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.

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.

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.

The Potential of JAG Ligands as Therapeutic Targets and Predictive Biomarkers in Multiple Myeloma.

The NOTCH ligands JAG1 and JAG2 have been correlated in vitro with multiple myeloma (MM) cell proliferation, drug resistance, self-renewal and a pathological crosstalk with the tumor microenvironment resulting in angiogenesis and osteoclastogenesis. These findings suggest that a therapeutic approach targeting JAG ligands might be helpful for the care of MM patients and lead us to explore the role of JAG1 and JAG2 in a MM in vivo model and primary patient samples. JAG1 and JAG2 protein expression represents a common feature in MM cell lines; therefore, we assessed their function through JAG1/2 conditional silencing in a MM xenograft model. We observed that JAG1 and JAG2 showed potential as therapeutic targets in MM, as their silencing resulted in a reduction in the tumor burden. Moreover, JAG1 and JAG2 protein expression in MM patients was positively correlated with the presence of MM cells in patients' bone marrow biopsies. Finally, taking advantage of the Multiple Myeloma Research Foundation (MMRF) CoMMpass global dataset, we showed that JAG2 gene expression level was a predictive biomarker associated with patients' overall survival and progression-free survival, independently from other main molecular or clinical features. Overall, these results strengthened the rationale for the development of a JAG1/2-tailored approach and the use of JAG2 as a predictive biomarker in MM.

GPER1 Activation Exerts Anti-Tumor Activity in Multiple Myeloma.

G protein-coupled estrogen receptor 1 (GPER1) activation is emerging as a promising therapeutic strategy against several cancer types. While GPER targeting has been widely studied in the context of solid tumors, its effect on hematological malignancies remains to be fully understood. Here, we show that GPER1 mRNA is down-regulated in plasma cells from overt multiple myeloma (MM) and plasma cell leukemia patients as compared to normal donors or pre-malignant conditions (monoclonal gammopathy of undetermined significance and smoldering MM); moreover, lower GPER1 expression associates with worse overall survival of MM patients. Using the clinically applicable GPER1-selective agonist G-1, we demonstrate that the pharmacological activation of GPER1 triggered in vitro anti-MM activity through apoptosis induction, also overcoming the protective effects exerted by bone marrow stromal cells. Noteworthy, G-1 treatment reduced in vivo MM growth in two distinct xenograft models, even bearing bortezomib-resistant MM cells. Mechanistically, G-1 upregulated the miR-29b oncosuppressive network, blunting an established miR-29b-Sp1 feedback loop operative in MM cells. Overall, this study highlights the druggability of GPER1 in MM, providing the first preclinical framework for further development of GPER1 agonists to treat this malignancy.

Lysin (K)-specific demethylase 1 inhibition enhances proteasome inhibitor response and overcomes drug resistance in multiple myeloma.

BACKGROUND: Multiple myeloma (MM) is an incurable plasma cell malignancy, accounting for approximately 1% of all cancers. Despite recent advances in the treatment of MM, due to the introduction of proteasome inhibitors (PIs) such as bortezomib (BTZ) and carfilzomib (CFZ), relapses and disease progression remain common. Therefore, a major challenge is the development of novel therapeutic approaches to overcome drug resistance, improve patient outcomes, and broaden PIs applicability to other pathologies. METHODS: We performed genetic and drug screens to identify new synthetic lethal partners to PIs, and validated candidates in PI-sensitive and -resistant MM cells. We also tested best synthetic lethal interactions in other B-cell malignancies, such as mantle cell, Burkitt's and diffuse large B-cell lymphomas. We evaluated the toxicity of combination treatments in normal peripheral blood mononuclear cells (PBMCs) and bone marrow stromal cells (BMSCs). We confirmed the combo treatment' synergistic effects ex vivo in primary CD138+ cells from MM patients, and in different MM xenograft models. We exploited RNA-sequencing and Reverse-Phase Protein Arrays (RPPA) to investigate the molecular mechanisms of the synergy. RESULTS: We identified lysine (K)-specific demethylase 1 (LSD1) as a top candidate whose inhibition can synergize with CFZ treatment. LSD1 silencing enhanced CFZ sensitivity in both PI-resistant and -sensitive MM cells, resulting in increased tumor cell death. Several LSD1 inhibitors (SP2509, SP2577, and CC-90011) triggered synergistic cytotoxicity in combination with different PIs in MM and other B-cell neoplasms. CFZ/SP2509 treatment exhibited a favorable cytotoxicity profile toward PBMCs and BMSCs. We confirmed the clinical potential of LSD1-proteasome inhibition in primary CD138+ cells of MM patients, and in MM xenograft models, leading to the inhibition of tumor progression. DNA damage response (DDR) and proliferation machinery were the most affected pathways by CFZ/SP2509 combo treatment, responsible for the anti-tumoral effects. CONCLUSIONS: The present study preclinically demonstrated that LSD1 inhibition could provide a valuable strategy to enhance PI sensitivity and overcome drug resistance in MM patients and that this combination might be exploited for the treatment of other B-cell malignancies, thus extending the therapeutic impact of the project.

