Debora Soncini

Aim: Extracellular vesicles (EVs) mediate cell-to-cell communication and co-contribute in cancer progression and modulation of immune responses. The aim of this work was to characterize the phenotype and function of EVs isolated from the peripheral blood (PB) of multiple myeloma (MM) patients, with particular attention to adenosinergic ectoenzymes. Methods: EVs were isolated by centrifugation of PB plasma samples from newly diagnosed (ND) MM patients and healthy donors (HD). EVs were characterized by nanoparticle tracking analysis (NTA). Flow cytometry was used to assess the expression of CD138 (a tumor-associated antigen), nicotinamide phosphoribosyltransferase/pre-B cell colony enhancing factor (NAMPT/PBEF1), and adenosinergic ectoenzymes CD38, CD39, CD203a, and CD73. Functional studies were performed by assessing T-cell proliferation in vitro using carboxyfluorescein succinimidyl ester (CFSE) dilution and flow cytometry, with or without EVs to the culture. Results: The PB EVs size was comparable between MM patients and HDs, whereas EV concentration was significantly higher in MM patients. EVs from MM patients expressed higher levels of CD138, PBEF, CD38, and CD73 than those from HDs. In addition, CD138+ EVs from MM patients expressed higher levels of PBEF, CD38, CD39, and CD73 than CD138- counterparts. Finally, MM-derived EVs inhibited T-cell proliferation in vitro. This inhibition correlated with the expression of CD138 and CD203a. Conclusion: This study provides the first phenotypic and functional characterization of EVs obtained from the PB of MM patients. The presence of functional adenosinergic molecules may represent a novel immune-escape mechanism operating at distant sites. Nonetheless, further studies are required to define specific contributions of adenosinergic ectoenzymes and other immune-regulatory mechanisms.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas genome editing has advanced from an experimental tool to a clinically validated therapeutic platform in hematology. Landmark successes in inherited blood diseases including sickle cell disease, β-thalassemia, and severe combined immunodeficiency, have demonstrated that precise and durable genetic modifications can be safely and effectively implemented in human hematopoietic cells, positioning hematology at the forefront of translational genome editing. Beyond monogenic disease, CRISPR-based approaches are transforming both the biological understanding and treatment of hematologic malignancies by enabling systematic interrogation of cancer dependencies, functional mapping of genetic vulnerabilities, and mechanism-driven target validation, including in vivo and immune-relevant contexts. In parallel, therapeutic applications are emerging through the development of engineered cellular therapies, including edited autologous and allogeneic immune effector cells designed to enhance antitumor efficacy, persistence, and immune evasion. This review synthesizes recent CRISPR-based advances across benign and malignant hematologic diseases. We compare major editing modalities, including nuclease-mediated disruption, base editing, prime editing, and CRISPR-based transcriptional modulation, and highlight key preclinical studies alongside emerging clinical trial data. We also discuss translational challenges that currently limit broader clinical adoption, including delivery and manufacturing scalability, off-target and genotoxicity risks, tumor and immune heterogeneity, and the long-term durability and fitness of edited cell populations. Finally, we outline priorities for the next phase of the field, emphasizing how continued innovation in CRISPR technologies may enable increasingly precise, durable, and mechanism-informed therapeutic strategies in hematology.

Despite transformative therapeutic advances, multiple myeloma (MM) remains an incurable malignancy characterized by sequential relapses and progressive treatment resistance. Patients with heavily pretreated relapsed or refractory MM continue to face limited therapeutic options and poor outcomes. Melflufen (melphalan flufenamide) is a peptide–drug conjugate that enhances intracellular delivery of alkylating agents via aminopeptidase-mediated activation. Early clinical studies demonstrated encouraging activity in advanced MM, leading to accelerated approval by the U.S. Food and Drug Administration in 2021. However, results from the phase III OCEAN trial raised concerns regarding overall survival, ultimately resulting in withdrawal of the drug from the U.S. market. In this review, we examine the biological rationale, clinical development, and regulatory trajectory of melflufen, and critically reassess its role within the evolving therapeutic landscape of MM. While the negative survival findings observed in the phase III OCEAN trial substantially limited the clinical development of melflufen, the biological principles underlying enzyme-targeted intracellular drug delivery may still provide relevant insights for biomarker-driven therapeutic strategies and future peptide–drug conjugate development in MM.

