Quantification of cyclin D1 and D2 proteins in multiple myeloma identifies different expression patterns from those revealed by gene expression profiling.

Upregulation of a cyclin D gene determined by expression microarrays is an almost universal event in multiple myeloma (MM), but this finding has not been properly confirmed at the protein level. For this reason, we carried out a quantitative analysis of cyclin D proteins using a capillary electrophoresis nanoimmunoassay in newly diagnosed MM patients. Exclusive expression of cyclin D1 and D2 proteins was detected in 54 of 165 (33%) and 30 of 165 (18%) of the MM patients, respectively. Of note, cyclin D1 or D2 proteins were undetectable in 41% of the samples. High levels of cyclin D1 protein were strongly associated with the presence of t(11;14) or 11q gains. Cyclin D2 protein was detected in all the cases bearing t(14;16), but in only 24% of patients with t(4;14). The presence of cyclin D2 was associated with shorter overall survival (hazard ratio =2.14; P=0.017), although patients expressing cyclin D2 protein, but without 1q gains, had a favorable prognosis. In conclusion, although one of the cyclins D is overexpressed at the mRNA level in almost all MM patients, in approximately half of the patients this does not translate into detectable protein. This suggests that cyclins D could not play an oncogenic role in a proportion of patients with MM (clinicaltrials gov. identifier: NCT01916252).

Transcriptional signature of TP53 biallelic inactivation identifies a group of multiple myeloma patients without this genetic condition but with dismal outcome.

Biallelic inactivation of TP53 has been included in the definition of double-hit (DH) multiple myeloma (MM), which entails an ominous prognosis. However, this condition, or even the presence of high-risk cytogenetic abnormalities, cannot accurately capture the 15%-20% of the MM population with a median overall survival below 24 months. This prompted us to look for other MM patients who might have transcriptional characteristics similar to those with DH-TP53. In the present study, we analysed RNA-seq, whole-genome and whole-exome sequencing data from 660 newly diagnosed MM (NDMM) patients from the MMRF (Multiple Myeloma Research Foundation) CoMMpass study to characterize the transcriptional signature of TP53 double-hit (DH-TP53) MM. We found 78 genes that were exclusively deregulated in DH-TP53 patients. A score based on these genes identified a group of 50 patients who shared the same transcriptional profile (DH-TP53-like group) whose prognosis was particularly unfavourable [median overall survival (OS) < 2 years], despite not harbouring the biallelic inactivation of TP53. The prognostic value of the DH-TP53 score was externally validated using gene expression data from 850 NDMM patients analysed by microarrays. Furthermore, our DH-TP53 score refined the traditional prognostic stratification of MM patients according to the cytogenetic abnormalities and International Staging System (ISS).

RNA sequencing identifies novel regulated IRE1-dependent decay targets that affect multiple myeloma survival and proliferation.

BACKGROUND: IRE1 is an unfolded protein response (UPR) sensor with kinase and endonuclease activity. It plays a central role in the endoplasmic reticulum (ER) stress response through unconventional splicing of XBP1 mRNA and regulated IRE1-dependent decay (RIDD). Multiple myeloma (MM) cells are known to exhibit an elevated level of baseline ER stress due to immunoglobulin production, however RIDD activity has not been well studied in this disease. In this study, we aimed to investigate the potential of RNA-sequencing in the identification of novel RIDD targets in MM cells and to analyze the role of these targets in MM cells. METHODS: In vitro IRE1-cleavage assay was combined with RNA sequencing. The expression level of RIDD targets in MM cell lines was measured by real-time RT-PCR and Western blot. RESULTS: Bioinformatic analysis revealed hundreds of putative IRE1 substrates in the in vitro assay, 32 of which were chosen for further validation. Looking into the secondary structure of IRE1 substrates, we found that the consensus sequences of IRF4, PRDM1, IKZF1, KLF13, NOTCH1, ATR, DICER, RICTOR, CDK12, FAM168B, and CENPF mRNAs were accompanied by a stem-loop structure essential for IRE1-mediated cleavage. In fact, we show that mRNA and protein levels corresponding to these targets were attenuated in an IRE1-dependent manner by treatment with ER-stress-inducing agents. In addition, a synergistic effect between IMiDs and ER-stress inducers was found. CONCLUSION: This study, using RNA sequencing, shows that IRE1 RNase has a broad range of mRNA substrates in myeloma cells and demonstrates for the first time that IRE1 is a key regulator of several proteins of importance in MM survival and proliferation.

Genetic Abnormalities in Multiple Myeloma: Prognostic and Therapeutic Implications.

Some genetic abnormalities of multiple myeloma (MM) detected more than two decades ago remain major prognostic factors. In recent years, the introduction of cutting-edge genomic methodologies has enabled the extensive deciphering of genomic events in MM. Although none of the alterations newly discovered have significantly improved the stratification of the outcome of patients with MM, some of them, point mutations in particular, are promising targets for the development of personalized medicine. This review summarizes the main genetic abnormalities described in MM together with their prognostic impact, and the therapeutic approaches potentially aimed at abrogating the undesirable pathogenic effect of each alteration.