Euchromatic Histone Lysine Methyltransferase 2 Inhibition Enhances Carfilzomib Sensitivity and Overcomes Drug Resistance in Multiple Myeloma Cell Lines.

Proteasome inhibitors (PIs) are extensively used for the therapy of multiple myeloma. However, patients continuously relapse or are intrinsically resistant to this class of drugs. In addition, adverse toxic effects such as peripheral neuropathy and cardiotoxicity could arise. Here, to identify compounds that can increase the efficacy of PIs, we performed a functional screening using a library of small-molecule inhibitors covering key signaling pathways. Among the best synthetic lethal interactions, the euchromatic histone-lysine N-methyltransferase 2 (EHMT2) inhibitor UNC0642 displayed a cooperative effect with carfilzomib (CFZ) in numerous multiple myeloma (MM) cell lines, including drug-resistant models. In MM patients, EHMT2 expression correlated to worse overall and progression-free survival. Moreover, EHMT2 levels were significantly increased in bortezomib-resistant patients. We demonstrated that CFZ/UNC0642 combination exhibited a favorable cytotoxicity profile toward peripheral blood mononuclear cells and bone-marrow-derived stromal cells. To exclude off-target effects, we proved that UNC0642 treatment reduces EHMT2-related molecular markers and that an alternative EHMT2 inhibitor recapitulated the synergistic activity with CFZ. Finally, we showed that the combinatorial treatment significantly perturbs autophagy and the DNA damage repair pathways, suggesting a multi-layered mechanism of action. Overall, the present study demonstrates that EHMT2 inhibition could provide a valuable strategy to enhance PI sensitivity and overcome drug resistance in MM patients.

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.

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.

Therapeutic activation of G protein-coupled estrogen receptor 1 in Waldenström Macroglobulinemia.

Activating G protein-coupled estrogen receptor 1 (GPER1) is an attractive therapeutic strategy for treating a variety of human diseases including cancer. Here, we show that GPER1 is significantly upregulated in tumor cells from different cohorts of Waldenström Macroglobulinemia (WM) patients compared to normal B cells. Using the clinically applicable GPER1-selective small-molecule agonist G-1 (also named Tespria), we found that pharmacological activation of GPER1 leads to G2/M cell cycle arrest and apoptosis both in vitro and in vivo in animal models, even in the context of the protective bone marrow milieu. Activation of GPER1 triggered the TP53 pathway, which remains actionable during WM progression. Thus, this study identifies a novel therapeutic target in WM and paves the way for the clinical development of the GPER1 agonist G-1.

DIS3 mutations in multiple myeloma impact the transcriptional signature and clinical outcome.

DIS3 gene mutations occur in roughly 10% of patients with multiple myeloma (MM); furthermore, DIS3 expression can be affected by monosomy 13 and del(13q), which occur in approximately 40% of MM cases. Despite several reports on the prevalence of DIS3 mutations, their contribution to the pathobiology of MM remains largely unknown. We took advantage of the large public CoMMpass dataset to investigate the spectrum of DIS3 mutations in MM and its impact on the transcriptome and clinical outcome. We found that the clinical relevance of DIS3 mutations strictly depended on the co-occurrence of del(13q). In particular, bi-allelic DIS3 lesions significantly affected progression-free survival, independently of other predictors of poor clinical outcome, while mono-allelic events mostly affected overall survival. As expected, DIS3 mutations affect the MM transcriptome involving cellular processes and signaling pathways associated with RNA metabolism, and the deregulation of a large number of long non-coding RNA, among which we identified five distinct transcripts as independent predictors of poorer overall survival and nine of worse progression-free survival, with two (AC015982.2 and AL445228.3) predicting both unfavorable outcomes. These findings strongly prompt further studies investigating the relevance of these long non-coding RNA in MM.

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.

Mechanisms of Immune Evasion in Multiple Myeloma: Open Questions and Therapeutic Opportunities.

Multiple myeloma (MM) is the second most common hematologic malignancy, characterized by a multi-step evolutionary path, which starts with an early asymptomatic stage, defined as monoclonal gammopathy of undetermined significance (MGUS) evolving to overt disease in 1% of cases per year, often through an intermediate phase known as "smoldering" MM (sMM). Interestingly, while many genomic alterations (translocation, deletions, mutations) are usually found at early stages, they are not sufficient (alone) to determine disease evolution. The latter, indeed, relies on significant "epigenetic" alterations of different normal cell populations within the bone marrow (BM) niche, including the "evasion" from immune-system control. Additionally, MM cells could "educate" the BM immune microenvironment (BM-IM) towards a pro-inflammatory and immunosuppressive phenotype, which ultimately leads to disease evolution, drug resistance, and patients' worse outcome. Indeed, it is not a case that the most important drugs for the treatment of MM include immunomodulatory agents (thalidomide, lenalidomide, and pomalidomide) and monoclonal antibodies (daratumumab, isatuximab, and elotuzumab). On these bases, in this review, we describe the most recent advances in the comprehension of the role of the different cells composing the BM-IM, and we discuss the potential molecular targets, which could represent new opportunities to improve current treatment strategies for MM patients.

