High Wnt Research Articles

P53 terminates the regenerative fetal-like state after colitis-associated injury.

Cells that lack p53 signaling frequently occur in ulcerative colitis (UC) and are considered early drivers in UC-associated colorectal cancer (CRC). Epithelial injury during colitis is associated with transient stem cell reprogramming from the adult, homeostatic to a "fetal-like" regenerative state. Here, we use murine and organoid-based models to study the role of Trp53 during epithelial reprogramming. We find that p53 signaling is silent and dispensable during homeostasis but strongly up-regulated in the epithelium upon DSS-induced colitis. While in WT cells this causes termination of the regenerative state, crypts that lack Trp53 remain locked in the highly proliferative, regenerative state long-term. The regenerative state in WT cells requires high Wnt signaling to maintain elevated levels of glycolysis. Instead, Trp53 deficiency enables Wnt-independent glycolysis due to overexpression of rate-limiting enzyme PKM2. Our study reveals the context-dependent relevance of p53 signaling specifically in the injury-induced regenerative state, explaining the high abundance of clones lacking p53 signaling in UC and UC-associated CRC.

Abstract CT101: Final results of the first-in-human study of the porcupine (PORCN) inhibitor zamaporvint (RXC004) in patients with advanced solid tumors

Abstract Background Dysregulated Wnt signalling drives several cancers through effects on proliferation, fibrosis and immune evasion. RXC004 is a novel oral small molecule inhibitor of protein-serine O-palmitoyltransferase (PORCN), which is required for the post-translational modification and activation of Wnt ligands. RXC004 has potential for efficacy in upstream Wnt pathway activated tumours: ~5% pancreatic cancer and ~8% microsatellite stable colorectal (CRC) cancer with RNF43 mutations or RSPO fusions, and tumors with high Wnt ligand expression. Methods In this Phase I study of RXC004 (NCT03447470), Module 1 investigated continuous monotherapy dosing, Module 2 investigated combination with nivolumab 480mg s.c. Q4W and Module 3 investigated intermittent monotherapy dosing (2 weeks on 1 week off). Patients received denosumab 120 mg s.c Q4W to prevent loss of bone mineral density (BMD), a known effect of Wnt inhibition. The primary objectives were safety and tolerability. Secondary objectives were PK and RECIST response. PD markers included on-treatment changes in: Wnt pathway and fibrosis biomarkers in paired skin biopsies; circulating immune subsets by flow cytometry; cytokine changes by Luminex, and changes in circulating-tumor DNA (ctDNA) levels by ddPCR. Results In Module 1, 25 patients (pts) with unselected advanced solid tumors (AST) received RXC004 between 0.5mg and 10mg QD. The RP2D was 2mg QD; at this dose the most common AEs were fatigue, diarrhoea, dysgeusia and decreased appetite; no grade 4/5 AEs or bone fragility events were reported whilst 1 pt had a RXC004-related grade 3 AE, 3 pts (50%) had a dose interruption and 1 had a dose reduction. The median T1/2 of RXC004 was 14.5h. 5 pts with upstream Wnt pathway activated tumors had stable disease (SD), in 1 case for 26 weeks with sustained reduction in ctDNA and no BMD loss. In Module 2, 14 patients with unselected AST received RXC004 at 1mg QD or 1.5mg QD with nivolumab. The AE profile was similar to Module 1. The RP2D was 1.5mg QD; at this dose 3 pts had a RXC004-related grade 3 AE, 6 pts (75%) had a dose interruption and 3 had a dose reduction, and 4 pts had SD. PK was similar to Module 1. In Module 3, 7 patients with upstream Wnt-activated AST received RXC004 2mg QD, 2 weeks on, 1 week off. The AE profile was similar to Modules 1 and 2. There were no RXC004-related Grade 3 AEs, 2 pts (29%) had a dose interruption and there were no dose reductions. PK and PD analyses confirmed time off target. 2 pts had SD; 1 pt with RSPO fusion small intestinal adenocarcinoma remained on treatment for 36 weeks, with sustained reduction in ctDNA and no BMD loss. Conclusions In patients with AST, RXC004 was safe and tolerated at doses up to 2mg QD as monotherapy and 1.5mg QD in combination with nivolumab, demonstrating differential disease control in upstream Wnt pathway activated tumours. Denosumab prevented loss of BMD. Intermittent RXC004 dosing was well tolerated with no detrimental impact on efficacy. Citation Format: Natalie Cook, Sarah Blagden, Juanita Lopez, Debashis Sarker, Alastair Greystoke, Noor MD Haris, Farasat Kazmi, Kaiser Anam, Ana Ortego Franco, Rille Pihlak, Louise Goodwin, David Wilson, Caroline Phillips, Jane Robertson, Helen Timmis, Craig Tilston, Simon A. Woodcock, Ruth Plummer. Final results of the first-in-human study of the porcupine (PORCN) inhibitor zamaporvint (RXC004) in patients with advanced solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT101.

