PARP1 trapping activates cGAS-STING pathway to induce immunogenic cell death in multiple myeloma Giada Juli, Domenica Ronchetti, Stefania Signorelli, Sara Squillacioti, Nicoletta Polerà, Anna Maienza, Michelangelo Vocaturo, Antonio Galvano, Elisa Taiana, Pierosandro Tagliaferri, Pierfrancesco Tassone, Daniele Caracciolo Cancer Cell International, 2026 BACKGROUND: Alternative Non-Homologous End Joining (Alt-NHEJ) DNA repair is considered a major player in cancer genomic instability. Here, we investigated cGAS-STING pathway as crucial node in the interplay between Alt-NHEJ repair and immune response, in the aim to discover novel therapeutic vulnerability in Multiple Myeloma (MM). METHODS: In silico analyses were performed by querying publicly available MM datasets (GSE66293 and CoMMpass). Anti-proliferative activity was evaluated by CellTiter-Glo, while flow cytometry analysis was used to determine the apoptotic process, cell cycle, phagocytosis, micronuclei detection, Calreticulin and T-cell activation markers. Protein expression was detected by western blot of whole or fractioned protein extracts. RESULTS: By interrogating public MM datasets, a significant correlation between hyperactivation of cGAS-STING mRNA signature and poor PFS and OS in MM was observed. Indeed, Gene Set Enrichment Analysis (GSEA) showed enrichment of DNA repair, TNFA signaling and oxidative phosphorylation in patients with cGAS-STING activation patients, associated to higher mRNA expression of DNA Ligase 3 (LIG3) and PARP1. On this basis, we evaluated the activity of Alt-NHEJ inhibitor Talazoparib (PARP1-inhibitor) on MM cell lines, focusing on their capability to modulate cGAS-STING pathway. We first detected a significant reduction of cell proliferation and the induction of apoptosis following Talazoparib treatment, which in turn induced DNA damage response and cell cycle blockade, and finally cGAS-STING pathway activation as result of PARP1-trapping into chromatin. Next, by performing co-culture experiments with healthy donor's peripheral blood mononuclear cells (PBMCs), we finally demonstrated the induction of immunogenic cell death, which was abrogated in cGAS-knockout cells, underscoring the pathway's functional relevance. CONCLUSION: Taken together, our findings indicate that Alt-NHEJ inhibitors are potential immune-stimulating agents for MM with hyperactivation of cGAS-STING pathway, coherently with our working hypothesis.
Identification of ceRNA Regulatory Networks Driven by the lncRNA NEAT1 in Multiple Myeloma Domenica Ronchetti, Valentina Traini, Ilaria Silvestris, Giuseppina Fabbiano, Andrea Devecchi, Federica Torricelli, Noemi Puccio, Ilaria Craparotta, Marco Bolis, Roberto Piva, Antonino Neri, Luca Agnelli, Francesco Passamonti, Niccolò Bolli, Elisa Taiana Journal of Cellular and Molecular Medicine, 2026 The lncRNA NEAT1 is overexpressed in multiple myeloma (MM) plasma cells and plays a key role in MM pathogenesis. NEAT1 is involved in ceRNA network in several cancers; however, data in MM are virtually absent. This study identified a NEAT1‐driven ceRNA network involving 96 miRNAs and 40 target genes, selected as concurrently downregulated in NEAT1‐KD AMO1 cells and upregulated in NEAT1‐overexpressing AMO1 cells (AMO1‐OVX). The co‐expression of NEAT1 and the targets was validated in MM patients (GSE116294, GSE13591, GSE6477, CoMMpass), and in NEAT1‐KD NCI‐H929, LP1, and KMS27 cell lines, showing for all targets a consistent downregulation, resembling that of NEAT1. The functional implication of the ceRNA network was explored by functional enrichment analyses of the 40 targets, identifying 78 significant gene sets, 17 of which were found significantly enriched by GSEA analysis in at least one experimental condition among NEAT1‐KD LP1, NCI‐H929, and KMS27 cells, AMO1‐OVX cells, or the extreme quartiles of NEAT1 expression in the CoMMpass dataset. Noteworthy, the cell cycle gene set was validated in 5 out of 6 conditions tested, suggesting that in MM the impact of NEAT1 upregulation on the cell cycle, experimentally demonstrated in our earlier publications, may be attributable, at least partially, to ceRNA mechanisms.
