Among colorectal cancers (CRC), DNA mismatch repair-deficient (dMMR) tumors exhibit high mutational burden, increased immunogenicity, and better responsiveness to immune checkpoint blockade, when compared to MMR-proficient (pMMR) tumors. As a result, conventional T cells have been extensively studied in this disease, while the function of gδ T cells is understudied. Here, we report on a novel dMMR CRC mouse model whose cancer progression relies on IL-17A-producing gδ T cells. Deletion of Mlh1 in mouse intestinal epithelial cells resulted in tumors with a long latency period and high mutational burden. Mlh1 -deficient tumors were characterized by inflammatory transcriptional signatures. IL-17A-producing gδ T cells were the most abundant T cell in these tumors, and deletion or inhibition of these cells extended survival of tumor-bearing mice, indicative of their pro-tumorigenic role. Interfering with gδ T cells or IL-17A increased CD8 T cell and reduced neutrophil infiltration into tumors. Mlh1 -deficient tumors were unresponsive to anti-PD-1 immunotherapy. Resistance to this immunotherapy was facilitated by IL-17A-producing gδ T cells. However, tumors were sensitive to COX-2 inhibition, as celecoxib extended survival and reduced tumor-associated gδ T cells. Analysis of human CRC indicated that gδ T cells are more frequent in dMMR than pMMR and exhibit activation of IL-17 pathway. Tumor-associated macrophages exhibited the highest expression of PTGS2 , mirroring observations in mice. This study demonstrates that IL-17A-producing gδ T cells under regulation of COX-2 play an important role in dMMR CRC progression, revealing potential new targets for immunotherapy-resistant dMMR tumors.

Knockdown of Cdx1, Cdx2 and Hnf4a fails to influence organoid morphology or expression of Btnl genes

Aberrant Wnt/β-catenin signaling is a hallmark of hepatocellular carcinoma (HCC), with activating CTNNB1 mutations contributing to oncogenesis, immune evasion, and resistance to therapy. Up to 40% of HCC cases are driven by activating mutations in β-catenin or other components of the canonical Wnt pathway, which are associated with primary resistance to standard-of-care therapies. Transcriptional activation of Wnt target genes requires β-catenin recruitment via transducin beta-like protein 1 (TBL1). Tegavivint, an investigational small molecule currently in clinical studies for HCC (NCT05797805), inhibits the β-catenin–TBL1 interaction. While tegavivint has demonstrated selective activity in CTNNB1-mutant desmoid tumors, it is also known to disrupt both wild-type and mutant β-catenin interactions in other tumor contexts. Here, we evaluated how Wnt pathway mutation status shapes cellular phenotype and response to tegavivint using in vitro and in vivo HCC models. We profiled three HCC cell lines with distinct CTNNB1 statuses: HUH7 (wild-type), HUH6 (G34V), and HepG2 (exon 3 deletion). Baseline pathway activation, phenotypic features, and response to tegavivint were assessed. Apoptosis was measured by Annexin V staining, cell viability via CellTiter-Glo, and cell death kinetics using real-time Sytox Green imaging on the IncuCyte platform. Wnt target gene expression (AXIN2, GLUL, LGR5, NOTUM) was quantified by qPCR. In vivo, orthotopic HepG2 xenografts were established in NOD/SCID mice and treated with tegavivint (50 mg/kg BIW for 3 weeks). Tumor growth was monitored by ultrasound. Post-treatment analyses included serum alpha-fetoprotein (AFP) and serum protein profiling via Luminex, and immunohistochemistry for β-catenin, TBL1, and additional markers. Tegavivint treatment led to cell death in CTNNB1 mutant cell lines and a significant tumor growth inhibition in vivo, accompanied by transcriptional suppression of β-catenin targets (AXIN2, GLUL, LGR5, NOTUM) and re-expression of hepatic differentiation genes. Luminex analysis revealed upregulation of immune-modulatory proteins, suggesting a shift in the tumor immune microenvironment. In our ongoing clinical study, patient samples collected pre- and post-dosing with tegavivint were evaluated for mutational status using the FoundationOne® Liquid CDx assay and for changes in serum proteins using Luminex. Mutations in the Wnt/beta-catenin pathway were assessed for correlation with clinical activity, along with preliminary analyses of associations between clinical response and changes in serum protein biomarkers. These findings demonstrate that Wnt pathway mutation status shapes HCC phenotype and predicts sensitivity to tegavivint. CTNNB1-mutant models showed enhanced responses, including growth inhibition, differentiation, and transcriptomic reprogramming. These data support the use of Wnt/β-catenin pathway inhibitors as a precision medicine strategy in CTNNB1-mutant HCC and highlight the need for further studies in immune-competent models and biomarker-enriched clinical trials. Ciara Curran, Toshiyasu Suzuki, Aundrietta D. Duncan, Stephen K. Horrigan, Thomas G. Bird. Activity of Tegavivint in Hepatocellular Carcinoma with Aberrant Wnt/β-catenin Signaling and Evaluation of Biomarker Response [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr B135.

