Hypoxia-driven regulatory mechanisms play a critical role in tumor progression and therapeutic resistance in hepatocellular carcinoma (HCC), yet hypoxia-responsive microRNAs (HRMs) remain incompletely characterized. This study aimed to identify HRMs in canine HCC to evaluate their diagnostic potential and translational relevance to human disease. Next-generation sequencing of two canine HCC cell lines under normoxic and hypoxic conditions identified 332 and 321 differentially expressed miRNAs, respectively. Integrating these with tumor tissue data revealed 11 HRMs, featuring consistent upregulation of cfa-miR-210 and cfa-miR-34a, which was validated via RT-qPCR in hypoxic cells and clinical tissues. Both miRNAs were significantly elevated in plasma-derived extracellular vesicles (EVs), highlighting their value as promising circulating biomarkers (AUC 1.00 for miR-210; 0.98 for miR-34a). Target gene and pathway analyses identified shared regulatory nodes, including TGIF2 and SPRED1; and enrichment of MAPK, Ras, PI3K-Akt, and Rap1 signaling, broadly linked to hypoxia adaptation, cellular metabolism, and stress-response signaling. Cross-species comparison with human HCC datasets showed that, while miR-210 associates with poor prognosis in human HCC, miR-34a exhibits tumor-suppressive features. These findings define complementary HRM programs in canine HCC, reflecting conserved adaptive and stress-limiting regulatory mechanisms with potential diagnostic and translational relevance to human HCC.

Anti-CD38 monoclonal antibodies have substantially improved outcomes in multiple myeloma (MM). Although daratumumab and isatuximab target the same antigen, accumulating evidence indicates that they differ in epitope recognition, biological activity, and immunomodulatory properties, suggesting these agents may not be therapeutically interchangeable. This review summarizes the molecular and immunological mechanisms underlying their distinct antitumor effects and their implications for treatment selection. Isatuximab binds near the catalytic site of CD38, resulting in potent enzymatic inhibition, enhanced antibody internalization, FOXM1 suppression, and reactive oxygen species-mediated cytotoxicity, which may preferentially target MM cells harboring 1q21 amplification. In contrast, daratumumab exerts prominent Fc-dependent immune effects, including trogocytosis-mediated downregulation of CD38 and VLA-4, suppression of cell adhesion-mediated drug resistance, and modulation of the immune microenvironment, potentially enhancing subsequent T-cell-redirecting therapies. We further discuss the relevance of these mechanistic differences to measurable residual disease, extramedullary disease, and sequencing with BCMA- and GPRC5D-directed immunotherapies. Finally, we propose a biology-guided treatment-selection model integrating genomic alterations, tumor biology, and immune remodeling to support precision medicine for patients with MM.

Caspase-8 and the NF-κB essential modulator (NEMO, also referred to as IKKγ) play critical roles in controlling TNF-α-induced cell death and survival in hepatocytes. The aim of this study was to generate a hepatocyte-derived cell line in which the Casp8 and Nemo genes can be conditionally inactivated simultaneously to investigate the significance of the corresponding signaling pathways. To this end, we induced hepatocellular carcinoma in Casp8f/fNemof/f mice using diethylnitrosamine and established an immortalized hepatoma cell line from explanted liver tumors, which is subsequently referred to as ΔCN60. ΔCN60 cells still retain floxed Casp8 and Nemo alleles, allowing for efficient Cre-mediated deletion to generate Casp8ΔNemoΔ derivatives. Loss of both Caspase-8 and NEMO inhibits cell proliferation, increases the expression of tumor and progenitor markers (AFP, CD133), reduces albumin expression, and blocks TNF-α-induced NF-κB p65 nuclear translocation. ΔCN60 Casp8ΔNemoΔ cells display altered sensitivity to prolonged TNF-α exposure, suggesting a potential shift in necroptosis-associated signaling pathways, although necroptotic cell death was not directly demonstrated. ΔCN60 serves as a versatile hepatoma model for investigating Caspase-8/NEMO-dependent TNF-α signaling and hepatocyte plasticity and may help prioritize future in vivo experiments by enabling selected mechanistic questions to be addressed first in vitro.

