Genome-wide CRISPR screening identifies tyrosylprotein sulfotransferase-2 as a target for augmenting anti-PD1 efficacy.

BACKGROUND: Immune checkpoint therapy (ICT) provides durable responses in select cancer patients, yet resistance remains a significant challenge, prompting the exploration of underlying molecular mechanisms. Tyrosylprotein sulfotransferase-2 (TPST2), known for its role in protein tyrosine O-sulfation, has been suggested to modulate the extracellular protein-protein interactions, but its specific role in cancer immunity remains largely unexplored. METHODS: To explore tumor cell-intrinsic factors influencing anti-PD1 responsiveness, we conducted a pooled loss-of-function genetic screen in humanized mice engrafted with human immune cells. The responsiveness of cancer cells to interferon-γ (IFNγ) was estimated by evaluating IFNγ-mediated induction of target genes, STAT1 phosphorylation, HLA expression, and cell growth suppression. The sulfotyrosine-modified target gene of TPST2 was identified by co-immunoprecipitation and mass spectrometry. The in vivo effects of TPST2 inhibition were evaluated using mouse syngeneic tumor models and corroborated by bulk and single-cell RNA sequencing analyses. RESULTS: Through in vivo genome-wide CRISPR screening, TPST2 loss-of-function emerged as a potential enhancer of anti-PD1 treatment efficacy. TPST2 suppressed IFNγ signaling by sulfating IFNγ receptor 1 at Y397 residue, while its downregulation boosted IFNγ-mediated signaling and antigen presentation. Depletion of TPST2 in cancer cells augmented anti-PD1 antibody efficacy in syngeneic mouse tumor models by enhancing tumor-infiltrating lymphocytes. RNA sequencing data revealed TPST2's inverse correlation with antigen presentation, and increased TPST2 expression is associated with poor prognosis and altered cancer immunity across cancer types. CONCLUSIONS: We propose TPST2's novel role as a suppressor of cancer immunity and advocate for its consideration as a therapeutic target in ICT-based treatments.

Graphislactone A, a Fungal Antioxidant Metabolite, Reduces Lipogenesis and Protects against Diet-Induced Hepatic Steatosis in Mice.

Graphislactone A (GPA), a secondary metabolite derived from a mycobiont found in the lichens of the genus Graphis, exhibits antioxidant properties. However, the potential biological functions and therapeutic applications of GPA at the cellular and animal levels have not yet been investigated. In the present study, we explored the therapeutic potential of GPA in mitigating non-alcoholic fatty liver disease (NAFLD) and its underlying mechanisms through a series of experiments using various cell lines and animal models. GPA demonstrated antioxidant capacity on a par with that of vitamin C in cultured hepatocytes and reduced the inflammatory response induced by lipopolysaccharide in primary macrophages. However, in animal studies using an NAFLD mouse model, GPA had a milder impact on liver inflammation while markedly attenuating hepatic steatosis. This effect was confirmed in an animal model of early fatty liver disease without inflammation. Mechanistically, GPA inhibited lipogenesis rather than fat oxidation in cultured hepatocytes. Similarly, RNA sequencing data revealed intriguing associations between GPA and the adipogenic pathways during adipocyte differentiation. GPA effectively reduced lipid accumulation and suppressed lipogenic gene expression in AML12 hepatocytes and 3T3-L1 adipocytes. In summary, our study demonstrates the potential application of GPA to protect against hepatic steatosis in vivo and suggests a novel role for GPA as an underlying mechanism in lipogenesis, paving the way for future exploration of its therapeutic potential.

HNF4α is a therapeutic target that links AMPK to WNT signalling in early-stage gastric cancer.

