Glutamine is a non-essential amino acid that is critical for cell growth. However, the differential metabolism of l-glutamine in metastatic versus primary colorectal cancer (CRC) has not been evaluated adequately. Differential expression of glutamine-related genes was determined in primary versus metastatic CRC. Univariate Cox regression and hierarchical clustering were used to generate a gene signature for prognostication. Untargeted metabolomics and ¹⁸O based fluxomics were used to identify differential metabolite levels and energy turnover in the paired primary (SW480) and metastatic (SW620) CRC cells. Western blot and qRT-PCR were used to validate differential gene expression. Subcellular localization of E-cadherin was determined by immunocytochemistry. Lipid droplets were visualized with Nile Red. The GO term "Glutamine metabolism" was significantly enriched in metastatic versus primary tumors. Supporting this, SW620 cells showed decreased membrane localization of E-cadherin and increased motility upon l-Glutamine withdrawal. A glutamine related signature associated with worse prognosis was identified and validated in multiple datasets. A fluxomics assay revealed a slower TCA cycle in SW480 and SW620 cells upon l-Glutamine withdrawal. SW620 cells, however, could maintain high ATP levels. Untargeted metabolomics indicated the preferential metabolism of fatty acids in SW620 but not SW480 cells. Lipids were mainly obtained from the environment rather than by de novo synthesis. Metastatic CRC cells can display aberrant glutamine metabolism. We show for the first time that upon l-glutamine withdrawal, SW620 (but not SW480) cells were metabolically plastic and could metabolize lipids for survival and cellular motility.

Hepatocellular cancer (HCC) is a leading cause of cancer-related mortality worldwide, and chronic hepatitis B virus infection (CHB) has been a major risk factor for HCC development. The pathogenesis of HBV-related HCC has been a major focus revealing the interplay of a multitude of intracellular signaling pathways, yet the precise mechanisms and their implementations to clinical practice remain to be elucidated. This study utilizes publicly available transcriptomic data from the livers of CHB patients in order to identify a population with a higher risk of malignant transformation. We report the identification of a novel list of genes (PCM1) which can generate clear transcriptomic sub-groups among HBV-infected livers. PCM1 includes genes related to cell cycle activity and liver cancer development. In addition, markers of inflammation, M1 macrophages and gamma delta T cell infiltration are present within the signature. Genes within PCM1 are also able to differentiate HCC from normal liver, and some genes within the signature are associated with poor prognosis of HCC at the mRNA level. The analysis of the immunohistochemical stainings validated that proteins coded by a group of PCM1 genes were overexpressed in liver cancer, while minimal or no expression was detected in normal liver. Altogether, our findings suggest that PCM1 can be developed into a clinically applicable method to identify CHB patients with a higher risk of HCC development.

Background: Molecular biomarkers that predict disease progression can help identify tumor subtypes and shape treatment plans. In this study, we aimed to identify robust biomarkers of prognosis in gastric cancer based on transcriptomic data obtained from primary gastric tumors. Methods: Microarray, RNA sequencing, and single-cell RNA sequencing-based gene expression data from gastric tumors were obtained from public databases. Freshly frozen gastric tumors (n = 42) and matched FFPE (formalin-fixed, paraffin-embedded) (n = 40) tissues from a Turkish gastric cancer cohort were used for quantitative real-time PCR and immunohistochemistry-based assessments of gene expression, respectively. Results: A novel list of 20 prognostic genes was identified and used for the classification of gastric tumors into two major tumor subgroups with differential stromal gene expression (“Stromal-UP” (SU) and “Stromal-DOWN” (SD)). The SU group had a more mesenchymal profile with an enrichment of extracellular matrix-related gene sets and a poor prognosis compared to the SD group. Expression of the genes within the signature correlated with the expression of mesenchymal markers ex vivo. A higher stromal content in FFPE tissues was associated with shorter overall survival. Conclusions: A stroma-rich, mesenchymal subgroup among gastric tumors identifies an unfavorable clinical outcome in all cohorts tested.