Impact of Btnl1 and Btnl6 expression on Vgamma7+ cells and the tumor microenvironment

Clinicopathological characteristics of colon cancer patient cohorts.

Deletion of Apc in organoids increases WNT target genes

Disruption of WNT gradient in normal intestinal villi reduces gamma delta T cells

Identification of a WNT responsive element (WRE) in the HNF4G genomic locus.

Correlation between gut-specific transcription factors, gamma delta T cell density and BTNL genes in human tumors

WNT expression is independent of BTN3A1 and BTN2A1

Hepatocellular carcinoma (HCC), the most common form of primary liver cancer, is a leading cause of cancer-related mortality worldwide1,2. HCC occurs typically from a background of chronic liver disease, caused by a spectrum of predisposing conditions. Tumour development is driven by the expansion of clones that accumulate progressive driver mutations3, with hepatocytes the most likely cell of origin2. However, the landscape of driver mutations in HCC is broadly independent of the underlying aetiologies4. Despite an increasing range of systemic treatment options for advanced HCC, outcomes remain heterogeneous and typically poor. Emerging data suggest that drug efficacies depend on disease aetiology and genetic alterations5,6. Exploring subtypes in preclinical models with human relevance will therefore be essential to advance precision medicine in HCC7. Here we generated a suite of genetically driven immunocompetent in vivo and matched in vitro HCC models. Our models represent multiple features of human HCC, including clonal origin, histopathological appearance and metastasis. We integrated transcriptomic data from the mouse models with human HCC data and identified four common human-mouse subtype clusters. The subtype clusters had distinct transcriptomic characteristics that aligned with the human histopathology. In a proof-of-principle analysis, we verified response to standard-of-care treatment and used a linked in vitro-in vivo pipeline to identify a promising therapeutic candidate, cladribine, that has not previously been linked to HCC treatment. Cladribine acts in a highly effective subtype-specific manner in combination with standard-of-care therapy.

TPS4192 Background: β-catenin mutations are present in up to 40% of aHCC pts. Elevated nuclear β-catenin expression levels correlate with poor responses to standard of care and β-catenin regulates both tumor metabolism and the immune microenvironment. Tegavivint is a first-in-class small molecule inhibitor of TBL1, a novel downstream Wnt-signaling pathway target. Tegavivint binds to TBL1 in the β-catenin pocket, disrupting the formation of the activation complex necessary for oncogenic activity, and enabling degradation of free nuclear β-catenin. The safety, clinical activity, and PK/PD of tegavivint was demonstrated through a proof-of-concept study in pts with desmoid tumors and is being studied through multiple Investigator Initiated Trials in AML (NCT04874480), pediatric solid tumors (NCT04851119), NSCLC (NCT04780568), and lymphoma (NCT05755087). Tegavivint was also studied in preclinical mouse models of β-cateninexon3 mutant HCC and the H22 syngeneic HCC model. In these models tegavivint decreased Wnt target gene expression and enhanced CD3+ T-cell infiltration in liver tumors. Tegavivint treatment of established β-cateninexon3 activated tumors resulted in reduced tumor growth and burden. Based on these promising preclinical results, the following phase 1/2 exploratory study was designed. Methods: This is a phase 1/2 study of tegavivint in pts with aHCC to characterize safety, PK/PD, and preliminary antitumor activity (NCT05797805). Eligibility includes aHCC pts ≥18 years old, with AXIN1or CTNNB1 mutation for all pts, except those in the single agent dose escalation; have BCLC Stage C or Stage B disease not amendable to local therapy or curative approaches, have Child-Pugh class A or B7 liver score, and must have received at least one prior line of systemic therapy. This study will be conducted in 2 parts. First, tegavivint will be administered as a single agent in a dose escalation/optimization and subsequent dose expansion cohort. Upon completion of the dose escalation via a 3+3 design, two dose levels will be selected for the dose selection optimization to determine the recommended phase 2 dose (RP2D) for use in the dose expansion. If sufficient clinical benefit is observed, the combination of tegavivint plus pembrolizumab will be explored in the second part of the study in aHCC pts previously treated with a PD-1/PD-L1 inhibitor. Tegavivint will be administered intravenously weekly. Primary objective is safety and secondary objectives are preliminary efficacy and PK/PD. The first patient began treatment in October of 2023 and the first dose escalation cohort was completed in January. Three institutions are open for enrollment; 5 other sites are pending site activation; all sites are in the United States and Canada. Clinical trial information: NCT05797805 .