EXPLORING THE TRANSCRIPTIONAL PROFILE OF DIS3 CAN UNRAVEL NEW TARGETING STRATEGIES IN MULTIPLE MYELOMA Christian Boni Haematologica, 2026 Introduction. Multiple myeloma (MM) is a rare hematologic malignancy marked by neoplastic transformation of plasma cells (PCs) that are characterized by deep genomic instability. Karyotype abnormalities are considered early events in MM, whereas gene mutations arise later and are related to disease progression. Mutations of DIS3 were found in 10% of MM patients and were preferentially localized within the major ribonuclease (RNB) domain affecting its catalytic activity. In addition, del(13q) found in 40% of MM cases, impact the expression of DIS3. This gene encodes a highly conserved 3′–5′ exoribonuclease associated with the RNA-exosome complex essential for RNA turnover. Here, we characterized the transcriptional landscape induced by DIS3 silencing in an MM cell line and exploited the resultant signature to identify putative synergistic compounds.Methods. DIS3 silencing was performed using LNA-gapmeR designed for gymnosis. Transcriptome profiling by bulk RNA-seq of DIS3-silenced NCI-H929 cells versus gapmeR scrambled as controls was carried out on an Illumina NextSeq 500; DESeq2 pipeline was used for differentially expressed genes analysis. Immunofluorescence experiments were performed using anti α-Tubulin 488-conjugated for mitosis and S9.6 antibody for R-loop detection.Results. Differential expression analysis of RNA-seq identified 809 significantly deregulated genes (FDR<0.05), particularly 492 and 317 genes were found upregulated and downregulated respectively. GO bioprocess analysis of downregulated genes were enriched in microtubule and kinetochore assembly, promoting regulatory role of DIS3 in mitotic spindle organization. Interestingly, upregulated genes were predominantly associated with antiviral immune response and interferon-mediated signaling, a pattern possibly due to RNA accumulation. Additionally, immunofluorescence staining of DIS3 silenced cells showed increased R-Loop formation. To identify synergistic agents consistent with DIS3KD signature, the CMap query analysis revealed a strong association with microtubule-targeting inhibitors. Filanesib (ARRY-520) is a selective kinesin inhibitor of EG5/KIF11 that is fundamental mitotic kinesin necessary for spindle orientation. It has been previously studied for MM therapy—either as monotherapy or in combination—within phase II clinical trials. Filanesib treatment negatively affected cell viability in MM cell lines harboring del(13q) compared to bi-allelic WT counterparts. Furthermore, co-treatment with Filanesib and DIS3 LNA-gapmer produced a synergistic cytostatic response.Conclusions. Our rusults suggest that DIS3KD perturbs mitotic spindle dynamics and fosters genomic instability in MM, consistent with prior evidence that DIS3 depletion increases G0/G1-phase accumulation and compromises centrosome formation. In addition, the synergy observed with a microtubule-targeting agent could encourage future investigation to improve MM treatment strategies.
DISSECTION OF NEAT1’S ROLE IN TRANSCRIPTIONAL REGULATION REVEALS ACTIONABLE TARGETS IN HIGH-RISK MULTIPLE MYELOMA Noemi Puccio Haematologica, 2026 Introduction. LncRNAs emerged as a key element of genome regulation, driving multiple myeloma (MM) progression and therapy resistance. Among them, NEAT1 has been described as overexpressed in MM patients, promoting malignant PC proliferation. Besides its conventional function in the assembly of paraspeckles, we recently demonstrated that NEAT1 is directly involved in transcriptional control. In this study, we deepen our understanding of NEAT1’s mechanistic function in shaping the activity of transcriptional bodies, providing the rationale for targeted therapeutic intervention.Methods. We used an RNA-seq approach in NEAT1 KD and in CRISPRa NEAT1 overexpressing AMO-1 cell line to derive a NEAT1 transcriptomic signature. Unsupervised clustering analysis in the CoMMpass dataset was used to validate the clinical relevance of the NEAT1 gene program. Computational approaches were used to predict a list of transcriptional regulators of NEAT1’s signature. RNA immunoprecipitation (RIP) and RNA-FISH combined with immunofluorescence (IF) were used to confirm the in silico prediction in AMO-1 and NCI-H929 cell lines. Chromatin immunoprecipitation (ChIP) was employed to validate the involvement of NEAT1 in the transcriptional apparatus. High-throughput (HT) drug screening in NEAT1 KD cells was used to identify small compounds that interfere with NEAT1-dependent transcriptional activity. Rescue experiments were performed using NEAT1 overexpressing cells.Results. Transcriptomic analysis in NEAT1 KD and in NEAT1 overexpressing AMO-1 cell line revealed 378 NEAT1 targets. Unsupervised clustering analysis based on their expression segregated CoMMpass patients into two distinct groups, displaying high or low NEAT1 transcriptional activity. The cluster with the high NEAT1 transcriptional program showed reduced survival and was enriched in high-risk cytogenetic lesions, including 1q gain/amp and del(17)p. Noticeably, computational analysis predicted FOXM1 and CDK9 as upstream regulators of the NEAT1 program, both components MMB:FOXM1 transcriptional apparatus, which controls the expression of G2/M genes. Consistently, we highlighted that 60% of NEAT1 target genes harbor a Cell cycle homology region (CHR) motif, recognized by MMB:FOXM1 complex. In vitro molecular validation confirmed co-localization of NEAT1 and FOXM1 condensates in MM cells, and direct binding of NEAT1-FOXM1/NEAT1-CDK9. Additionally, we demonstrated that NEAT1 KD results in a reduced occupancy of FOXM1 at the promoters of CHR genes. HT drug screening revealed a synthetic lethal interaction between NEAT1 depletion and CDK9 inhibition, whereas its overexpression confers resistance to CDK9 blockade, confirming the interplay between NEAT1-CDK9 to sustain the mitotic gene program.Conclusions. These findings demonstrate that NEAT1 coordinates the expression of mitotic genes through the interaction with FOXM1 and CDK9, providing a mechanistic rationale for targeted interventions in high-risk patients.
