Investigation of the oncogenic role of MAF in multiple myeloma
Creators: name: Katsarou, Alexia Despoina
Publisher: name: Imperial College London
Year: 2020

description: Multiple myeloma (MM) is an incurable malignancy of bone marrow plasma cells (PC), affecting over 5,600 new patients per year in the UK. Translocation of the transcription factor MAF to the IgH enhancer, t(14;16), is a myeloma-initiating event in 3-5% of MM cases. As well as in t(14;16), MAF is over-expressed in another 30% of MM, mostly in association with the MMSET t(4;14) translocation. The mechanisms by which aberrant expression of MAF promotes myelomagenesis, including the oncogenic transcriptome it regulates as well as the regulatory regions it impacts are unknown. In this work, with reference to the normal PC, and using both primary myeloma cells and myeloma cell lines, I have applied a multi-omics approach to define the MAF regulome and determine the impact of ectopic MAF expression on MM cells. First, analysis of the MAF transcriptome in primary myeloma, as compared to normal PC, revealed upregulated genes that are shared with other MM subgroups or are MAF-unique. Having established MAF as a t(14;16) myeloma cell dependency using knock down assays, I obtained and integrated the transcriptome following MAF depletion with the MAF cistrome using RNA-seq and ChIP-seq respectively. This identified 2,112 genes directly bound and regulated by MAF, the majority of which were predicted to be activated by the transcription factor. Intersecting these direct MAF targets with the genes differentially upregulated in MAF primary MM cells, revealed a core transcriptional MAF programme involved in cell cycle regulation and cell to cell interactions such as cell adhesion, migration, cytokine and chemokine signaling pathways. Identification of the MAF cistrome revealed that MAF preferentially binds enhancers, prompting further exploration of the MAF-associated distal regulatory genome. Compared to normal PC, t(14;16) primary MM cells displayed enhanced chromatin accessibility as assessed by ATAC-seq, with 33% of those regions being over-accessible uniquely in the MAF MM subgroup. Indeed, the chromatin accessibility profile of MAF MM was able to distinctly cluster the samples separately from other genetic MM groups. Remarkably, a quarter of the over-accessible MAF regions were also bound by the MAF (in total 1,661 regions), consistent with a role of MAF in the regulation of chromatin accessibility. Juxtaposing these regions against chromatin mark maps of the MAF translocated MM.1S cell line and normal PC revealed 44% (726/1,661) of these regions, acquire activatory marks in MAF-translocated cells having been inactive in normal PC and the majority also inactive throughout B cell development. This finding suggested the possible implication of MAF in the activation of these regions. Indeed, following overexpression of MAF in a MAF-negative myeloma cell line, over 50% of these regions acquired enhanced accessibility, demonstrating MAF as an activator of specific regulatory regions in t(14;16) MM. This enabled identification of MAF-activated enhancers, such as in the case of a CCR1 enhancer peak, validated to be a CCR1 regulatory region by CRISPRi. Finally, using ChIP-seq data and chromatin proteomics I find IRF4 to be co-bound in 70-80% of MAF-bound genomic regions, suggesting possible cooperation as well as cross-regulation between the two transcription factors. In addition, several other proteins are identified as candidate partners of the MAF interactome. In conclusion, this work has provided a comprehensive description of the role of oncogenic MAF in MM and given insights into events related to ectopic MAF expression in PC occurring during the process of myelomagenesis. It has revealed the full spectrum of directly MAF-regulated genes and the consequent cellular pathways affected in MM. In addition, it has established MAF’s role in modifying the PC regulatory genome by activating specific regulatory regions, and has enabled the identification of enhancers regulating key MAF-dependent genes, critical for myeloma cell biology.