Abstract 3163: (Phospho)proteomic analysis reveals vulnerabilities in refractory metastatic colorectal cancer

Abstract Proteomic networks are crucial for cellular homeostasis maintenance and, indeed, their aberrant regulation is implicated in cancer development and progression. Understanding alterations underlying these pathways, provides a way to design therapeutic interventions in human cancer. Improvements in mass spectrometry analysis have unlocked our potential to unveil cancer vulnerabilities. Here, we performed (phospho)proteomic analysis and ex-vivo drug testing on a cohort of refractory metastatic colorectal cancer (mCRC) tissues and matched patients-Derived Organoids (PDO) to identify new putative druggable biomarkers. The comparison between the proteome and phosphoproteome of 10 mCRCs tissues with their matched PDOs normalized to a pool of 6 colon mucosa tissues highlighted a strong overall correlation (R= 0.57 and R=0.6, respectively) suggesting a good level of similarity. Applying GSEA on our proteomic dataset, we identified up-regulation in MYC, cell cycle and MTORC1 signaling pathways in mCRC tissues and PDO, while kinase enrichment analysis highlighted an increase in Casein Kinase II (CKII) activity, an important cell cycle regulator. Our ex-vivo experiments showed that silmitasertib-mediated CKII inhibition reduced the cell viability in our PDO models, but with an overall modest effect. Analysis revealed that, even though not statistically significant, high Wnt signaling score correlated with an higher resistance to silmitasertib treatment (R=0.61; p=0.08). Previous works highlighted that MAPK activity is increased after silmitasertib treatment and responsible for resistance mechanisms to CKII inhibition. Strikingly, the combined trametinib-mediated MEK blockade and CKII blockade resulted in a synergistic interaction leading to enhanced antitumor activity, especially in PDO harboring high Wnt (R=0.74; p=0.022) and cell cycle (R=0.69; p=0.04) pathway activity. Moreover, the combined treatment strongly reduced S6 activity and RAPTOR expression, overcoming the significant effect exerted by the single agents. Surprisingly, none of them altered baseline MAPK activity, suggesting that silmitasertib and trametinib synergize their activity via inactivating the MTORC1-S6 axis in an ERK-independent manner. We are currently integrating genomic and transcriptomic data to better characterize the observed drug synergy. Taken together, our findings propose the silmitasertib and trametinib combination as a new therapeutic opportunity for refractory mCRC patients and strengthen the use of PDO as a suitable model for drug screening. Citation Format: Mattia Marinucci, Gina Faye Boot, Lara Zaidi, Cinzia Esposito, Mairene Coto Llerena, Savas Soysal, Otto Kolmar, Charlotte K Y Ng, Salvatore Piscuoglio. (Phospho)proteomic analysis reveals vulnerabilities in refractory metastatic colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3163.

Lung endothelium exploits susceptible tumor cell states to instruct metastatic latency

In metastasis, cancer cells travel around the circulation to colonize distant sites. Due to the rarity of these events, the immediate fates of metastasizing tumor cells (mTCs) are poorly understood while the role of the endothelium as a dissemination interface remains elusive. Using a newly developed combinatorial mTC enrichment approach, we provide a transcriptional blueprint of the early colonization process. Following their arrest at the metastatic site, mTCs were found to either proliferate intravascularly or extravasate, thereby establishing metastatic latency. Endothelial-derived angiocrine Wnt factors drive this bifurcation, instructing mTCs to follow the extravasation–latency route. Surprisingly, mTC responsiveness towards niche-derived Wnt was established at the epigenetic level, which predetermined tumor cell behavior. Whereas hypomethylation enabled high Wnt activity leading to metastatic latency, methylated mTCs exhibited low activity and proliferated intravascularly. Collectively the data identify the predetermined methylation status of disseminated tumor cells as a key regulator of mTC behavior in the metastatic niche.

LncRNA-CCAT5-mediated crosstalk between Wnt/β-Catenin and STAT3 signaling suggests novel therapeutic approaches for metastatic gastric cancer with high Wnt activity.

