miRNA Expression in Multiple Myeloma as Predictive Model of Response to Bortezomib.

Bortezomib therapeutic efficacy is well established in multiple myeloma (MM) however response to this therapy remains difficult to predict with resistant disease observed in nearly 20% of MM patients. Through DNA microarrays, predictive models of response to stem cell transplant and Bortezomib were reported correlating mRNA expression data with disease outcomes and response to therapy. MicroRNAs (miRNAs) are a key class of small, non-coding RNA molecules that modulate post-transcriptional regulation of gene expression and were recently described to be involved in deregulation of gene expression in many cancers including MM. Little evidence however is available concerning the role of miRNA expression in the prediction of response to Bortezomib in MM. We aimed to assess the expression of miRNAs in a panel of Bortezomib highly sensitive and relatively resistant MM cell lines as well as primary MM cells and identify miRNA expression patterns that are associated with response to Bortezomib.

We have used miRNA microarrays (Affymetrix miRNA GeneChip) as well as liquid phase Luminex microbead miRNA profiling (Flexmir, Luminex) to profile miRNA expression in MM cell lines (MM1S, KMS11, INA6, U266) and sorted CD138+ bone marrow PCs from MM patients prior to treatment with Bortezomib (n=5; 3 sensitive and 2 resistant) and PCs from a healthy normal donor (n=1). The MM cell lines included in this analysis were classified as sensitive (S) or resistant (R) based on their Bortezomib IC50 at 48 hours (IC50 for MM1S and KMS11 ∼ 5 nM versus INA6 and U266 ∼ 20nM). For the microarray studies the hybridization signal values for the multiple probes for each miRNA were obtained and normalized with the use of miRNA QC tool (Affymetrix) and analyzed using Partek Genomics Suite software. Thereafter, filters were applied to identify the miRNA probes whose normalized signal were at least 2 folds differentially expressed between sensitive (MM1S) and resistant (INA6) cell lines with a P value < 0.05 (ANOVA) and a FDR of 0.05. Bortezomib sensitive (n=3) and resistant (n=2) primary MM samples were subjected to the same miRNA array analysis and filtering. Liquid phase Luminex microbead miRNA profiling (FlexmiR) was used for the confirmation (MM1S and INA6) and validation of the array results in other MM cell lines KMS11 (IC50 5nM) and U266 (IC50 20nM).

Using Affymetrix miRNA GeneChip we identified 22 differentially expressed miRNA with overexpression of miR-155, miR-342-3p, miR-181a and b, miR-128, miR-20b and downregulation miR-let-7b, miR-let-7i, miR-let-7d, miR-let-7c, miR-222, miR-221, miR-23a, miR-27a and miR-29a in bortezomib relatively resistant (INA6) versus sensitive (MM1S) cell line. These results were confirmed in INA6 and MM1S cells with the use of Luminex microbead miRNA profiling and validated to be similarly differentially expressed between KMS11 (sensitive) and relatively U266 (resistant) cell lines. Furthermore, TargetScan algorithms and Ingenuity Pathway Analysis software were used to identify predicted miRNAs-targeted mRNAs or potentially regulated networks and included genes involved in cell cycle regulation, cell growth, apoptosis and ubiquitin-conjugation pathways. Lastly to further investigate the clinical relevance of miRNAs in MM in terms of prediction of response and outcome to Bortezomib, we correlated miRNA expression profile of sorted CD138+ bone marrow PCs from Bortezomib sensitive (n=3) and resistant (n=2) MM patients with their response to therapy. Unsupervised analysis of the data revealed that the Bortezomib sensitive MM patients clustered with MM1S cell line while resistant patients segregated into the INA6 cluster.

In summary, we have described a MM miRNA signature, which includes miRNAs that modulate the expression of proteins critical to myeloma pathogenesis and is predictive of response to Bortezomib. Further validation of this miRNA signature in a larger cohort of Bortezomib-treated MM patients is ongoing.

Stewart:Glaxo-Smith-Kline: Research Funding.