Long non-coding RNA NEAT1 is the core structural component of the nuclear paraspeckle (PS) organe... more 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...
Multiple myeloma is a malignancy of terminally differentiated plasma cells, characterized by an e... more Multiple myeloma is a malignancy of terminally differentiated plasma cells, characterized by an extreme genetic heterogeneity that poses great challenges for its successful treatment. Due to antibody overproduction, MM cells depend on the precise regulation of the protein degradation systems. Despite the success of PIs in MM treatment, resistance and adverse toxic effects such as peripheral neuropathy and cardiotoxicity could arise. To this end, the use of rational combinatorial treatments might allow lowering the dose of inhibitors and therefore, minimize their side-effects. Even though the suppression of different cellular pathways in combination with proteasome inhibitors have shown remarkable anti-myeloma activities in preclinical models, many of these promising combinations often failed in clinical trials. Substantial progress has been made by the simultaneous targeting of proteasome and different aspects of MM-associated immune dysfunctions. Moreover, targeting deranged metabo...
Simple Summary Genomic instability (GI) plays an important role in the pathobiology of multiple m... more Simple Summary Genomic instability (GI) plays an important role in the pathobiology of multiple myeloma (MM) by promoting the acquisition of several tumor hallmarks. Molecular determinants of GI in MM are continuously emerging and will be herein discussed, with specific regard to non-coding RNAs. Targeting non-coding RNA molecules known to be involved in GI indeed provides novel routes to dampen such oncogenic mechanisms in MM. Abstract Multiple myeloma (MM) is a complex hematological malignancy characterized by abnormal proliferation of malignant plasma cells (PCs) within a permissive bone marrow microenvironment. The pathogenesis of MM is unequivocally linked to the acquisition of genomic instability (GI), which indicates the tendency of tumor cells to accumulate a wide repertoire of genetic alterations. Such alterations can even be detected at the premalignant stages of monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM) and, overall, c...
Because of its role in the regulation of the cell cycle, DNA damage response, apoptosis, DNA repa... more Because of its role in the regulation of the cell cycle, DNA damage response, apoptosis, DNA repair, cell migration, autophagy, and cell metabolism, the TP53 tumor suppressor gene is a key player for cellular homeostasis. TP53 gene is mutated in more than 50% of human cancers, although its overall dysfunction may be even more frequent. TP53 mutations are detected in a lower percentage of hematological malignancies compared to solid tumors, but their frequency generally increases with disease progression, generating adverse effects such as resistance to chemotherapy. Due to the crucial role of P53 in therapy response, several molecules have been developed to re-establish the wild-type P53 function to mutant P53. PRIMA-1 and its methylated form PRIMA-1Met (also named APR246) are capable of restoring the wild-type conformation to mutant P53 and inducing apoptosis in cancer cells; however, they also possess mutant P53-independent properties. This review presents the activities of PRIMA-...
Bone remodeling is uncoupled in the multiple myeloma (MM) bone marrow niche, resulting in enhance... more Bone remodeling is uncoupled in the multiple myeloma (MM) bone marrow niche, resulting in enhanced osteoclastogenesis responsible of MM-related bone disease (MMBD). Several studies have disclosed the mechanisms underlying increased osteoclast formation and activity triggered by the various cellular components of the MM bone marrow microenvironment, leading to the identification of novel targets for therapeutic intervention. In this regard, recent attention has been given to non-coding RNA (ncRNA) molecules, that finely tune gene expression programs involved in bone homeostasis both in physiological and pathological settings. In this review, we will analyze major signaling pathways involved in MMBD pathophysiology, and report emerging evidence of their regulation by different classes of ncRNAs.
The biological impact of long non-coding RNAs (lncRNAs) in multiple myeloma (MM) is becoming an e... more 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...