Background: Recent evidence suggests that the antidepressant vortioxetine (Vx) inhibits the growth of glioblastoma (GBM), the most aggressive primary malignant tumor of the CNS. We used five patient-derived GBM cell lines to confirm the anti-GBM effect of Vx and explore its mechanism(s) of action. Methods: We performed in silico analysis, PCR, TUNEL assay, colony assay, and proliferation assay. Results: Vx potently inhibited GBM cell growth, and showed efficacy at concentrations of 0.1 μM that roughly correspond to therapeutic concentrations of Vx in major depression. Other antidepressants, i.e., fluoxetine and duloxetine, inhibited GBM cell growth only at high concentrations. Computational analysis showed that at least three receptor targets of Vx (5-HT7, 5-HT1D, and 5-HT1B) were expressed at moderate/high levels in GBM. The 5-HT1B receptor transcript was found in all GBM cell lines and was the only detectable Vx target in three of the five cell lines. Blocking 5-HT1B with SB224289 abolished the anti-GBM effect of Vx, even in cells expressing other Vx. Conclusions: These findings demonstrate that therapeutic concentrations of Vx inhibit GBM cell proliferation and suggest that this action may be mediated, at least in part, by 5-HT1B receptor activation.

Metastasis remains the principal cause of cancer mortality, with the lungs representing one of the most frequent and clinically challenging sites. The tumor microenvironment, particularly tumor-associated macrophages (TAMs), plays a pivotal role in establishing and maintaining metastatic niches. Yet, how host ketone bodies influence the immune microenvironment to govern metastatic progression is poorly defined.

Nuclear receptor coactivator 3 (NCOA3) is associated with various cancers, but its function and mechanism in glioblastoma multiforme (GBM) are still unclear. Bioinformatics analysis, in vitro cell experiments (NCOA3 silencing (si-NCOA3) or NCOA3 small-molecule inhibitor SI-2), in vivo animal models, and metabolic level detection were used to elucidate the activity of NCOA3 in GBM. The data revealed that GBM tissues had NCOA3 overexpression, which was linked with poor prognosis. It regulates pathways related to glycolysis, the cell cycle, and immunosuppression. Functionally, si-NCOA3/SI-2 suppressed GBM cell proliferation and migration. In vivo, sh-NCOA3/SI-2 demonstrated anti-glioma effects. Metabolically, treatment with si-NCOA3/SI-2 reduced glucose uptake, pyruvate and lactate production, ATP levels, and glycolysis-related enzyme expression in GBM cells. Combination therapy with SI-2 and TMZ enhanced GBM cell sensitivity to TMZ. Single-cell RNA sequencing revealed high NCOA3 expression in glioma stem cells (GSCs). si-NCOA3 inhibited GSCs proliferation and self-renewal while reducing the expression of Nestin and SOX2. NCOA3 is an oncogene in GBM. In mechanism, NCOA3 promotes GBM progression by enhancing the Warburg effect. In addition, NCOA3 is also highly expressed in GSCs and significantly promotes their proliferation and self-renewal ability. NCOA3 may represent a promising therapeutic target for GBM.