Although the constitutively activated Wnt/β-catenin signaling pathway plays vital roles in gastric cancer (GC) progression, few Wnt inhibitors are approved for clinical use. Additionally, the clinical significance of long non-coding RNAs (lncRNAs) in GC intraperitoneal dissemination (IPD) remains elusive. Here, we investigated the function and therapeutic potential of Wnt-transactivated lncRNA, colon cancer-associated transcript 5 (CCAT5), in GC metastasis. LncRNA-sequencing assay was performed to document abundance changes of lncRNAs induced by Wnt family member 3A (Wnt3a) and degradation-resistant β-catenin (S33Y mutated) in ascites-derived GC cells with low Wnt activity. Luciferase reporter, Chromatin immunoprecipitation (ChIP)-re-ChIP assays were performed to determine how CCAT5 was transcribed. The clinical significance of CCAT5 was examined in 2 cohorts of GC patients. The biological function of CCAT5 was investigated through gain- and loss-of-function studies. The molecular mechanism was explored through RNA-sequencing, mass spectrometry, and CRISPR/Cas9-knocknout system. The therapeutic potential of CCAT5 was examined through RNAi-based cell xenograft model and patient-derived xenograft (PDX) model of IPD. We identified a novel Wnt-regulated lncRNA, CCAT5, which was transactivated by the β-catenin/transcription factor 3 (TCF3) complex. CCAT5 was significantly upregulated in GC and predicted poor prognosis. Functional studies confirmed the promotive role of CCAT5 in GC growth and metastasis. Mechanistically, CCAT5 bound to the C-end domain of signal transducer and activator of transcription 3 (STAT3) and blocks Src homology 2 domain-containing protein tyrosine phosphatase 1 (SHP-1)-mediated STAT3Y705 dephosphorylation, leading to STAT3 nuclear entry and transactivation, thus accelerating GC progression. Furthermore, we demonstrated that both Wnt3a and β-catenin acted as activator of STAT3 signaling pathway, and the interplay between CCAT5 and STAT3 was functionally essential for Wnt-drived STAT3 signaling and tumor evolution. Finally, we revealed in vivo si-CCAT5 selectively attenuated growth and metastasis of Wnthigh GC, but not Wntlow GC. The combination of si-CCAT5 and oxaliplatin displayed obvious synergistic therapeutic effects on Wnthigh PDX mice. We identified a novel Wnt-transactivated lncRNA, CCAT5. Our study revealed a mechanism of STAT3 signaling regulation via canonical Wnt signaling and the functional significance of CCAT5 as critical mediator. We provided conceptual advance that lncRNAs serve as therapeutic targets reversing GC progression.

Region-specific Wnt signaling responses promote gastric polyp formation in patients with familial adenomatous polyposis.

Germline adenomatous polyposis coli (APC) mutation in patients with familial adenomatous polyposis (FAP) promotes gastrointestinal polyposis, including the formation of frequent gastric fundic gland polyps (FGPs). In this study, we investigated how dysregulated Wnt signaling promotes FGPs and why they localize to the corpus region of the stomach. We developed a biobank of FGP and surrounding nonpolyp corpus biopsies and organoids from patients with FAP for comparative studies. Polyp biopsies and polyp-derived organoids exhibited enhanced Wnt target gene expression. Polyp-derived organoids with intrinsically upregulated Wnt signaling showed poor tolerance to further induction, suggesting that high Wnt restricts growth. Targeted genomic sequencing revealed that most gastric polyps did not arise via APC loss of heterozygosity. Studies in genetic mouse models demonstrated that heterozygous Apc loss increased epithelial cell proliferation in the corpus but not the antrum, while homozygous Apc loss was not maintained in the corpus yet induced hyperproliferation in the antrum. Our findings suggest that heterozygous APC mutation in patients with FAP may be sufficient to drive polyp formation in the corpus region while subsequent loss of heterozygosity to further enhance Wnt signaling is not tolerated. This finding contextualizes the abundant yet benign nature of gastric polyps in FAP patient corpus compared with the rare, yet adenomatous polyps in the antrum.

G protein-coupled receptor 17 is regulated by WNT pathway during oligodendrocyte precursor cell differentiation

G protein-coupled receptor 17 (GPR17) and the WNT pathway are critical players of oligodendrocyte (OL) differentiation acting as essential timers in developing brain to achieve fully-myelinating cells. However, whether and how these two systems are related to each other is still unknown. Of interest, both factors are dysregulated in developing and adult brain diseases, including white matter injury and cancer, making the understanding of their reciprocal interactions of potential importance for identifying new targets and strategies for myelin repair. Here, by a combined pharmacological and biotechnological approach, we examined regulatory mechanisms linking WNT signaling to GPR17 expression in OLs. We first analyzed the relative expression of mRNAs encoding for GPR17 and the T cell factor/Lymphoid enhancer-binding factor-1 (TCF/LEF) transcription factors of the canonical WNT/β-CATENIN pathway, in PDGFRα+ and O4+ OLs during mouse post-natal development. In O4+ cells, Gpr17 mRNA level peaked at post-natal day 14 and then decreased concomitantly to the physiological uprise of WNT tone, as shown by increased Lef1 mRNA level. The link between WNT signaling and GPR17 expression was further reinforced in vitro in primary PDGFRα+ cells and in Oli-neu cells. High WNT tone impaired OL differentiation and drastically reduced GPR17 mRNA and protein levels. In Oli-neu cells, WNT/β-CATENIN activation repressed Gpr17 promoter activity through both putative WNT response elements (WRE) and upregulation of the inhibitor of DNA-binding protein 2 (Id2). We conclude that the WNT pathway influences OL maturation by repressing GPR17, which could have implications in pathologies characterized by dysregulations of the OL lineage including multiple sclerosis and oligodendroglioma.