DIS3 gene mutations (DIS3mts) occur in roughly 10% of multiple myeloma (MM) patients; furthermore... more DIS3 gene mutations (DIS3mts) occur in roughly 10% of multiple myeloma (MM) patients; furthermore, DIS3 expression could be affected by monosomy 13 or del(13q), which occur in approximately 40% of MM cases. Despite several reports on the prevalence of DIS3mts, their contribution to the pathobiology of MM remains largely unknown. We took advantage of the large public CoMMpass dataset to investigate the spectrum of DIS3mts in MM and its impact on the transcriptome and clinical outcome. We found that DIS3mts clinical relevance strictly depended on del(13q) co-occurrence. In particular, bi-allelic DIS3 lesions significantly affected PFS, independently from other predictors of poor clinical outcome, while mono-allelic events mostly impacted OS. As expected, DIS3mts affect MM transcriptome involving cellular processes and signaling pathways associated with RNA metabolism, and the deregulation of a large number of lncRNAs, among which we identified five distinct transcripts as independent ...
Despite the fact that next-generation sequencing approaches, in particular RNA sequencing, provid... more 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.
The human genome contains thousands of long noncoding RNAs (lncRNAs), even outnumbering protein-c... more The human genome contains thousands of long noncoding RNAs (lncRNAs), even outnumbering protein-coding genes. These molecules can play a pivotal role in the development and progression of human disease, including cancer, and are susceptible to therapeutic intervention. Evidence of biologic function, however, is still missing for the vast majority of them. Both loss-of-function (LOF) and gain-of-function (GOF) studies are therefore necessary to advance our understanding of lncRNA networks and programs driving tumorigenesis. Here, we describe a protocol to perform lncRNA's LOF or GOF studies in multiple myeloma (MM) cells, using CRISPR interference (CRISPRi) or CRISPR activation (CRISPRa) technologies, respectively. These approaches have many advantages, including applicability to large-scale genetic screens in mammalian cells and possible reversibility of modulating effects; moreover, CRISPRa offers the unique opportunity to enhance lncRNA expression at the site of transcription, with relevant biologic implications.
Despite substantial advancements have been achieved in the identification of long noncoding RNA (... more 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.
Mechanisms underlying the pathophysiology of primary Plasma Cell Leukemia (pPCL) and intramedulla... more 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 deregulat...
The mitochondrial quality control network includes several epigenetically-regulated genes involve... more 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-de...
Multiple myeloma (MM) is an incurable plasma cell malignancy arising primarily within the bone ma... more Multiple myeloma (MM) is an incurable plasma cell malignancy arising primarily within the bone marrow (BM). During MM progression, different modifications occur in the tumor cells and BM microenvironment, including the angiogenic shift characterized by the increased capability of endothelial cells to organize a network, migrate and express angiogenic factors, including vascular endothelial growth factor (VEGF). Here, we studied the functional outcome of the dysregulation of Notch ligands, Jagged1 and Jagged2, occurring during disease progression, on the angiogenic potential of MM cells and BM stromal cells (BMSCs). Jagged1–2 expression was modulated by RNA interference or soluble peptide administration, and the effects on the MM cell lines’ ability to induce human pulmonary artery cells (HPAECs) angiogenesis or to indirectly increase the BMSC angiogenic potential was analyzed in vitro; in vivo validation was performed on a zebrafish model and MM patients’ BM biopsies. Overall, our r...
Nuclear paraspeckle assembly transcript 1 (NEAT1) is a long non-coding RNA (lncRNA) reported to b... more 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.
The biological impact of long non-coding RNAs (lncRNAs) in multiple myeloma (MM) is becoming an i... more 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 path...
The biological role and therapeutic potential of long non-coding RNAs (lncRNAs) in chronic lympho... more 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 re...
