DIS3 depletion in multiple myeloma causes extensive perturbation in cell cycle progression and centrosome amplification.

DIS3 gene mutations occur in approximately 10% of patients with multiple myeloma (MM); furthermore, DIS3 expression can be affected by monosomy 13 and del(13q), found in roughly 40% of MM cases. Despite the high incidence of DIS3 mutations and deletions, the biological significance of DIS3 and its contribution to MM pathogenesis remain poorly understood. In this study we investigated the functional role of DIS3 in MM, by exploiting a loss-of-function approach in human MM cell lines. We found that DIS3 knockdown inhibits proliferation in MM cell lines and largely affects cell cycle progression of MM plasma cells, ultimately inducing a significant increase in the percentage of cells in the G0/G1 phase and a decrease in the S and G2/M phases. DIS3 plays an important role not only in the control of the MM plasma cell cycle, but also in the centrosome duplication cycle, which are strictly co-regulated in physiological conditions in the G1 phase. Indeed, DIS3 silencing leads to the formation of supernumerary centrosomes accompanied by the assembly of multipolar spindles during mitosis. In MM, centrosome amplification is present in about a third of patients and may represent a mechanism leading to genomic instability. These findings strongly prompt further studies investigating the relevance of DIS3 in the centrosome duplication process. Indeed, a combination of DIS3 defects and deficient spindle-assembly checkpoint can allow cells to progress through the cell cycle without proper chromosome segregation, generating aneuploid cells which ultimately lead to the development of MM.

Activation of long non-coding RNA NEAT1 leads to survival advantage of multiple myeloma cells by supporting a positive regulatory loop with DNA repair proteins.

Long non-coding RNA NEAT1 is the core structural component of the nuclear paraspeckle (PS) organelles and it has been found to be deregulated in multiple myeloma (MM) patients. Experimental evidence indicated that NEAT1 silencing negatively impacts proliferation and viability of MM cells, both in vitro and in vivo, suggesting a role in DNA damage repair (DDR). In order to elucidate the biological and molecular relevance of NEAT1 upregulation in MM disease we exploited the CRISPR/Cas9 synergistic activation mediator genome editing system to engineer the AMO-1 MM cell line and generate two clones that para-physiologically transactivate NEAT1 at different levels. NEAT1 overexpression is associated with oncogenic and prosurvival advantages in MM cells exposed to nutrient starvation or a hypoxic microenvironment, which are stressful conditions often associated with more aggressive disease phases. Furthermore, we highlighted the NEAT1 involvement in virtually all DDR processes through, at least, two different mechanisms. On one side NEAT1 positively regulates the posttranslational stabilization of essential PS proteins, which are involved in almost all DDR systems, thus increasing their availability within cells. On the other hand, NEAT1 plays a crucial role as a major regulator of a molecular axis that includes ATM and the catalytic subunit of DNA-PK kinase proteins, and their direct targets pRPA32 and pCHK2. Overall, we provided novel important insightsthe role of NEAT1 in supporting MM cells adaptation to stressful conditions by improving the maintenance of DNA integrity. Taken together, our results suggest that NEAT1, and probably PS organelles, could represent a potential therapeutic target for MM treatment.

The pleiotropic nature of NONO, a master regulator of essential biological pathways in cancers.

NONO is a member of the Drosophila behavior/human splicing (DBHS) family of proteins. NONO is a multifunctional protein that acts as a "molecular scaffold" to carry out versatile biological activities in many aspects of gene regulation, cell proliferation, apoptosis, migration, DNA damage repair, and maintaining cellular circadian rhythm coupled to the cell cycle. Besides these physiological activities, emerging evidence strongly indicates that NONO-altered expression levels promote tumorigenesis. In addition, NONO can undergo various post-transcriptional or post-translational modifications, including alternative splicing, phosphorylation, methylation, and acetylation, whose impact on cancer remains largely to be elucidated. Overall, altered NONO expression and/or activities are a common feature in cancer. This review provides an integrated scenario of the current understanding of the molecular mechanisms and the biological processes affected by NONO in different tumor contexts, suggesting that a better elucidation of the pleiotropic functions of NONO in physiology and tumorigenesis will make it a potential therapeutic target in cancer. In this respect, due to the complex landscape of NONO activities and interactions, we highlight caveats that must be considered during experimental planning and data interpretation of NONO studies.

