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At ACMG, Mechanism for Common Breast Cancer Locus Proposed From Functional Analysis

NEW YORK – At the American College of Medical Genetics and Genomics annual clinical genetics meeting on Wednesday, an investigator from Cedars-Sinai Medical Center presented findings from a regulatory and functional validation study of a proposed breast cancer susceptibility site on chromosome 6, highlighting a strategy that may help to understand susceptibility mechanisms more broadly.

Along with high-penetrance, but rare, risk variants in cancer susceptibility genes such as BRCA1/2, TP53, and PTEN, investigators are tapping into common, low-penetrance and intermediate, moderate-penetrance alleles to continue unraveling heritable or familial forms of the disease — efforts that can be improved by bringing in non-coding regulatory, functional, and clinical clues, explained Saeideh Torabi Dalivandan, a graduate student in Simon Gayther's bioinformatics and functional genomics lab at Cedars-Sinai.

"While rare mutations in DNA double-strand break genes like BRCA1, BRCA2, and PALB2 confer high- to moderate-penetrance risk to a subset group of breast cancers, these only account for around 20 percent of hereditary breast cancer," she said, noting that genome-wide association studies have identified many more common contributors to breast cancer risk.

As part of a session on Cancer Genetics and Therapeutics, Dalivandan outlined a validation pipeline and functional strategy that she and her colleagues used to interrogate a breast cancer-associated locus at 6q23.1, as well as its potential mechanistic ties to breast cancer.

That risk locus was among more than 150 genomic sites linked to breast cancer during a GWAS involving some 109,900 cases and more than 88,900 unaffected control individuals, she explained, and included 43 candidate causal variants picked up by fine mapping. The nearest gene, L3MBTL3, codes for a chromatin-interacting transcriptional repressor.

By connecting insights from GWAS to biological processes the play a role in breast cancer development, she added, it may eventually be possible to improve the strategies used to prevent, screen for, diagnose, or treat the disease.

Even so, "there remains a huge bottleneck in translating the GWAS data to help patients," Dalivandan said. "That is mainly because most of the GWAS variants map to the non-coding genome and, therefore, assigning a direct function to them is not easy."

For their 6q23.1 analysis, investigators at Cedars-Sinai Medical Center, the University of Bristol, and Dana-Farber Cancer Institute turned to epigenetic regulatory variant annotation, along with interaction clues generated with circularized chromosome conformation capture (4C) sequencing, to prioritize possible contributors to breast cancer susceptibility at chromosome 6q23.1.

"Integration of these three datasets allows us to first prioritize the risk SNPs in that locus, and also to confirm the potential target gene at that risk locus," she explained.

In the case of 6q23.1, for example, the team noted that breast cancer-associated SNPs have been linked to lower-than-usual levels of L3MBTL3 in data from the Genotype-Tissue Expression Project, while L3MBTL3 expression in general appeared to be slightly reduced in breast cancer transcriptome data from the Cancer Genome Atlas project.

When the researchers used 4C-seq in normal cells and in breast cancer cells to search for informative L3MBTL3 promoter interactions, they highlighted three SNPs: one in the L3MBTL3 promoter and two 4C-interacting SNPs at predicted breast cancer-related transcription factor binding sites.

After focusing in on the three most promising SNPs in the region, the team turned to cell proliferation, colony formation, and cell migration assays — coupled with cell line experiments, transcriptome profiling, and pathway analyses — to tease out mechanistic contributions to cancer risk and development.

Based on their CRISPR-Cas9-based gene editing and transcriptome sequencing experiments on cell lines with or without L3MBTL3, for example, the researchers found that levels of cell adhesion and differentiation genes appeared to notch down in the absence of the L3MBTL3.

More broadly, reduced L3MBTL3 expression also seemed to boost activity by genes involved in a tumor invasion and metastasis-related process known as the epithelial-to-mesenchymal transition (EMT), consistent with a proposed role for L3MBTL3 in degrading a DNA methyltransferase enzyme involved in EMT.

On the other hand, bumping up L3MBTL3 levels in breast cancer led to cell lines with more muted colony formation capabilities, hinting that the gene may code for a previously unappreciated tumor suppressor.

Based on their results so far, the investigators are planning to explore cell invasion patterns in cells treated with a small molecule inhibitor that targets L3MBTL3 or related pathways.

"If we know the mechanism [behind GWAS associations], that helps us to come up with a small molecule that either directly targets the gene of interest or another target in that pathway," Dalivandan said, noting that the broader pipeline is being applied in a high-throughput manner to other loci identified by GWAS.

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