NOTCH1 is a relevant gene for outcome in chronic lymphocytic leukemia (CLL), being recurrently ta... more NOTCH1 is a relevant gene for outcome in chronic lymphocytic leukemia (CLL), being recurrently targeted by coding mutations deleting the PEST (proline [P], glutamic acid [E], serine [S], threonine [T]) domain (involved in NOTCH1 degradation) and thus generating a more stable protein whose accumulation is associated with poor prognosis. Recently, Puente et al identified one recurrent (chr9:136495700T>C) and two additional less frequent mutations (136495691T>G and 136495693T>C) affecting NOTCH1 30-untranslated region (UTR) in 5.6% of cases. By causing aberrant splicing events, these variants led to PEST domain deletion and were associated with short time to first treatment (TTFT) and poor overall survival, similarly to coding mutations. In a previous study, we investigated the NOTCH1 mutational hotspot (c.7541_7542delCT) in a large series of early stage Binet A CLL enrolled in the O-CLL1 multicenter prospective observational trial (clinicaltrials.gov: NCT00917540) including to date 523 patients. Here, we deepened NOTCH1 mutational characterization in this cohort by sequencing the 30-UTR of the gene and expanding the analysis of c.7541_7542delCT to several additional patients. Next-generation sequencing of NOTCH1 30-UTR was performed in 502 patients including 146 with clinical monoclonal B cell lymphocytosis (MBL) at a median depth of coverage of 555x. Details concerning trial design and experimental procedures are available in Supporting Information. Two variants were identified in 24/502 cases (4.8%), whose characteristics are summarized in Supporting Information Table S1. Twenty-one patients carried the 136495700T>C substitution, at variant allele frequencies (VAF) from 0.6% to 46.7%, and three the 136495693T>C variant (0.8% < VAF < 28.9%). NOTCH1 30UTR mutations were positively associated with IGHV germline configuration and ZAP70 expression (Fisher's test P value = .0051 and 0.046, respectively). To investigate the presence of the aberrant transcript generated by the splicing between the cryptic donor site in the coding portion of exon 34 (9:136496228-9) and the acceptor site created by mutations in 30-UTR,2 we performed reverse transcriptionpolymerase chain reaction (RT-PCR) in 14 cases carrying the 136495700T>C substitution with available RNA material. By developing a transcript-specific RT-PCR assay using a boundary-spanning primer overlapping the newly created splicing acceptor site (Supporting Information Figure S1), we were able to detect the aberrantly spliced transcript in all mutated patients, consistently with their VAFs in genomic DNA (Supporting Information Figure S2). We then completed the previous screening of the O-CLL1 series for NOTCH1 mutation hotspot (c.7541_7542delCT) by analyzing 133 additional patients with available DNA, of whom 17 tested positive. Therefore, based on these and previous data, 65/510 (12.7%) patients harbored the c.7541_7542delCT coding mutation. Notably, we found that NOTCH1 coding and 30-UTR mutations co-occurred in seven patients (Fisher's test P value = .023). We subsequently tested the prognostic power for TTFT of 30UTR mutations in 487/502 patients with available follow-up. In addition, we re-evaluated the prognostic value of the c.7541_7542delCT coding mutation in predicting TTFT. This re-evaluation was prompted by the significantly higher number of evaluable cases in the O-CLL1 trial and by the longer follow-up compared with the previous report. With regard to the c.7541_7542delCT mutation, the median TTFT was significantly shorter in mutated than in wild-type cases (Figure 1A), even when considering MBL and CLL separately (Supporting Information Figure S3A,B). Concerning 30-UTR mutation, only a trend towards significance was observed when all the patients were considered (Figure 1B); however, if considered separately, the occurrence of 30-UTR mutation was associated with a significantly shorter TTFT in CLL group (Supporting Information Figure S4A,B). Furthermore, when 30-UTR variants were stratified based upon the VAF cutoff value of 10%, all mutated patients (MBL or CLL) at VAF ≥ 10% had a significantly shorter TTFT, while the curve of mutated patients at lower VAFs overlapped with that of wild-type group (Figure 1C). When forced into a multivariate Cox model together with CD38 and ZAP-70 expression, β2M levels, Rai stage, MBL status, IGHV mutational status, chromosomal abnormalities, and SF3B1 mutational status (each of which resulted significant in a Cox univariate analysis; Supporting Information Figure S5), NOTCH1 30-UTR mutation at VAF ≥ 10% failed to maintain its independent prognostic power, even after excluding MBL cases from the analysis. This was probably due to the tight association of this mutation with other poor prognosis predictors, that is, IGHV UM status, CD38 positivity, ZAP-70 positivity, del (17p) or del(11q). In the same way we tested NOTCH1 coding mutations, which in the current setting maintained an independent prognostic impact on…
The biological role and therapeutic potential of long non-coding RNAs (lncRNAs) in multiple myelo... more 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.
