1 blockade in NASH-HCC allografts. CONCLUSIONS: We identified that YTHDF1 promotes NASH-HCC tumorigenesis via EZH2-IL-6 signaling, which recruits and activates MDSCs to cause cytotoxic CD8+ T-cell dysfunction. YTHDF1 may be a novel therapeutic target to improve responses to anti-PD-1 immunotherapy in NASH-HCC. IMPACT AND IMPLICATIONS: YTHDF1, a N6-methyladenosine reader, is upregulated in patients with non-alcoholic steatohepatitis (NASH)-associated hepatocellular carcinoma (HCC); however, its role in modulating the tumor immune microenvironment in NASH-HCC remains unclear. Here, we show that Ythdf1 mediates immunosuppression in NASH-HCC and that targeting YTHDF1 in combination with immune checkpoint blockade elicits robust antitumor immune responses. Our findings suggest novel therapeutic targets for potentiating the efficacy of immune checkpoint blockade in NASH-HCC and provide the rationale for developing YTHDF1 inhibitors for the treatment of NASH-HCC.

MLK4 promotes glucose metabolism in lung adenocarcinoma through CREB-mediated activation of phosphoenolpyruvate carboxykinase and is regulated by KLF5.

MLK4, a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family, has been implicated in cancer progression. However, its role in lung adenocarcinoma has not been characterized. Here, we showed that MLK4 was overexpressed in a significant subset of lung adenocarcinoma, associated with a worse prognosis, and exerted an oncogenic function in vitro and in vivo. Bioinformatics analyses of clinical datasets identified phosphoenolpyruvate carboxykinase 1 (PCK1) as a novel target of MLK4. We validated that MLK4 regulated PCK1 expression at transcriptional level, by phosphorylating the transcription factor CREB, which in turn mediated PCK1 expression. We further demonstrated that PCK1 is an oncogenic factor in lung adenocarcinoma. Given the importance of PCK1 in the regulation of cellular metabolism, we next deciphered the metabolic effects of MLK4. Metabolic and mass spectrometry analyses showed that MLK4 knockdown led to significant reduction of glycolysis and decreased levels of glycolytic pathway metabolites including phosphoenolpyruvate and lactate. Finally, the promoter analysis of MLK4 unravelled a binding site of transcription factor KLF5, which in turn, positively regulated MLK4 expression in lung adenocarcinoma. In summary, we have revealed a KLF5-MLK4-PCK1 signalling pathway involved in lung tumorigenesis and established an unusual link between MAP3K signalling and cancer metabolism.