Clear cell renal cell carcinoma (ccRCC) exhibits substantial molecular and clinical heterogeneity, contributing to variable disease progression and therapeutic outcomes. We previously identified a de-clear cell differentiation (DCCD) tumor state characterized by loss of canonical clear-cell features and aggressive clinical behavior. This study aimed to translate DCCD-associated biology into a prognostic framework and investigate its underlying molecular basis. Using TCGA-KIRC as the training cohort, we developed a five-gene prognostic signature comprising COL7A1, IGFN1, AJAP1, SMIM24, and ADGRV1, which consistently stratified patient survival across multiple public and institutional cohorts. Comparison with SSIGN, Leibovich, and ClearCode34-like models demonstrated that the DCCD signature provided complementary prognostic information. High-risk tumors exhibited enhanced proliferative and invasive programs, increased IL6-JAK-STAT3 pathway activity, and an immune-excluded microenvironment. Associations with treatment response were also observed in retrospective therapeutic cohorts. Integrated analyses of bulk and single-cell transcriptomics, spatial transcriptomics, tissue microarrays, CPTAC proteomics, and molecular interaction networks identified SMIM24 as an epithelial-associated factor linked to favorable outcomes and preserved metabolic programs. Functional experiments showed that SMIM24 overexpression suppressed migration, invasion, three-dimensional spheroid invasion, and clonogenic growth in ccRCC cells, accompanied by reduced JAK1 and STAT3 phosphorylation and decreased PD-L1 expression. Pharmacological activation of STAT3 with Colivelin partially restored PD-L1 expression in SMIM24-overexpressing cells. Collectively, these findings support the reproducibility of the DCCD-associated prognostic framework and identify SMIM24 as a candidate suppressor of ccRCC aggressiveness and PD-L1 expression, potentially acting in part through reduced STAT3 activation.

R-CHOP-treated diffuse large B-cell lymphoma (DLBCL) shows heterogeneous outcomes. High-risk relapsed/refractory (R/R) patients, especially activated B-cell (ABC) and double-/triple-hit subtypes, have poor prognosis, highlighting an urgent need to uncover the underlying aggressive molecular mechanisms. Epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) properties drive its invasion, metastasis, and drug resistance. This study investigated the regulatory role and mechanism of Hedgehog (Hh) pathway key factor GLI1 in DLBCL. Immunohistochemistry detected GLI1, Bmi1, and SOX2 expression in DLBCL tissues. DLBCL cell lines (HBL-1, SUDHL-2) were treated with pan-GLI inhibitor GANT61, or transfected with GLI1 overexpression/silencing plasmids combined with Bmi1 intervention. CCK-8, flow cytometry, RT-qPCR, Western blot, Co-IP, tumor sphere, and immunofluorescence assays were performed. In vivo xenograft experiments were conducted to verify GANT61's anti-tumor effect. GLI1 was aberrantly activated in DLBCL tissues and positively correlated with Bmi1/SOX2. GANT61 inhibited DLBCL cell proliferation, induced G0/G1 arrest and apoptosis, reversed EMT, and reduced CSC-related molecules. Co-IP confirmed GLI1-Bmi1 interaction. GLI1 overexpression enhanced proliferation, EMT, and CSC properties, while GLI1 silencing exerted opposite effects. Bmi1 inhibitor reversed GLI1's pro-tumor effects, and Bmi1 agonist partially restored GLI1 silencing-induced inhibition. In vivo, GANT61 significantly reduced tumor volume/weight in HBL-1 xenografts with good tolerability. GLI1 modulates EMT-like plasticity and stem cell traits in DLBCL via direct protein interaction with Bmi1, confirming a functional regulatory connection between these two molecules that forms the core Hh-GLI1-Bmi1 regulatory pathway. Targeting this axis provides a novel therapeutic strategy for DLBCL. Key words: Hedgehog signaling pathway; Diffuse large B-cell lymphoma (DLBCL); Epithelial-mesenchymal transition (EMT); Cancer stem cell properties; GLI1-Bmi1 axis; GANT61; Xenograft model.

Population-based genomic newborn screening identifying newborns at risk for early-onset cancers has not been evaluated. Here, within a Michigan birth cohort (1987-2020), we identify all children developing a solid or central nervous system malignancy by age 8 years (n = 1948). We perform targeted sequencing of 11 cancer predisposition genes using archived newborn dried blood spot DNA. We find pathogenic or likely-pathogenic germline variants (P/LP) in 6.8% of cases (n = 132): RB1 (n = 69), TP53 (n = 24), SMARCB1 (n = 8), WT1 (n = 7), RET (n = 6), SUFU (n = 6), PTCH1 (n = 4), DICER1 (n = 4), APC (n = 3) and PHOX2B (n = 1). We show approximately 1/27,000 newborns develop an early-onset malignancy with an associated pathogenic or likely-pathogenic variant. Germline variant prevalence is 100% in medullary thyroid carcinoma, 40% in retinoblastoma, and 11-30% across five additional diagnoses, with strong gene-tumor specificity (p < 0.001). P/LP variants are rare in comparison datasets from healthy newborns and gnomAD. Our data support newborn screening for selected cancer-risk genes.