Cellular senescence is not only associated with ageing but also impacts physiological and pathological processes, such as embryonic development and wound healing. Factors secreted by senescent cells affect their microenvironment and can induce spreading of senescence locally. Acute severe liver disease is associated with hepatocyte senescence and frequently progresses to multi-organ failure. Why the latter occurs is poorly understood. Here we demonstrate senescence development in extrahepatic organs and associated organ dysfunction in response to liver senescence using liver injury models and genetic models of hepatocyte-specific senescence. In patients with severe acute liver failure, we show that the extent of hepatocellular senescence predicts disease outcome, the need for liver transplantation and the occurrence of extrahepatic organ failure. We identify the TGFβ pathway as a critical mediator of systemic spread of senescence and demonstrate that TGFβ inhibition in vivo blocks senescence transmission to other organs, preventing liver senescence induced renal dysfunction. Our results highlight the systemic consequences of organ-specific senescence, which, independent of ageing, contributes to multi-organ dysfunction.

Abstract Background Inflammatory Bowel Disease-associated colorectal cancer (IBDCRC) is a known and serious complication of Inflammatory Bowel Disease (IBD) affecting the colon. However, relatively little is known about the pathogenesis of IBD-associated colorectal cancer in comparison with its sporadic cancer counterpart. Mutations in Dock2 occur in around 10% of IBD-associated colorectal cancers. Methods We used an APC-mediated mouse model of inflammation-associated tumourigenesis. VilApcfl/+Dock2tm1b/tm1b and VilApcfl/+ (control) mice were subjected to chronic dextran sodium sulphate (DSS) colitis and aged. After tamoxifen induction, mice were given two seven-day rounds of 0.5% DSS, interspersed with normal drinking water, and then aged to 47 days before culling for analysis. Additionally in vitro, Apc deficient, tumourigenic colonic organoids were generated by deleting Apc from wild type and Dock2tm1a/tm1a colonic organoids using CRISPR/Cas9. Results We found that loss of Dock2 increases tumourigenesis (both in terms of tumour number and tumour burden) via immune dysregulation in an APC-mediated model of inflammation-induced tumourigenesis in vivo. The increased tumourigenesis observed is associated with a CD3 and gamma delta (γδ) T-positive immune cell infiltrate, an interferon gamma signature, and a significant upregulation of Ido1 (which catalyses the conversion of amino acid tryptophan to kynurenine). In tumours from VilApcfl/+Dock2tm1b/tm1b mice, tryptophan is reduced whilst the metabolite xanthurenate is increased, demonstrating IDO activity. Separately in vitro, we showed that epithelial IDO1 is induced by administration of interferon gamma. We demonstrated a source of this interferon gamma as γδ T cells, which are increased in tumours of mice lacking Dock2. Critically, inhibition of IDO1 (with 1-L-MT) abrogated the increased tumourigenesis observed as a result of Dock2 loss in mice. Conclusion Overall, we have identified a role for γδ T cells in promoting tumour initiation via Ido1 expression, using a Dock2 model to accentuate this phenotype.Further work is now needed to both fully characterise this novel pathway in IBDCRC, and establish how immune dysregulation promotes tumourigenesis, but it opens exciting potential for a therapeutic strategy in preventing cancer as a result of Inflammatory Bowel Disease.

Inflammatory Bowel Disease-associated colorectal cancer (IBD-CRC) is a known and serious complication of Inflammatory Bowel Disease (IBD) affecting the colon. However, relatively little is known about the pathogenesis of IBD-associated colorectal cancer in comparison with its sporadic cancer counterpart. Here, we investigated the function of Dock2, a gene mutated in ~10% of IBD-associated colorectal cancers that encodes a guanine nucleotide exchange factor (GEF). Using a genetically engineered mouse model of IBD-CRC, we found that whole body loss of Dock2 increases tumourigenesis via immune dysregulation. Dock2-deficient tumours displayed increased levels of IFNγ-associated genes, including the tryptophan metabolising, immune modulatory enzyme, IDO1, when compared to Dock2-proficient tumours. This phenotype was driven by increased IFNγ-production in T cell populations, which infiltrated Dock2-deficient tumours, promoting IDO1 expression in tumour epithelial cells. We show that IDO1 inhibition delays tumourigenesis in Dock2 knockout mice, and we confirm that this pathway is conserved across species as IDO1 expression is elevated in human IBD-CRC and in sporadic CRC cases with mutated DOCK2. Together, these data demonstrate a previously unidentified tumour suppressive role of DOCK2 that limits IFNγ-induced IDO1 expression and cancer progression, opening potential new avenues for therapeutic intervention.

