Targeting the H3K4 demethylase KDM5B/JARID1B affects the metabolic landscape and malignant phenotype of melanoma cells

Melanoma is one of the most heterogeneous tumors among all cancers. Despite the development of novel therapeutic approaches including targeted and immune checkpoint therapy, most of the patients with advanced melanoma develop resistance and tumor progression after a few months. Over recent years, next to purely genetically driven cancer models, phenotypic heterogeneity models arose suggesting that there is a dynamic switch between different phenotypes allowing cells to functionally escape from therapy. In previous work of our group, a small subpopulation of multi-drug-resistant tumorrepopulating melanoma cells has been identified that was characterized by high expression of the histone H3K4 demethylase KDM5B (also known as JARID1B) and high dependence on oxidative phosphorylation (OXPHOS) leading to a higher production of ATP irrespective of the cells genetic background. In my thesis, I showed that the expression of KDM5B correlates with the expression of various metabolic gene signatures. In detail, the analysis of human melanoma transcriptomes published in The Cancer Genome Atlas (TCGA; from whole melanoma tumors) and of melanoma single cell RNA sequencing results of the Tirosh data set showed that 154 genes significantly overlapped with the expression of KDM5B. 29 of the identified genes (18.9%) encoded for central metabolic proteins. Functionally, 7 out of the 29 metabolic genes were associated with mitochondrial processes and energy metabolism, or are involved in the control of the intracellular redox capacity. The results of my liquid-chromatography-high-resolution-mass-spectrometry (LCHRMS) profiling unraveled the metabolome of the multi-drug resistant KDM5B-driven melanoma cell phenotype. I set up a novel and fast protocol for standardized, high sensitive LC-HRMS of melanoma cells based on functional modulation of the KDM5B expression phenotype by shRNA or doxycycline-induced overexpression. Within the KDM5B-dependent metabolome, I discovered a significant and highly specific regulation of 11 intracellular metabolites and 9 secreted metabolites in the supernatant. In detail, the detected metabolites in KDM5Bhigh cells contribute to an enhanced glutathione-dependent redox capacity, reflect a shift from glycolysis towards the pentose phosphate pathway (PPP), and show metabolic signatures that are characterized by upregulation of oxidative (mitochondrial) bioenergetic pathways. Functionally, as confirmed by physiological activity assays (e.g. Seahorse or GSH/GSSG Glo™ Assay), overexpression of KDM5B in melanoma cells led to broadening of their oxidative metabolism from mainly glutamine-dependent to additionally glucose- and fatty acid-utilizing, upregulation of the pentose phosphate pathway as a source of anti-oxidant NADPH, and maintenance of a high GSH/GSSG ratio. Finally, chemical KDM inhibition (GSK-J1, PBIT, or 2,4-PDCA) significantly regulated the expression of metabolic key enzymes (e.g. glutaminase). My results showed that KDM inhibition decreased proliferation of melanoma cells in a 2D cell culture model and significantly reduced colony formation and invasion in 3D models. Most importantly, similar effects were observed when KDM inhibitors were applied to BRAF-inhibitor resistant melanoma cells. Thus, targeting KDM5B could represent a superior way of modulating the metabolome and malignant cell behavior in melanoma. As soon as better bioavailable compounds are on hand, pre-clinical in vivo models must be performed to further assess the full potential of KDM inhibition in the context of melanoma drug-resistance and systemic tumor progression.