DISSECTING THE CLINICAL AND BIOLOGICAL RELEVANCE OF DIS3 GENE IN MULTIPLE MYELOMA

Multiple myeloma (MM) is a B cell malignancy characterized by abnormal proliferation of plasma cells (PCs) within the bone marrow. MM is characterized by a wide clinical spectrum ranging from the presumed asymptomatic pre-malignant condition called monoclonal gammopathy of undetermined significance (MGUS), to extra-medullary plasma cell leukemia (PCL). Notably, MM is characterized by a deep genomic instability involving ploidy, structural rearrangements and a high range of mutations involving both putative oncogenes and tumor suppressor genes that may explain its clinical heterogeneity. Half of MM tumors are hyperdiploid, associated with non-random trisomies of odd chromosomes and low prevalence of chromosomal translocations involving the immunoglobulin heavy chain locus (IGH) on chromosome 14q32, whereas the remaining tumors are non hyperdiploid and characterized by the prevalence of IGH translocations affecting several oncogenic loci. Such a large heterogeneous genomic pattern may represent the basis for the abnormal transcriptional expression profile associated with MM. Despite the remarkable improvements in treatment and patient care, MM remains an incurable disease. Over the past years, our and other research groups have provided valuable information on coding and non-coding transcripts aberrantly expressed in MM by expression profiling analyses based on microarray or RNAseq approaches. These studies have been of high relevance to better understand the biology of the disease, to identify of novel prognostic and predictive biomarkers and putative therapeutic targets. DIS3 (chr.13q22.1 localization) is a conserved exoribonuclease and catalytic subunit of the exosome, a protein complex involved in the 3' to 5' degradation and processing of different species of RNA. Recently, aberrant expression of DIS3 has been found to be implicated in a range of different cancers. Notably, DIS3 is recurrently mutated in multiple myeloma (MM) patients. Most of the identified mutations are predominantly missense variants localized in the ribonucleolytic domain (RNB), mainly abolishing the exoribonucleolytic activity and are often accompanied by loss of heterozygosity (LOH) or biallelic inactivation due to 13q14 deletion. Furthermore, it has been reported in the literature that the inactivity or incorrect activity of DIS3 is also associated with the regulation of non-coding transcripts, as well as long non-coding RNA (lncRNA). Long non-coding RNAs (lncRNAs) are a large class of non-coding RNAs involved in many physiological cellular and genomic processes as well as in carcinogenesis, cancer metastasis and invasion. The knowledge of the role of lncRNAs in MM is progressively expanding. Our group provided recent evidence based on microarray and RNA seq analyses of deregulated patterns of lncRNA expression in MM showing that they may be specifically associated with distinct molecular types of the malignancy. In a paper published during the first years of the project, it has been shown that MM patients carrying Dis3 gene mutations are associated with a distinct transcriptional signature characterized by many non-coding transcripts, mainly lncRNAs. Firstly, our interest focused on investigating the deregulation of transcripts (including coding and non-coding portion) associated with DIS3 mutations in MM by analyzing RNA-Seq transcriptional profiles in a proprietary publicly available dataset and CoMMpass study, including approximately 1000 MM patients at diagnosis. We demonstrated that DIS3 mutations clinical relevance strictly depended on del(13q) co-occurrence. We observed that the bi-allelic DIS3 lesions significantly affected PFS (Progression Free Survival) and overall survival (OS). As expected, DIS3 mutations affect MM transcriptome involving cellular processes and signaling pathways associated with RNA metabolism. We found the downregulation of gene sets related to oxidative phosphorylation, RNA or amino acid metabolism, translation, and mitotic spindle checkpoint. Overall, our comprehensive assessment of the clinical and transcriptional consequences of DIS3 mutations or its deletion in MM strongly indicates that they may play an important role in the mechanisms of transformation and progression of MM. For these reasons, during the last year of my PhD program I focalized the efforts on the functional investigation of DIS3 putative role as potential therapeutic target in MM. To this aim I took advantage of the experimental silencing strategy based on the use of LNA-gapmeR technology and the gymnotic delivery of the in-house designed DIS3-specific antisense oligonucleotide in multiple myeloma cell lines (HMCLs). The results obtained show that DIS3 silencing decreases cell growth, increases the percentage of apoptosis, reducing the oncogenic potential of MM cell lines. Indeed, interference with DIS3 expression reveals an important perturbation of the cell cycle distribution accompanied by mitotic defects. I investigated the possibility of translating these results in combination with an available drug currently in clinical trial, such as ARRY-520, an inhibitor that interferes with the correct organization of microtubules. I found that the effect of DIS3 KD sensitizes MM cell lines in combination with ARRY-520, leading to a mitotic catastrophe. These results suggest that DIS3 could be an important target for the future therapeutic approach in MM disease.