Immunodeficiency 50 (IMD50) Cell Models for Research

Immunodeficiency 50 (IMD50) is an ultra-rare primary immunodeficiency disorder caused by biallelic mutations in the PMS2 gene, a key component of the DNA mismatch repair (MMR) system. The exact prevalence is unknown, but it is estimated to affect fewer than 1 in 1,000,000 individuals worldwide (WHO, 2023). IMD50 is characterized by early-onset colorectal cancer, hematological malignancies, and features of constitutional mismatch repair deficiency (CMMRD) syndrome. Without treatment, the prognosis is poor, with a median survival of less than 20 years. The 5-year survival for associated cancers varies by type, but for colorectal cancer it is approximately 50% (NCI, 2020).

IMD50 serves as an ideal model for studying DNA mismatch repair mechanisms, cancer predisposition, and immune system dysfunction. The disease is driven by well-defined genetic alterations in PMS2, making it amenable to CRISPR-based modeling. Public datasets, such as those from TCGA and COSMIC, provide extensive genomic and transcriptomic data on MMR-deficient tumors, enabling mechanistic studies. Open questions include the role of PMS2 in immune cell development and the potential for targeted therapies exploiting MMR deficiency.

The primary pathway disrupted in IMD50 is the DNA mismatch repair (MMR) pathway, which is essential for correcting replication errors. The steps involved are:

1. Recognition of mismatched bases by the MutSα complex (MSH2-MSH6) or MutSβ (MSH2-MSH3).

2. Recruitment of the MutLα complex (MLH1-PMS2) to the lesion.

3. Excision of the error-containing DNA strand by exonucleases.

4. Resynthesis and ligation by DNA polymerase and ligase.

Loss of PMS2 function leads to microsatellite instability (MSI) and a hypermutation phenotype, promoting tumorigenesis.

Data from TCGA and COSMIC.

In addition to MMR, several signaling pathways are deregulated in IMD50-associated tumors:

• • Wnt/β-catenin pathway: Mutations in APC, CTNNB1, or AXIN2 lead to constitutive activation, promoting cell proliferation.
• • MAPK pathway: BRAF or KRAS mutations are common, driving uncontrolled growth.
• • PI3K/AKT pathway: PIK3CA mutations or PTEN loss activate survival signals.
• • p53 pathway: TP53 mutations impair apoptosis and cell cycle arrest.

These pathways interact with MMR deficiency to accelerate tumor progression.

Organoids derived from patient tissues retain the genetic and phenotypic features of IMD50 and are valuable for drug testing and personalized medicine.

• • Patient-derived xenografts (PDX): Implantation of patient tumors into immunodeficient mice, preserving tumor heterogeneity.
• • Genetically engineered mouse models (GEMM): Pms2 knockout mice develop lymphomas and gastrointestinal tumors, recapitulating human disease.
• • Induced models: Use of carcinogens or CRISPR to introduce MMR deficiency in mice.

CRISPR-Cas9 technology enables the creation of isogenic cell lines with precise PMS2 knockout or knock-in mutations. For example, a PMS2 knockout in HCT116 cells (which already have MLH1 deficiency) can be used to study combined MMR defects. Alternatively, a PMS2 point mutation knock-in in a wild-type cell line can model specific patient mutations. These sequence-verified models are commercially available and accelerate research by providing consistent, reproducible systems for functional studies.

Gene-edited cell lines are used to validate the function of PMS2 and other MMR genes. For instance, knocking out PMS2 in a wild-type cell line leads to increased mutation frequency and MSI, confirming its role in MMR. Knock-in of a pathogenic PMS2 variant can assess its impact on protein function and cellular phenotypes.

Isogenic pairs (e.g., PMS2 knockout vs. wild-type) are used in high-throughput screens to identify compounds that selectively kill MMR-deficient cells. These models also help study resistance mechanisms to chemotherapeutics like 5-fluorouracil and cisplatin, which are commonly used in colorectal cancer treatment.

CRISPR-based synthetic lethality screens can identify genes that are essential only in MMR-deficient cells. For example, inhibition of DNA polymerase β or PARP1 has been shown to be synthetic lethal with MMR deficiency, providing potential therapeutic targets and biomarkers for patient stratification.