BACKGROUND: Worldwide, gastric cancer (GC) is the fourth most common malignancy and the most common cancer in East Asia. Development of targeted therapies for this disease has focused on a few known oncogenes but has had limited effects. OBJECTIVE: To determine oncogenic mechanisms and novel therapeutic targets specific for GC by identifying commonly dysregulated genes from the tumours of both Asian-Pacific and Caucasian patients. METHODS: We generated transcriptomic profiles of 22 Caucasian GC tumours and their matched non-cancerous samples and performed an integrative analysis across different GC gene expression datasets. We examined the inhibition of commonly overexpressed oncogenes and their constituent signalling pathways by RNAi and/or pharmacological inhibition. RESULTS: Hepatocyte nuclear factor-4α (HNF4α) upregulation was a key signalling event in gastric tumours from both Caucasian and Asian patients, and HNF4α antagonism was antineoplastic. Perturbation experiments in GC tumour cell lines and xenograft models further demonstrated that HNF4α is downregulated by AMPKα signalling and the AMPK agonist metformin; blockade of HNF4α activity resulted in cyclin downregulation, cell cycle arrest and tumour growth inhibition. HNF4α also regulated WNT signalling through its target gene WNT5A, a potential prognostic marker of diffuse type gastric tumours. CONCLUSIONS: Our results indicate that HNF4α is a targetable oncoprotein in GC, is regulated by AMPK signalling through AMPKα and resides upstream of WNT signalling. HNF4α may regulate 'metabolic switch' characteristic of a general malignant phenotype and its target WNT5A has potential prognostic values. The AMPKα-HNF4α-WNT5A signalling cascade represents a potentially targetable pathway for drug development.

Improving gastric cancer preclinical studies using diverse in vitro and in vivo model systems.

BACKGROUND: "Biomarker-driven targeted therapy," the practice of tailoring patients' treatment to the expression/activity levels of disease-specific genes/proteins, remains challenging. For example, while the anti-ERBB2 monoclonal antibody, trastuzumab, was first developed using well-characterized, diverse in vitro breast cancer models (and is now a standard adjuvant therapy for ERBB2-positive breast cancer patients), trastuzumab approval for ERBB2-positive gastric cancer was largely based on preclinical studies of a single cell line, NCI-N87. Ensuing clinical trials revealed only modest patient efficacy, and many ERBB2-positive gastric cancer (GC) patients failed to respond at all (i.e., were inherently recalcitrant), or succumbed to acquired resistance. METHOD: To assess mechanisms underlying GC insensitivity to ERBB2 therapies, we established a diverse panel of GC cells, differing in ERBB2 expression levels, for comprehensive in vitro and in vivo characterization. For higher throughput assays of ERBB2 DNA and protein levels, we compared the concordance of various laboratory quantification methods, including those of in vitro and in vivo genetic anomalies (FISH and SISH) and xenograft protein expression (Western blot vs. IHC), of both cell and xenograft (tissue-sectioned) microarrays. RESULTS: The biomarker assessment methods strongly agreed, as did correlation between RNA and protein expression. However, although ERBB2 genomic anomalies showed good in vitro vs. in vivo correlation, we observed striking differences in protein expression between cultured cells and mouse xenografts (even within the same GC cell type). Via our unique pathway analysis, we delineated a signaling network, in addition to specific pathways/biological processes, emanating from the ERBB2 signaling cascade, as a potential useful target of clinical treatment. Integrated analysis of public data from gastric tumors revealed frequent (10 - 20 %) amplification of the genes NFKBIE, PTK2, and PIK3CA, each of which resides in an ERBB2-derived subpathway network. CONCLUSION: Our comprehensive bioinformatics analyses of highly heterogeneous cancer cells, combined with tumor "omics" profiles, can optimally characterize the expression patterns and activity of specific tumor biomarkers. Subsequent in vitro and in vivo validation, of specific disease biomarkers (using multiple methodologies), can improve prediction of patient stratification according to drug response or nonresponse.

Targeting the m6A RNA methyltransferase METTL3 attenuates the development of kidney fibrosis.

Kidney fibrosis is a major mechanism underlying chronic kidney disease (CKD). N6-methyladenosine (m6A) RNA methylation is associated with organ fibrosis. We investigated m6A profile alterations and the inhibitory effect of RNA methylation in kidney fibrosis in vitro (TGF-ß-treated HK-2 cells) and in vivo (unilateral ureteral obstruction [UUO] mouse model). METTL3-mediated signaling was inhibited using siRNA in vitro or the METTL3-specific inhibitor STM2457 in vivo and in vitro. In HK-2 cells, METTL3 protein levels increased in a dose- and time-dependent manner along with an increase in the cellular m6A levels. In the UUO model, METTL3 expression and m6A levels were significantly increased. Transcriptomic and m6A profiling demonstrated that epithelial-to-mesenchymal transition- and inflammation-related pathways were significantly associated with RNA m6A methylation. Genetic and pharmacologic inhibition of METTL3 in HK-2 cells decreased TGF-ß-induced fibrotic marker expression. STM2457-induced inhibition of METTL3 attenuated the degree of kidney fibrosis in vivo. Furthermore, METTL3 protein expression was significantly increased in the tissues of CKD patients with diabetic or IgA nephropathy. Therefore, targeting alterations in RNA methylation could be a potential therapeutic strategy for treating kidney fibrosis.

