Oligodontia-Colorectal Cancer Syndrome (ODCRCS) Cell Models for Research

Oligodontia-Colorectal Cancer Syndrome (ODCRCS) is an ultra-rare autosomal dominant disorder characterized by severe tooth agenesis (oligodontia) and a high predisposition to colorectal cancer (CRC). The exact prevalence is unknown; fewer than 50 families have been reported worldwide. The syndrome is caused by pathogenic variants in the AXIN2 gene (OMIM 608615). Colorectal cancer in ODCRCS typically presents at an early age (mean age ~48 years) and often with multiple polyps, resembling familial adenomatous polyposis (FAP) but without the classic extracolonic features. The lifetime risk of CRC in AXIN2 mutation carriers is estimated to be over 90% by age 70, based on limited cohort data. According to the WHO, colorectal cancer is the third most common cancer globally, with over 1.9 million new cases and 935,000 deaths in 2020. The 5-year survival for localized CRC is 91% (NCI SEER), but drops to 14% for distant-stage disease, underscoring the need for early detection and targeted therapies. ODCRCS offers a unique model to study the Wnt/β-catenin pathway, which is dysregulated in over 90% of sporadic CRCs.

ODCRCS is an ideal model for studying the molecular basis of Wnt signaling in colorectal carcinogenesis and tooth development. The syndrome provides a direct link between a single gene (AXIN2) and two distinct phenotypes, enabling mechanistic studies of tissue-specific Wnt pathway regulation. Public datasets such as TCGA (The Cancer Genome Atlas) and COSMIC (Catalogue of Somatic Mutations in Cancer) contain extensive mutation and expression data for AXIN2 in sporadic CRC, allowing comparative analyses between germline and somatic alterations. Open questions include the role of AXIN2 in tumor microenvironment, the potential for targeted therapies (e.g., tankyrase inhibitors), and the development of biomarkers for early CRC detection in at-risk individuals. Gene-edited cell models with AXIN2 knockout or knock-in mutations are essential for functional validation and drug screening.

The primary pathway affected in ODCRCS is the Wnt/β-catenin signaling pathway. AXIN2 is a scaffold protein in the β-catenin destruction complex, which also includes APC, GSK3β, and CK1α. Loss-of-function mutations in AXIN2 lead to stabilization of β-catenin and constitutive transcriptional activation of Wnt target genes.

• • Key steps in the pathway:

1. In the absence of Wnt ligand, the destruction complex phosphorylates β-catenin, marking it for ubiquitination and proteasomal degradation.

2. In ODCRCS, mutant AXIN2 fails to properly assemble the destruction complex, leading to reduced β-catenin degradation.

3. Stabilized β-catenin translocates to the nucleus and binds TCF/LEF transcription factors.

4. This activates oncogenes such as MYC and CCND1, promoting cell proliferation and inhibiting apoptosis.

Other pathways implicated in CRC progression include the MAPK/ERK pathway (via KRAS/BRAF mutations) and the PI3K/AKT pathway (via PIK3CA mutations), which often co-occur with Wnt pathway alterations.

Data from TCGA PanCancer Atlas and COSMIC. Frequencies are approximate and vary by cohort.

The Wnt/β-catenin pathway is the central hub in ODCRCS, but it interacts with multiple other signaling networks:

• • Wnt/β-catenin: Key nodes include AXIN2, APC, CTNNB1 (β-catenin), TCF7L2, MYC, CCND1.
• • MAPK/ERK: KRAS, BRAF, MEK, ERK – often activated downstream of Wnt in CRC.
• • PI3K/AKT: PIK3CA, PTEN, AKT, mTOR – promotes cell survival and proliferation.
• • TGF-β/SMAD: SMAD4, TGFBR2 – frequently mutated in CRC, leading to evasion of growth suppression.
• • Notch signaling: Involved in intestinal stem cell maintenance; cross-talk with Wnt.

These networks converge to drive tumorigenesis, making them attractive targets for combination therapies.

Organoids derived from normal intestinal epithelium or tumors retain the genetic and phenotypic characteristics of the tissue of origin. They can be used to study AXIN2 mutations in a 3D context, allowing for more physiologically relevant drug testing and CRISPR editing.

• • Patient-derived xenografts (PDX): Immunodeficient mice engrafted with tumor tissue from ODCRCS patients, preserving the tumor microenvironment and genetic heterogeneity.
• • Genetically engineered mouse models (GEMM): Conditional Axin2 knockout mice (e.g., Axin2fl/fl; Villin-Cre) that develop intestinal polyps, recapitulating the human phenotype.
• • Induced models: Use of azoxymethane (AOM) and dextran sulfate sodium (DSS) to induce CRC in mice, which can be combined with Axin2 mutations to study synergy.
• • Organoid-derived xenografts: Orthotopic implantation of gene-edited organoids into mice for in vivo validation.

CRISPR-Cas9 gene editing enables the creation of isogenic cell lines with precise AXIN2 mutations, either knockouts (loss-of-function) or knock-ins (patient-specific variants). These models are essential for studying the functional consequences of AXIN2 alterations in a controlled genetic background. For example, an AXIN2 knockout in HCT116 cells (which have wild-type AXIN2) can be used to assess the impact on Wnt signaling and drug sensitivity. Similarly, introducing a patient-specific AXIN2 mutation into a normal intestinal epithelial cell line can model the early stages of tumorigenesis. Commercially available, sequence-verified gene-edited cell lines accelerate research by providing ready-to-use models, but they must be validated for off-target effects and phenotypic stability. These models are widely used for drug discovery, target validation, and functional genomics.

• • Gene-edited cell lines with AXIN2 knockout or knock-in mutations are used to:
• • Validate the role of AXIN2 in Wnt pathway regulation by measuring β-catenin levels and target gene expression.
• • Identify synthetic lethal partners of AXIN2 mutations through CRISPR screens, revealing potential therapeutic targets.
• • Study the interaction of AXIN2 with other CRC-associated genes (e.g., APC, KRAS) using combinatorial editing.
• • Elucidate the tissue-specific effects of AXIN2 mutations by comparing intestinal and dental epithelial cell lines.

• • Isogenic pairs (wild-type vs. AXIN2 knockout) are powerful tools for drug screening:
• • They allow high-throughput screening of compounds that selectively kill AXIN2-deficient cells while sparing wild-type cells.
• • Resistance mechanisms can be studied by exposing AXIN2 knockout cells to increasing concentrations of Wnt pathway inhibitors (e.g., tankyrase inhibitors) and identifying secondary mutations.
• • Combination therapies targeting both Wnt and MAPK pathways can be evaluated in isogenic backgrounds to optimize treatment regimens.

CRISPR-based synthetic lethality screens in AXIN2-deficient cells can identify genes whose loss is lethal only in the mutant context. These genes may serve as biomarkers for patient stratification or as novel drug targets. Additionally, gene-edited cells can be used to identify secreted proteins or exosomal markers that reflect AXIN2 status, aiding in non-invasive early detection of CRC in ODCRCS patients.