Long non-coding RNA NEAT1 is the core structural component of the nuclear paraspeckle (PS) organe... more 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...
Multiple myeloma is a malignancy of terminally differentiated plasma cells, characterized by an e... more Multiple myeloma is a malignancy of terminally differentiated plasma cells, characterized by an extreme genetic heterogeneity that poses great challenges for its successful treatment. Due to antibody overproduction, MM cells depend on the precise regulation of the protein degradation systems. Despite the success of PIs in MM treatment, resistance and adverse toxic effects such as peripheral neuropathy and cardiotoxicity could arise. To this end, the use of rational combinatorial treatments might allow lowering the dose of inhibitors and therefore, minimize their side-effects. Even though the suppression of different cellular pathways in combination with proteasome inhibitors have shown remarkable anti-myeloma activities in preclinical models, many of these promising combinations often failed in clinical trials. Substantial progress has been made by the simultaneous targeting of proteasome and different aspects of MM-associated immune dysfunctions. Moreover, targeting deranged metabo...
Simple Summary Genomic instability (GI) plays an important role in the pathobiology of multiple m... more Simple Summary Genomic instability (GI) plays an important role in the pathobiology of multiple myeloma (MM) by promoting the acquisition of several tumor hallmarks. Molecular determinants of GI in MM are continuously emerging and will be herein discussed, with specific regard to non-coding RNAs. Targeting non-coding RNA molecules known to be involved in GI indeed provides novel routes to dampen such oncogenic mechanisms in MM. Abstract Multiple myeloma (MM) is a complex hematological malignancy characterized by abnormal proliferation of malignant plasma cells (PCs) within a permissive bone marrow microenvironment. The pathogenesis of MM is unequivocally linked to the acquisition of genomic instability (GI), which indicates the tendency of tumor cells to accumulate a wide repertoire of genetic alterations. Such alterations can even be detected at the premalignant stages of monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM) and, overall, c...
Because of its role in the regulation of the cell cycle, DNA damage response, apoptosis, DNA repa... more Because of its role in the regulation of the cell cycle, DNA damage response, apoptosis, DNA repair, cell migration, autophagy, and cell metabolism, the TP53 tumor suppressor gene is a key player for cellular homeostasis. TP53 gene is mutated in more than 50% of human cancers, although its overall dysfunction may be even more frequent. TP53 mutations are detected in a lower percentage of hematological malignancies compared to solid tumors, but their frequency generally increases with disease progression, generating adverse effects such as resistance to chemotherapy. Due to the crucial role of P53 in therapy response, several molecules have been developed to re-establish the wild-type P53 function to mutant P53. PRIMA-1 and its methylated form PRIMA-1Met (also named APR246) are capable of restoring the wild-type conformation to mutant P53 and inducing apoptosis in cancer cells; however, they also possess mutant P53-independent properties. This review presents the activities of PRIMA-...
Bone remodeling is uncoupled in the multiple myeloma (MM) bone marrow niche, resulting in enhance... more Bone remodeling is uncoupled in the multiple myeloma (MM) bone marrow niche, resulting in enhanced osteoclastogenesis responsible of MM-related bone disease (MMBD). Several studies have disclosed the mechanisms underlying increased osteoclast formation and activity triggered by the various cellular components of the MM bone marrow microenvironment, leading to the identification of novel targets for therapeutic intervention. In this regard, recent attention has been given to non-coding RNA (ncRNA) molecules, that finely tune gene expression programs involved in bone homeostasis both in physiological and pathological settings. In this review, we will analyze major signaling pathways involved in MMBD pathophysiology, and report emerging evidence of their regulation by different classes of ncRNAs.
The biological impact of long non-coding RNAs (lncRNAs) in multiple myeloma (MM) is becoming an e... more 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...
