Publications by authors named "Rossella Paolini"

67 Publications

Efficacy of idelalisib and rituximab in relapsed/refractory chronic lymphocytic leukemia treated outside of clinical trials. A report of the Gimema Working Group.

Hematol Oncol 2021 Mar 19. Epub 2021 Mar 19.

Department of Medical Sciences, Hematology Section, University of Ferrara, Cona - Ferrara, Italy.

Because the efficacy of new drugs reported in trials may not translate into similar results when used in the real-life, we analyzed the efficacy of idelalisib and rituximab (IR) in 149 patients with relapsed/refractory chronic lymphocytic leukemia treated at 34 GIMEMA centers. Median progression-free survival (PFS) and overall survival were 22.9 and 44.5 months, respectively; performance status (PS) ≥2 and ≥3 previous lines of therapy were associated with shorter PFS and overall survival (OS). 48% of patients were on treatment at 12 months; the experience of the centers (≥5 treated patients) and PS 0-1 were associated with a significantly longer treatment duration (p = 0.015 and p = 0.002, respectively). TP53 disruption had no prognostic significance. The overall response rate to subsequent treatment was 49.2%, with median OS of 15.5 months and not reached in patients who discontinued, respectively, for progression and for toxicity (p < 0.01). Treatment breaks ≥14 days were recorded in 96% of patients and adverse events mirrored those reported in trials. In conclusion, this real-life analysis showed that IR treatment duration was longer at experienced centers, that the ECOG PS and ≥3 lines of previous therapy are strong prognostic factor and that the overall outcome with this regimen was superimposable to that reported in a randomized trial.
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http://dx.doi.org/10.1002/hon.2861DOI Listing
March 2021

SAMHD1 phosphorylation and cytoplasmic relocalization after human cytomegalovirus infection limits its antiviral activity.

PLoS Pathog 2020 09 28;16(9):e1008855. Epub 2020 Sep 28.

Department of Molecular Medicine, "Sapienza" University of Rome, Rome, Italy.

SAMHD1 is a host restriction factor that functions to restrict both retroviruses and DNA viruses, based on its nuclear deoxynucleotide triphosphate (dNTP) hydrolase activity that limits availability of intracellular dNTP pools. In the present study, we demonstrate that SAMHD1 expression was increased following human cytomegalovirus (HCMV) infection, with only a modest effect on infectious virus production. SAMHD1 was rapidly phosphorylated at residue T592 after infection by cellular cyclin-dependent kinases, especially Cdk2, and by the viral kinase pUL97, resulting in a significant fraction of phosho-SAMHD1 being relocalized to the cytoplasm of infected fibroblasts, in association with viral particles and dense bodies. Thus, our findings indicate that HCMV-dependent SAMHD1 cytoplasmic delocalization and inactivation may represent a potential novel mechanism of HCMV evasion from host antiviral restriction activities.
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http://dx.doi.org/10.1371/journal.ppat.1008855DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7544099PMC
September 2020

FcεRI Signaling in the Modulation of Allergic Response: Role of Mast Cell-Derived Exosomes.

Int J Mol Sci 2020 Jul 30;21(15). Epub 2020 Jul 30.

Department of Molecular Medicine, "Sapienza" University of Rome, Laboratory Affiliated to Istituto Pasteur Italia-Fondazione Cenci Bolognetti, Viale Regina Elena 291, 00161 Rome, Italy.

Mast cells (MCs) are immune cells that act as environment resident sentinels playing a crucial role in Th2-mediated immune responses, including allergic reactions. Distinguishing features of MCs are the presence of numerous cytoplasmic granules that encapsulate a wide array of preformed bio-active molecules and the constitutive expression of the high affinity receptor of IgE (FcεRI). Upon FcεRI engagement by means of IgE and multivalent antigens, aggregated receptors trigger biochemical pathways that ultimately lead to the release of granule-stored and newly synthesized pro-inflammatory mediators. Additionally, MCs are also able to release exosomes either constitutively or upon stimulation. Exosomes are nanosized vesicles of endocytic origin endowed with important immunoregulatory properties, and represent an additional way of intercellular communication. Interestingly, exosomes generated upon FcεRI engagement contain co-stimulatory and adhesion molecules, lipid mediators, and MC-specific proteases, as well as receptor subunits together with IgE and antigens. These findings support the notion that FcεRI signaling plays an important role in influencing the composition and functions of exosomes derived by MCs depending on their activation status.
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http://dx.doi.org/10.3390/ijms21155464DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7432241PMC
July 2020

Bone Marrow Stromal Cell-Derived IL-8 Upregulates PVR Expression on Multiple Myeloma Cells via NF-kB Transcription Factor.

Cancers (Basel) 2020 Feb 13;12(2). Epub 2020 Feb 13.

Department of Molecular Medicine, Istituto Pasteur-Fondazione Cenci Bolognetti, Sapienza University of Rome, 00161 Rome, Italy.

Bone marrow stromal cells (BMSCs) strongly contribute to multiple myeloma (MM) progression, promoting the survival and growth of malignant plasma cells (PCs). However, the possible impact of these cells on the immune-mediated recognition of MM cells remains largely unknown. DNAM-1 activating receptor plays a prominent role in NK cell anti-MM response engaging the ligands poliovirus receptor (PVR) and nectin-2 on malignant PCs. Here, we analysed the role of MM patient-derived BMSCs in the regulation of PVR expression. We found that BMSCs enhance PVR surface expression on MM cells and promote their NK cell-mediated recognition. PVR upregulation occurs at transcriptional level and involves NF-kB transcription factor activation by BMSC-derived soluble factors. Indeed, overexpression of a dominant-negative mutant of IKBα blocked PVR upregulation. IL-8 plays a prominent role in these mechanisms since blockade of CXCR1/2 receptors as well as depletion of the cytokine via RNA interference prevents the enhancement of PVR expression by BMSC-derived conditioned medium. Interestingly, IL-8 is associated with stromal microvesicles which are also required for PVR upregulation via CXCR1/CXCR2 signaling activation. Our findings identify BMSCs as regulators of NK cell anti-MM response and contribute to define novel molecular pathways involved in the regulation of PVR expression in cancer cells.
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http://dx.doi.org/10.3390/cancers12020440DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072437PMC
February 2020

CD155: A Multi-Functional Molecule in Tumor Progression.

