Leiomyoma Cell Models for Research

Leiomyomas, also known as uterine fibroids, are the most common benign tumors of the female reproductive tract, affecting up to 70-80% of women by age 50 (WHO, 2022). Although benign, they cause significant morbidity including heavy menstrual bleeding, pelvic pain, and reproductive dysfunction. The global burden is substantial, with millions of women seeking treatment annually. In the United States, fibroids are the leading indication for hysterectomy, with an estimated cost of $5.9-34.4 billion per year (NCI, 2021). Risk factors include age, race (higher incidence in African American women), obesity, and family history. Unlike malignant tumors, the 5-year survival is essentially 100%, but quality of life is severely impacted. Research focuses on understanding the molecular drivers to develop non-surgical therapies.

Leiomyomas are ideal for studying benign tumorigenesis, hormone-dependent growth, and the role of somatic mutations in tumor initiation. They exhibit distinct molecular subtypes based on driver mutations (e.g., MED12, HMGA2, FH), providing a unique opportunity to dissect genotype-phenotype correlations. Public datasets such as TCGA (though limited for benign tumors) and GEO provide transcriptomic and genomic data. Open questions include the exact mechanisms of hormone signaling, the role of the extracellular matrix, and the development of targeted therapies that avoid surgery. Gene-edited cell models are crucial for functional validation of these mutations.

Leiomyomas are driven by several key pathways, though they are benign, they share some signaling aberrations with cancer. The major pathways include:

• • MED12/ Wnt/β-catenin pathway: MED12 mutations (found in ~70% of fibroids) lead to dysregulation of Wnt/β-catenin signaling, promoting cell proliferation and fibrosis.
• • HMGA2 overexpression: Rearrangements at 12q15 cause HMGA2 overexpression, which alters chromatin architecture and gene expression, driving cell growth.
• • FH (fumarate hydratase) deficiency: Loss-of-function mutations in FH lead to accumulation of fumarate, which inhibits prolyl hydroxylases, stabilizing HIF1α and promoting pseudo-hypoxic responses.
• • Hormonal signaling (estrogen/progesterone): Estrogen and progesterone receptors drive tumor growth via paracrine and autocrine loops, involving growth factors like TGF-β and EGF.

Data from TCGA (fibroid samples) and COSMIC (somatic mutations).

Key signaling networks deregulated in leiomyomas include:

• • Wnt/β-catenin: MED12 mutations lead to β-catenin accumulation and TCF/LEF transcription, promoting proliferation and fibrosis.
• • MAPK/ERK: Growth factor signaling (EGF, PDGF) activates MAPK, driving cell cycle progression.
• • PI3K/AKT/mTOR: FH deficiency and hormonal signaling activate this pathway, promoting survival and growth.
• • TGF-β/SMAD: Overactive TGF-β signaling increases extracellular matrix production, a hallmark of fibroids.
• • HIF1α pathway: In FH-deficient tumors, HIF1α stabilization leads to metabolic reprogramming and angiogenesis.

Organoids derived from patient fibroids retain the genetic and phenotypic heterogeneity, making them valuable for drug testing and studying tumor-stroma interactions. However, they are more complex to culture and less amenable to high-throughput screening than cell lines.

Animal models for leiomyoma include:

• • Patient-derived xenografts (PDX): Immunodeficient mice implanted with patient fibroid tissue or cells. They preserve the tumor microenvironment but are costly and time-consuming.
• • Genetically engineered mouse models (GEMM): Mice with conditional MED12 mutations or FH deletion in uterine smooth muscle cells. They recapitulate fibroid development but require breeding and have long latency.
• • Induced models: Treatment with estrogen/progesterone in immunodeficient mice to promote growth of implanted human fibroid cells. Useful for studying hormonal dependence.

CRISPR-based gene editing has revolutionized leiomyoma research by enabling the creation of isogenic cell lines that differ only in a specific genetic alteration. For example, introducing the MED12 G44D mutation into a wild-type uterine smooth muscle cell line (e.g., HuLM) creates a pair of isogenic lines that can be used to study the mutation's effect on proliferation, signaling, and drug response. Similarly, knocking out FH or overexpressing HMGA2 can model those subtypes. These models are commercially available from various sources and are sequence-verified to ensure specificity. They provide a controlled system to dissect the functional consequences of driver mutations, screen for targeted therapies, and validate biomarkers. Using isogenic pairs eliminates confounding genetic background, making them superior to comparing different cell lines.

Gene-edited cell lines are essential for functional genomics studies. For example, a MED12 knockout line can be used to identify downstream target genes via RNA-seq, revealing the Mediator complex's role in gene regulation. Similarly, a FH knockout line can be used to study the metabolic consequences of fumarate accumulation, such as HIF1α stabilization and altered mitochondrial function. These models allow researchers to validate candidate genes from GWAS or expression studies and to perform CRISPR screens to identify synthetic lethal partners.

Isogenic cell line pairs (e.g., MED12 mutant vs. wild-type) are powerful tools for drug screening. They can be used to identify compounds that selectively inhibit the mutant cells, providing a therapeutic window. For example, screening a library of kinase inhibitors against MED12 mutant cells may reveal dependencies on specific signaling pathways. Additionally, gene-edited cells can be used to model resistance to existing therapies, such as hormonal treatments, by introducing mutations that confer resistance. This helps in developing next-generation drugs.

CRISPR-based synthetic lethality screens in leiomyoma cell lines can identify genes that are essential only in the presence of a specific mutation (e.g., MED12 mutation). This approach can uncover novel therapeutic targets and biomarkers. For instance, a screen in FH-deficient cells may reveal a dependency on the antioxidant response pathway, suggesting NRF2 inhibitors as potential drugs. Gene-edited cells also enable the identification of secreted proteins that could serve as non-invasive biomarkers for fibroid progression or recurrence.