The role of KDM5B in creating synthetic vulnerabilities in combination with radiotherapy in melanoma cells

Cutaneous melanoma (CM) is the most aggressive skin cancer with a high metastatic potential and despite advances in treatment, declining survival rates are mainly attributed to therapy resistance, particularly in advanced stages. The challenge of resistance to therapies aimed at the MAPK pathway and immune checkpoint inhibitors are on the rise, influenced by various factors within the tumor microenvironment (TME). KDM5B, a histone demethylase that is often overexpressed in melanoma, plays a key role in developing resistance to treatments and promoting tumor progression. Additionally, hypoxia within the TME exacerbates treatment challenges by fostering aggressive cell characteristics. The effectiveness of radiotherapy is diminished due to the heterogeneity of tumors and the development of resistance, emphasizing the need for novel treatment strategies. This study investigated the function of KDM5B in mechanisms of resistance linked to hypoxia and assess the potential of new combination therapies to improve treatment outcomes for melanoma. In this study the effects of cycling hypoxia on melanoma cell lines were investigated, specifically WM3734 naïve and WM3734 KDM5BOE, which had been generated by a former PhD student, Safa Larafa, by subjecting them to repeated cycles of hypoxia-reoxygenation (Hx10 and Hx20) or maintaining them under normoxic conditions (Ctrl), mimicking the conditions of cycling hypoxia in the TME. Colony formation assays demonstrated that Hx10 cells exhibited greater survival and clonogenic abilities following irradiation compared to Hx20 and control cells, suggesting that Hx10 cells developed enhanced survival capabilities. Additionally, cell cycle analysis indicated that Hx10 cells had a more proliferative response to radiation exposure, while Hx20 cells showed increased G2/M arrest, indicating heightened susceptibility to radiation, hence excessive cycles of anoxia-reoxygenation lead to increased vulnerability. The study also highlighted KDM5B’s radioprotective function, as increased levels of KDM5B were found in the more resistant Hx10 cells especially in the KDM5BOE cells. The naïve cells appeared to be more sensitive than those with KDM5B overexpression. Immunofluorescence analysis of γH2AX foci revealed less residual IR-induced DNA damage in Hx10 cells compared to Hx20 cells, confirming KDM5B's influence on radioresistance. Furthermore, treatment combinations with PI3K and AKT inhibitors alongside radiation revealed that these inhibitors improved cytotoxic effects of IR, particularly in the radiation resistant Hx10 cell lines with increased KDM5B and PI3K levels. The findings of this thesis confirm our hypothesis that KDM5B enhances radioresistance and hypoxia tolerance in melanoma cells and suggests that its modulation could provide new therapeutic strategies for overcoming treatment resistance. In conclusion, an initial characterization of hypoxia/reoxygenation-tolerant melanoma cell lines without or with KDM5B overexpression was performed, and PI3K/Akt as potential therapeutic target was identified. Additional research is necessary to understand how KDM5B and adaptation to cycling hypoxia influence acquired treatment resistance.