The role of transcription factors in multiple myeloma

Abstract

Multiple myeloma (MM) is an incurable cancer of antibody secreting plasma cells. Approximately 1700 people are diagnosed and 900 people die from MM in Australia annually. During my PhD, I have interrogated the function of key transcription factors in MM, with the ultimate aim of translating discoveries into improvements in patient care. The immunomodulatory drugs (IMiDs) are a cornerstone of current MM therapy. Our understanding of their underlying mechanism of action has advanced significantly in recent years, with the findings that they lead to the Cereblon dependent, proteasomal degradation of the transcription factors IKAROS and AIOLOS. However, why their loss results in MM cell death remains unclear. I have used CRISPR-Cas9 genome editing, to delete IKZF1/IKAROS and IKZF3/AIOLOS in human MM cell lines to gain further insight into their downstream gene regulatory networks. Inactivation of either factor alone recapitulates the cell intrinsic action of the IMiDs, including induction of a cell cycle arrest and apoptosis, as well as striking overlap of transcriptional changes. Importantly IKAROS and AIOLOS repress the expression of interferon stimulated genes (ISGs), including CD38, and their loss led to the activation of an interferon-like response, contributing to MM cell death. IKAROS/AIOLOS repressed CD38 expression through interaction with the nucleosome remodelling and deacetylase complex in MM. IMiD induced loss of IKAROS or treatment with interferon resulted in an upregulation of CD38 surface expression on MM cells, priming for daratumumab induced NK cell mediated antibody-dependent cellular cytotoxicity. The transcription factor IRF4 is essential for the survival of both normal and MM plasma cells. Furthermore its expression is almost exclusively limited to the immune compartment and specifically up regulated in both normal and malignant PCs, making it a highly attractive therapeutic target in MM and plasma cell driven autoimmune diseases. However, it is currently not known why IRF4 loss results in MM cell death. I have used a similar CRISPR-Cas9 and RNA sequencing approach to address this question. This screen led to the identification of 86 core transcriptional target genes whose expression either decreases or increases upon IRF4 deletion, and includes a number of genes likely contributing to both the survival and proliferative defect that ensues. This included two related pro-apoptotic BH3-only proteins whose expression is normally repressed by IRF4, Bcl2 Modifying Factor (BMF) and Bcl2-like protein 11 (BCL2L11), encoding BIM. Importantly, deletion of either BH3-only protein led to enhanced survival upon IRF4 inactivation. Thus, IRF4 maintains MM survival in part through repression of the proapoptotic molecules BMF and BIM. Finally, through performing RNA sequencing on primary patient samples, I have constructed a transcriptional roadmap of human B cell terminal differentiation to plasma cells and transformation to multiple myeloma. These studies have enabled new insights into both normal B cell ontogeny as well as the pathogenesis of multiple myeloma. Furthermore analysis of this data in conjunction with large external clinical MM datasets and genome wide large volume cancer cell line CRISPR-Ca9 dependency screens, have identified novel poor risk prognostic predictors and potential future therapeutic targets.