Int J Mol Sci 2020 Jan 30;21(3). Epub 2020 Jan 30.

Department of Molecular Medicine, "Sapienza" University of Rome, Laboratory affiliated to Istituto Pasteur Italia-Fondazione Cenci Bolognetti, "Viale Regina Elena 291, 00161 Rome, Italy.

CD155 is an adhesion molecule belonging to the Nectin/Nectin-like family often overexpressed on tumor cells and involved in many different processes such as cell adhesion, migration and proliferation. In contrast to these pro-tumorigenic functions, CD155 is also a ligand for the activating receptor DNAM-1 expressed on cytotoxic lymphocytes including Natural Killer (NK) cells and involved in anti-tumor immune response. However, during tumor progression inhibitory receptors for CD155 are up-regulated on the surface of effector cells, contributing to an impairment of their cytotoxic capacity. In this review we will focus on the roles of CD155 as a ligand for the activating receptor DNAM-1 regulating immune surveillance against cancer and as pro-oncogenic molecule favoring tumor proliferation, invasion and immune evasion. A deeper understanding of the multiple roles played by CD155 in cancer development contributes to improving anti-tumor strategies aimed to potentiate immune response against cancer.
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http://dx.doi.org/10.3390/ijms21030922DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7037299PMC
January 2020

Post-translational Mechanisms Regulating NK Cell Activating Receptors and Their Ligands in Cancer: Potential Targets for Therapeutic Intervention.

Front Immunol 2019 31;10:2557. Epub 2019 Oct 31.

Department of Molecular Medicine, Sapienza University of Rome, Laboratory Affiliated to Istituto Pasteur Italia-Fondazione Cenci Bolognetti, Rome, Italy.

Efficient clearance of transformed cells by Natural Killer (NK) cells is regulated by several activating receptors, including NKG2D, NCRs, and DNAM-1. Expression of these receptors as well as their specific "induced self" ligands is finely regulated during malignant transformation through the integration of different mechanisms acting on transcriptional, post-transcriptional, and post-translational levels. Among post-translational mechanisms, the release of activating ligands in the extracellular milieu through protease-mediated cleavage or by extracellular vesicle secretion represents some relevant cancer immune escape processes. Moreover, covalent modifications including ubiquitination and SUMOylation also contribute to negative regulation of NKG2D and DNAM-1 ligand surface expression resulting either in ligand intracellular retention and/or ligand degradation. All these mechanisms greatly impact on NK cell mediated recognition and killing of cancer cells and may be targeted to potentiate NK cell surveillance against tumors. Our mini review summarizes the main post-translational mechanisms regulating the expression of activating receptors and their ligands with particular emphasis on the contribution of ligand shedding and of ubiquitin and ubiquitin-like modifications in reducing target cell susceptibility to NK cell-mediated killing. Strategies aimed at inhibiting shedding of activating ligands and their modifications in order to preserve ligand expression on cancer cells will be also discussed.
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http://dx.doi.org/10.3389/fimmu.2019.02557DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6836727PMC
October 2020

Immune complexes exposed on mast cell-derived nanovesicles amplify allergic inflammation.

Allergy 2020 05 28;75(5):1260-1263. Epub 2019 Nov 28.

Department of Molecular Medicine, Laboratory affiliated to Istituto Pasteur Italia-Fondazione Cenci Bolognetti, "Sapienza" University of Rome, Rome, Italy.

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http://dx.doi.org/10.1111/all.14103DOI Listing
May 2020

Activation of liver X receptor up-regulates the expression of the NKG2D ligands MICA and MICB in multiple myeloma through different molecular mechanisms.

FASEB J 2019 08 24;33(8):9489-9504. Epub 2019 May 24.

Department of Molecular Medicine, Sapienza University of Rome, Rome, Italy.

NK cells have an important role in immunosurveillance of multiple myeloma (MM) progression, and their activity is enhanced by combination therapies able to regulate the expression of specific activating ligands. Liver X receptors (LXRs) are nuclear receptors and important regulators of intracellular cholesterol and lipid homeostasis. Moreover, they have regulatory roles in both cancer and immune response. Indeed, they can regulate inflammation and innate and acquired immunity. Furthermore, LXR activation directly acts in cancer cells (, prostate, breast, melanoma, colon cancer, hepatocarcinoma, glioblastoma, and MM) that show an accumulation of cholesterol and alteration of LXR-mediated metabolic pathways. Here, we investigated the role of LXR and cholesterol on the expression of the NK cell-activating ligands major histocompatibility complex class I chain-related molecule A and B (MICA and MICB) in MM cells. The results shown in this work indicate that MM cells are responsive to LXR activation, which induces changes in the intracellular cholesterol content. These changes correlate with an enhanced expression of MICA and MICB in human MM cell lines and in primary malignant plasma cells, 2 ligands of the NK group 2D receptor (NKG2D)/CD314 activating receptor expressed in cytotoxic lymphocytes, rendering MM cells more sensitive to recognition, degranulation, and killing by NK cells. Mechanistically, we observed that LXR activation regulates MICA and MICB expression at different levels: MICA at the transcriptional level, enhancing promoter activity, and MICB by inhibiting its degradation in lysosomes. The present study provides evidence that activation of LXR, by enhancing NKG2D ligand expression, can promote NK cell-mediated cytotoxicity and suggests a novel immune-mediated mechanism involving modulation of intracellular cholesterol levels in cancer cells.-Bilotta, M. T., Abruzzese, M. P., Molfetta, R., Scarno, G., Fionda, C., Zingoni, A., Soriani, A., Garofalo, T., Petrucci, M. T., Ricciardi, M. R., Paolini, R., Santoni, A., Cippitelli, M. Activation of liver X receptor up-regulates the expression of the NKG2D ligands MICA and MICB in multiple myeloma through different molecular mechanisms.
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http://dx.doi.org/10.1096/fj.201900319RDOI Listing
August 2019

The homeobox transcription factor MEIS2 is a regulator of cancer cell survival and IMiDs activity in Multiple Myeloma: modulation by Bromodomain and Extra-Terminal (BET) protein inhibitors.