Opposing roles for TGFβ- and BMP-signaling during nascent alveolar differentiation in the developing human lung

Alveolar type 2 (AT2) cells function as stem cells in the adult lung and aid in repair after injury. The current study aimed to understand the signaling events that control differentiation of this therapeutically relevant cell type during human development. Using lung explant and organoid models, we identified opposing effects of TGFβ- and BMP-signaling, where inhibition of TGFβ- and activation of BMP-signaling in the context of high WNT- and FGF-signaling efficiently differentiated early lung progenitors into AT2-like cells in vitro. AT2-like cells differentiated in this manner exhibit surfactant processing and secretion capabilities, and long-term commitment to a mature AT2 phenotype when expanded in media optimized for primary AT2 culture. Comparing AT2-like cells differentiated with TGFβ-inhibition and BMP-activation to alternative differentiation approaches revealed improved specificity to the AT2 lineage and reduced off-target cell types. These findings reveal opposing roles for TGFβ- and BMP-signaling in AT2 differentiation and provide a new strategy to generate a therapeutically relevant cell type in vitro.

Differential sensitivity to Wnt signaling gradients in human gastric organoids derived from corpus and antrum.

Wnt signaling regulates gastric stem cell proliferation and differentiation. Although similar Wnt gradients exist within the corpus and antrum of the human stomach, there are striking differences in gland architecture and disease manifestation that suggest Wnt may differentially regulate progenitor cell function in each compartment. In this study, we tested sensitivities to Wnt activation in human gastric corpus and antral organoids to determine whether progenitor cells have region-specific differences in Wnt responsiveness. Human patient-matched corpus and antral organoids were grown in the presence of varying concentrations of the Wnt pathway activator CHIR99021 to assess regional sensitivity to Wnt signaling on growth and proliferation. Corpus organoids were further studied to understand how high Wnt affected cellular differentiation and progenitor cell function. A lower concentration of CHIR99021 stimulated peak growth in corpus organoids compared with patient-matched antral organoids. Supramaximal Wnt signaling levels in corpus organoids suppressed proliferation, altered morphology, reduced surface cell differentiation, and increased differentiation of deep glandular neck and chief cells. Surprisingly, corpus organoids grown in high CHIR99021 had enhanced organoid forming potential, indicating that progenitor cell function was maintained in these nonproliferative, deep glandular cell-enriched organoids. Passaging high-Wnt quiescent organoids into low Wnt rescued normal growth, morphology, and surface cell differentiation. Our findings suggest that human corpus progenitor cells have a lower threshold for optimal Wnt signaling than antral progenitor cells. We demonstrate that Wnt signaling in the corpus regulates a bimodal axis of differentiation, with high Wnt promoting deep glandular cell differentiation and suppressing proliferation while simultaneously promoting progenitor cell function.NEW & NOTEWORTHY This study demonstrates that human gastric corpus organoids have a lower Wnt signaling threshold to drive optimal growth relative to patient-matched antral organoids. Paradoxically, supramaximal Wnt levels suppress corpus organoid proliferation, yet promote differentiation toward deep glandular cell types while simultaneously enhancing progenitor cell function. These findings provide novel insights into how Wnt signaling differentially regulates homeostasis in the human gastric corpus and antrum and contextualizes patterns of Wnt activation diseases.

CNSC-02. INVESTIGATING THE ROLE OF THE WNT SIGNALING TRANSDUCTION PATHWAY IN GLIOMA BLOOD-BRAIN-BARRIER INTEGRITY

Abstract The WNT signaling transduction pathway has been linked to cancer stem cell self-renewal, differentiation, and blood brain barrier (BBB) development. Previous studies have shown that WNT pathway inhibition increased BBB permeability, specifically in WNT medulloblastoma. Glioblastoma, an aggressive malignant tumor with poor prognosis, has also been shown to have high WNT expression and variable BBB permeability. We hypothesize that activating WNT pathway inhibition in glioblastoma stem cells (GSCs) will inhibit glioma progression, increase BBB permeability within the tumor microenvironment, and enhance overall treatment responsiveness. We transduced primary pediatric derived glioma stem cells with overexpressing vectors for WNT inhibitors (DKK1 or WIF1). To characterize effects of DKK1 or WIF1 overexpression, we evaluated cell migration patterns via RTCA xCelligence, cell cycle progression via Annexin 5 assays, and cell viability via cell-titer glo. Brain endothelial cell integrity was evaluated with the treatment of CHIR99021, WNT pathway activator, or GSC condition media with immunoblotting for junctional protein expression (Claudin-5, Claudin-3, Occludin, ZO-1, VE-Cadherin). Verification of WNT inhibitory overexpressing GSCs for DKK1 and WIF1 was performed using RNAseq, proteomics, and immunoblotting. DKK1-GSCs demonstrated a statistically significant impairment in the migratory slope compared to empty vector GSCs. Cell cycle analysis also revealed that DKK1-GSCs were arrested in G0/1 phase while WIF1-GSCs were arrested in S/G2M phase. CHIR99021 endothelial treatment resulted in increased secretion of WNT inhibitors DKK1 and SFRP1 and resultantly, there was a decrease in junctional protein expression. Overall, WNT inhibitor overexpressing GSCs exhibited unique cellular patterns and indirectly disrupted endothelial cell integrity. Future studies will evaluate orthotopic WNT inhibitor overexpressing transplant rodent models to determine BBB integrity, drug permeability, model survival, and chemotherapeutic treatment response. By enhancing our understanding of the WNT pathway in GSCs, we anticipate understanding the use of future therapeutic WNT pathway targeting to increase chemotherapeutic responsiveness and improve glioblastoma prognosis.