Hepatocellular carcinoma (HCC), the most common form of primary liver cancer, is a leading cause of cancer related mortality worldwide. HCC occurs typically from a background of chronic liver disease, caused by a spectrum of predisposing conditions. Tumour development is driven by the expansion of clones that accumulated progressive driver mutations, with hepatocytes the most likely cell of origin. However, the landscape of driver mutations in HCC is independent of the underlying aetiologies. Despite an increasing range of systemic treatment options for advanced HCC outcomes remain heterogeneous and typically poor. Emerging data suggest that drug efficacies depend on disease aetiology and genetic alterations. Exploring subtypes in preclinical models with human relevance will therefore be essential to advance precision medicine in HCC. We generated over twenty-five new genetically-driven in vivo and in vitro HCC models. Our models represent multiple features of human HCC, including clonal origin, histopathological appearance, and metastasis to distant organs. We integrated transcriptomic data from the mouse models with human HCC data and identified four common human-mouse subtype clusters. The subtype clusters had distinct transcriptomic characteristics that aligned with histopathology. In a proof-of-principle analysis, we verified response to standard of care treatment and used a linked in vitro-in vivo pipeline to identify a promising therapeutic candidate, cladribine, that has not been linked to HCC treatment before. Cladribine acts in a highly effective subtype-specific manner in combination with standard of care therapy.

Activation of β-catenin decreases butyrophilin-like molecule expression. A and B, Correlation between indicated genes as determined by TempO-seq and γδ T-cell density determined by IHC in the Scotland cohort. Units on axes are normalized read counts × 103. Each dot represents one tumor (n = 77 left and 82 right). P and r values determined by Pearson correlation. C, Correlation between CTNNB1 or SOX9 expression and BTNL3 or BTNL8 expression in the Marisa cohort (28). Units on axes are normalized counts × 103. Each dot represents one tumor (n = 258). P and r values determined by Pearson correlation. D, Images of organoids derived from indicated mouse models taken 4 days after tamoxifen treatment. E, Fold change in expression levels of indicated genes in organoids from various genotypes measured at indicated days after tamoxifen treatment. Each dot represents one organoid derived from one mouse (n = 3). F, Fold change in expression levels of indicated genes in WT organoids treated with 3 or 10 μmol/L CHIR-99021 for indicated days. Each dot represents one organoid derived from one mouse (n = 3). Data are presented as mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (one-way ANOVA followed by Dunnett post hoc test).

Inhibition of β-catenin transcriptional activity increases expression of HNF4A, HNF4G, and butyrophilin-like molecules. A, Vγ7+ cell viability in cocultures with CT26 or CT26-B1/6 cells. Each dot represents one paired biological replicate (n = 4). B, CD25 expression by Vγ7+ cells in cocultures with CT26 or CT26-B1/6 cells. Each dot represents one paired biological replicate (n = 4). C, CT26 and CT26-B1/6 cancer cell death using flow cytometry after coculture with Vγ7+ cell as indicated. Each dot represents one biological replicate (n = 4). D, Kaplan–Meier survival analysis of doxycycline-treated CT26 and CT26-B1/6 tumor-bearing mice (n = 4 CT26, 5 CT26-B1/6) using the log-rank test. E, γδ T-cell numbers in tumors from doxycycline-treated CT26 and CT26-B1/6 tumor-bearing mice. Each dot represents one mouse (n = 4 CT26, 5 CT26-B1/6). F and H, Images taken from serially stained sections of indicated protein in tumors from indicated mouse models (n = 3–4; scale bar, 500 μm. G and I, Images of Trdc expression in tumors from indicated mouse models; scale bar, 500 μm. γδ T-cell numbers in tumors. Each dot represents one mouse. Data are presented as mean ± SD. *, P < 0.05; **, P < 0.01 (paired t test or unpaired t test or one-way ANOVA followed by Tukey post hoc test).