NONO SHAPES MULTIPLE MYELOMA PROGRESSION THROUGH PARASPECKLE-DEPENDENT AND INDEPENDENT PATHWAYS Elisa Taiana Haematologica, 2026 Introduction: Multiple myeloma (MM) is an incurable cancer caused by malignant proliferation of bone marrow plasma cells. The lncRNA NEAT1, the scaffold of paraspeckle (PS), promotes MM progression by regulating DNA repair and cell survival. NONO, which stabilizes NEAT1 and supports PSs biogenesis, is upregulated in MM and associated with poor survival. In addition to its essential role within PSs, NONO may also have functions beyond PSs. This study aims to define how NONO shapes the transcriptomic landscape of MM plasma cells, both through its PS-associated and independent mechanisms.Methods. RNA was extracted from NONO-KD, NEAT1-KD, and scramble AMO1 and LP1 human MM cells lines (HMCL). RNA-seq libraries were prepared following Illumina Stranded TotalRNA PrepLigation with Ribo-zero Plus protocol (Illumina). Sequencing was performed on Illumina Novaseq 6000 S2 cartridge. CoMMpass data were retrieved from the Interim Analysis 15a (MMRF_CoMMpass_IA15a).Results. To investigate the role of NONO in both PS-related and independent pathways, we compared transcriptomic data from NONO-KD and NEAT1-KD AMO1 and LP1 cells. Overlapping pathways suggested PS-related functions. Silencing NEAT1 or NONO led to significant downregulation of gene sets involved in chromatin modification, as well as WNT/β-catenin and NOTCH signalling pathways. Consistently, RNAseq analysis of NEAT1-overexpressing AMO1 cells revealed positive modulation of the same pathways. Further validation came from GSEA of CoMMpass samples stratified by NONO expression, comparing the two extreme quartiles.Since NONO is essential for protecting NEAT1 from degradation, its silencing results in a marked downregulation of NEAT1 expression levels, thus impacting the transcriptome of NONO-KD cells in a NEAT1-dependent manner. As a result, all the pathways modulated in the NONO-KD HMCLs were also confirmed in the NEAT1-KD samples, making it impossible to identify any pathways regulated by NONO independently of PSs. However, the analysis of data from the extreme quartile of NONO in the CoMMpass dataset identified pathways absent in NEAT1-KD HMCLs, suggesting PS-independent roles for NONO in RNA processing, RNA trafficking, mitochondrial biogenesis, and cell-cell communication. To validate pathways selectively regulated by NONO independently of PS, we focused on its role in intercellular communication and adhesion. Functionally, HUVECs cultured in conditioned media from NONO-KD AMO-1 or NCI-H929 cells showed disrupted VE-cadherin localization and expression, indicating that the NONO-dependent MM secretome can alter endothelial adhesion. These results reveal a potential PS-independent role for NONO in modulating the tumor microenvironment.Conclusions. This study highlights the multifaceted transcriptional roles of NONO in MM, revealing both PS-dependent and -independent functions, and underscores its potential impact on tumor microenvironment regulation and disease progression.