Transcription factor SNAI1 guides plasticity and invasiveness in cancer. Using a complete SNAI1 knockout in mesenchymal, triple-negative breast cancer cells, unbiased genome-wide transcriptomic analysis revealed a marked under-expression of integrin-based adhesion and endocytic components. Utilizing this knockout cell model, complementary breast cancer cell models and functional screening of multiple differentially expressed genes, we found that the pioneering transcription factor FOXA1, whose expression is repressed by SNAI1, associates with several key mediators of the cellular phenotype. FOXA1 represses the small GTPase ARF6 and its exchange factor PSD4. In addition, some of the integrin and matrix metalloproteinase genes are regulated by the transcriptional FOXA1 signal. Accordingly, SNAI1 knockout cells presented poor adhesion to collagen type I or fibronectin, formed defective invadopodia and focal adhesions with weakened FAK/SRC signaling. SNAI1 knockout cells performed ineffective receptor-mediated internalization, including nanoparticle and extracellular vesicle (EV) uptake, exhibited reduced lysosomal content, lacked multivesicular bodies enriched in intraluminal vesicles and showed decreased EV secretion. Gain-of-function experiments demonstrated that SNAI1 has an impact on the PSD4/ARF6 signaling module, using FOXA1 as an intermediate factor to regulate EV release by tumor cells. We propose that the SNAI1-FOXA1 transcriptional mechanism operates at the level of membrane and vesicular trafficking control, which interlinks cell plasticity, adhesion and invasiveness through the extracellular environment, with the associated process of EV secretion.

Mefatinib, a novel second-generation epidermal growth factor (EGFR) tyrosine kinase inhibitor that has shown promising antitumor activity in targeting non-small cell lung cancer (NSCLC) with common and uncommon EGFR-activating mutations. In this phase III, randomized, double-blind trial in China, 336 eligible patients with advanced nonsquamous NSCLC harboring EGFR L858R or exon 19 deletion (ex19del) were assigned (2:1) to receive either mefatinib (60 mg daily, n = 223) or gefitinib (250 mg daily, n = 113). The primary endpoint was progression-free survival (PFS), assessed by an independent review committee (IRC). The trial is registered with chinadrugtrials.org.cn (CTR20192297). After a median follow-up of 15.9 months for mefatinib and 18.5 months for gefitinib, mefatinib demonstrated a significantly longer median IRC-assessed PFS compared to gefitinib (13.7 vs. 9.7 months; hazard ratio [HR] = 0.68; 95% confidence intervals [CI]: 0.53-0.87; p = 0.002). The 30-month overall survival rate was 60.2% for mefatinib and 54.3% for gefitinib. Patients with EGFR ex19del had comparable PFS for both treatment arms (p > 0.100), whereas patients with EGFR L858R had significantly longer median PFS when treated with mefatinib than gefitinib (13.7 vs 8.3 months HR = 0.55 [95% CI: 0.38-0.78]; p = 0.001). Patients with EGFR L858R had a 30-month overall survival rate of 56.6% with mefatinib and 43.7% with gefitinib. Treatment-related adverse events ≥grade 3 were reported in 45.7% of the mefatinib group and 24.8% of the gefitinib group. No new safety signals were observed for mefatinib. Mefatinib demonstrated superior efficacy to gefitinib with a similar tolerability profile in the first-line treatment of EGFR-mutated advanced NSCLC.

