Co-inhibition of Notch1 and ChK1 triggers genomic instability and melanoma cell death increasing the lifespan of mice bearing melanoma brain metastasis.

backgroundMelanoma brain metastases (MBM) are a leading cause of death in patients with advanced disease. MBM treatment relay on targeted and immunotherapy and on stereotactic radiosurgery as gold standard. Life expectancy has improved significantly with these therapies however, targeted therapy is short lived and only about half of the patients respond to immunotherapy, while radiation is limited by melanoma cells intrinsic resistance to DNA damage. New therapeutic approaches are therefore needed to treat MBM. Here we investigate a new role of Notch1 in genomic instability and demonstrate that blockade of both Notch1 and the DNA repair factor ChK1 causes extensive DNA damage and tumor cell death increasing survival in MBM bearing mice.

methodsAnti-Notch1 (anti-N1) was previously described. Prexaserib, a ChK1 inhibitor, is currently in clinical trials. K457 and A375 melanoma cells were used. RNA sequencing was performed in K457 cells treated with anti-N1 and Gene Set Enrichment Analysis performed. DNA damage was evaluated by a DNA fiber assay to assess replication fork speed; and γH2AX foci count and neutral comet assay to quantify double strand breaks. Cell survival was evaluated by trypan blue and a colony formation assay. Luciferase expressing A375 cells were orthotopically inoculated in the right cerebral cortex of athymic nude mice, for in vivo evaluation of a therapy with anti-N1 and prexasertib. Survival was assessed by Kaplan-Meyer survival curves and significance assessed by a Log-rank test.

resultsNotch1 blockade caused genomic instability by reducing histone availability, leading to DNA replication stress and DNA damage. This in turn, resulted in the activation of the DNA Damage Response pathway ATR/ChK1 to counter the damage. Co-inhibition of Notch1, via anti-N1, and ChK1, via prexasertib (prex), exacerbated DNA damage increasing melanoma cell death. Importantly, combination anti-N1/prex significantly improved survival of mice bearing MBMs.

conclusionsA therapy with anti-N1/prexasertib could represent a novel treatment strategy, alone or in combination with current treatment regimens, for melanoma brain metastases.