Clonal hematopoiesis is clonally unrelated to multiple myeloma and is associated with specific microenvironmental changes Marta Lionetti, Margherita Scopetti, Antonio Matera, Akihiro Maeda, Alessio Marella, Francesca Lazzaroni, Giancarlo Castellano, Sonia Fabris, Stefania Pioggia, Silvia Lonati, Alfredo Marchetti, Alessandra Cattaneo, Marta Tornese, Antonino Neri, Claudia Leoni, Loredana Pettine, Valentina Traini, Ilaria Silvestris, Marzia Barbieri, Giuseppina Fabbiano, Domenica Ronchetti, Elisa Taiana, Claudio De Magistris, Matteo Claudio Da Via', Francesco Passamonti, Niccolò Bolli Blood, 2025 Multiple myeloma (MM) initiation is dictated by genomic events. However, its progression from asymptomatic stages to an aggressive disease that ultimately fails to respond to treatments is also dependent on changes of the tumor microenvironment (TME). Clonal hematopoiesis of indeterminate potential (CHIP) is a prevalent clonal condition of the hematopoietic stem cell whose presence is causally linked to a more inflamed microenvironment. Here, we show in 106 patients with MM that CHIP is frequently co-existing with MM at diagnosis, associates with a more advanced R-ISS stage, higher age and shows a non-significant trend towards lower median hemoglobin. In our cohort the two conditions do not share a clonal origin. Single cell RNA-sequencing in 16 MM patients highlights significant TME changes when CHIP is present: decreased naïve T cells, a pro-inflammatory TME, decreased antigen-presenting function by dendritic cells and expression of exhaustion markers in CD8 cells. Inferred interactions between cell types in CHIP-positive TME suggested that especially monocytes, T cells and clonal plasma cells may have a prominent role in mediating inflammation, immune evasion and pro-survival signals in favor of MM cells. Altogether, our data show that, in the presence of CHIP, the TME of MM at diagnosis is significantly disrupted in line with what usually seen in more advanced disease, with potential translational implications. Our data highlight the relevance of this association and prompt for further studies on the modifier role of CHIP in the MM TME.
Combinatorial strategies targeting NEAT1 and AURKA as new potential therapeutic options for multiple myeloma Noemi Puccio, Gloria Manzotti, Elisabetta Mereu, Federica Torricelli, Domenica Ronchetti, Michela Cumerlato, Ilaria Craparotta, Laura Di Rito, Marco Bolis, Valentina Traini, Veronica Manicardi, Valentina Fragliasso, Yvan Torrente, Nicola Amodio, Niccolò Bolli, Elisa Taiana, Alessia Ciarrochi, Roberto Piva, Antonino Neri Haematologica, 2024 Multiple myeloma (MM) is a dreadful disease, marked by the uncontrolled proliferation of clonal plasma cells (PCs) within the bone marrow (BM). MM is characterized by a highly heterogeneous clinical and molecular background, supported by severe genomic alterations. Important deregulation of long non-coding RNAs (lncRNAs) expression has been reported in MM patients, influencing progression and therapy resistance. NEAT1 is a lncRNA essential for nuclear paraspeckles and involved in gene expression regulation. We showed that NEAT1 supports MM proliferation making this lncRNA an attractive therapeutic candidate. Here, we used a combinatorial strategy integrating transcriptomic and computational approaches with functional high-throughput drug screening, to identify compounds that synergize with NEAT1 inhibition in restraining MM cells growth. AUKA inhibitors were identified as top-scoring drugs in these analyses. We showed that the combination of NEAT1 silencing and AURKA inhibitors in MM profoundly impairs microtubule organization and mitotic spindle assembly, finally leading to cell death. Analysis of the large publicly CoMMpass dataset showed that in MM patients AURKA expression is strongly associated with reduced progression-free (p < 0.0001) and overall survival probability (p < 0.0001) and patients displaying high expression levels of both NEAT1 and AURKA have a worse clinical outcome. Finally, using RNA-sequencing data from NEAT1 knockdown (KD) MM cells, we identified the AURKA allosteric regulator TPX2 as a new NEAT1 target in MM and as a mediator of the interplay between AURKA and NEAT1, therefore providing a possible explanation of the synergistic activity observed upon their combinatorial inhibition.
Inference of genomic lesions from single-cell RNA-seq in myeloma improves functional intraclonal and interclonal analysis Francesca Lazzaroni, Antonio Matera, Alessio Marella, Akihiro Maeda, Giancarlo Castellano, Alfredo Marchetti, Sonia Fabris, Stefania Pioggia, Ilaria Silvestris, Domenica Ronchetti, Silvia Lonati, Giuseppina Fabbiano, Valentina Traini, Elisa Taiana, Laura Porretti, Federico Colombo, Claudio De Magistris, Margherita Scopetti, Marzia Barbieri, Loredana Pettine, Federica Torricelli, Antonino Neri, Francesco Passamonti, Marta Lionetti, Matteo Claudio Da Vià, Niccolò Bolli Blood Advances, 2024 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 immunoglobulin heavy-chain 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 PCs derived from 5 patients with SMM/MM and unbiasedly identified clonal and polyclonal PCs. 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 scRNA-seq, 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 Domenica Ronchetti, Valentina Traini, Ilaria Silvestris, Giuseppina Fabbiano, Francesco Passamonti, Niccolò Bolli, Elisa Taiana Cancer Gene Therapy, 2024 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.
