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Researchers Generate Catalog of Kinase Response to DNA-Damaging Agents

NEW YORK (GenomeWeb) – Researchers have developed a catalog of how kinases respond to DNA-damaging agents, such as ones used in cancer treatment.

DNA-damaging agents are often used to treat cancer as they lead to cell death, either by targeting DNA itself or proteins involved in DNA repair and related processes. Kinases have a number of roles following DNA damage, including sensing lesions and regulating cell cycle progression, and are also often deregulated in cancers.

Researchers led by Joanna Loizou from the CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences treated more than 300 cell lines deficient in certain kinases with 10 different DNA-damaging agents to map their drug-specific sensitivities and resistance. As they wrote in Cell Reports, the researchers found new synthetic lethal and gene-drug interactions and a potential role for MARK3 in DNA damage response.

"Our data suggest that cancers with inactivating mutations in kinases, including MARK3, are particularly vulnerable to alkylating chemotherapeutic agents," the researchers wrote in their paper.

Loizou and her colleagues used CRISPR/Cas9 to target 313 kinases in the human cell line HAP1 to generate lines lacking kinases involved in various parts of the DNA damage response. They then exposed these kinase-deficient cell lines to 10 compounds that inflict different types of DNA damage and initiate different DNA repair response pathways. The lines were exposed to four different concentrations of these drugs and their cellular survival was determined after three days.

Most of the cell lines responded as the researchers expected based on previous reports in the literature. For instance, they noted that PRKDC-deficient cells were the most sensitive to etoposide and doxorubicin — both DNA double-strand-break-inducing drugs — but that ABL1-deficient cells were resistant to those drugs.

When they clustered the cell lines by their sensitivity to those 10 drugs, the researchers found that they grouped into three clusters marked by their sensitivity to carmustine, hydroxyurea, and DNA double-strand-break-inducing drugs.

The researchers then focused on the cluster marked by sensitivity to the alkylating agent carmustine. A Gene Ontology analysis noted that this cluster was enriched for terms related to alkylating or crosslinking and cellular response to hydrogen peroxide, among others.

Using a fluorescence-activated cell sorting-based phenotypic assay, the researchers measured DNA damage, apoptosis, cell cycle phase, and proliferation in cell lines lacking MARK3, PRKACA, CSNK1G1, PNCK, DYRK4, or EPHB6 after treatment with either a DNA alkylating to DNA crosslinking agent. These lines, they noted, showed the strongest unreported synthetic lethal interaction with carmustine. From this, they found that the cellular sensitivity to carmustine was largely due to alkylation-induced synthetic lethality.

They then sought to validate these interactions by reconstituting the wild-type genes in MARK3, PNCK, DYRK4, and EPHB6 knockout cell lines. They found that the sensitive and resistant phenotypes could be corrected by expressing the missing kinase.

The researchers noted that mutations within EPHB6 and MARK3 are common among cancers, appearing in 42 percent and 31 percent of cancers, respectively, according to The Cancer Genome Atlas.

Using a phosophoproteomics approach, they investigated potential MARK3 substrates to find that it affects the phosphorylation of proteins involved in the cellular response to DNA damage. For this, the researchers treated wild-type and MARK3-deficient cells with the monofunctional alkylating agent temozolomide (TMZ), which is often used instead of carmustine.

Treatment with TMZ, the researchers found, downregulated phosphorylation sites in MARK3-deficient cells. This suggests TMZ could be an effective treatment for cancers lacking MARK3, they added.