Cell Death Dis 2019 04 11;10(4):324. Epub 2019 Apr 11.

Department of Molecular Medicine, Sapienza University of Rome, Rome, Italy.

The transcription factor Myeloid Ecotropic Insertion Site 2 (MEIS2) has been identified as a cellular substrate of the E3-ubiquitin ligase complex CRL4-cereblon (CRBN) in crystal structure and by biochemical screen. Emerging evidence suggests that IMiDs can block MEIS2 from binding to CRBN facilitating the subsequent activation of a CRBN-E3-ubiquitin ligase activity and proteasome-mediated degradation of critical substrates regulators of Multiple Myeloma (MM) cell survival and proliferation. Bromodomain and Extra-Terminal (BET) family of proteins are important epigenetic regulators involved in promoting gene expression of several oncogenes, and many studies have revealed important anticancer activities mediated by BET inhibitors (BETi) in hematologic malignancies including MM. Here, we investigated MEIS2 in MM, the role of this protein as a modulator of IMiDs activity and the ability of BETi to inhibit its expression. Our observations indicate that inhibition of MEIS2 in MM cells by RNA interference correlates with reduced growth, induction of apoptosis and enhanced efficacy of different anti-MM drugs. In addition, MEIS2 regulates the expression of Cyclin E/CCNE1 in MM and induction of apoptosis after treatment with the CDK inhibitor Seliciclib/Roscovitine. Interestingly, modulation of MEIS2 can regulate the expression of NKG2D and DNAM-1 NK cell-activating ligands and, importantly, the activity of IMiDs in MM cells. Finally, BETi have the ability to inhibit the expression of MEIS2 in MM, underscoring a novel anticancer activity mediated by these drugs. Our study provides evidence on the role of MEIS2 in MM cell survival and suggests therapeutic strategies targeting of MEIS2 to enhance IMiDs anti-myeloma activity.
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http://dx.doi.org/10.1038/s41419-019-1562-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6459881PMC
April 2019

Lenalidomide in Pretreated Patients with Diffuse Large B-Cell Lymphoma: An Italian Observational Multicenter Retrospective Study in Daily Clinical Practice.

Oncologist 2019 09 2;24(9):1246-1252. Epub 2019 Apr 2.

Institute of Hematology, University of Bologna, Bologna, Italy

Background: Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma subtype, and approximately 50% of the patients are >60 years of age. Patients with relapsed/refractory (rr) disease have a poor prognosis with currently available treatments. Lenalidomide is available in Italy for patients with rrDLBCL based on a local disposition of the Italian Drug Agency.

Subjects, Materials, And Methods: An observational retrospective study was conducted in 24 Italian hematology centers with the aim to improve information on effectiveness and safety of lenalidomide use for rrDLBCL in real practice.

Results: One hundred fifty-three patients received lenalidomide for 21/28 days with a median of four cycles. At the end of therapy, there were 36 complete responses (23.5%) and 9 partial responses with an overall response rate (ORR) of 29.4%. In the elderly (>65 years) subset, the ORR was 33.6%. With a median follow-up of 36 months, median overall survival was reached at 12 months and median disease-free survival was not reached at 62 months. At the latest available follow-up, 29 patients are still in response out of therapy. Median progression-free survivals differ significantly according to age (2.5 months vs. 9.5 in the younger vs. elderly group, respectively) and to disease status at the latest previous therapy (15 months for relapsed patients vs. 3.5 for refractory subjects). Toxicities were manageable, even if 30 of them led to an early drug discontinuation.

Conclusion: Lenalidomide therapy for patients with rrDLBCL is effective and tolerable even in a real-life context, especially for elderly patients.

Implications For Practice: Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma, and approximately 50% of the patients are >60 years of age. Patients with relapsed/refractory (rr) disease have a poor prognosis, reflected by the remarkably short life expectancy of 12 months with currently available treatments. The rrDLBCL therapeutic algorithm is not so well established because data in the everyday clinical practice are still poor. Lenalidomide for patients with rrDLBCL is effective and tolerable even in a real-life context, especially for elderly patients.
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http://dx.doi.org/10.1634/theoncologist.2018-0603DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6738312PMC
September 2019

The Ubiquitin-proteasome pathway regulates Nectin2/CD112 expression and impairs NK cell recognition and killing.

Eur J Immunol 2019 06 27;49(6):873-883. Epub 2019 Mar 27.

Department of Molecular Medicine, "Sapienza" University of Rome, Laboratory affiliated to Istituto Pasteur Italia-Fondazione Cenci Bolognetti, Rome, Italy.