Novel PORCN inhibitor WHN-88 targets Wnt/β-catenin pathway and prevents the growth of Wnt-driven cancers

Wnt/β-catenin signaling pathway is a classical and crucial oncogenic pathway in many carcinomas, and Porcupine (PORCN) is an O-acyltransferase, which is indispensable and highly specific for catalyzing palmitoylation of Wnt ligands and facilitating their secretion and biofunction. Targeting PORCN provides a promising approach to specifically cure Wnt-driven cancers from the root. In this study, we designed series of pyridonyl acetamide compounds, and discovered a novel PORCN inhibitor WHN-88 with a unique di-iodinated pyridone structural fragment, which is significantly different from the reported inhibitors. We demonstrated that WHN-88 effectively abolished palmitoylation of Wnt ligands and prevented their secretion and the subsequent Wnt/β-catenin signaling transduction. Further experiments showed that, at well-tolerated doses, WHN-88 remarkably suppressed the spontaneous occurrence and growth of MMTV-Wnt1 murine breast tumors. Consistently, WHN-88 also notably restrained the progress of xenografted Wnt-driven human tumors, including PA-1 teratocarcinoma with high autocrine Wnt signaling and Aspc-1 pancreatic carcinoma with Wnt-sensitizing RNF43 mutation. Additionally, we disclosed that WHN-88 inhibited cancer cell stemness obviously. Together, we verified WHN-88 is a novel PORCN inhibitor with potent efficacy against the Wnt-driven cancers. Our findings enriched the structural types of PORCN inhibitors, and facilitated the development and application of PORCN inhibiting therapy in clinic.

Intravital imaging of Wnt/β-catenin and ATF2-dependent signalling pathways during tumour cell invasion and metastasis.

Wnt signalling has been implicated as a driver of tumour cell metastasis, but less is known about which branches of Wnt signalling are involved and when they act in the metastatic cascade. Here, using a unique intravital imaging platform and fluorescent reporters, we visualised β-catenin/TCF-dependent and ATF2-dependent signalling activities during human cancer cell invasion, intravasation and metastatic lesion formation in the chick embryo host. We found that cancer cells readily shifted between states of low and high canonical Wnt activity. Cancer cells that displayed low Wnt canonical activity showed higher invasion and intravasation potential in primary tumours and in metastatic lesions. In contrast, cancer cells showing low ATF2-dependent activity were significantly less invasive both at the front of primary tumours and in metastatic lesions. Simultaneous visualisation of both these reporters using a double-reporter cell line confirmed their complementary activities in primary tumours and metastatic lesions. These findings might inform the development of therapies that target different branches of Wnt signalling at specific stages of metastasis.

Regulation of the THRA gene, encoding the thyroid hormone nuclear receptor TRα1, in intestinal lesions.

The THRA gene, encoding the thyroid hormone nuclear receptor TRα1, is expressed in an increasing gradient at the bottom of intestinal crypts, overlapping with high Wnt and Notch activities. Importantly, THRA is upregulated in colorectal cancers, particularly in the high-Wnt molecular subtype. The basis of this specific and/or altered expression pattern has remained unknown. To define the mechanisms controlling THRA transcription and TRα1 expression, we used multiple in vitro and ex vivo approaches. Promoter analysis demonstrated that transcription factors important for crypt homeostasis and altered in colorectal cancers, such as transcription factor 7-like 2 (TCF7L2; Wnt pathway), recombining binding protein suppressor of hairless (RBPJ; Notch pathway), and homeobox protein CDX2 (epithelial cell identity), modulate THRA activity. Specifically, although TCF7L2 and CDX2 stimulated THRA, RBPJ induced its repression. In-depth analysis of the Wnt-dependent increase showed direct regulation of the THRA promoter in cells and of TRα1 expression in murine enteroids. Given our previous results on the control of the Wnt pathway by TRα1, our new results unveil a complex regulatory loop and synergy between these endocrine and epithelial-cell-intrinsic signals. Our work describes, for the first time, the regulation of the THRA gene in specific cell and tumor contexts.

Axin2/Conductin Is Required for Normal Haematopoiesis and T Lymphopoiesis.

The development of T lymphocytes in the thymus and their stem cell precursors in the bone marrow is controlled by Wnt signaling in strictly regulated, cell-type specific dosages. In this study, we investigated levels of canonical Wnt signaling during hematopoiesis and T cell development within the Axin2-mTurquoise2 reporter. We demonstrate active Wnt signaling in hematopoietic stem cells (HSCs) and early thymocytes, but also in more mature thymic subsets and peripheral T lymphocytes. Thymic epithelial cells displayed particularly high Wnt signaling, suggesting an interesting crosstalk between thymocytes and thymic epithelial cells (TECs). Additionally, reporter mice allowed us to investigate the loss of Axin2 function, demonstrating decreased HSC repopulation upon transplantation and the partial arrest of early thymocyte development in Axin2Tg/Tg full mutant mice. Mechanistically, loss of Axin2 leads to supraphysiological Wnt levels that disrupt HSC differentiation and thymocyte development.