Inference of genomic lesions from single-cell RNAseq in myeloma improves functional intra- and inter-clonal analysis.

Smoldering Multiple Myeloma (SMM) is an asymptomatic plasma cell (PC) neoplasm that may evolve with variable frequency into multiple myeloma (MM). SMM is initiated by chromosomal translocations involving the IgH locus or by hyperdiploidy and evolves through acquisition of additional genetic lesions. In this scenario, we aimed at establishing a reliable analysis pipeline to infer genomic lesions from transcriptomic analysis, by combining single-cell RNA sequencing (scRNA-seq) with B-cell receptor sequencing and copy- number abnormality (CNA) analysis to identify clonal PCs at the genetic level along their specific transcriptional landscape. We profiled 20,465 bone marrow (BM) PCs derived from five SMM/MM patients and unbiasedly identified clonal and polyclonal plasma cells. Hyperdiploidy, t(11;14) and t(6;14) were identified at the scRNA level by analysis of chimeric reads. Subclone functional analysis was improved by combining transcriptome with CNA analysis. As examples, we illustrate the different functional properties of a light chain escape subclone in SMM, and of different B-cell and PC subclones in a patient affected by Wäldenstrom Macroglobulinemia and SMM. Overall, our data provide a proof of principle for inference of clinically relevant genotypic data from scRNAseq, which in turn will refine functional annotation of the clonal architecture of PC dyscrasias.

Expression levels of NONO, a nuclear protein primarily involved in paraspeckles function, are associated with several deregulated molecular pathways and poor clinical outcome in multiple myeloma.

PURPOSE: The NONO protein belongs to the multifunctional family of proteins that can bind DNA, RNA and proteins. It is located in the nucleus of most mammalian cells and can affect almost every step of gene regulation. Dysregulation of NONO has been found in many types of cancer; however, data regarding its expression and relevance in Multiple Myeloma (MM) are virtually absent. METHODS: We took advantage of a large cohort of MM patients enrolled in the Multiple Myeloma Research Foundation CoMMpass study to elucidate better the clinical and biological relevance of NONO expression in the context of the MM genomic landscape and transcriptome. RESULTS: NONO is overexpressed in pathological samples compared to normal controls. In addition, higher NONO expression levels are significant independent prognostic markers of worse clinical outcome in MM. Our results indicate that NONO deregulation may play a pathogenetic role in MM by affecting cell cycle, DNA repair mechanisms, and influencing translation by regulating ribosome biogenesis and assembly. Furthermore, our data suggest NONO involvement in the metabolic reprogramming of glucose metabolism from respiration to aerobic glycolysis, a phenomenon known as the 'Warburg Effect' that supports rapid cancer cell growth, survival, and invasion. CONCLUSION: These findings strongly support the need of future investigations for the understanding of the mechanisms of deregulation and the biological role and activity of NONO in MM.

Circulating and progenitor endothelial cells are abnormal in patients with different types of von Willebrand disease and correlate with markers of angiogenesis.