Nectin2 is a member of immunoglobulin-like cell adhesion molecules and plays a prominent role in the establishment of adherens and tight junctions. It is also upregulated on the surface of tumor and virus-infected cells where it functions as a ligand for the activating receptor CD226, thus contributing to cytotoxic lymphocyte-mediated recognition and killing of damaged cells. Little is currently known about the regulation of Nectin2 expression and, in particular, whether posttranscriptional and posttranslational mechanisms are involved. Here, we analyzed Nectin2 expression on a panel of human tumor cell lines and primary cultures and we found that Nectin2 is mainly expressed in cytoplasmic pools. Moreover, we demonstrated that ubiquitination of Nectin2 promotes its degradation and is responsible for protein intracellular retention. Indeed, inhibition of the ubiquitin pathway results in increased Nectin2 surface expression and enhances tumor cell susceptibility to NK cell cytotoxicity. Our results demonstrate a previously unknown mechanism of Nectin2 regulation revealing that the ubiquitin pathway represents a potential target of intervention in order to increase susceptibility to NK cell-mediated lysis.
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http://dx.doi.org/10.1002/eji.201847848DOI Listing
June 2019

Translating the anti-myeloma activity of Natural Killer cells into clinical application.

Cancer Treat Rev 2018 Nov 10;70:255-264. Epub 2018 Oct 10.

Department of Molecular Medicine, Sapienza University of Rome, Laboratory affiliated to Istituto Pasteur Italia - Fondazione Cenci Bolognetti, Rome, Italy; IRCCS NEUROMED, Pozzilli (IS), Italy.

Natural Killer cells (NK) are innate effector cells with a critical role in immunosurveillance against different kinds of cancer cells, including Multiple Myeloma (MM). However, the number and/or function of these lymphocytes are strongly reduced during MM progression and in advanced clinical stages. A better understanding of the mechanisms controlling both MM and NK cell biology have greatly contributed to develop novel and combined therapeutic strategies in the treatment of this incurable hematologic malignancy. These include approaches to reverse the immunosuppressive MM microenvironment or potentiate the natural or antibody-dependent cellular cytotoxicity (ADCC) of NK cells. Moreover, chemotherapeutic drugs or specific monoclonal antibodies (mAbs) can render cancer cells more susceptible to NK cell-mediated recognition and lysis; direct enhancement of NK cell function can be obtained by means of immunomodulatory drugs, cytokines and blocking mAbs targeting NK cell inhibitory receptors. Finally, adoptive transfer of ex-vivo expanded and genetically manipulated NK cells is also a promising therapeutic tool for MM. Here, we review current knowledge on complex mechanisms affecting NK cell activity during MM progression. We also discuss recent advances on innovative approaches aimed at boosting the functions of these cytotoxic innate lymphocytes. In particular, we focus our attention on recent preclinical and clinical studies addressing the therapeutic potential of different NK cell-based strategies for the management of MM.
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http://dx.doi.org/10.1016/j.ctrv.2018.10.005DOI Listing
November 2018

Abnormal regulation of BCR signalling by c-Cbl in chronic lymphocytic leukaemia.

Oncotarget 2018 Aug 14;9(63):32219-32231. Epub 2018 Aug 14.

Department of Medicine, Hematology and Clinical Immunology Branch, University School of Medicine, Padua, Italy.

Abnormalities of molecules involved in signal transduction pathways are connected to Chronic Lymphocytic Leukemia (CLL) pathogenesis and a critical role has been already ascribed to B-Cell Receptor (BCR)-Lyn axis. E3 ubiquitin ligase c-Cbl, working together with adapter protein CIN85, controls the degradation of protein kinases involved in BCR signaling. To investigate cell homeostasis in CLL, we studied c-Cbl since in normal B cells it is involved in the ubiquitin-dependent Lyn degradation and in the down-regulation of BCR signaling. We found that c-Cbl is overexpressed and not ubiquitinated after BCR engagement. We observed that c-Cbl did not associate to CIN85 in CLL with respect to normal B cells at steady state, nor following BCR engagement. c-Cbl association to Lyn was not detectable in CLL after BCR stimulation, as it happens in normal B cells. In some CLL patients, c-Cbl is constitutively phosphorylated at Y731 and in the same subjects, it associated to regulatory subunit p85 of PI3K. Moreover, c-Cbl is constitutive associated to Cortactin in those CLL patients presenting Cortactin overexpression and bad prognosis. These results support the hypothesis that c-Cbl, rather than E3 ligase activity, could have an adaptor function in turn influencing cell homeostasis in CLL.
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http://dx.doi.org/10.18632/oncotarget.25951DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6114956PMC
August 2018

Italian real life experience with ibrutinib: results of a large observational study on 77 relapsed/refractory mantle cell lymphoma.

Oncotarget 2018 May 4;9(34):23443-23450. Epub 2018 May 4.

Unit of Hematology, University of Padova, Padova, Italy.

Although sometimes presenting as an indolent lymphoma, mantle cell lymphoma (MCL) is an aggressive disease, hardly curable with standard chemo-immunotherapy. Current approaches have greatly improved patients' outcomes, nevertheless the disease is still characterized by high relapse rates. Before approval by EMA, Italian patients with relapsed/refractory MCL were granted ibrutinib early access through a Named Patient Program (NPP). An observational, retrospective, multicenter study was conducted. Seventy-seven heavily pretreated patients were enrolled. At the end of therapy there were 14 complete responses and 14 partial responses, leading to an overall response rate of 36.4%. At 40 months overall survival was 37.8% and progression free survival was 30%; disease free survival was 78.6% at 4 years: 11/14 patients are in continuous complete response with a median of 36 months of follow up. Hematological toxicities were manageable, and main extra-hematological toxicities were diarrhea (9.4%) and lung infections (9.0%). Overall, 4 (5.2%) atrial fibrillations and 3 (3.9%) hemorrhagic syndromes occurred. In conclusions, thrombocytopenia, diarrhea and lung infections are the relevant adverse events to be clinically focused on; regarding effectiveness, ibrutinib is confirmed to be a valid option for refractory/relapsed MCL also in a clinical setting mimicking the real world.
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http://dx.doi.org/10.18632/oncotarget.25215DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5955107PMC
May 2018

NKG2D and Its Ligands: "One for All, All for One".