Selected Articles from This Issue

Despite previous assumptions that constitutively high Wnt signaling promotes colorectal cancer in each stage of disease, recent studies have demonstrated that reduced Wnt signaling is associated with colorectal cancer invasiveness and poor prognoses. However, little work has been done to elucidate how diminished Wnt signaling underlies aggressive colorectal cancer phenotypes. Chen and colleagues used genetic manipulation strategies and orthotopic mouse models to show that abrogating Wnt signaling increases colorectal cancer invasiveness and decreases immune responsiveness in vivo. Disrupting Wnt signaling by targeting Wnt coreceptor LDL receptor-related protein 6 (LRP6) with CRISPR/Cas9 or transducing a dominant negative version of Wnt transcriptional effector lymphoid enhancer binding factor 1 (LEF1) increases tumor submucosal invasion and dissemination in mice. Using RNA-Seq, the authors revealed that reducing Wnt signaling elevates expression of markers associated with stemness and a mesenchymal phenotype, while repressing expression of immune-stimulatory molecules and collagens. The authors used TCGA data to demonstrate that the low Wnt signaling expression profile corresponds with poor survival in colorectal cancer patients, underscoring the potential clinical relevance of dampened Wnt signaling in colorectal cancer progression.Cholangiocarcinoma (CCA) is an aggressive biliary tract cancer for which effective therapeutics are lacking. Prostaglandin E2 (PGE2)-mediated inflammation is a critical component of CCA carcinogenesis and progression. 15-hydroxyprostaglandin dehydrogenase (15-PGDH) chemically modifies and inactivates PGE2, but its expression is often diminished in CCA. In their work, Zhang and colleagues leveraged public data to show that expression of histone methyltransferase G9a negatively correlates with 15-PGDH expression. The authors validated the G9a-15-PGDH relationship in a mouse model of CCA in which the Notch intracellular domain and constitutively active Akt are expressed via hydrodynamic injection of sleeping beauty transposase-based plasmids. shRNA-mediated G9a silencing increases 15-PGDH expression in CCA cell lines, limiting cell proliferation in vitro and tumor growth in vivo. ChIP assays demonstrated that G9a is recruited to the 15-PGDH promoter by the Myc/Max/E-box complex, which prevents signal transducer and activator of transcription 4 (STAT4) from binding the promoter and inducing 15-PGDH expression. The work presented elucidates a previously unknown mechanism underlying CCA development and progression and reveals a novel potential target for CCA therapeutic strategies.Neurofibromatosis type 2 is characterized by growth of benign nervous system tumors, such as schwannomas, which can lead to deafness, brain stem compression, and death. Neurofibromatosis type 2 is caused by mutations in tumor suppressor gene NF2. Previously, Cho and colleagues demonstrated that NF2 loss promotes elevated TβR1 signaling, resulting in phosphorylation and degradation of tumor suppressor Raf kinase inhibitor protein (RKIP). While TGF-β inhibitors halt schwannoma growth, they are not ideal therapeutics given schwannomas occur around adolescence and TGF-β is required for development. Here, Cho and colleagues developed a novel TβR1 inhibitor, Nf18001, that abrogates TβR1-mediated RKIP degradation while maintaining canonical TGF-β signaling. The authors used human and mouse schwannoma cell lines to demonstrate that Nf18001 disrupts TβR1-RKIP interactions to sustain RKIP expression and lower NF2-mutant cell viability while not influencing normal fibroblast survival. Nf18001 arrests cell cycle progression in schwannoma cells, and augments Schwann cell differentiation marker expression while diminishing expression of markers associated with cell stemness. Nf18001 also displayed efficacy in a schwannoma allograft mouse model, where treatment slowed tumor growth.The pro-tumorigenic role of signal transducer and activator of transcription 3 (STAT3) in multiple myeloma has been established, but therapeutically targeting STAT3 has proven challenging. Similarly, given its canonical role in DNA damage repair, checkpoint kinase 1 (Chk1) inhibition has been tested in anti-cancer therapeutic regimens, but with ambiguous results. In their study, Zhou and colleagues employed a gene expression array to demonstrate that Chk1 inhibition diminishes expression of oncogenic STAT3 target genes in multiple myeloma cells, suggesting Chk1 inhibition may also effectively inhibit STAT3 activity. Subsequent work showed that Chk1 directly binds to STAT3 and phosphorylates it at tyrosine 705, enabling STAT3 dimerization and DNA binding. Chk1 inhibition — pharmacologically and via shRNA silencing — diminishes STAT3 tyrosine 705 phosphorylation and inhibits STAT3 DNA binding, reducing expression of STAT3 transcriptional targets. Chk1 inhibition abrogates STAT3 tyrosine 705 phosphorylation in IL-6–dependent, -independent, and bortezomib-resistant cells, as well as in patient primary myeloma mononuclear cells and multiple myeloma cells implanted into mice, suggesting Chk1 inhibition may provide therapeutic efficacy against multiple myeloma through a STAT3-related mechanism.