von Willebrand disease (VWD) is the most common inherited bleeding disorder and is caused by quantitative or qualitative defects of von Willebrand factor (VWF). VWF, synthesized by endothelium and megakaryocytes (MK), circulates in plasma and is present in subendothelium and platelets. Circulating endothelial cells (CEC) and progenitor endothelial cells (EPC) have been recently proposed as markers of peripheral and bone marrow-derived angiogenesis. To evaluate the association of CEC/EPC with known inherited defects of cellular and circulating VWF, we have measured the number of CEC/EPC together with cytokines involved in angiogenesis in different VWD types. A group of 74 patients was composed by the following VWD types: VWD1 (n = 22), VWD2A (n = 9), VWD2B (n = 19), VWD2M (n = 17), and VWD3 (n = 7). Healthy individuals (n = 20) were used as controls. CEC (CD146(+) , CD31(+) , and CD45(-) ) and EPC (CD34(+) , CD133(+) , and CD45(-) ) were evaluated by flow cytometry. Circulating serum levels of VEGF, E-selectin, P-selectin, EPO, and TPO were determined by ELISA. CEC, VEGF, E-selectin, and EPO were higher and EPC lower in VWD patients than in controls (P < 0.01). Among the five groups of VWD patients and controls, a significant difference was found for CEC (one-way ANOVA: P = 0.005), EPC (P = 0.001), E-Selectin (P < 0.0001), EPO (P = 0.021), and TPO (P = 0.004): the latter was high in VWD3 patients. In VWD1, we found an inverse relationship between CEC and VWF:Ag levels (P = 0.048; R(2) = 0.19). Based on these data, CEC are increased in VWD and are associated with the high levels of cytokines involved in angiogenesis (up-regulation). EPC are decreased, suggesting down-regulation of bone marrow-derived angiogenesis in VWD.

Dissecting the Biological Relevance and Clinical Impact of lncRNA MIAT in Multiple Myeloma.

The biological impact of long non-coding RNAs (lncRNAs) in multiple myeloma (MM) is becoming an essential aspect of the investigation, which may contribute to understanding the disease's complex pathobiology, providing novel potential therapeutic targets. Herein, we investigated the expression pattern and the clinical relevance of the lncRNA MIAT in MM, taking advantage of the publicly available CoMMpass database. MIAT expression in MM is highly heterogeneous and significantly associated with specific molecular lesions frequently occurring in MM. Transcriptome analyses of MM PCs from patients included in the CoMMpass database indicated a potential involvement of MIAT in different signaling pathways and ribosome biogenesis and assembly. These findings suggest that MIAT deregulation may play a pathogenetic role in MM by affecting both proliferation pathways and, indirectly, the translational process. Although MIAT expression levels seem not to be significantly associated with clinical outcome in multivariate analyses, high MIAT expression levels are associated with bortezomib resistance, this suggesting that MIAT targeting could overcome drug resistance in MM. These findings strongly prompt for further studies investigating the significance of MIAT in MM.

Transcriptomic Analysis in Multiple Myeloma and Primary Plasma Cell Leukemia with t(11;14) Reveals Different Expression Patterns with Biological Implications in Venetoclax Sensitivity.

Mechanisms underlying the pathophysiology of primary Plasma Cell Leukemia (pPCL) and intramedullary multiple myeloma (MM) need to be further elucidated, being potentially relevant for improving therapeutic approaches. In such a context, the MM and pPCL subgroups characterized by t(11;14) deserve a focused investigation, as the presence of the translocation is mainly associated with sensitivity to venetoclax. Herein, we investigated a proprietary cohort of MM and pPCL patients, focusing on the transcriptional signature of samples carrying t(11;14), whose incidence increases in pPCL in association with an unfavorable outcome. In addition, we evaluated the expression levels of the BCL2-gene family members and of a panel of B-cell genes recently reported to be associated with sensitivity to venetoclax in MM. Moreover, transcriptional analysis of lncRNAs in the two clinical settings led to the identification of several differentially expressed transcripts, among which the SNGH6 deregulated lncRNA might be relevant in the pathogenesis and prognosis of pPCL with t(11;14). Overall, our data suggest that MMs and pPCLs with t(11;14) might be responsive to venetoclax based on different molecular programs, prompting further studies to elucidate better novel potential predictive biomarkers.

NEAT1 Long Isoform Is Highly Expressed in Chronic Lymphocytic Leukemia Irrespectively of Cytogenetic Groups or Clinical Outcome.