Front Immunol 2018 12;9:476. Epub 2018 Mar 12.

Department of Molecular Medicine, Sapienza University of Rome, Laboratory Affiliated to Istituto Pasteur Italia-Fondazione Cenci Bolognetti, Rome, Italy.

The activating receptor NKG2D is peculiar in its capability to bind to numerous and highly diversified MHC class I-like self-molecules. These ligands are poorly expressed on normal cells but can be induced on damaged, transformed or infected cells, with the final NKG2D ligand expression resulting from multiple levels of regulation. Although redundant molecular mechanisms can converge in the regulation of all NKG2D ligands, different stimuli can induce specific cellular responses, leading to the expression of one or few ligands. A large body of evidence demonstrates that NK cell activation can be triggered by different NKG2D ligands, often expressed on the same cell, suggesting a functional redundancy of these molecules. However, since a number of evasion mechanisms can reduce membrane expression of these molecules both on virus-infected and tumor cells, the co-expression of different ligands and/or the presence of allelic forms of the same ligand guarantee NKG2D activation in various stressful conditions and cell contexts. Noteworthy, NKG2D ligands can differ in their ability to down-modulate NKG2D membrane expression in human NK cells supporting the idea that NKG2D transduces different signals upon binding various ligands. Moreover, whether proteolytically shed and exosome-associated soluble NKG2D ligands share with their membrane-bound counterparts the same ability to induce NKG2D-mediated signaling is still a matter of debate. Here, we will review recent studies on the NKG2D/NKG2D ligand biology to summarize and discuss the redundancy and/or diversity in ligand expression, regulation, and receptor specificity.
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http://dx.doi.org/10.3389/fimmu.2018.00476DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5890157PMC
June 2019

False-Positive 18F-FDG PET/CT Due to Filgrastim That Induced Extramedullary Liver Hematopoiesis in a Burkitt Lymphoma.

Clin Nucl Med 2018 Apr;43(4):e130-e131

We report a case of Burkitt lymphoma with largely extranodal disease localizations at staging. Chemotherapy was given, thus obtaining a complete metabolic response in all previous disease sites as shown at a control PET, however associated to the appearance of new focal uptake areas in the liver; these findings were confirmed at US and MRI. Chemotherapy determined also neutropenia that was treated by filgrastim, followed by a prompt and important medullary response. Liver biopsy revealed extramedullary hematopoiesis, probably filgrastim induced. Filgrastim administration may cause false-positive findings in the liver at FDG PET.
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http://dx.doi.org/10.1097/RLU.0000000000001998DOI Listing
April 2018

Innate immune activating ligand SUMOylation affects tumor cell recognition by NK cells.

Sci Rep 2017 09 5;7(1):10445. Epub 2017 Sep 5.

Department of Molecular Medicine, "Sapienza" University of Rome, Laboratory affiliated to Istituto Pasteur Italia - Fondazione Cenci Bolognetti, "Viale Regina Elena 291, 00161, Rome, Italy.

Natural Killer cells are innate lymphocytes involved in tumor immunosurveillance. They express activating receptors able to recognize self-molecules poorly expressed on healthy cells but up-regulated upon stress conditions, including transformation. Regulation of ligand expression in tumor cells mainly relays on transcriptional mechanisms, while the involvement of ubiquitin or ubiquitin-like modifiers remains largely unexplored. Here, we focused on the SUMO pathway and demonstrated that the ligand of DNAM1 activating receptor, PVR, undergoes SUMOylation in multiple myeloma. Concurrently, we found that PVR is preferentially located in intracellular compartments in human multiple myeloma cell lines and malignant plasma cells and that inhibition of the SUMO pathway promotes its translocation to the cell surface, increasing tumor cell susceptibility to NK cell-mediated cytolysis. Our findings provide the first evidence of an innate immune activating ligand regulated by SUMOylation, and confer to this modification a novel role in impairing recognition and killing of tumor cells.
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http://dx.doi.org/10.1038/s41598-017-10403-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5585267PMC
September 2017

Regulation of NKG2D-Dependent NK Cell Functions: The Yin and the Yang of Receptor Endocytosis.

Int J Mol Sci 2017 Aug 2;18(8). Epub 2017 Aug 2.

Department of Molecular Medicine, Sapienza University of Rome, Laboratory Affiliated to Istituto Pasteur Italia-Fondazione Cenci Bolognetti, Viale Regina Elena 291, 00161 Rome, Italy.

Natural-killer receptor group 2, member D (NKG2D) is a well characterized natural killer (NK) cell activating receptor that recognizes several ligands poorly expressed on healthy cells but up-regulated upon stressing stimuli in the context of cancer or viral infection. Although NKG2D ligands represent danger signals that render target cells more susceptible to NK cell lysis, accumulating evidence demonstrates that persistent exposure to ligand-expressing cells causes the decrease of NKG2D surface expression leading to a functional impairment of NKG2D-dependent NK cell functions. Upon ligand binding, NKG2D is internalized from the plasma membrane and sorted to lysosomes for degradation. However, receptor endocytosis is not only a mechanism of receptor clearance from the cell surface, but is also required for the proper activation of signalling events leading to the functional program of NK cells. This review is aimed at providing a summary of current literature relevant to the molecular mechanisms leading to NKG2D down-modulation with particular emphasis given to the role of NKG2D endocytosis in both receptor degradation and signal propagation. Examples of chronic ligand-induced down-regulation of NK cell activating receptors other than NKG2D, including natural cytotoxicity receptors (NCRs), DNAX accessory molecule-1 (DNAM1) and CD16, will be also discussed.
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http://dx.doi.org/10.3390/ijms18081677DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5578067PMC
August 2017

Targeted therapy in severe asthma today: focus on immunoglobulin E.

