Peutz-Jeghers Syndrome (PJS) Cell Models for Research

Peutz-Jeghers Syndrome (PJS) is a rare autosomal dominant disorder with an estimated incidence of 1 in 50,000 to 200,000 live births (WHO, 2023). It is characterized by hamartomatous polyps in the gastrointestinal tract and mucocutaneous pigmentation. The cumulative risk of developing cancer by age 70 is 85% (NCI, 2023), with a 5-year survival rate for associated cancers (e.g., colorectal, pancreatic, breast) varying by stage, ranging from 90% for localized disease to less than 20% for metastatic disease (NCI SEER, 2023). Key risk factors include germline mutations in the STK11 gene (also known as LKB1), which are found in over 90% of PJS patients (NCBI Gene, 2023).

PJS provides an ideal model for studying tumor suppressor gene function, particularly STK11/LKB1, which regulates cellular metabolism, polarity, and energy sensing. The disease offers a clear genotype-phenotype correlation, enabling mechanistic studies of cancer initiation and progression. Public datasets, such as TCGA and COSMIC, provide extensive genomic and transcriptomic data for STK11-mutant tumors, facilitating bioinformatics analysis. Open questions include the role of STK11 in immune evasion and the development of targeted therapies for STK11-deficient cancers.

The loss of STK11/LKB1 function leads to dysregulation of multiple pathways:

1. AMPK signaling: LKB1 phosphorylates and activates AMPK, a key energy sensor. Loss of LKB1 reduces AMPK activity, leading to uncontrolled cell growth under metabolic stress.

2. mTOR pathway: LKB1-AMPK negatively regulates mTORC1. Loss of LKB1 results in constitutive mTORC1 activation, promoting protein synthesis and cell proliferation.

3. Wnt/β-catenin pathway: LKB1 can modulate Wnt signaling; its loss enhances β-catenin transcriptional activity, contributing to tumorigenesis.

4. Cell polarity and migration: LKB1 regulates cell polarity via PAR-1 and other substrates; its loss disrupts tissue architecture and promotes invasion.

Key signaling networks affected in PJS:

• • AMPK/mTOR axis: Loss of LKB1 leads to AMPK inactivation and mTORC1 hyperactivation, promoting anabolic metabolism and cell growth.
• • MAPK/ERK pathway: KRAS mutations activate RAF/MEK/ERK signaling, driving proliferation and survival.
• • PI3K/AKT pathway: PIK3CA mutations enhance AKT signaling, supporting cell survival and metabolism.
• • Wnt/β-catenin pathway: β-catenin stabilization leads to transcriptional activation of oncogenes like MYC and cyclin D1.
• • Immune regulation: STK11 loss is associated with reduced PD-L1 expression and altered cytokine secretion, affecting anti-tumor immunity.

Organoids derived from PJS patient tissues retain STK11 mutations and recapitulate the intestinal polyp phenotype, offering a more physiologically relevant model for drug testing and mechanistic studies.

• • Patient-derived xenografts (PDX): Implantation of PJS tumor tissue into immunodeficient mice preserves the STK11 mutation and tumor microenvironment, useful for preclinical drug evaluation.
• • Genetically engineered mouse models (GEMM): Conditional knockout of Stk11 in intestinal epithelium (e.g., Villin-Cre) leads to polyp formation, mimicking PJS pathology.
• • Inducible models: Doxycycline-inducible shRNA targeting Stk11 allows temporal control of gene silencing, enabling study of early tumorigenesis.
• • Xenograft models: Injection of STK11-mutant human cell lines (e.g., Capan-1) into mice to study tumor growth and metastasis.

CRISPR-Cas9 technology enables the creation of isogenic cell models with precise STK11 knockouts or knock-ins. For example, HCT116 cells with STK11 knockout (STK11-/-) are generated by introducing frameshift mutations via guide RNAs targeting exon 1 or exon 3. Similarly, KRAS G12D knock-in models can be created to study the cooperative effects of STK11 loss and KRAS activation. These gene-edited models are commercially available from various sources, are sequence-verified, and are free of off-target effects, ensuring reproducibility. They are essential for dissecting the molecular consequences of STK11 mutations and for screening targeted therapies.

Gene-edited cell models are used to validate the function of STK11 and other genes in PJS. For example, STK11 knockout cells show increased mTORC1 activity and enhanced cell proliferation under low glucose conditions, confirming the role of LKB1 in energy sensing. Knock-in of STK11 into knockout cells restores AMPK phosphorylation, providing a rescue control. These models also enable genome-wide CRISPR screens to identify synthetic lethal partners of STK11 loss, such as ARID1A or SMARCA4, which are potential therapeutic targets.

Isogenic pairs (STK11 wild-type vs. knockout) are used in high-throughput drug screens to identify compounds that selectively kill STK11-deficient cells. For instance, mTOR inhibitors (e.g., rapamycin) show enhanced efficacy in STK11 knockout cells. Resistance models are generated by chronically exposing STK11 knockout cells to drugs, allowing identification of resistance mechanisms, such as upregulation of PI3K/AKT signaling. These models are crucial for developing combination therapies to overcome resistance.

CRISPR screens using STK11 knockout cells can identify genes whose loss is synthetically lethal with STK11 deficiency, serving as potential biomarkers for patient stratification. For example, a screen may reveal that STK11-mutant cells are dependent on the oxidative phosphorylation pathway, suggesting that inhibitors of mitochondrial complex I (e.g., metformin) could be repurposed. Additionally, gene-edited models help identify secreted proteins or metabolites that serve as non-invasive biomarkers for early detection of PJS-associated cancers.