Résumé
Background:
Multiple myeloma (MM) is a clonal plasma cell malignancy that remains incurable, even in the age of novel agents. Also, certain subsets of patients have high‐risk features linked with dismal outcome. Therefore, the lack of effective therapeutic options remains an unmet medical need. In recent years, epigenetic and DNA repair pathway alterations were identified to play an important role in MM disease.
Aims:
Our aim is to identify novel targets linked with epigenetics and DNA repair pathways in high‐risk phenotypes of multiple myeloma patients using bio‐informatic tools. Subsequent we aim to validate these targets as a potential novel treatment strategy for high‐risk multiple myeloma patients.
Methods:
We analyzed the prognostic significance of different epigenetic and DNA repair target genes using publically available gene‐expression data of MM patients, including the CoMMpass study (MMRF). As such, we were able to withhold the protein arginine methyltransferase PRMT5 as playing a role in high‐risk disease and relapse. As such, PRMT5 was identified as an interesting prognostic and therapeutic target. Druggability was evaluated in OPM2, JJN3, AMO1 and XG7 human myeloma cell lines using the PRMT5‐inhibitor EPZ015938. Apoptosis and cell cycle assays were performed using AnnexinV/7AAD‐ and PI‐staining, respectively. Effects on gene transcription and protein expression were evaluated with RNA‐sequencing and western blot,respectively.
Results:
By associating gene expression with survival, we identified a signature of epigenetic and DNA repair genes that is linked to poor prognosis in the TT2/3 cohort and in the CoMMpass cohort. The protein arginine methyltransferase 5 (PRMT5) was identified as a potential novel target. Decreased progression‐free survival was seen in CoMMpass study patients with high PRMT5‐expression (112.7 vs 189.9 weeks, p = 0.003). PRMT5‐inhibition in MM cell lines resulted in decreased cell growth. Cell cycle analysis revealed a significant increase of JJN3 and OPM2 cells in G2/M phase, as well as a decrease in S‐phase in OPM2 cells. No major difference upon treatment in XG‐7 cells were observed besides an increase in subG1 cells. PRMT5 inhibition arrested AMO1 cells arrested in G0/G1. These effects correlated with increased AnnexinV‐positivity, p53 phosphorylation and the absence of a DNA damage response. XG‐7 cells appeared more sensitive compared to JJN3, AMO1 and OPM2 cells. Apoptosis was associated with cleavage of PARP, caspase 3 and 9 proteins. Analysis of differential gene expression upon treatment, using RNAseq, revealed an important role for PRMT5 in regulating alternative splicing, nonsense‐mediated decay, DNA repair and PI3K/mTOR‐signaling, irrespective of cell line type.
Summary/Conclusion:
We have identified that PRMT5 is involved in high‐risk disease and plays an important role in MM cell growth and survival. Analysis of the transcriptome after PRMT5 inhibition reveals the importance of alternative splicing and PI3K/mTOR signalling, providing evidence that these pathways are implicated in high‐risk MM disease. The role of PRMT5 in MM pathogenesis and treatment strategies thus warrants further investigation.