Gut bacteria-derived 3-phenylpropionylglycine mitigates adipocyte differentiation of 3T3-L1 cells by inhibiting adiponectin-PPAR pathway.

BACKGROUND: Gut microbiota provide numerous types of metabolites that humans cannot produce and have a huge influence on the host metabolism. Accordingly, gut bacteria-derived metabolites can be employed as a resource to develop anti-obesity and metabolism-modulating drugs. OBJECTIVE: This study aimed to examine the anti-adipogenic effect of 3-phenylpropionylglycine (PPG), which is a glycine conjugate of bacteria-derived 3-phenylpropionic acid (PPA). METHODS: The effect of PPG on preadipocyte-to-adipocyte differentiation was evaluated in 3T3-L1 differentiation models and the degree of the differentiation was estimated by Oil red O staining. The molecular mechanisms of the PPG effect were investigated with transcriptome analyses using RNA-sequencing and quantitative real-time PCR. RESULTS: PPG suppressed lipid droplet accumulation during the adipogenic differentiation of 3T3-L1 cells, which is attributed to down-regulation of lipogenic genes such as acetyl CoA carboxylase 1 (Acc1) and fatty acid synthase (Fasn). However, other chemicals with chemical structures similar to PPG, including cinnamoylglycine and hippuric acid, had little effect on the lipid accumulation of 3T3-L1 cells. In transcriptomic analysis, PPG suppressed the expression of adipogenesis and metabolism-related gene sets, which is highly associated with downregulation of the peroxisome proliferator-activated receptor (PPAR) signaling pathway. Protein-protein association network analysis suggested adiponectin as a hub gene in the network of genes that were differentially expressed genes in response to PPG treatment. CONCLUSION: PPG inhibits preadipocyte-to-adipocyte differentiation by suppressing the adiponectin-PPAR pathway. These data provide a potential candidate from bacteria-derived metabolites with anti-adipogenic effects.

Augmentation of the RNA m6A reader signature is associated with poor survival by enhancing cell proliferation and EMT across cancer types.

N6-Methyladenosine (m6A) RNA modification plays a critical role in the posttranscriptional regulation of gene expression. Alterations in cellular m6A levels and m6A-related genes have been reported in many cancers, but whether they play oncogenic or tumor-suppressive roles is inconsistent across cancer types. We investigated common features of alterations in m6A modification and m6A-related genes during carcinogenesis by analyzing transcriptome data of 11 solid tumors from The Cancer Genome Atlas database and our in-house gastric cancer cohort. We calculated m6A writer (W), eraser (E), and reader (R) signatures based on corresponding gene expression. Alterations in the W and E signatures varied according to the cancer type, with a strong positive correlation between the W and E signatures in all types. When the patients were divided according to m6A levels estimated by the ratio of the W and E signatures, the prognostic effect of m6A was inconsistent according to the cancer type. The R and especially the R2 signatures (based on the expression of IGF2BPs) were upregulated in all cancers. Patients with a high R2 signature exhibited poor prognosis across types, which was attributed to enrichment of cell cycle- and epithelial-mesenchymal transition-related pathways. Our study demonstrates common features of m6A alterations across cancer types and suggests that targeting m6A R proteins is a promising strategy for cancer treatment.

Targeting antioxidant enzymes enhances the therapeutic efficacy of the BCL-XL inhibitor ABT-263 in KRAS-mutant colorectal cancers.