Chronic HBV infection remains incurable in most patients despite long-term nucleos(t)ide analog (NUC) therapy. Interferon-γ (IFN-γ) is a cornerstone of antiviral immunity, but its in situ source and status in the human liver remain unknown. We aimed to define the primary source of IFN-γ in healthy liver and determine how this axis is altered in stably suppressed NUC-HBV. Multiplex immunofluorescence for CD3, CD56, CXCR6, and IFN-γ was performed on liver biopsies from patients with NUC-HBV (n = 9) and healthy donors (n = 7), with whole-slide scanning and AI-based segmentation for unbiased in situ cell quantification. Single-cell RNA sequencing (scRNAseq) was conducted on paired blood and liver fine-needle aspirates (FNAs) from NUC-HBV (liver n = 9, blood n = 18) and integrated with control datasets (liver n = 5, blood n = 9). Differential gene expression was used for transcriptional readout, and cell-cell interaction analysis mapped altered signaling networks. In healthy liver, CXCR6-positive natural killer (CXCR6+NK) cells were the dominant IFN-γ producers (24.2%; IQR 14.1-67.2%). In NUC-HBV, these cells were reduced (675 vs. 1,918; p = 0.012) and rarely expressed IFN-γ (0.2%; IQR 0.0-1.4%), despite normal alanine aminotransferase and absence of fibrosis. To validate transcriptionally, we performed scRNAseq on FNA and paired blood. A single, liver-restricted CXCR6+NK cluster was identified, and showed selective downregulation of IFNG and chemokines (XCL1, CCL3, CCL4), while cytotoxic genes (GZMB, PRF1) were preserved. Interaction analysis revealed reduced pro-inflammatory signaling and enrichment of transforming growth factor-beta-associated pathways. CXCR6+NK frequency correlated with serum HBsAg (p = 0.037) but was unchanged after 24 weeks of NUC in a longitudinal dataset. Long-term NUC therapy does not restore intrahepatic IFN-γ. Loss and transcriptional reprogramming of CXCR6+NK may contribute to a stable, altered immune state, representing a target for immune-based HBV cure strategies. Effective antiviral therapy for chronic HBV suppresses viral replication but does not provide cure, indicating persistent defects in intrahepatic antiviral immunity. By combining protein-level analysis of liver tissue from healthy living donors and NUC-treated HBV liver biopsies with single-cell RNA-seq of fine-needle aspiration-derived immune cells from clinically stable, long-term NUC-treated patients, we directly examined IFN-γ regulation in the human liver-an immune compartment that is largely inaccessible in this patient group. We demonstrate that IFN-γ is produced in situ predominantly by CXCR6+NK cells in healthy liver and is selectively reduced in the liver of NUC-treated patients, despite normal alanine aminotransferase levels, absence of fibrosis, and lack of inflammatory or exhaustion signatures, while no corresponding defect is observed in blood. These findings identify a liver-restricted, non-exhaustion-driven impairment of NK-cell function and suggest that future therapeutic strategies should focus on restoring intrahepatic immunity-potentially via modulation of TGF-β-associated pathways or CXCR6+NK cell IFN-γ production.

T-lineage acute lymphoblastic leukemia (T-ALL) lacks effective targeted therapies, with poor outcomes in relapsed/refractory (R/R) disease. HOXAhigh T-ALL is biologically aggressive and often resistant to standard therapy. Menin inhibitors, recently approved for KMT2A-rearranged leukemias, may be effective in T-ALL, but biomarkers of response remain undefined. This study aims to evaluate the efficacy of menin inhibition in T-ALL and identify molecular predictors of sensitivity. We tested menin inhibitors (ziftomenib, revumenib, and VTP50469) in 14 primary T-ALL samples and 8 cell lines, representing HOXAhigh and HOXAlow genotypes. In vitro sensitivity assays, xenograft mouse models, transcriptomics, proteomics, and phosphoproteomics were used to characterize drug response. MEF2C modulation experiments and combination studies with cyclin-dependent kinase (CDK) 1/2 and ERK1/2 inhibitors were performed in vitro and in vivo. Menin inhibitors suppressed leukemic growth in a subset of HOXAhigh and HOXAlow primary human T-ALL samples. Similarly, ziftomenib was effective in reducing tumor burden in xenografts without major toxicity. Upon treatment, we observed downregulation of canonical menin targets (HOXA, MEIS1, and MEF2C) and upregulation of T-cell differentiation programs. Phosphoproteomic studies identified MEF2C S222 phosphorylation—mediated by CDK1/2 and ERK1/2—as a predictor of ziftomenib sensitivity in T-ALL. MEF2C overexpression promoted proliferation and ziftomenib resistance, whereas its knockdown impaired growth. Ziftomenib synergized with CDK1/2 and ERK1/2 inhibitors in vitro and improved survival in xenografted mice. In conclusion, a subset of T-ALL, defined by high p-MEF2C S222, is sensitive to menin inhibition. Combining ziftomenib with CDK or ERK inhibition offers synergistic efficacy, supporting biomarker-driven clinical trials of this strategy in R/R T-ALL.

Gene network maps showing fold-changes in the genes involved in down-regulated (A) or up-regulated (B) pathways in T-ALL primary cells treated with ziftomenib.