Investigating the Role of HIF-1 and HIF-2 Transcription Factors in Multiple Myeloma

Abstract

Multiple myeloma (MM) is an incurable haematological malignancy of bone marrow plasma cells (PCs) that presents with symptoms such as increased bone marrow angiogenesis, osteolytic bone lesions, bone pain and hypercalcaemia. Once in the marrow, MM PCs localise to hypoxic niches and upregulate the Hypoxia Inducible Factors (HIF-1α and HIF-2α) to facilitate survival and disease pathogenesis. Whilst the HIFs have been shown to contribute to MM tumour growth, angiogenesis and osteolysis, the majority of data has focused on HIF-1α. As such, HIF-2α remains largely understudied and under-considered in current approaches to disease prognosis and treatment. In this study, a lentiviral all-in-one Tet-inducible vector system was used to overexpress either HIF-1α or HIF-2α in murine 5TGM1 MM cells. Initial experiments in vivo using these cell lines in the established 5TGM1/C57BL/KaLwRij MM mouse model demonstrated comparable disease dissemination and tumour burden between uninduced stable 5TGM1 cells and control cells. Of particular interest, pilot experiments suggested there was a trend towards increased tumour burden in mice overexpressing HIF-1α or HIF-2α as detected by in vivo bioluminescence. Subsequent studies identified fundamental issues with transgene stability, showing a loss of expression over time in the stable cell lines, and demonstrated that they were unsuitable for use in the 4 week MM disease model. An alternative strategy was developed to knock out either HIF-1α or HIF-2α in the 5TGM1 MM cells using CRISPR-Cas9 technology. With this strategy, MM cell lines lacking HIF-2α were successfully generated, characterised and used in in vivo experiments. C57BL/KaLwRij mice injected with 5TGM1 HIF-2α knockout cells showed a significant delay and overall reduction or, in some instances, a complete lack of tumour dissemination and disease development compared to wild type control 5TGM1 cells. Together, the data presented in this dissertation demonstrate that HIF-2α is a critical contributing factor in MM disease progression in vivo, and suggest that HIF-2α has therapeutic potential in MM. This study has also generated valuable cell lines and methodologies for a more extensive comparative analysis of the specific roles of both HIF-1α and HIF-2α in MM.