Identification Of Novel Alternative Splice Variants Of Sirtuins In Multiple Myeloma: Therapeutic Implications

Alternative splicing (AS) is a normal epigenetic event with a critical role in the regulation of gene expression. Previous studies showed increased AS in Multiple Myeloma (MM) cells, suggesting the need to assess both expression level of genes and post translational modifications, mediating overall gene function. The NAD-dependent deacetylases Sirtuins (SIRTs), mammalian homologues of the yeast Sir2, modulate various biological processes including metabolism, cell survival, development, chromatin dynamics, or DNA repair. Recent microarray profiling data using newly diagnosed patients with MM, suggests clinical relevance of such deacetylases since their level predicts for both progression free and overall survival. Among SIRTs family membersSIRT-5, SIRT-6 and SIRT-7 transcript levels positively correlated with disease progression (from MGUS to active MM). These studies provide the rationale for further examining the biological processes including epigenetic changes, mutations, or AS events that contribute to aberrant expression of SIRTs in MM.

Purified RNA from MM cell lines, newly diagnosed MM patient cells, as well as peripheral blood mononuclear cells (PBMCs) from normal healthy donors was subjected to SIRT expression analysis. Specifically, SIRTs-specific primers were developed and optimized using standard RT-PCR conditions. RNA integrity was confirmed using Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an endogenous control. Aberrant splicing of SIRT-5, SIRT-6, and SIRT-7 was confirmed by cloning and sequencing, followed by analysis of their expression patterns in MM patients versus normal PBMCs.

We found that SIRTs genes are frequently miss-spliced in MM patients. To our knowledge, this is the first report showing SIRTs miss-splicing event in MM. Through cloning and sequencing analysis, we identified novel spliced isoforms of SIRT-5, SIRT-6 and SIRT-7; these occurred as a result of aberrant AS within exon-12, exon-4 and and exon-5, respectively. Specifically, exon skipping was noted in SIRT-6 and SIRT-7 variants, via cryptic 5 prime or 3 prime splice sites on exon 12 and/or through partial retention of an intron created SIRT-5 variants. The novel spliced forms were widely expressed in MM cell lines and primary cells, without significance occurrence in normal PBMCs. Our preliminary data show that even though these novel isoforms exhibit reduced deacetylase activity versus full-length variants, this characteristic may impart distinct functional outcome. Finally, in support of above studies, our analysis of MM patient samples suggest that AS among SIRTs is associated with poor clinical outcome in MM patients.

In the current study, we have identified novel transcript variants of SIRT-5, SIRT-6 and SIRT-7 in MM cells. These aberrant isoforms allow for generating transcripts that encode for dysfunctional proteins, which in turn, may contribute to the genetic heterogeneity in MM. Ongoing studies are delineating the function of these newly identified splice variants of SIRTs and their association with MM progression. Overall, our studies will provide basis for utilizing SIRTs variants as prognostic markers and/or as novel therapeutic targets in MM.

Hideshima:Acetylon Pharmaceuticals: Consultancy. Chauhan:Vivolux: Consultancy.