SUMOylation Potentiates ZIC Protein Activity to Influence Murine Neural Crest Cell Specification.

The mechanisms of neural crest cell induction and specification are highly conserved among vertebrate model organisms, but how similar these mechanisms are in mammalian neural crest cell formation remains open to question. The zinc finger of the cerebellum 1 (ZIC1) transcription factor is considered a core component of the vertebrate gene regulatory network that specifies neural crest fate at the neural plate border. In mouse embryos, however, Zic1 mutation does not cause neural crest defects. Instead, we and others have shown that murine Zic2 and Zic5 mutate to give a neural crest phenotype. Here, we extend this knowledge by demonstrating that murine Zic3 is also required for, and co-operates with, Zic2 and Zic5 during mammalian neural crest specification. At the murine neural plate border (a region of high canonical WNT activity) ZIC2, ZIC3, and ZIC5 function as transcription factors to jointly activate the Foxd3 specifier gene. This function is promoted by SUMOylation of the ZIC proteins at a conserved lysine immediately N-terminal of the ZIC zinc finger domain. In contrast, in the lateral regions of the neurectoderm (a region of low canonical WNT activity) basal ZIC proteins act as co-repressors of WNT/TCF-mediated transcription. Our work provides a mechanism by which mammalian neural crest specification is restricted to the neural plate border. Furthermore, given that WNT signaling and SUMOylation are also features of non-mammalian neural crest specification, it suggests that mammalian neural crest induction shares broad conservation, but altered molecular detail, with chicken, zebrafish, and Xenopus neural crest induction.

Good Neighbors: The Niche that Fine Tunes Mammalian Intestinal Regeneration.

The intestinal epithelium undergoes continuous cellular turnover, making it an attractive model to study tissue renewal and regeneration. Intestinal stem cells (ISCs) can both self-renew and differentiate along all epithelial cell lineages. Decisions about which fate to pursue are controlled by a balance between high Wnt signaling at the crypt bottom, where Lgr5 + ISCs reside, and increasing bone morphogenetic protein (BMP) levels toward the villus, where differentiated cells are located. Under stress conditions, epithelial cells in the intestine are quite plastic, with dedifferentiation, the reversal of cell fate from a differentiated cell to a more stem-like cell, allowing for most mature epithelial cell types to acquire stem cell-like properties. The ISC niche, mainly made up of mesenchymal, immune, enteric neuronal, and endothelial cells, plays a central role in maintaining the physiological function of the intestine. Additionally, the immune system and the microbiome play an essential role in regulating intestinal renewal. The development of various mouse models, organoid co-cultures and single-cell technologies has led to advances in understanding signals emanating from the mesenchymal niche. Here, we review how intestinal regeneration is driven by stem cell self-renewal and differentiation, with an emphasis on the niche that fine tunes these processes in both homeostasis and injury conditions.

517MO Phase I study of the porcupine (PORCN) inhibitor RXC004 in patients with advanced solid tumours

Dysregulated Wnt signalling drives several cancers through effects on proliferation, fibrosis and immune evasion. RXC004 is a novel small molecule inhibitor of the protein-serine O-palmitoyltransferase, PORCN, which is required for post-translational modification of Wnt ligands and downstream signaling. RXC004 has potential for monotherapy efficacy in Wnt pathway activated tumours: ∼5% pancreatic and ∼ 8% microsatellite stable colorectal (CRC) cancers with RNF-43 mutations or R-Spondin fusions, and thymus and biliary tract (BTC) cancers with high Wnt ligand activity. This open label, 3+3 dose escalation study was one module of a multi-modular adaptive design protocol (NCT03447470). Following a single dose with a 7-day washout, patients received RXC004 once daily in 21-day cycles. The primary objectives were to assess safety and tolerability and define a recommended phase 2 dose of RXC004. Secondary objectives were PK and RECIST response. Pharmacodynamic markers included skin Wnt pathway (Axin2) suppression. Between 05/02/2018 and 21/06/2021, 25 patients with advanced cancers received RXC004. The first patient, who received 10mg daily, discontinued due to diarrhoea and an asymptomatic clavicle fracture was found on follow up. The half-life (T1/2) and exposure were higher than predicted from preclinical models. The study was paused and restarted at 0.5mg. Subsequently, 1mg, 1.5mg, 2mg and 3mg doses were evaluated. Patients received denosumab 120 mg s.c monthly to prevent bone adverse events [AEs]. For doses <3mg, the most common treatment related AEs were fatigue, nausea, dysgeusia, vomiting and anorexia. No grade 4/5 AEs or bone fragility events were reported. Exposure was dose proportional and median T1/2 was 14.5h. Exposures above the preclinical model IC50 were observed at all doses, as was Axin2 suppression. Five patients, all with Wnt pathway activated tumours [2 BTC, 1 Thymus, 1 mutRNF43 CRC and 1 RSPO fusion CRC] had stable disease, in one case for up to 26 weeks. In patients with unselected cancers, RXC004 was safe and tolerated at doses up to 2mg/day, supporting phase 2 development in selected patients with Wnt pathway activated tumours. Studies will open in 2021.