The biological role and therapeutic potential of long non-coding RNAs (lncRNAs) in chronic lymphocytic leukemia (CLL) are still open questions. Herein, we investigated the significance of the lncRNA NEAT1 in CLL. We examined NEAT1 expression in 310 newly diagnosed Binet A patients, in normal CD19+ B-cells, and other types of B-cell malignancies. Although global NEAT1 expression level was not statistically different in CLL cells compared to normal B cells, the median ratio of NEAT1_2 long isoform and global NEAT1 expression in CLL samples was significantly higher than in other groups. NEAT1_2 was more expressed in patients carrying mutated IGHV genes. Concerning cytogenetic aberrations, NEAT1_2 expression in CLL with trisomy 12 was lower with respect to patients without alterations. Although global NEAT1 expression appeared not to be associated with clinical outcome, patients with the lowest NEAT1_2 expression displayed the shortest time to first treatment; however, a multivariate regression analysis showed that the NEAT1_2 risk model was not independent from other known prognostic factors, particularly the IGHV mutational status. Overall, our data prompt future studies to investigate whether the increased amount of the long NEAT1_2 isoform detected in CLL cells may have a specific role in the pathology of the disease.

Long non-coding RNA NEAT1 targeting impairs the DNA repair machinery and triggers anti-tumor activity in multiple myeloma.

The biological role and therapeutic potential of long non-coding RNAs (lncRNAs) in multiple myeloma (MM) are still open questions. Herein, we investigated the functional significance of the oncogenic lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) in MM. Our study demonstrates that NEAT1 expression level is higher in MM than in the majority of hematological malignancies. NEAT1 silencing by novel LNA-gapmeR antisense oligonucleotide inhibits MM cell proliferation and triggers apoptosis in vitro and in vivo murine MM model as well. By transcriptome analyses, we found that NEAT1 targeting downregulates genes involved in DNA repair processes including the Homologous Recombination pathway, which in turn results in massive DNA damage. These findings may explain the synergistic impact on apoptosis observed in MM cell lines co-treated with inhibitors of both NEAT1 and PARP. The translational significance of NEAT1 targeting is further underlined by its synergistic effects with the most common drugs administered for MM treatment, including bortezomib, carfilzomib, and melphalan. Overall, NEAT1 silencing is associated with a chemo-sensitizing effect of both conventional and novel therapies, and its targeting could therefore represent a promising strategy for novel anti-MM therapeutic options.

Bone marrow glycophorin-positive erythroid cells of myelodysplastic patients responding to high-dose rHuEPO therapy have a different gene expression pattern from those of nonresponders.

The main clinical problems of low-risk patients with myelodysplastic syndromes (MDS), as defined by the International Prognostic Scoring System, are infections and the need for frequent transfusions due to ineffective myelopoiesis and peripheral blood cytopenia. Promising results in treating MDS-related anemia have been obtained using high-dose recombinant human erythropoietin (rhEPO). To evaluate the molecular basis of the response to rhEPO, we used commercially available macro-arrays to investigate gene expression profiles in the glycophorin-expressing (Gly+) bone marrow (BM) erythroid cells of five responders (ERs) and five non-responders (ENRs) to rhEPO treatment. The cells were separated by means of positive selection using an immunomagnetic procedure, after which flow cytometry showed that their purity was more than 97% in all cases. The array data were validated by means of real time RT-PCR. The results showed that the genes responsible for proliferation/differentiation and DNA repair/stability were repressed in the BM Gly+ erythroid cells of the ENRs, but almost normally expressed in the ERs. Furthermore, the expression of genes involved in signal transduction suggested that the activity of the MAPK signaling pathway is inhibited in ERs. The different gene expression profiles of ERs and ENRs may provide a basis for early gene testing as a means of predicting the response to rhEPO of MDS patients with low endogenous EPO levels.

Effects of simulated altitude (normobaric hypoxia) on cardiorespiratory parameters and circulating endothelial precursors in healthy subjects.