Cancer chemotherapeutic drugs exert cytotoxic effects by modulating intracellular reactive oxygen species (ROS) levels. However, whether ROS modulates the efficacy of targeted therapeutics remains poorly understood. Previously, we reported that upregulation of the anti-apoptotic protein, BCL-XL, by KRAS activating mutations was a potential target for KRAS-mutant colorectal cancer (CRC) treatment. Here, we demonstrated that the BCL-XL targeting agent, ABT-263, increased intracellular ROS levels and targeting antioxidant pathways augmented the therapeutic efficacy of this BH3 mimetic. ABT-263 induced expression of genes associated with ROS response and increased intracellular ROS levels by enhancing mitochondrial superoxide generation. The superoxide dismutase inhibitor, 2-methoxyestradiol (2-ME), exhibited synergism with ABT-263 in KRAS-mutant CRC cell lines. This synergistic effect was attributed to the inhibition of mTOR-dependent translation of the anti-apoptotic MCL-1 protein via caspase 3-mediated cleavage of AKT and S6K. In addition, combination treatment of ABT-263 and 2-ME demonstrated a synergistic effect in in vivo patient-derived xenografts harboring KRAS mutations. Our data suggest a novel role for ROS in BH3 mimetic-based targeted therapy and provide a novel strategy for treatment of CRC patients with KRAS mutations.

CRISPR screens identify a novel combination treatment targeting BCL-XL and WNT signaling for KRAS/BRAF-mutated colorectal cancers.

Metastatic or recurrent colorectal cancer (CRC) patients require systemic chemotherapy, but the therapeutic options of targeted agents remain limited. CRC patients with KRAS or BRAF gene mutations exhibit a worse prognosis and are resistant to anti-EGFR treatment. Previous studies have shown that the expression of anti-apoptotic protein BCL-XL is increased in CRC patients with KRAS/BRAF mutations, suggesting BCL-XL as a therapeutic target for this subgroup. Here, we performed genome-wide CRISPR/Cas9 screens of cell lines with KRAS mutations to investigate the factors required for sensitivity to BCL-XL inhibitor ABT-263 using single-guide RNAs (sgRNAs) that induce loss-of-function mutations. In the presence of ABT-263, sgRNAs targeting negative regulators of WNT signaling (resulting in WNT activation) were enriched, whereas sgRNAs targeting positive regulators of WNT signaling (resulting in WNT inhibition) were depleted in ABT-263-resistant cells. The activation of WNT signaling was highly associated with an increased expression ratio of anti- to pro-apoptotic BCL-2 family genes in CRC samples. Genetic and pharmacologic inhibition of WNT signaling using ß-catenin short hairpin RNA or TNIK inhibitor NCB-0846, respectively, augmented ABT-263-induced cell death in KRAS/BRAF-mutated cells. Inhibition of WNT signaling resulted in transcriptional repression of the anti-apoptotic BCL-2 family member, MCL1, via the functional inhibition of the ß-catenin-containing complex at the MCL1 promoter. In addition, the combination of ABT-263 and NCB-0846 exhibited synergistic effects in in vivo patient-derived xenograft (PDX) models with KRAS mutations. Our data provide a novel targeted combination treatment strategy for the CRC patient subgroup with KRAS or BRAF mutations.

Correlation of Nasal Fluid Biomarkers and Symptoms in Patients with Persistent Allergic Rhinitis.

OBJECTIVES: This study investigated whether the biomarkers present in nasal fluid reflect the severity of symptoms in patients with persistent allergic rhinitis (PAR). METHODS: We enrolled 29 PAR patients complaining of nasal symptoms and testing positive to skin prick test. Patients' total nasal symptom score (TNSS) was measured and their nasal lavage fluid (NALF) was collected. The levels of biomarkers including Clara cell protein 16 (CC16), tryptase, and interleukin 5 (IL-5) in NALF were determined via enzyme-linked immunosorbent assay (ELISA). RESULTS: PAR patients were classified into persistent mild and persistent moderate-to-severe groups according to the Allergic Rhinitis and its Impact on Asthma (ARIA) guidelines. The CC16 alone was significantly negatively correlated with TNSS (P < .05). Further, the CC16 level was significantly lower in persistent moderate-to-severe group than persistent mild group of patients (P < .05). CONCLUSIONS: The levels of CC16 alone among several NALF biomarkers showed an inverse correlation with symptoms of PAR patients.