DIS3 gene mutations (DIS3mts) occur in roughly 10% of multiple myeloma (MM) patients; furthermore... more DIS3 gene mutations (DIS3mts) occur in roughly 10% of multiple myeloma (MM) patients; furthermore, DIS3 expression could be affected by monosomy 13 or del(13q), which occur in approximately 40% of MM cases. Despite several reports on the prevalence of DIS3mts, their contribution to the pathobiology of MM remains largely unknown. We took advantage of the large public CoMMpass dataset to investigate the spectrum of DIS3mts in MM and its impact on the transcriptome and clinical outcome. We found that DIS3mts clinical relevance strictly depended on del(13q) co-occurrence. In particular, bi-allelic DIS3 lesions significantly affected PFS, independently from other predictors of poor clinical outcome, while mono-allelic events mostly impacted OS. As expected, DIS3mts affect MM transcriptome involving cellular processes and signaling pathways associated with RNA metabolism, and the deregulation of a large number of lncRNAs, among which we identified five distinct transcripts as independent ...
Despite the fact that next-generation sequencing approaches, in particular RNA sequencing, provid... more 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.
The human genome contains thousands of long noncoding RNAs (lncRNAs), even outnumbering protein-c... more The human genome contains thousands of long noncoding RNAs (lncRNAs), even outnumbering protein-coding genes. These molecules can play a pivotal role in the development and progression of human disease, including cancer, and are susceptible to therapeutic intervention. Evidence of biologic function, however, is still missing for the vast majority of them. Both loss-of-function (LOF) and gain-of-function (GOF) studies are therefore necessary to advance our understanding of lncRNA networks and programs driving tumorigenesis. Here, we describe a protocol to perform lncRNA's LOF or GOF studies in multiple myeloma (MM) cells, using CRISPR interference (CRISPRi) or CRISPR activation (CRISPRa) technologies, respectively. These approaches have many advantages, including applicability to large-scale genetic screens in mammalian cells and possible reversibility of modulating effects; moreover, CRISPRa offers the unique opportunity to enhance lncRNA expression at the site of transcription, with relevant biologic implications.
Despite substantial advancements have been achieved in the identification of long noncoding RNA (... more 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.
Mechanisms underlying the pathophysiology of primary Plasma Cell Leukemia (pPCL) and intramedulla... more 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 deregulat...
The mitochondrial quality control network includes several epigenetically-regulated genes involve... more 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-de...
Multiple myeloma (MM) is an incurable plasma cell malignancy arising primarily within the bone ma... more Multiple myeloma (MM) is an incurable plasma cell malignancy arising primarily within the bone marrow (BM). During MM progression, different modifications occur in the tumor cells and BM microenvironment, including the angiogenic shift characterized by the increased capability of endothelial cells to organize a network, migrate and express angiogenic factors, including vascular endothelial growth factor (VEGF). Here, we studied the functional outcome of the dysregulation of Notch ligands, Jagged1 and Jagged2, occurring during disease progression, on the angiogenic potential of MM cells and BM stromal cells (BMSCs). Jagged1–2 expression was modulated by RNA interference or soluble peptide administration, and the effects on the MM cell lines’ ability to induce human pulmonary artery cells (HPAECs) angiogenesis or to indirectly increase the BMSC angiogenic potential was analyzed in vitro; in vivo validation was performed on a zebrafish model and MM patients’ BM biopsies. Overall, our r...
Nuclear paraspeckle assembly transcript 1 (NEAT1) is a long non-coding RNA (lncRNA) reported to b... more 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.
The biological impact of long non-coding RNAs (lncRNAs) in multiple myeloma (MM) is becoming an i... more 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 path...
The biological role and therapeutic potential of long non-coding RNAs (lncRNAs) in chronic lympho... more 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 re...