BACKGROUND: Circulating Endothelial Precursors (PB-EPCs) are involved in the maintenance of the endothelial compartment being promptly mobilized after injuries of the vascular endothelium, but the effects of a brief normobaric hypoxia on PB-EPCs in healthy subjects are scarcely studied. METHODS: Clinical and molecular parameters were investigated in healthy subjects (n = 8) in basal conditions (T0) and after 1 h of normobaric hypoxia (T1), with Inspiratory Fraction of Oxygen set at 11.2% simulating 4850 mt of altitude. Blood samples were obtained at T0 and T1, as well as 7 days after hypoxia (T2). RESULTS: In all studied subjects we observed a prompt and significant increase in PB-EPCs, with a return to basal value at T2. The induction of hypoxia was confirmed by Alveolar Oxygen Partial Pressure (PAO2) and Spot Oxygen Saturation decreases. Heart rate increased, but arterial pressure and respiratory response were unaffected. The change in PB-EPCs percent from T0 to T1 was inversely related to PAO2 at T1. Rapid (T1) increases in serum levels of hepatocyte growth factor and erythropoietin, as well as in cellular PB-EPCs-expression of Hypoxia Inducible Factor-1alpha were observed. CONCLUSION: In conclusion, the endothelial compartment seems quite responsive to standardized brief hypoxia, possibly important for PB-EPCs activation and recruitment.

Endothelial colony forming capacity is related to C-reactive protein levels in healthy subjects.

The majority of clinical studies on endothelial progenitor cells (EPCs) focuses on the role of these cells in cardiovascular diseases and no systematic studies exist regarding their variations in healthy subjects. In order to define the burden of angiogenesis in physiological conditions we assessed the frequency of peripheral blood endothelial colonies (PB-ECs) and their relation with other factors possibly involved in their function such as high-sensitivity C-reactive protein (hs-CRP), endothelial cell-specific mitogen factor (VEGF) and tissue inhibitor of metalloproteinases-1 (TIMP-1) in a highly selected healthy population. A PB sample was obtained from 37/47 healthy subjects (age 40.2+/-15.0yrs; M/F 15/22) without known cardiovascular risk factors. The serum level of hs-CRP, VEGF, TIMP-1, the frequency of PB-ECs by clonogenic assay, and the number of early EPCs and late EPCs by flow cytometry analysis were evaluated. PB-ECs were formed by 40.5% of studied subjects with a mean of 0.40+/-0.82 colonies/10(6) cells. The differences in the frequency of colony formation between genders were not statistically significant. The subjects with PB-ECs were characterized by higher values of hs-CRP, when compared with those not forming colonies, 0.276+/-0.230 vs 0.095+/-0.077 mg/l (p=0.003) respectively, and of VEGF, 328.3+/-162.9 vs 202.68+/-118.53 pg/ml (p=0.02). No significant differences were found in TIMP-1 values. The EPC clonogenic potential seems to be related to hs-CRP and VEGF levels even in healthy population supporting the concept that these mediators are involved in physiological ECs function.

Simvastatin reduces endothelial activation and damage but is partially ineffective in inducing endothelial repair in systemic sclerosis.

OBJECTIVE: To investigate whether statins may improve endothelial function in systemic sclerosis (SSc) by evaluating the effects of simvastatin on vasculogenesis [indicated by the expansion of circulating endothelial progenitor cells (EPC)] and the markers of vascular injury in the peripheral blood of patients with SSc. METHODS: Twenty SSc patients with normal cholesterol concentrations and 20 hypercholesterolemic subjects were allocated to receive 20 mg/day simvastatin for 12 weeks. Peripheral blood samples were collected before and 12 weeks after initiation of treatment, and 4 weeks after discontinuation. Five-parameter, 3-color flow cytometry was performed with a FacScan to enumerate EPC and mature circulating endothelial cells (CEC). Levels of soluble E-selectin, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, interleukin 6, and endothelin-1 were assessed by commercial ELISA. RESULTS: Simvastatin treatment significantly increased EPC in the hypercholesterolemic group, but failed to improve the EPC levels in the SSc patients, mainly in patients with late disease. Baseline levels of CEC were significantly higher in SSc patients compared with controls and at the end of the treatment they were significantly decreased. Regarding other markers of endothelial activation, we found that all the cytokine levels decreased in a statistically significant manner in the treated patients. CONCLUSION: Treatment with simvastatin results in rapid and significant improvement of measures of endothelial activation, suggesting a potential role of statins in the treatment of peripheral vascular disease in SSc. The lack of effect on increase of EPC confirms our previous findings of a defective endothelial stem cell recruitment in the bone marrow of SSc patients. This could indicate that the potential effectiveness of statins in SSc could mainly be ascribed to their effectiveness in modulating endothelial activation mechanisms.