Cancer stem cells (CSCs) are tumor cell subsets with self-renewal, multilineage differentiation, and tumor-initiating capacity that sustain cancer initiation, progression, metastasis, and relapse. Targeting CSCs therefore represents a promising route to improve the durability of cancer treatment. However, translation of this approach into routine care has been slow because of the biological complexity and clinical constraints. This review discusses current concepts of CSC origin and plasticity, the criteria used to define CSCs across different tumor types, and the marker systems as well as high-resolution technologies that are used to track CSC states. Developmental pathways, growth factor and cytokine cascades, as well as microenvironmental and stress responses that control CSC maintenance and therapy resistance are explored with a focus on their tractability as drug targets. We then discuss mechanisms through which CSCs escape chemotherapy, radiotherapy, and targeted agents. We review current efforts to use these pathways in designing small molecules, antibodies, cellular therapies, and vaccines aimed at CSC compartments. Heterogeneity within and between tumors, dynamic interconversion between CSC and non-CSC states, and support from specialized niches are considered as major barriers for clinical trial design, biomarker development, and response assessment. Emerging single-cell, spatial, and lineage tracing approaches, together with organoid and ex vivo platforms, are reviewed as tools that can bridge preclinical models and patient samples and guide the development of CSC-directed combination regimens. The goal is to outline translational principles that can guide future strategies for integrating CSC-focused interventions with established therapies to improve long-term disease control.

KRAS mutations are highly prevalent in pancreatic cancer and are critical for epithelial reprogramming during tumor initiation. In this issue of Developmental Cell, Grimont et al.1 demonstrate that the three most common KRAS mutations differentially activate downstream signaling pathways, resulting in distinct capacities to develop pancreatic pre-neoplastic lesions.

Comprehensive genomic profiling (CGP) is increasingly adopted in the management of patients affected by GI cancers. However, the applicability, performance, and clinical utility of CGP in the real-world setting are still undefined.

The molecular analysis of urine cell-free DNA offers a noninvasive tool to advanced bladder cancer (BC) management. Assessment of somatic copy number aberration (SCNA) and DNA methylation analysis have emerged as promising approaches for BC detection. Here, we developed an integrated analysis to assess both SCNA and targeted methylation changes from the same template molecules, which we named the integrated sequencing-based copy number and methylation analysis in urine (iSECURE) method.

Pulmonary large-cell neuroendocrine carcinoma (LCNEC) is a rare yet highly aggressive subtype of non-small cell lung carcinoma (NSCLC) with limited therapeutic options. We conduct a comprehensive proteogenomic analysis of LCNEC using tumors and paired normal adjacent tissues from 107 patients (81 pure LCNEC and 26 combined LCNEC). APOBEC mutational signatures strongly correlate processes of tumor initiation and immune suppression, and KEAP1 mutations correlate with metabolic reprogramming in LCNEC combined with NSCLC. A conflicting relationship is observed between neuroendocrine and immune phenotypes. We identify three LCNEC subtypes with unique prognosis features, microenvironment dysregulation, genetic alterations, and potential therapeutic targets. Interleukin-33 (IL-33) emerges as a critical therapeutic biomarker associated with enhanced T cell infiltration and antitumor activity. We further optimize recombinant IL-33 (rIL33) with site-directed mutagenesis and develop PEGylated rIL33, demonstrating its prolonged circulation time in cynomolgus monkeys and superior immune agonist activity in mouse models. Overall, this study offers insights into LCNEC biology and provides promising innovative immunoagonist therapy strategies for lung cancer.

Myasthenia gravis (MG) presents a clinical challenge where autoantibody titers poorly predict disease severity, and thymectomy provides inconsistent benefits. We hypothesized that thymic B cells acquire survival mechanisms that bypass canonical tolerance checkpoints, enabling persistence independent of antigen-specific selection. Using single-cell RNA sequencing, V(D)J repertoire analysis, and spatial transcriptomics to generate the largest MG atlas to date (237,661 cells), we identified a tolerance checkpoint swap in MG pathogenesis. Pathological class-switched B cells within thymic germinal centers exhibited reduced antigen presentation (CD74 and CD1C) and diminished CD40 costimulation while up-regulating TNFRSF17 to engage BAFF-driven survival. This shift allows polyclonal autoreactive B cells to evade stringent selection and seed peripheral sites. T follicular helper cells supported this reprogramming via increased TNFSF13B and CHGB expression, with the latter facilitating dopamine-mediated synaptic acceleration. These findings offer insight into clinical paradoxes in MG and point to the BAFF-BCMA axis as a potential therapeutic target and biomarkers for disease activity.