Drug Des Devel Ther 2017 29;11:1979-1987. Epub 2017 Jun 29.

Cardio Thoracic and Vascular Department, Pathophysiology Unit, University of Pisa, Pisa, Italy.

Asthma is a complex chronic inflammatory disease of multifactorial etiology. International guidelines increasingly recognize that a standard "one size fits all" approach is no longer an effective approach to achieve optimal treatment outcomes, and a number of disease phenotypes have been proposed for asthma, which has the potential to guide treatment decisions. Among the many asthma phenotypes, allergic asthma represents the widest and most easily recognized asthma phenotype, present in up to two-thirds of adults with asthma. Immunoglobulin E (IgE) production is the primary and key cause of allergic asthma leading to persistent symptoms, exacerbations and a poor quality of life. Therefore, limiting IgE activity upstream could stop the entire allergic inflammation cascade in IgE-mediated allergic asthma. The anti-IgE treatment omalizumab has an accepted place in the management of severe asthma (Global Initiative for Asthma [GINA] step 5) and represents the first (and, currently, only) targeted therapy with a specific target in severe allergic asthma. This review summarizes current knowledge of the mechanisms and pathogenesis of severe asthma, examines the actual role of IgE in asthma and the biological rationale for targeting IgE in allergic asthma and reviews the data for the efficacy and safety of omalizumab in the treatment of severe asthma. Current knowledge of the role of IgE in asthma, extensive clinical trial data and a decade of use in clinical practice has established omalizumab as a safe and effective targeted therapy for the treatment of patients with severe persistent IgE-mediated allergic asthma.
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http://dx.doi.org/10.2147/DDDT.S130743DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5500555PMC
April 2018

Epstein-Barr Virus-Positive Mucocutaneous Ulcer Mimicking Rectal Carcinoma at 18F-FDG PET/CT.

Clin Nucl Med 2017 Aug;42(8):645-646

From the Departments of *Nuclear Medicine and PET, †Hematology, ‡Pathology, and §Radiation Oncology, Santa Maria della Misericordia Hospital, Rovigo, Italy; and ∥Department of Radiology, University of Southern California, Los Angeles, CA.

We report focally intense F-FDG PET/CT rectal activity (SUVmax = 25) with a horseshoe distribution in an 81-year-old man with B-cell chronic lymphocytic leukemia and suspected Richter transformation. While imaging findings were typical for rectal adenocarcinoma, histology revealed Epstein-Barr virus-positive mucocutaneous ulcer.
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http://dx.doi.org/10.1097/RLU.0000000000001725DOI Listing
August 2017

3D Microfluidic model for evaluating immunotherapy efficacy by tracking dendritic cell behaviour toward tumor cells.

Sci Rep 2017 04 24;7(1):1093. Epub 2017 Apr 24.

Department of Oncology and Molecular Medicine, Istituto Superiore di Sanità, 00161, Rome, Italy.

Immunotherapy efficacy relies on the crosstalk within the tumor microenvironment between cancer and dendritic cells (DCs) resulting in the induction of a potent and effective antitumor response. DCs have the specific role of recognizing cancer cells, taking up tumor antigens (Ags) and then migrating to lymph nodes for Ag (cross)-presentation to naïve T cells. Interferon-α-conditioned DCs (IFN-DCs) exhibit marked phagocytic activity and the special ability of inducing Ag-specific T-cell response. Here, we have developed a novel microfluidic platform recreating tightly interconnected cancer and immune systems with specific 3D environmental properties, for tracking human DC behaviour toward tumor cells. By combining our microfluidic platform with advanced microscopy and a revised cell tracking analysis algorithm, it was possible to evaluate the guided efficient motion of IFN-DCs toward drug-treated cancer cells and the succeeding phagocytosis events. Overall, this platform allowed the dissection of IFN-DC-cancer cell interactions within 3D tumor spaces, with the discovery of major underlying factors such as CXCR4 involvement and underscored its potential as an innovative tool to assess the efficacy of immunotherapeutic approaches.
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http://dx.doi.org/10.1038/s41598-017-01013-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5430848PMC
April 2017

Obinutuzumab-mediated high-affinity ligation of FcγRIIIA/CD16 primes NK cells for IFNγ production.

Oncoimmunology 2017;6(3):e1290037. Epub 2017 Feb 10.

Department of Experimental Medicine, Laboratorio Pasteur Italia Fondazione Cenci Bolognetti, Sapienza University , Rome, Italy.

Natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC), based on the recognition of IgG-opsonized targets by the low-affinity receptor for IgG FcγRIIIA/CD16, represents one of the main mechanisms by which therapeutic antibodies (mAbs) mediate their antitumor effects. Besides ADCC, CD16 ligation also results in cytokine production, in particular, NK-derived IFNγ is endowed with a well-recognized role in the shaping of adaptive immune responses. Obinutuzumab is a glycoengineered anti-CD20 mAb with a modified crystallizable fragment (Fc) domain designed to increase the affinity for CD16 and consequently the killing of mAb-opsonized targets. However, the impact of CD16 ligation in optimized affinity conditions on NK functional program is not completely understood. Herein, we demonstrate that the interaction of NK cells with obinutuzumab-opsonized cells results in enhanced IFNγ production as compared with parental non-glycoengineered mAb or the reference molecule rituximab. We observed that affinity ligation conditions strictly correlate with the ability to induce CD16 down-modulation and lysosomal targeting of receptor-associated signaling elements. Indeed, a preferential degradation of FcεRIγ chain and Syk kinase was observed upon obinutuzumab stimulation independently from CD16-V158F polymorphism. Although the downregulation of FcεRIγ/Syk module leads to the impairment of cytotoxic function induced by NKp46 and NKp30 receptors, obinutuzumab-experienced cells exhibit an increased ability to produce IFNγ in response to different stimuli. These data highlight a relationship between CD16 aggregation conditions and the ability to promote a degradative pathway of CD16-coupled signaling elements associated to the shift of NK functional program.
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http://dx.doi.org/10.1080/2162402X.2017.1290037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5384385PMC
February 2017

Genotoxic stress modulates the release of exosomes from multiple myeloma cells capable of activating NK cell cytokine production: Role of HSP70/TLR2/NF-kB axis.

