Toll-like receptor signaling in multiple myeloma cells promotes the expression of pro-survival genes B-cell lymphoma 2 and MYC and modulates the expression of B-cell maturation antigen.

Infections are common in plasma cell cancer multiple myeloma (MM) due to disease-related immune deficiencies and cancer treatment. Myeloma cells express Toll-like receptors (TLRs), and TLR activation has been shown to induce proliferative and pro-survival signals in cancer cells. MM is a complex and heterogeneous disease, and expression levels of TLRs as well as downstream signaling components are likely to differ between patients. Here, we show that in a large cohort of patients, TLR1, TLR4, TLR6, TLR9, and TLR10 are the most highly expressed in primary CD138+ cells. Using an MM cell line expressing TLR4 and TLR9 as a model, we demonstrate that TLR4 and TLR9 activation promoted the expression of well-established pro-survival and oncogenes in MM such as MYC, IRF4, NFKB, and BCL2. TLR4 and TLR9 activation inhibited the efficacy of proteasome inhibitors bortezomib and carfilzomib, drugs used in the treatment of MM. Inhibiting the autophagosome-lysosome protein degradation pathway by hydroxychloroquine (HCQ) diminished the protective effect of TLR activation on proteasome inhibitor-induced cytotoxicity. We also found that TLR signaling downregulated the expression of TNFRSF17, the gene encoding for B-cell maturation antigen (BCMA). MYC, BCL2, and BCL2L1 were upregulated in approximately 50% of primary cells, while the response to TLR signaling in terms of TNFRSF17 expression was dichotomous, as an equal fraction of patients showed upregulation and downregulation of the gene. While proteasome inhibitors are part of first-line MM treatment, several of the new anti-MM immune therapeutic drugs target BCMA. Thus, TLR activation may render MM cells less responsive to commonly used anti-myeloma drugs.

Diet-induced obesity reduces bone marrow T and B cells and promotes tumor progression in a transplantable Vk*MYC model of multiple myeloma.

Obesity is associated with an increased risk of developing multiple myeloma (MM). The molecular mechanisms causing this association is complex and incompletely understood. Whether obesity affects bone marrow immune cell composition in multiple myeloma is not characterized. Here, we examined the effect of diet-induced obesity on bone marrow immune cell composition and tumor growth in a Vk*MYC (Vk12653) transplant model of multiple myeloma. We find that diet-induced obesity promoted tumor growth in the bone marrow and spleen and reduced the relative number of T and B cells in the bone marrow. Our results suggest that obesity may reduce MM immune surveillance and thus may contribute to increased risk of developing MM.

IL-32 is induced by activation of toll-like receptors in multiple myeloma cells.

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.

Comprehensive small RNA-sequencing of primary myeloma cells identifies miR-105-5p as a predictor of patient survival.

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.

Paired miRNA- and messenger RNA-sequencing identifies novel miRNA-mRNA interactions in multiple myeloma.

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.