NOTCH1 is a relevant gene for outcome in chronic lymphocytic leukemia (CLL), being recurrently ta... more NOTCH1 is a relevant gene for outcome in chronic lymphocytic leukemia (CLL), being recurrently targeted by coding mutations deleting the PEST (proline [P], glutamic acid [E], serine [S], threonine [T]) domain (involved in NOTCH1 degradation) and thus generating a more stable protein whose accumulation is associated with poor prognosis. Recently, Puente et al identified one recurrent (chr9:136495700T>C) and two additional less frequent mutations (136495691T>G and 136495693T>C) affecting NOTCH1 30-untranslated region (UTR) in 5.6% of cases. By causing aberrant splicing events, these variants led to PEST domain deletion and were associated with short time to first treatment (TTFT) and poor overall survival, similarly to coding mutations. In a previous study, we investigated the NOTCH1 mutational hotspot (c.7541_7542delCT) in a large series of early stage Binet A CLL enrolled in the O-CLL1 multicenter prospective observational trial (clinicaltrials.gov: NCT00917540) including to date 523 patients. Here, we deepened NOTCH1 mutational characterization in this cohort by sequencing the 30-UTR of the gene and expanding the analysis of c.7541_7542delCT to several additional patients. Next-generation sequencing of NOTCH1 30-UTR was performed in 502 patients including 146 with clinical monoclonal B cell lymphocytosis (MBL) at a median depth of coverage of 555x. Details concerning trial design and experimental procedures are available in Supporting Information. Two variants were identified in 24/502 cases (4.8%), whose characteristics are summarized in Supporting Information Table S1. Twenty-one patients carried the 136495700T>C substitution, at variant allele frequencies (VAF) from 0.6% to 46.7%, and three the 136495693T>C variant (0.8% < VAF < 28.9%). NOTCH1 30UTR mutations were positively associated with IGHV germline configuration and ZAP70 expression (Fisher's test P value = .0051 and 0.046, respectively). To investigate the presence of the aberrant transcript generated by the splicing between the cryptic donor site in the coding portion of exon 34 (9:136496228-9) and the acceptor site created by mutations in 30-UTR,2 we performed reverse transcriptionpolymerase chain reaction (RT-PCR) in 14 cases carrying the 136495700T>C substitution with available RNA material. By developing a transcript-specific RT-PCR assay using a boundary-spanning primer overlapping the newly created splicing acceptor site (Supporting Information Figure S1), we were able to detect the aberrantly spliced transcript in all mutated patients, consistently with their VAFs in genomic DNA (Supporting Information Figure S2). We then completed the previous screening of the O-CLL1 series for NOTCH1 mutation hotspot (c.7541_7542delCT) by analyzing 133 additional patients with available DNA, of whom 17 tested positive. Therefore, based on these and previous data, 65/510 (12.7%) patients harbored the c.7541_7542delCT coding mutation. Notably, we found that NOTCH1 coding and 30-UTR mutations co-occurred in seven patients (Fisher's test P value = .023). We subsequently tested the prognostic power for TTFT of 30UTR mutations in 487/502 patients with available follow-up. In addition, we re-evaluated the prognostic value of the c.7541_7542delCT coding mutation in predicting TTFT. This re-evaluation was prompted by the significantly higher number of evaluable cases in the O-CLL1 trial and by the longer follow-up compared with the previous report. With regard to the c.7541_7542delCT mutation, the median TTFT was significantly shorter in mutated than in wild-type cases (Figure 1A), even when considering MBL and CLL separately (Supporting Information Figure S3A,B). Concerning 30-UTR mutation, only a trend towards significance was observed when all the patients were considered (Figure 1B); however, if considered separately, the occurrence of 30-UTR mutation was associated with a significantly shorter TTFT in CLL group (Supporting Information Figure S4A,B). Furthermore, when 30-UTR variants were stratified based upon the VAF cutoff value of 10%, all mutated patients (MBL or CLL) at VAF ≥ 10% had a significantly shorter TTFT, while the curve of mutated patients at lower VAFs overlapped with that of wild-type group (Figure 1C). When forced into a multivariate Cox model together with CD38 and ZAP-70 expression, β2M levels, Rai stage, MBL status, IGHV mutational status, chromosomal abnormalities, and SF3B1 mutational status (each of which resulted significant in a Cox univariate analysis; Supporting Information Figure S5), NOTCH1 30-UTR mutation at VAF ≥ 10% failed to maintain its independent prognostic power, even after excluding MBL cases from the analysis. This was probably due to the tight association of this mutation with other poor prognosis predictors, that is, IGHV UM status, CD38 positivity, ZAP-70 positivity, del (17p) or del(11q). In the same way we tested NOTCH1 coding mutations, which in the current setting maintained an independent prognostic impact on…
The biological role and therapeutic potential of long non-coding RNAs (lncRNAs) in multiple myelo... more 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.
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Papers by Elisa Taiana