Oncoimmunology 2017;6(3):e1279372. Epub 2017 Jan 13.

Department of Molecular Medicine - Pasteur Italia Laboratory, Sapienza University of Rome , Rome, Italy.

Exosomes are a class of nanovesicles formed and released through the late endosomal compartment and represent an important mode of intercellular communication. The ability of anticancer chemotherapy to enhance the immunogenic potential of malignant cells mainly relies on the establishment of the immunogenic cell death (ICD) and the release of damage-associated molecular patterns (DAMPs). Here, we investigated whether genotoxic stress could promote the release of exosomes from multiple myeloma (MM) cells and studied the immunomodulatory properties they exert on NK cells, a major component of the antitumor immune response playing a key role in the immunosurveillance of MM. Our findings show that melphalan, a genotoxic agent used in MM therapy, significantly induces an increased exosome release from MM cells. MM cell-derived exosomes are capable of stimulating IFNγ production, but not the cytotoxic activity of NK cells through a mechanism based on the activation of NF-κB pathway in a TLR2/HSP70-dependent manner. Interestingly, HSP70 exosomes are primarily found in the bone marrow (BM) of MM patients suggesting that they might have a crucial immunomodulatory action in the tumor microenvironment. We also provide evidence that the CD56 NK cell subset is more responsive to exosome-induced IFNγ production mediated by TLR2 engagement. All together, these findings suggest a novel mechanism of synergism between chemotherapy and antitumor innate immune responses based on the drug-promotion of nanovesicles exposing DAMPs for innate receptors.
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http://dx.doi.org/10.1080/2162402X.2017.1279372DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5384384PMC
January 2017

Plasma matrix metalloprotease 9 correlates with blood lymphocytosis, leukemic cell invasiveness, and prognosis in B-cell chronic lymphocytic leukemia.

Tumour Biol 2017 Feb;39(2):1010428317694325

2 Department of Medicine, Azienda ULSS 18 Rovigo, Rovigo, Italy.

The complex biology underlying chronic lymphocytic leukemia cell migration and tissue invasiveness is not yet completely understood and might provide novel predictive markers and therapeutic targets. A total of 36 patients out of treatment from at least 3 months were enrolled and followed up for a median period of 44.2 months (range: 4.4-99.2). Matrix metalloprotease 9 and tissue inhibitor of metalloproteases 1 plasma levels and production/release from lymphoid cells were measured by zymography and enzyme-linked immunosorbent assay (ELISA) analysis. Malignant and normal lymphocyte mobility and matrix-degradation capability were studied using a Boyden chamber system, with and without autologous plasma. Free matrix metalloprotease 9 plasma levels were related with blood lymphocytosis, especially in more advanced stages (p = 0.003), and higher concentrations were associated with an increased disease progression risk (hazard ratio = 9.0, 95% confidence interval = 1.5-13.8). Leukemic cells expressed and secreted very little matrix metalloprotease 9. On the contrary, normal lymphocytes derived from the same leukemic patients showed matrix metalloprotease 9 intracellular levels that were lower in subjects with higher blood lymphocytosis (p = 0.024) and more advanced stages (p = 0.03); the released quantities were inversely associated with matrix metalloprotease 9 plasma concentrations (p = 0.035). Leukemic cells had a reduced spontaneous mobility and matrix-degradation capability that were stimulated by autologous plasma (p = 0.001) and normal lymphocytes (p = 0.005), respectively. Matrix metalloprotease 9 affected cell invasiveness depending on concentration and disease stage. In conclusion, chronic lymphocytic leukemia cells have a reduced mobility, matrix-degradation capability, and matrix metalloprotease 9 production compared to their own autologous normal lymphocytes. They are exposed to matrix metalloprotease 9 of prevalently systemic origin whose higher levels are associated with both leukemic and normal lymphocyte accumulation in the peripheral blood and have a negative prognostic value.
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http://dx.doi.org/10.1177/1010428317694325DOI Listing
February 2017

p38 MAPK differentially controls NK activating ligands at transcriptional and post-transcriptional level on multiple myeloma cells.

Oncoimmunology 2017;6(1):e1264564. Epub 2016 Dec 2.

Department of Molecular Medicine-Istituto Pasteur Italia Fondazione Cenci Bolognetti, Sapienza University of Rome, Rome, Italy; Neuromed I.R.C.C.S.-Istituto Neurologico Mediterraneo, Pozzilli (IS), Italy.

The mechanisms that regulate the expression of the NKG2D and DNAM-1 activating ligands are only partially known, but it is now widely established that their expression is finely regulated at transcriptional, post-transcriptional and post-translational level, and involve numerous stress pathways depending on the type of ligand, stressor, and cell context. We show that treatment of Multiple Myeloma (MM) cells with sub-lethal doses of Vincristine (VCR), an anticancer drug that inhibits the assembly of microtubules, stimulates the expression of NKG2D and DNAM-1 activating ligands, rendering these cells more susceptible to NK cell-mediated killing. Herein, we focused our attention on the identification of the signaling pathways leading to surface expression of ULBP-1, and to MICA and PVR upregulation on VCR-treated MM cells, both at protein and mRNA levels. We found that p38MAPK differentially regulates drug-dependent ligand upregulation at transcriptional and post-transcriptional level. More specifically, we observed that ULBP-1 expression is attributable to both increased transcriptional activity mediated by ATM-dependent p53 activation, and enhanced mRNA stability; while the p38-activated E2F1 transcription factor regulates MICA and PVR mRNA expression. All together, our findings reveal a previously unrecognized activity of VCR as anticancer agent, and indicate that in addition to its established ability to arrest cell growth, VCR can also modulate the expression of NKG2D and DNAM-1 activating ligand on tumor cells and thus promoting NK cell-mediated immunosurveillance.
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http://dx.doi.org/10.1080/2162402X.2016.1264564DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5283620PMC
December 2016

Antiplatelet therapy in patients with glucose-6-phosphate dehydrogenases deficiency after percutaneous coronary intervention: A reappraisal for clinical and interventional cardiologists.

