Sir Peter MacCallum Department of Oncology - Theses
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ItemInvestigating mechanisms of sensitivity and resistance to immunomodulatory thalidomide analogues in multiple myeloma( 2021)Multiple myeloma (MM) is an incurable plasma cell neoplasm. The incorporation of immunomodulatory imide drugs (IMiDs) – thalidomide, lenalidomide and pomalidomide - into clinical practice has improved survival and quality of life of MM patients. However, most patients develop therapeutic resistance to IMiDs, representing a significant clinical problem. IMiDs exert anti-MM effects by engaging Cereblon (CRBN), a ligand of the E3 ligase complex CUL4-DDB1-RBX1. By binding CRBN, IMiDs promote ubiquitination and proteasomal degradation of important MM cell transcription factors, IKZF1 (Ikaros) and IKZF3 (Aiolos). Degradation of such neosubstrates has also been shown to augment an anti-cancer immune response by activation of cytotoxic effector cells against MM plasma cells. Finally, it has been demonstrated that IMiDs modulate events occurring within the bone marrow microenvironment with negative impact on the MM-sustaining niche. Despite these recent advances in the characterisation of IMiD mechanism of action, the precise molecular events underpinning the anti-MM activity of IMiDs remain incompletely understood. Further insight is required to improve clinical responses to IMiDs and overcome therapy resistance in MM. In this work, genome-wide approaches such as RNA-sequencing and CRISPR-based functional genomics screening were employed to dissect the molecular mechanisms of IMiD-sensitivity and -resistance in models of acquired resistance to lenalidomide. RNA-sequencing revealed a strong interferon-like transcriptional response and increased transcription of genes downstream the MAPK pathway, together with downregulation of MYC-responsive genes, following IMiD treatment of IMiD-sensitive MM cells. Conversely, IMiD-resistant cells displayed reduced transcriptional changes following IMiD exposure as a likely consequence of CRBN downregulation and subsequent attenuation of neosubstrate degradation. Genome-wide CRISPR screening demonstrated that loss of CRBN and other genes regulating protein degradation, such as subunits of the COP9 signalosome, but also loss of genes that are not implicated in protein turnover, such as NCOR1 and EDC4, is sufficient to cause resistance to IMiDs in IMiD-sensitive cells. CRIPSR screening also demonstrated that resensitisation of IMiD-resistant cells to IMiDs can occur through loss of ATXN7, TOP2B and other genes implicated in several distinct biological processes, such as glucose metabolism, cell cycle progression and RNA processing. Dexrazoxane, a pan-TOP2 inhibitor and TOP2B-selective degrader, was discovered to possess anti-proliferative properties and combinatorial activity with lenalidomide in certain MM cell lines. To develop a deeper understanding of the biology of CRBN and IMiDs, proximity labelling with BioID2 was employed to characterise the interactome of CRBN in the presence and absence of an IMiD. Co-treatment of BioID2-CRBN-expressing OPM2 cells with IMiDs and/or the proteasome inhibitor bortezomib, followed by liquid chromatography mass spectrometry proteomics, allowed identification of known CRBN interactors (COP9 signalosome subunits) and neosubstrates (IKZF1, IKZF3 and CK1alpha). This approach enabled further characterisation of CRBN-associated proteome and identification of new CRBN and CRBN-IMiD interacting partners, such as MEF2C, DVL1, DVL2, MYH9, PRRC2C and EDC4. This data led to the hypothesis that CRBN may be implicated in several novel biological processes, such as RNA processing and protein translation regulation. Additionally, some of the newly identified interacting partners may modulate the MM niche in the bone marrow and may help understand the occurrence of side effects to IMiD therapy. This work has further defined transcriptional signatures of IMiD treatment and of acquired IMiD-resistance. TOP2B was found to be a potential druggable vulnerability of MM cells with acquired IMiD-resistance. The unbiased study of the CRBN-interacting proteome has helped broaden the knowledge on its biological activities both in presence and absence of an IMiD. Collectively, this thesis provides new and important insight into IMiD mechanism of action and further expands knowledge of IMiD biology.