Summary Interleukin-32 (IL-32) is a nonclassical cytokine expressed in cancers, inflammatory diseases, and infections. Its expression is regulated by two different oxygen sensing systems; HIF1α and cysteamine dioxygenase (ADO), indicating that IL-32 may be involved in the response to hypoxia. We here demonstrate that endogenously expressed, intracellular IL-32 interacts with components of the mitochondrial respiratory chain and promotes oxidative phosphorylation. Knocking out IL-32 in three myeloma cell lines reduced cell survival and proliferation in vitro and in vivo . High-throughput transcriptomic and MS-metabolomic profiling of IL-32 KO cells revealed that cells depleted of IL-32 had perturbations in metabolic pathways, with accumulation of lipids, pyruvate precursors, and citrate. IL-32 was expressed in a subgroup of myeloma patients with inferior survival, and primary myeloma cells expressing IL-32 had a gene signature associated with immaturity, proliferation, and oxidative phosphorylation. In conclusion, we demonstrate a previously unrecognized role of IL-32 in the regulation of plasma cell metabolism.

Multiple myeloma (MM) is an incurable cancer of terminally differentiated plasma cells that proliferate in the bone marrow. miRNAs are promising biomarkers for risk stratification in MM and several miRNAs are shown to have a function in disease pathogenesis. However, to date, surprisingly few miRNA-mRNA interactions have been described for and functionally validated in MM. In this study, we performed miRNA-seq and mRNA-seq on CD138 + cells isolated from bone marrow aspirates of 86 MM patients to identify novel interactions between sRNAs and mRNAs. We detected 9.8% significantly correlated miRNA-mRNA pairs of which 5.17% were positively correlated and 4.65% were negatively correlated. We found that miRNA-mRNA pairs that were predicted by in silico target-prediction algorithms were more negatively correlated than non-target pairs, indicating functional miRNA targeting and that correlation between miRNAs and mRNAs from patients can be used to identify miRNA-targets. mRNAs for negatively correlated miRNA-mRNA target pairs were associated with gene ontology terms such as autophagy, protein degradation and endoplasmic stress response, reflecting important processes in MM. Targets for two specific miRNAs, miR-125b-5p and miR-365b-3p, were functionally validated in MM cell line transfection experiments followed by RNA-sequencing and qPCR. In summary, we identified functional miRNA-mRNA target pairs by correlating miRNA and mRNA data from primary MM cells. We identified several target pairs that are of potential interest for further studies. The data presented here may serve as a hypothesis-generating knowledge base for other researchers in the miRNA/MM field. We also provide an interactive web application that can be used to exploit the miRNA-target interactions as well as clinical parameters associated to these target-pairs.

IL-32 is a non-classical cytokine expressed in cancers, inflammatory diseases and infections. IL-32 can have both extracellular and intracellular functions, and its receptor is not identified. We here demonstrate that endogenously expressed, intracellular IL-32 binds to components of the mitochondrial respiratory chain and promotes oxidative phosphorylation. Knocking out IL-32 in malignant plasma cells significantly reduced survival and proliferation in vitro and in vivo. High throughput transcriptomic and MS-metabolomic profiling of IL-32 KO cells revealed that loss of IL-32 leads to profound perturbations in metabolic pathways, with accumulation of lipids, pyruvate precursors and citrate, indicative of reduced mitochondrial function. IL-32 is expressed in a subgroup of multiple myeloma patients with an inferior prognosis. Primary myeloma cells expressing IL-32 were characterized by a plasma cell gene signature associated with immune activation, proliferation and oxidative phosphorylation. We propose a novel concept for regulation of metabolism by an intracellular cytokine and identify IL-32 as an endogenous growth and survival factor for malignant plasma cells. IL-32 is a potential prognostic biomarker and a treatment target in multiple myeloma.

Background Small RNAs (sRNAs), a heterogenous group of non-coding RNAs, are emerging as promising molecules for cancer patient risk stratification and as players in tumour pathogenesis. Here, we have studied microRNAs (miRNAs) and other sRNAs in relation to survival and disease severity in multiple myeloma. Methods We comprehensively characterised sRNA expression in multiple myeloma patients by performing sRNA-sequencing on myeloma cells isolated from bone marrow aspirates of 86 myeloma patients. The sRNA expression profiles were correlated with the patients’ clinical data to investigate associations with survival and disease subgroups, by using cox proportional hazards (coxph) -models and limma-voom, respectively. A publicly available sRNA dataset was used as external validation (n = 151). Results We show that multiple miRNAs are differentially expressed between ISS Stage I and III. Interestingly, we observed the downregulation of seven different U2 spliceosomal RNAs, a type of small nuclear RNAs in severe disease stages. Further, by a discovery-based approach, we identified miRNA miR-105-5p as a predictor of poor overall survival (OS) in multiple myeloma. Multivariate analysis showed that miR-105-5p predict OS independently of established disease markers. Conclusions Overexpression of miR-105-5p in myeloma cells correlates with reduced OS, potentially improving prognostic risk stratification in multiple myeloma.

Multiple myeloma (MM) is a hematological cancer characterized by accumulation of malignant plasma cells in the bone marrow. The patients are immune suppressed and suffer from recurrent and chronic infections. Interleukin-32 is a non-conventional, pro-inflammatory cytokine expressed in a subgroup of MM patients with a poor prognosis. IL-32 has also been shown to promote proliferation and survival of the cancer cells. Here we show that activation of toll-like receptors (TLRs) promotes expression of IL-32 in MM cells through NFκB activation. In patient-derived primary MM cells, IL-32 expression is positively associated with expression of TLRs. Furthermore, we found that several TLR genes are upregulated from diagnosis to relapse in individual patients, predominantly TLRs sensing bacterial components. Interestingly, upregulation of these TLRs coincides with an increase in IL-32. Taken together, these results support a role for IL-32 in microbial sensing in MM cells and suggest that infections can induce expression of this pro-tumorigenic cytokine in MM patients.