Bioinformatics Pipeline to Analyze lncRNA Arrays.

Despite the fact that next-generation sequencing approaches, in particular RNA sequencing, provide deep genome-wide expression data that allow both careful annotations/mapping of long noncoding RNA (lncRNA) molecules and de-novo sequencing, lncRNA expression studies by microarray is a still cost-effective procedure that could allow to have a landscape of the most characterized lncRNA species. However, microarray design does not always correctly address the overlap between coding and noncoding samples to discriminate between the original transcript source. In order to overcome this issue, in this chapter we present a bioinformatics pipeline that enables accurate annotation of GeneChip® microarrays, to date the most commonly adopted among the commercial solutions. Overall, this approach holds two main advantages, a gain in specificity of transcript detection and the adaptability to the whole panel of GeneChip® arrays.

In Vitro Silencing of lncRNAs Using LNA GapmeRs.

Despite substantial advancements have been achieved in the identification of long noncoding RNA (lncRNA) molecules, many challenges still remain into their functional characterization. Loss-of-function approaches are needed to study oncogenic lncRNAs, which appear more difficult to knock down by RNA interference as compared to mRNAs. In this chapter, we present a protocol based on the use of a novel class of antisense oligonucleotides, named locked nucleic acid (LNA) GapmeRs, to inhibit the oncogenic lncRNA NEAT1 in multiple myeloma cells. Overall, this approach holds many advantages, including its possible independence from delivery reagents as well as the capability to knock down lncRNAs even in hard-to-transfect suspension cells, like hematopoietic cells.

Epigenetic Regulation of Mitochondrial Quality Control Genes in Multiple Myeloma: A Sequenom MassARRAY Pilot Investigation on HMCLs.

The mitochondrial quality control network includes several epigenetically-regulated genes involved in mitochondrial dynamics, mitophagy, and mitochondrial biogenesis under physiologic conditions. Dysregulated expression of such genes has been reported in various disease contexts, including cancer. However, their expression pattern and the possible underlying epigenetic modifications remain to be defined within plasma cell (PC) dyscrasias. Herein, we compared the mRNA expression of mitochondrial quality control genes from multiple myeloma, plasma cell leukemia patients and human myeloma cell lines (HMCLs) with healthy plasma cells; moreover, by applying the Sequenom MassARRAY EpiTYPER technology, we performed a pilot investigation of their CpG methylation status in HMCLs. Overall, the results provided indicate dysregulated expression of several mitochondrial network's genes, and alteration of the CpG methylation profile, underscoring novel potential myeloma biomarkers deserving in-depth functional investigation in the future.

LncRNA NEAT1 in Paraspeckles: A Structural Scaffold for Cellular DNA Damage Response Systems?

Nuclear paraspeckle assembly transcript 1 (NEAT1) is a long non-coding RNA (lncRNA) reported to be frequently deregulated in various types of cancers and neurodegenerative processes. NEAT1 is an indispensable structural component of paraspeckles (PSs), which are dynamic and membraneless nuclear bodies that affect different cellular functions, including stress response. Furthermore, increasing evidence supports the crucial role of NEAT1 and essential structural proteins of PSs (PSPs) in the regulation of the DNA damage repair (DDR) system. This review aims to provide an overview of the current knowledge on the involvement of NEAT1 and PSPs in DDR, which might strengthen the rationale underlying future NEAT1-based therapeutic options in tumor and neurodegenerative diseases.

Expression Pattern and Biological Significance of the lncRNA ST3GAL6-AS1 in Multiple Myeloma.

The biological impact of long non-coding RNAs (lncRNAs) in multiple myeloma (MM) is becoming an important aspect of investigation, which may contribute to the understanding of the complex pathobiology of the disease whilst also providing novel potential therapeutic targets. Herein, we investigated the expression pattern and the biological significance of the lncRNA ST3 beta-galactoside alpha-2,3 sialyltransferase 6 antisense RNA 1 (ST3GAL6-AS1) in MM. We documented a high ST3GAL6-AS1 expression level in MM compared to normal plasma cells (PCs) or other hematological malignancies. Transcriptome analyses of MM PCs from patients included in the CoMMpass database indicated a potential involvement of ST3GAL6-AS1 in MAPK signaling and ubiquitin-mediated proteolysis pathways. ST3GAL6-AS1 silencing by LNA-gapmeR antisense oligonucleotides inhibits cell proliferation and triggers apoptosis in MM cell line. Notably, ST3GAL6-AS1 silencing in vitro displayed the down-regulation of the MAPK pathway and protein ubiquitination. These data suggest that ST3GAL6-AS1 deregulation may play a pathogenetic role in MM by affecting both proliferation pathways and circuits fundamental for PC survival. However, ST3GAL6-AS1 expression levels seem not to be significantly associated with clinical outcome and its targeting appears to exert antagonistic effects with proteasome inhibitors used in MM. These findings strongly urge the need for further studies investigating the relevance of ST3GAL6-AS1 in MM.

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.