P02.10.A IDENTIFYING NOVEL HEXOKINASE-2 ASSOCIATED PROCESSES IN THE GLIOBLASTOMA TUMOUR MICROENVIRONMENT

Glioblastoma (GB) is the most common and aggressive type of adult brain tumour. With a median patient survival of only 16 months, new therapies for GB are desperately needed. Deregulation of cellular metabolism is a hallmark of cancers, where they upregulate glycolysis and preferentially ferment glucose to lactate (over pyruvate) in the ‘Warburg effect’. Upregulation of hexokinase 2 (HK2), which catalyses the first rate-limiting step of glycolysis, is key to Warburg metabolism. Several studies have shown HK2 to be selectively overexpressed in GB, while normal human brain tissue (NHBT) relies upon its isoform HK1. As a result, HK2-orientated therapies may selectively target GB tumours while limiting toxicity to normal metabolising healthy tissue. In this study we sought to identify HK2-associated tumour processes in GB which could serve as potential therapeutic co-targets. High HK2 expressing (>20-fold upregulation vs NHBT) established and patient-derived GB cell cultures (U87-MG, IN859) were identified by RT-qPCR. HK2 expression was knocked down in these cultures by transfection with targeted siRNA, while non-targeting siRNA was used as a negative control (NC). Expression profiles of HK2 knockdown (HK2KD) and NC cultures were compared using RNAseq. Differentially expressed genes (DEGs) found to be associated with HK2KD were analysed for gene ontology category enrichment (GOSeq) and protein-protein interactions (STRING). DEGs shared between HK2KD cultures were validated by RT-qPCR. HK2KD was associated with significantly (padj ≤0.05) enriched GO biological processes (BPs) in both U87-MG (117) and IN859 (109). BPs related to glycolysis or glucose metabolism were not enriched. Large clusters of enriched BPs related to inflammatory and immune response were prominent in both HK2KD-U87-MG and HK2KD-IN859. Notable members of these clusters included ‘regulation of neuroinflammatory response’, ‘activation of innate immune response’, and ‘response to bacterium’. Numerous enriched BPs linked to the recruitment of immune cells, such as ‘leukocyte migration’ and ‘granulocyte chemotaxis’, were also evident. STRING analyses further revealed clusters of interacting DEG-encoded cytokines (i.e. CSF2, IL1A/1B/6, TNF) and chemokines (i.e. CCL3, CXCL1/2/3/5/6/8) associated with HK2KD. Several angiogenesis related BPs were also enriched, which corresponded with the downregulation of the major angiogenic regulators HIF2A, VEGF and FGF2 in these HK2KD cultures. These findings highlight potential non-canonical roles for HK2 in regulating inflammatory, immune and angiogenesis related processes within the GB tumour microenvironment. Agents targeting HK2 may thus improve the efficacy of existing preclinical immuno/anti-angiogenic therapies against GB, which warrants further investigation.