Regulation of Wnt Signaling by FOX Transcription Factors in Cancer.

Simple SummaryCancer is caused by a breakdown of cell-to-cell communication, which results in the unrestricted expansion of cells within a tissue. In many cases, tumor growth is maintained by the continuous activation of cell signaling programs that normally drive embryonic development and wound repair. In this review article, I discuss how one of the largest human protein families, namely FOX proteins, controls the activity of the Wnt pathway, a major regulatory signaling cascade in developing organisms and adult stem cells. Evidence suggests that there is considerable crosstalk between FOX proteins and the Wnt pathway, which contributes to cancer initiation and progression. A better understanding of FOX biology may therefore lead to the development of new targeted treatments for many types of cancer.Aberrant activation of the oncogenic Wnt signaling pathway is a hallmark of numerous types of cancer. However, in many cases, it is unclear how a chronically high Wnt signaling tone is maintained in the absence of activating pathway mutations. Forkhead box (FOX) family transcription factors are key regulators of embryonic development and tissue homeostasis, and there is mounting evidence that they act in part by fine-tuning the Wnt signaling output in a tissue-specific and context-dependent manner. Here, I review the diverse ways in which FOX transcription factors interact with the Wnt pathway, and how the ectopic reactivation of FOX proteins may affect Wnt signaling activity in various types of cancer. Many FOX transcription factors are partially functionally redundant and exhibit a highly restricted expression pattern, especially in adults. Thus, precision targeting of individual FOX proteins may lead to safe treatment options for Wnt-dependent cancers.

Abstract 3182: Tumor immune microenvironment based molecular functional clustering reveals a prognostic signature that predicts overall survival in patients with gastric cancer

Abstract Gastric Cancer (GC) is the second leading cause of cancer-related mortality (9.7% of the total) and most patients with advanced disease will die within one year of diagnosis. GC is histologically classified into intestinal, diffuse and the mixed types, and into four molecular subtypes based on genetic profiling (i.e. microsatellite instable (MSI), EBV positive, chromosomal instable, and genomically stable). Although the molecular annotation is meaningful, the tumor immune microenvironment (TIME) has largely not been evaluated. Using an integrated collection of 11 public cohorts (n=2,270), we identified 5 unique GC TIME phenotypes by unsupervised clustering of gene expression signatures of RNA that describe the composition and biology of TIME and properties of malignant cells: two stromal-enriched subtypes - 1) Mesenchymal-wnt activated, high stroma activation and WNT signaling and 2) Fibrotic, only high stroma activation; two immune enriched subtypes 3) Inflamed non-fibrotic, high immune infiltration and low stromal compartment and 4) B-cell inflamed, high B cell activation; and 5) Immune Depleted, lowest immune and the highest malignant cell properties. The clusters were robust, being identified across all 11 datasets and across all stages of disease. Intestinal, diffuse, and mixed histologies were identified in each cluster. These clusters will be orthogonally validated using immunohistochemistry. The current molecular subtypes were represented in each of our TIME clusters, with some enrichment. Specifically, we found enrichment of MSI molecular subtype, characterized by hypermutation and microsatellite instability, in “Inflamed, non-fibrotic” cluster whereas the MSS/EMT subtype, associated with poor overall survival, was enriched in “Mesenchymal, wnt activated” cluster. We also saw specific enrichment of EBV positive tumors, known to have good prognosis, in both immune-enriched clusters. These results show a high concordance with the current TCGA/ACRG molecular subtypes of GC. These TIME clusters are prognostic in GC. The “Inflamed, non-fibrotic” cluster demonstrated a better overall and relapse free survival whereas stromal enriched clusters exhibited the worst (p&amp;lt;0.001, HR=2.27). Interestingly, the most aggressive “Mesenchymal, wnt activated” subtype was also enriched of metastatic samples. These results were confirmed using an independent validation cohort (n=231) from four other datasets. Additionally, comparison of matching on-treatment vs baseline biopsies from 7 patients treated with cabazitaxel further suggested that TIME changed upon treatment and, in some cases, was indicative of poor response to taxanes. We have defined and characterized the TIME for GC. The GC microenvironment is both prognostic for patient outcome and predictive of response to cytotoxic therapy. Citation Format: Prashant V. Thakkar, Olga Kudryashova, Daria Melikhova, Naira Samarina, Sandrine Degryse, Alexander Bagaev, Felix Frenkel, Svetlana Podsvirova, Dmitry Tychinin, Sandipto Sarkar, Rhonda K. Yantiss, Nathan Fowler, Manish A. Shah. Tumor immune microenvironment based molecular functional clustering reveals a prognostic signature that predicts overall survival in patients with gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3182.