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.

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.

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.

Single-Cell RNA Sequencing for the Detection of Clonotypic V(D)J Rearrangements in Multiple Myeloma.

Multiple myeloma (MM) has a highly heterogeneous genetic background, which complicates its molecular tracking over time. Nevertheless, each MM patient's malignant plasma cells (PCs) share unique V(D)J rearranged sequences at immunoglobulin loci, which represent ideal disease biomarkers. Because the tumor-specific V(D)J sequence is highly expressed in bulk RNA in MM patients, we wondered whether it can be identified by single-cell RNA sequencing (scRNA-seq). To this end we analyzed CD138+ cells purified from bone marrow aspirates of 19 samples with PC dyscrasias by both a standard method based on bulk DNA and by an implementation of the standard 10x Genomics protocol to detect expressed V(D)J sequences. A dominant clonotype was easily identified in each sample, accounting on average for 83.65% of V(D)J-rearranged cells. Compared with standard methods, scRNA-seq analysis proved highly concordant and even more effective in identifying clonal productive rearrangements, by-passing limitations related to the misannealing of consensus primers in hypermutated regions. We next validated its accuracy to track 5 clonal cells with absolute sensitivity in a virtual sample containing 3180 polyclonal cells. This shows that single-cell V(D)J analysis may be used to find rare clonal cells, laying the foundations for functional single-cell dissection of minimal residual disease.

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.

Molecular Modelling of NONO and SFPQ Dimerization Process and RNA Recognition Mechanism.

NONO and SFPQ are involved in multiple nuclear processes (e.g., pre-mRNA splicing, DNA repair, and transcriptional regulation). These proteins, along with NEAT1, enable paraspeckle formation, thus promoting multiple myeloma cell survival. In this paper, we investigate NONO and SFPQ dimer stability, highlighting the hetero- and homodimer structural differences, and model their interactions with RNA, simulating their binding to a polyG probe mimicking NEAT1guanine-rich regions. We demonstrated in silico that NONO::SFPQ heterodimerization is a more favorable process than homodimer formation. We also show that NONO and SFPQ RRM2 subunits are primarily required for protein-protein interactions with the other DBHS protomer. Simulation of RNA binding to NONO and SFPQ, beside validating RRM1 RNP signature importance, highlighted the role of ß2 and ß4 strand residues for RNA specific recognition. Moreover, we demonstrated the role of the NOPS region and other protomer's RRM2 ß2/ß3 loop in strengthening the interaction with RNA. Our results, having deepened RNA and DBHS dimer interactions, could contribute to the design of small molecules to modulate the activity of these proteins. RNA-mimetics, able to selectively bind to NONO and/or SFPQ RNA-recognition site, could impair paraspeckle formation, thus representing a first step towards the discovery of drugs for multiple myeloma treatment.

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.

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.

CRISPR Interference (CRISPRi) and CRISPR Activation (CRISPRa) to Explore the Oncogenic lncRNA Network.

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.

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.

The Landscape of Signaling Pathways and Proteasome Inhibitors Combinations in Multiple Myeloma.

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 metabolic hubs could represent a new avenue to identify effective therapeutic combinations with PIs. Finally, epigenetic drugs targeting either DNA methylation, histone modifiers/readers, or chromatin remodelers are showing pleiotropic anti-myeloma effects alone and in combination with PIs. We envisage that the positive outcome of patients will probably depend on the availability of more effective drug combinations and treatment of early MM stages. Therefore, the identification of sensitive targets and aberrant signaling pathways is instrumental for the development of new personalized therapies for MM patients.

Genomic Instability in Multiple Myeloma: A "Non-Coding RNA" Perspective.

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, contribute to the acquisition of the malignant traits underlying disease progression. The molecular basis of GI remains unclear, with replication stress and deregulation of DNA damage repair pathways representing the most documented mechanisms. The discovery that non-coding RNA molecules are deeply dysregulated in MM and can target pivotal components of GI pathways has introduced a further layer of complexity to the GI scenario in this disease. In this review, we will summarize available information on the molecular determinants of GI in MM, focusing on the role of non-coding RNAs as novel means to tackle GI for therapeutic intervention.

Antitumor Effects of PRIMA-1 and PRIMA-1Met (APR246) in Hematological Malignancies: Still a Mutant P53-Dependent Affair?

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-1 and PRIMA-1Met/APR246 and describes their potential use in hematological malignancies.

Heterogeneity of TP53 Mutations and P53 Protein Residual Function in Cancer: Does It Matter?

The human TP53 locus, located on the short arm of chromosome 17, encodes a tumour suppressor protein which functions as a tetrameric transcription factor capable of regulating the expression of a plethora of target genes involved in cell cycle arrest, apoptosis, DNA repair, autophagy, and metabolism regulation. TP53 is the most commonly mutated gene in human cancer cells and TP53 germ-line mutations are responsible for the cancer-prone Li-Fraumeni syndrome. When mutated, the TP53 gene generally presents missense mutations, which can be distributed throughout the coding sequence, although they are found most frequently in the central DNA binding domain of the protein. TP53 mutations represent an important prognostic and predictive marker in cancer. The presence of a TP53 mutation does not necessarily imply a complete P53 inactivation; in fact, mutant P53 proteins are classified based on the effects on P53 protein function. Different models have been used to explore these never-ending facets of TP53 mutations, generating abundant experimental data on their functional impact. Here, we briefly review the studies analysing the consequences of TP53 mutations on P53 protein function and their possible implications for clinical outcome. The focus shall be on Chronic Lymphocytic Leukemia (CLL), which also has generated considerable discussion on the role of TP53 mutations for therapy decisions.

Jagged Ligands Enhance the Pro-Angiogenic Activity of Multiple Myeloma Cells.

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 results indicate that the MM-derived Jagged ligands (1) increase the tumor cell angiogenic potential by directly triggering Notch activation in the HPAECs or stimulating the release of angiogenic factors, i.e., VEGF; and (2) stimulate the BMSCs to promote angiogenesis through VEGF secretion. The observed pro-angiogenic effect of Notch activation in the BM during MM progression provides further evidence of the potential of a therapy targeting the Jagged ligands.

Non-Coding RNAs in Multiple Myeloma Bone Disease Pathophysiology.

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.