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BRAF Inhibitor Resistance Mutations Identified

NEW YORK (GenomeWeb) – Mutations in the cohesin complex genes STAG2 and STAG3 can lead to ERK signaling pathway changes, enabling resistance to BRAF inhibitors in BRAF-mutated melanoma, according to a new Nature Medicine study.

Researchers from the US and China did exome sequencing on a pretreatment melanoma sample from an individual treated with the BRAF inhibitor vemurafenib (Genentech's Zelboraf) and on a sample taken after the disease progressed around five months later. In the post-relapse sample, they saw the first glimpse of STAG2 pathway involvement in BRAF inhibitor resistance — a relationship they explored further in additional melanoma cases.

"The observation that mutations of STAG2 or STAG3 can lead to reactivation of the MAPK pathway was a novel and unexpected finding," senior author Bin Zheng, a cutaneous biology and dermatology researcher affiliated with Massachusetts General Hospital and Harvard Medical School, said in a statement. "We are now working to identify vulnerabilities in STAG2/3 mutant melanomas and discover new approaches to targeting these tumors, which we hope will provide new insight into both preventing BRAF inhibitor resistance and treating tumors that have become resistant."

BRAF inhibitors have been used to successfully treat melanomas containing mutations such as V600E that activate BRAF and ramp up activity of the MAP kinase signaling pathway, the team explained. But mutations to MAP kinase genes such as NRAS or NF1 can produce resistance to such treatments, as can alterations involving components of the PI3-kinase pathway.

And still more cases of BRAF inhibitor resistance — up to one-quarter of resistant melanomas — seem to involve yet-to-be-determined mechanisms, prompting the researchers to search for resistance-related mutations in other genes and pathways.

"Identifying biomarkers capable of predicting response to BRAF inhibitor drugs — such as vemurafenib and dabrafenib — will facilitate early identification of patients likely to benefit from treatment and enable monitoring the development of resistance, which would allow consideration of other treatment options prior to relapse," Zheng said.

For their study, he and his colleagues considered pre-vemurafenib treatment and post-relapse fresh-frozen paraffin-embedded samples from an individual with melanoma. Protein-coding portions of the genome were isolated with an Agilent SureSelect exon kit before exome sequencing at BGI.

In comparing the pre-treatment and post-relapse tumor samples to one another — and to a collection of significantly mutated genes identified in a pan-cancer analysis by the Cancer Genome Atlas — the team saw mutations to a single gene in the post-relapse tumor: the cohesin complex core subunit gene STAG2.

The researchers noted that STAG2 was mutated at low frequency prior to treatment, with mutation levels rising noticeably at relapse. They went on to do targeted sequencing of STAG2 and a paralog called STAG3 in BRAF inhibitor-resistance cell lines, including a cell line that was resistant to both BRAF- and MEK-inhibition.

Along with diminished STAG2 and STAG3 expression in multiple cell lines, their experiments uncovered a nonsense STAG2 mutation in one of the BRAF inhibitor-resistant cell lines.

Next, the team did a broader search for STAG2 or STAG3 mutations in V600 mutation-containing tumor samples from 45 individuals with metastatic melanoma who were treated with vemurafenib or the BRAF inhibitor Tafinlar (dabrafenib) from Novartis, including 14 individuals with early, acquired drug resistance.

In half a dozen cases, the analysis identified STAG3 mutations in post-relapse tumors that were not present prior to treatment. Similarly, the researchers found several examples of tumors with acquired resistance to a BRAF inhibitor or BRAF- and MEK-inhibitors that had reduced STAG2 or STAG3 expression.

Their subsequent cell line experiments provided further evidence of roles for STAG2 and STAG3 in the drug sensitivity or resistance of BRAF-mutant melanoma lines by helping to re-activate the MEK-ERK signaling pathway.

"Our findings not only reinforce the concept that reactivation of ERK signaling represents a major resistance mechanism of BRAF pathway inhibition," the study's author wrote, "but they also reveal a previously unappreciated connection between STAG proteins and ERK signaling."