Remodeling of fatty acid metabolism is increasingly recognized as a critical feature of tumor progression, yet the contribution of very long-chain fatty acid (VLCFA) remains incompletely understood. The elongation of VLCFAs, which is mediated by four consecutive enzymes in the endoplasmic reticulum (ER), has been implicated in tumor progression. However, the molecular mechanisms underlying VLCFA elongation enzymes and their specific contributions to tumorigenesis remain largely elusive. Here, we demonstrate that trans-2-enoyl-CoA reductase (TECR) is upregulated in colorectal cancer (CRC). Structural and biochemical analyses revealed a conserved catalytic mechanism for TECR-mediated trans-2-enoyl-CoA reduction. Moreover, we show that TECR forms a stable complex with 3-hydroxyacyl-CoA dehydratase (HACD), to cooperatively drive VLCFA elongation. A unique U-shaped loop in HACD is critical for recognizing TECR. Disruption of the HACD-TECR interaction interface significantly suppresses CRC cell growth. Collectively, these findings elucidate the molecular mechanism of HACD-TECR-mediated VLCFA elongation and suggest a potential therapeutic strategy for CRC treatment by modulating VLCFA metabolism.

Acute myeloid leukemia (AML) is an aggressive hematological malignancy arising from hematopoietic stem and progenitor cells (HSPCs). Current treatments often fail to eradicate AML; therefore, new therapeutic strategies are essential. Here, we reveal that RNA terminal uridylyl transferase enzymes 4 and 7 (TUT4/7) are druggable therapeutic targets, whose genetic deletion suppresses AML growth, induces apoptosis, and improves the survival in leukemic mouse models. Notably, a preclinical TUT4/7 inhibitor promotes cell death in samples from patients with AML and synergizes with venetoclax. Mechanistically, TUT4/7 inactivation suppresses mevalonate pathway gene expression, compromising the cholesterol synthesis pathway. Current AML therapies often cause severe hematopoietic toxicity. Although Tut4/7 deletion results in inflammatory activation throughout the hematopoietic system, this is permissive to a normal life span and Tut4/7 deficiency does not compromise HSPC function. Together, these findings identify TUT4/7 as druggable targets, whose inactivation suppresses AML while sparing normal hematopoiesis. In combination with venetoclax, this represents a promising therapeutic strategy.

Acute myeloid leukemia/myelodysplastic syndromes (AML/MDSs) carrying p53 mutations are refractory to various standard therapies. Arsenic trioxide (ATO) may be effective in restoring function to p53 structural mutants. Here, we report that mutant p53 rescued by ATO treatment strengthened interferon responses triggered by the DNA hypomethylating agent decitabine by transactivating interferon regulatory factor 7 (IRF7) directly. Decitabine also increased the transactivation activity of ATO-rescued mutant p53 by inducing p53-serine-20 phosphorylation and blocking p53-inhibitory mouse double minute 2 homolog (MDM2). ATO and decitabine together killed p53-mutant AML cells and suppressed tumor growth in cell line-derived xenografts. In a first-in-human pilot clinical trial for testing the combination of ATO and decitabine (PANDA-T0 trial, NCT03855371), which enrolled five patients with AML/MDS harboring p53 structural mutations, the ATO and decitabine regimen produced manageable adverse events, and four of the five treated patients achieved complete remission at the level of the bone marrow, associated with p53 activation and interferon response. In 103 p53-mutant patients whose samples were deposited in Ruijin AML/MDS sample repository, 48 distinct p53 missense mutants were identified, 21 of which were classified as ATO and decitabine regimen applicable because of their competencies in activating p53 and interferon responses upon cotreatment. This study establishes an alternative treatment regimen for patients with p53-mutant AML/MDS and provides a proof-of-concept framework for p53-targeted therapy that differentiates between p53 mutations.