Identification of new therapeutic targets and drug repositioning in myelodysplastic syndromes using transcriptional profiling and connectivity mapping
Keywords: drug repurposing
connectivity map
myelodysplastic syndromes

The myelodysplastic syndromes (MDS) are a heterogeneous group of clonal haematopoietic stem cell (HSC) malignancies that are characterised by ineffective haematopoiesis leading to peripheral blood cytopenias at one or more lineages. There are few effective treatments for this disorder, and the identification of new therapeutic agents in MDS is urgently needed. Drug repurposing is a potential alternative to novel drug discovery by redirecting existing drugs for treating another disease, which dramatically reduces drug development time, cost, and safety risk. In the last few years, gene expression signatures have been identified and compared based on genome-wide similarity metrics, unveiling ‘connections’ among drugs, pathways and disease phenotype by matching corresponding signatures of disease and drug responses. Some bioinformatic database and tools, such as connectivity map (CMap), containing transcriptomics of cell lines treated with small molecules were developed, greatly facilitating the discovery of new therapeutic applications of existing compounds.
In this study, we performed CMap on lists of differentially expressed genes (DEG) obtained from the analysis of RNA-seq or microarray-based gene expression profiling (GEP) data generated from bone marrow CD34+ cells of MDS patients with splicing factor mutations or with advanced MDS (Refractory anaemia with excess blasts-2 subtype, RAEB-2 subtype). Compounds inducing a transcriptional response opposite to that of MDS cases were identified, which might be capable of reverting the gene expression signature, and hence may change the disease phenotype. The compounds were ranked by the absolute value of connectivity scores and were primarily screened by comprehensive literature review based on their Mode of Action (MoA) and toxicity profiles in the context of leukaemia, cancer and other diseases. Several top-ranking candidate drugs for repositioning to MDS were selected for in vitro experimental validation in leukaemia cell lines, patient sample derived induced pluripotent stem cells (iPSCs), and patient primary bone marrow cells. Celastrol and Withaferin A were identified from the CMap analysis of RNA-seq data set of splicing factor mutant MDS patients, and Parbendazole and Fenbendazole were identified by mapping a microarray-based MDS-RAEB-2 gene signature to the CMap database. These drugs markedly inhibited proliferation, arrested cell cycle, and induced apoptosis of leukaemia cells. Notably, our study provided evidence of the involvement of splicing pathway modulation in the anti-proliferative activity of these drugs in MDS and leukaemia cells. Higher cytotoxicity induced by Celastrol, Withaferin A, Parbendazole, and Fenbendazole treatment was observed in splicing factor mutant cells, including HUDEP2-U2AF1Q157 cells, K562-SF3B1K700E cells and K562-U2AF1S34F cells. Taken together, our data show that these compounds are promising candidates for repurposing to MDS and other related myeloid malignancies.