Hewett, DuncanVandyke, KateZannettino, AndrewParkinson, Hayley Bridget2025-07-222025-07-222025https://hdl.handle.net/2440/146224Multiple myeloma (MM) is an incurable malignancy characterised by the uncontrolled proliferation of plasma cells (PCs) in the bone marrow (BM). MM is a genetically heterogeneous disease with each patient’s PCs harbouring unique genetic mutations; however, the development of MM tumours is not only dependent on the underlying genetics but also on the selective pressures applied by the BM microenvironment. Another defining feature of MM is its ability to disseminate to multiple sites throughout the BM via the peripheral circulation. High numbers of circulating tumour cells are predictive of shorter time to progression, shorter time to relapse post MM treatment and worsened overall survival. Similar to tumour development, the process of dissemination is reliant on both underlying genetics and microenvironmental factors. The exact gene/protein dependencies which are influenced by the BM microenvironment, supporting both tumour formation and subsequent dissemination have yet to be fully identified. In this thesis the differences in the BM MM PC transcriptome between patients with high and low levels of circulating tumour PCs was first explored. In addition, a CRISPR-Cas9 knockout screen was performed in vivo to identify genes essential for MM tumour formation and dissemination. To investigate the transcriptomic differences between patients with high and low levels of circulating tumours MM PCs, MM PCs were collected from matched peripheral blood (PB) and aspirates from patients at the time of MM diagnosis. Transcriptomic analysis revealed that the BM MM PCs from patients with the highest level of PB PCs highly expressed the transcription factors MAF or MAFB and had high expression of MAF target genes. To investigate if MAF plays a role in dissemination attempts were made to knockout MAF in two MM cell lines; however, these attempts were unsuccessful. MAF was successfully moderately over expressed in two MM cell lines (OPM2, U266); however, there was no significant change in the RNA expression of MAF downstream targets. Despite this, the MAF over expressing OPM2 cells were used in an in vivo, disseminatory murine model of MM to identify if increased MAF expression led to increased disseminatory disease. No significant difference in tumour burden in the primary or disseminated tumours was observed, when comparing MAF over expressing cells to wild type cells. In conjunction, to identify novel genes involved in MM tumour formation and/or dissemination, CRISPR-Cas9 and the Bassik human apoptosis and cancer CRISPR knockout library was used in an in vivo murine model of disseminatory MM. The human MM cell line OPM2 was transduced with the Bassik library which contains 31,324 sgRNAs that targeted 3,015 genes and a further 1,500 non- and safe-targeting control sgRNAs. The knockout cell-line (OPM2-Cas9-sgRNA) was injected into the tibiae of immunodeficient NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice and four weeks post-injection, primary tumours in the injected leg and disseminated tumours in the contralateral leg were isolated. sgRNA frequencies were assessed via next generation sequencing (NGS) and compared with the initial library and the injected cells using the MAGeCK algorithm. Due to the greatly diminished gene targeting and control sgRNA representation in the contralateral leg, statistical analysis could not be accurately performed using the MAGeCK algorithm, and it was decided that the focus of the project would be the formation of the primary tumour. The screen successfully identified 106 genes where CRISPR knockout had no significant effect in vitro, but were critical dependencies for tumour development, with sgRNAs significantly depleted (p<0.05) in vivo. To refine and validate this list of genes, a custom sgRNA library was designed targeting genes which had significantly changed sgRNA representation in the primary screen. The validation screen reidentified 20 genes where CRISPR knockout had no significant effect in vitro but were critical dependencies for tumour development, with sgRNAs significantly depleted (p<0.05) in vivo. Future studies should re-attempt to both knockout and overexpress MAF in MM cell lines, with the aim of inducing changes in the downstream targets. Investigation into the MAFs role in dissemination should then be re-performed. Similar experiments to those used to investigate MAFs role in MM should also be performed on the 20 novel genes identified to be critical tumour formation dependencies in vivo, to elucidate their role and therapeutic targetability in MM.enMultiple myelomatumour formationtumour disseminationfunctional genomic screentranscription factor MAFThesis