Cardiovasc Revasc Med 2017 Apr - May;18(3):226-229. Epub 2016 Nov 28.

Department of Cardiology, Rovigo General Hospital, Rovigo, Italy. Electronic address:

Glucose-6-phosphate dehydrogenase (G6PD) deficiency represents one of the most common erythrocyte enzymopathy. In the era of drug-eluting stents (DESs), the use of prolonged dual antiplatelet therapy (DAPT) with aspirin (ASA) and thienopyridine (clopidogrel or ticlopidine) has become mandatory in the treatment of patients with acute coronary syndromes (ACS) and/or after percutaneous coronary intervention (PCI). However, the use of ASA, and more in general of antiplatelet drugs in patients with G6PD deficiency remains controversial, also for the absence of specific guidelines and scientific evidences. In the present manuscript, we reviewed the few cases available in medical literature, regarding patients with G6PD deficiency treated with percutaneous coronary artery intervention (PCI) and DAPT, with the aim to discuss and clarify the optimal treatment in these patients.
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http://dx.doi.org/10.1016/j.carrev.2016.11.011DOI Listing
December 2017

Inhibition of bromodomain and extra-terminal (BET) proteins increases NKG2D ligand MICA expression and sensitivity to NK cell-mediated cytotoxicity in multiple myeloma cells: role of cMYC-IRF4-miR-125b interplay.

J Hematol Oncol 2016 12 1;9(1):134. Epub 2016 Dec 1.

Department of Molecular Medicine - Pasteur Italia Laboratory, Sapienza University of Rome, Viale Regina Elena 291, 00161, Rome, Italy.

Background: Anti-cancer immune responses may contribute to the control of tumors after conventional chemotherapy, and different observations have indicated that chemotherapeutic agents can induce immune responses resulting in cancer cell death and immune-stimulatory side effects. Increasing experimental and clinical evidence highlight the importance of natural killer (NK) cells in immune responses toward multiple myeloma (MM), and combination therapies able to enhance the activity of NK cells against MM are showing promise in treating this hematologic cancer. The epigenetic readers of acetylated histones bromodomain and extra-terminal (BET) proteins are critical regulators of gene expression. In cancer, they can upregulate transcription of key oncogenes such as cMYC, IRF4, and BCL-2. In addition, the activity of these proteins can regulate the expression of osteoclastogenic cytokines during cancer progression. Here, we investigated the effect of BET bromodomain protein inhibition, on the expression of NK cell-activating ligands in MM cells.

Methods: Five MM cell lines [SKO-007(J3), U266, RPMI-8226, ARP-1, JJN3] and CD138 MM cells isolated from MM patients were used to investigate the activity of BET bromodomain inhibitors (BETi) (JQ1 and I-BET151) and of the selective BRD4-degrader proteolysis targeting chimera (PROTAC) (ARV-825), on the expression and function of several NK cell-activating ligands (NKG2DLs and DNAM-1Ls), using flow cytometry, real-time PCR, transient transfections, and degranulation assays.

Results: Our results indicate that inhibition of BET proteins via small molecule inhibitors or their degradation via a hetero-bifunctional PROTAC probe can enhance the expression of MICA, a ligand of the NKG2D receptor, in human MM cell lines and primary malignant plasma cells, rendering myeloma cells more efficient to activate NK cell degranulation. Noteworthy, similar results were obtained using selective CBP/EP300 bromodomain inhibition. Mechanistically, we found that BETi-mediated inhibition of cMYC correlates with the upregulation of miR-125b-5p and the downregulation of the cMYC/miR-125b-5p target gene IRF4, a transcriptional repressor of MICA.

Conclusions: These findings provide new insights on the immuno-mediated antitumor activities of BETi and further elucidate the molecular mechanisms that regulate NK cell-activating ligand expression in MM.
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http://dx.doi.org/10.1186/s13045-016-0362-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5131470PMC
December 2016

Regulation of NKG2D Expression and Signaling by Endocytosis.

Trends Immunol 2016 11 22;37(11):790-802. Epub 2016 Sep 22.

Department of Molecular Medicine, Institute Pasteur-Fondazione Cenci Bolognetti, 'Sapienza' University of Rome, 00161, Rome, Italy. Electronic address:

NKG2D is an activating receptor that can bind to a large number of stress-induced ligands that are expressed in the context of cancer or viral infection. This receptor is expressed on many cytotoxic lymphocytes, and plays a crucial role in antitumor and antiviral immune responses. However, exposure to NKG2D ligand-expressing target cells promotes receptor endocytosis, ultimately leading to lysosomal receptor degradation and impairment of NKG2D-mediated functions. Interestingly, before being degraded, internalized receptors can signal from the endosomal compartment, leading to the appropriate activation of cellular functional programs. This review summarizes recent findings on ligand-induced receptor internalization, with particular emphasis on the role of endocytosis in the control of both NKG2D-mediated intracellular signaling and receptor degradation.
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http://dx.doi.org/10.1016/j.it.2016.08.015DOI Listing
November 2016