Generation and Transcriptomic Analysis of HIF-1Alpha and HIF-2Alpha Knockout 5TGM1 Multiple Myeloma Cells

Abstract

Multiple myeloma (MM) is a blood cancer characterised by the uncontrolled proliferation and dissemination of neoplastic plasma cells in the bone marrow (BM). As the BM is physiologically hypoxic, hypoxia may serve as a microenvironmental stimulus that drives MM disease development and progression by promoting a broad range of tumorigenic features like tumour growth, angiogenesis, metastasis and bone osteolysis. Major cellular responses and adaptation to hypoxia are mediated through Hypoxia-Inducible Factor (HIF) signalling. The HIFs are heterodimeric transcription factors that activate the transcription of hundreds of target genes in response to hypoxia. In most solid tumours, HIF signalling is co-opted to contribute to tumour survival and progression. However, the role of HIF signalling in blood cancers is less well understood, specifically the distinct roles of the major HIF-α isoforms. Given that MM PCs reside in the hypoxic BM, the HIFs are likely to play important roles in MM disease. HIF-isoform specific inhibitors are currently in development and are being tested at the clinical level, and therefore, it would of considerable interest to determine if these drugs can potentially be repurposed for MM treatment. This research project aimed to investigate the unique and overlapping roles of HIF-1α and HIF-2α in the 5TGM1 mouse MM cell lines, which are syngeneic with the C57BL/KaLwRij mouse model. In this thesis, HIF-1α and HIF-2α inducible knockout and knocked-out 5TGM1 cells were generated using a doxycycline-inducible CRISPR/Cas9 system. Subsequently, HIF-1α and HIF-2α knockout 5TGM1 cells were characterised at the DNA, RNA and protein levels. Lastly, the in-vitro transcriptomes of HIF-1α and HIF-2α knockout 5TGM1 cells cultured in normoxia and hypoxia were profiled by RNA-sequencing to identify HIF-1α and HIF-2α target genes. In the 5TGM1 cells, HIF-1α was shown to regulate a broad range of target genes in response to hypoxia, with prominent roles in metabolic programming, cell-survival and transcriptional regulation. In comparison, HIF-2α appears to be expressed in very low Overall, the data on 5TGM1 cells in this thesis indicate that the HIFs, especially HIF-1, are likely to play important roles to support MM tumour adaptation, survival and development in the hypoxic BM microenvironment. The generated HIF-1α and HIF-2α inducible knockout and knocked-out 5TGM1 cells in this study are valuable tools for the further investigation and comparative analysis of the roles of each HIF-α isoform in-vivo in the syngeneic KaLwRij mouse model.