In-vitro evaluation of Notch inhibition to enhance efficacy of radiation therapy in melanoma

Abstract Purpose The scope of radiation therapy is limited in melanoma. Using in-vitro melanoma models, we investigated a Notch signalling inhibitor as a radiosensitiser to explore its potential to improve the efficacy of radiation therapy to widen the clinical application of radiation therapy in melanoma. Methods and materials Melanoma cell lines A375, SKMEL28 and G361 were grown using standard tissue culture methods. Radiation was delivered with a clinical X-ray unit, and a gamma secretase inhibitor (GSI) RO4929097 was used to inhibit Notch signalling. Cell viability signal was used to calculate Loewe’s combination index to assess the interaction between radiation and RO4929097 and also the impact of scheduling of radiation and RO4929097 on synergy. Clonogenic assays were used to assess the clonogenic potential. An in-vitro 3D culture model, γ-H2AX and notch intra cellular domain (NICD) assays were used to interrogate potential underlying biological mechanisms of this approach. Scratch and transwell migration assays were used to assess cell migration. Results A375 and SKMEL28 cell lines showed consistent synergy for most single radiation doses examined with a tendency for better synergy with the radiation-first schedule (irradiation performed 24 hours before RO4929097 exposure). Clonogenic assays showed dose dependent reduction in colony numbers. Both radiation and RO4929097 reduced the size of melanospheres grown in 3D culture in-vitro where RO4929097 demonstrated a significant impact on the size of A375 and SKMEL28 melanospheres, indicating potential modulation of stem cell phenotype. Radiation induced γ-H2AX foci signal levels were reduced following exposure to RO4929097 with a tendency towards reduction in NICD levels for all the three cell lines. RO4929097 impaired both de novo and radiation enhanced cell migration. Conclusions We demonstrate Notch signalling inhibition with RO4929097 as a promising strategy to potentially improve the efficacy of radiation therapy in melanoma. This strategy warrants further validation in-vivo.