Despite transformative therapeutic advances, multiple myeloma (MM) remains an incurable malignancy characterized by sequential relapses and progressive treatment resistance. Patients with heavily pretreated relapsed or refractory MM continue to face limited therapeutic options and poor outcomes. Melflufen (melphalan flufenamide) is a peptide–drug conjugate that enhances intracellular delivery of alkylating agents via aminopeptidase-mediated activation. Early clinical studies demonstrated encouraging activity in advanced MM, leading to accelerated approval by the U.S. Food and Drug Administration in 2021. However, results from the phase III OCEAN trial raised concerns re-garding overall survival, ultimately resulting in withdrawal of the drug from the U.S. market. In this review, we examine the biological rationale, clinical development, and regulatory trajectory of melflufen, and critically reassess its role within the evolving therapeutic landscape of MM. The negative survival signal observed in OCEAN challenges the clinical viability of melflufen, yet also provides key insights into patient selection and mechanism-based drug delivery strategies. These observations support a selective, mechanism-based positioning of melflufen and argue for a broader shift from drug-centered evaluation to biology-driven therapeutic strategies.

Sirtuin 6 (SIRT6) is a member of the mammalian NAD⁺-dependent deac(et)ylase sirtuin family. SIRT6’s anti-inflammatory roles are emerging increasingly often in different diseases and cell types, including endothelial cells. In this study, the role of SIRT6 in pro-inflammatory conditions was investigated by engineering human umbilical vein endothelial cells to overexpress SIRT6 (SIRT6+ HUVECs). Our results showed that SIRT6 overexpression affected the levels of adhesion molecules and sustained megakaryocyte proliferation and proplatelet formation. Interestingly, the pro-inflammatory activation of the ATP/purinergic axis was reduced in SIRT6+ HUVECs. Specifically, the TNFα-induced release of ATP in the extracellular space and the increase in pannexin-1 hemichannel expression, which mediates ATP efflux, were hampered in SIRT6+ cells. Instead, NAD⁺ release and Connexin43 expression were not modified by SIRT6 levels. Moreover, the Ca²⁺ influx in response to ATP and the expression of the purinergic receptor P2X7 were decreased in SIRT6+ HUVECs. Contrary to extracellular ATP, extracellular NAD⁺ did not evoke pro-inflammatory responses in HUVECs. Instead, NAD⁺ administration reduced endothelial cell proliferation and motility and counteracted the TNFα-induced angiogenesis. Altogether, our data reinforce the view of SIRT6 activation as an anti-inflammatory approach in vascular endothelium.

Cancer cells fuel growth and energy demands by increasing their NAD⁺ biosynthesis dependency, which therefore represents an exploitable vulnerability for anti-cancer strategies. CD38 is a NAD⁺-degrading enzyme that has become crucial for anti-MM therapies since anti-CD38 monoclonal antibodies represent the backbone for treatment of newly diagnosed and relapsed multiple myeloma patients. Nevertheless, further steps are needed to enable a full exploitation of these strategies, including deeper insights of the mechanisms by which CD38 promotes tumorigenesis and its metabolic additions that could be selectively targeted by therapeutic strategies. Here, we present evidence that CD38 upregulation produces a pervasive intracellular-NAD⁺ depletion, which impairs mitochondrial fitness and enhances oxidative stress; as result, genetic or pharmacologic approaches that aim to modify CD38 surface-level prime MM cells to NAD⁺-lowering agents. The molecular mechanism underlying this event is an alteration in mitochondrial dynamics, which decreases mitochondria efficiency and triggers energetic remodeling. Overall, we found that CD38 handling represents an innovative strategy to improve the outcomes of NAD⁺-lowering agents and provides the rationale for testing these very promising agents in clinical studies involving MM patients.

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.

Genomic instability plays a pathological role in various malignancies, including acute myeloid leukemia, and thus represents potential therapeutic target. Recent studies demonstrate that SIRT6, a NAD+-dependent nuclear deacetylase, functions as genome-guardian by preserving DNA integrity in different tumor cells. Here, we demonstrate that also CD34+ blasts from Acute Myeloid Leukemia patients show ongoing DNA damage and SIRT6 overexpression. Indeed, we identified a poor-prognostic subset of patients, with widespread instability, which relies on SIRT6 to compensate for DNA-replication stress. As result, SIRT6 depletion compromises the ability of leukemia cells to repair DNA double-strand breaks that, in turn, increases their sensitivity to daunorubicin and Ara-C, both in vitro and in vivo. In contrast, low SIRT6 levels observed in normal CD34+ hematopoietic progenitors explain their weaker sensitivity to genotoxic stress. Intriguingly, we have identified DNA-PKcs and CtIP deacetylation as crucial for SIRT6-mediated DNA repair. Together, our data suggest that inactivation of SIRT6 in leukemia cells leads to DNA-repair mechanisms disruption, genomic instability and aggressive Acute Myeloid Leukemia. This synthetic lethal approach, enhancing DNA damage while concomitantly blocking repair responses, provides the rationale for the clinical evaluation of SIRT6 modulators in leukemia treatment.