Publications by authors named "Paolo Sportoletti"

59 Publications

Comparison of ibrutinib and idelalisib plus rituximab in real-life relapsed/resistant chronic lymphocytic leukemia cases.

Eur J Haematol 2020 Dec 30. Epub 2020 Dec 30.

Biothecnology Research Unit, AO of Cosenza, Cosenza, Italy.

Objectives: To compare the capacity of ibrutinib (IB) and idelalisib-rituximab (IDELA-R) of prolonging overall survival (OS) as in CLL patients, previously treated with chemotherapy only.

Methods: A real-life cohort of 675 cases has been identified and investigated in the database of the groups participating in the study.

Results: At an unadjusted univariate analysis, a significant death risk reduction was observed favoring IB (IDELA-R vs IB HR = 0.5, 95% CI = 0.36-0.71) although with some limitations due to the non-randomized and retrospective nature of the study and to the lower number of patients in the IDELA-R group (112 cases) related to the current prescribing practice. To overcome the potential problem of confounding by indication, we adjusted the association between the type of therapy and mortality for all variables significantly associated with OS at Cox univariate analysis. Furthermore, those variables, differently distributed between the two study groups, were introduced into the multivariate Cox model to improve the effectiveness of the analysis. By introducing all these variables into the multiple Cox regression model, we confirmed the protective effect of IB vs IDELA-R (HR = 0.67, 95% CI = 0.45-0.98, P = .04) independent of potential confounders.

Conclusions: Although our analysis presents some constraints, that is, the unavailability of additional potential confounders, and the retrospective nature of the study, this observation may be of help for the daily clinical practice, particularly in the absence of randomized trials comparing the two schedules.
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http://dx.doi.org/10.1111/ejh.13573DOI Listing
December 2020

Bcor deficiency perturbs erythro-megakaryopoiesis and cooperates with Dnmt3a loss in acute erythroid leukemia onset in mice.

Leukemia 2020 Nov 6. Epub 2020 Nov 6.

Centro di Ricerca Emato-Oncologica (CREO), University of Perugia, Perugia, 06132, Italy.

Recurrent loss-of-function mutations of BCL6 co-repressor (BCOR) gene are found in about 4% of AML patients with normal karyotype and are associated with DNMT3a mutations and poor prognosis. Therefore, new anti-leukemia treatments and mouse models are needed for this combinatorial AML genotype. For this purpose, we first generated a Bcor knockout mouse model characterized by impaired erythroid development (macrocytosis and anemia) and enhanced thrombopoiesis, which are both features of myelodysplasia/myeloproliferative neoplasms. We then created and characterized double Bcor/Dnmt3a knockout mice. Interestingly, these animals developed a fully penetrant acute erythroid leukemia (AEL) characterized by leukocytosis secondary to the expansion of blasts expressing c-Kit+ and the erythroid marker Ter119, macrocytic anemia and progressive reduction of the thrombocytosis associated with loss of Bcor alone. Transcriptomic analysis of double knockout bone marrow progenitors revealed that aberrant erythroid skewing was induced by epigenetic changes affecting specific transcriptional factors (GATA1-2) and cell-cycle regulators (Mdm2, Tp53). These findings prompted us to investigate the efficacy of demethylating agents in AEL, with significant impact on progressive leukemic burden and mice overall survival. Information gained from our model expands the knowledge on the biology of AEL and may help designing new rational treatments for patients suffering from this high-risk leukemia.
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http://dx.doi.org/10.1038/s41375-020-01075-3DOI Listing
November 2020

Clinical-Grade Expanded Regulatory T Cells Are Enriched with Highly Suppressive Cells Producing IL-10, Granzyme B, and IL-35.

Biol Blood Marrow Transplant 2020 12 19;26(12):2204-2210. Epub 2020 Sep 19.

Department of Medicine and Aging Sciences, University of Chieti-Pescara, Pescara, Italy; Department of Oncology Hematology, Pescara Hospital, Pescara, Italy. Electronic address:

In the setting of T cell-depleted, full-haplotype mismatched transplantation, adoptive immunotherapy with regulatory T cells (Tregs) and conventional T cells (Tcons) can prevent graft-versus-host disease (GVHD) and improve post-transplantation immunologic reconstitution and is associated with a powerful graft-versus-leukemia effect. To improve the purity and the quantity of the infused Tregs, good manufacturing practices (GMP)-compatible expansion protocols are needed. Here we expanded Tregs using an automated, clinical-grade protocol. Cells were extensively characterized in vitro, and their efficiency was tested in vivo in a mouse model. Tregs were selected by CliniMacs (CD4CD25, 94.5 ± 6.3%; FoxP3, 63.7 ± 11.5%; CD127, 20 ± 3%; suppressive activity, 60 ± 7%), and an aliquot of 100 × 10 was expanded for 14 days using the CliniMACS Prodigy System, obtaining 684 ± 279 × 10 cells (CD4CD25, 99.6 ± 0.2%; FoxP3, 82 ± 8%; CD127, 1.1 ± 0.8%; suppressive activity, 75 ± 12%). CD39 and CTLA4 expression levels increased from 22.4 ± 12% to 58.1 ± 13.3% (P < .05) and from 20.4 ± 6.7% to 85.4 ± 9.8% (P < .01), respectively. TIM3 levels increased from .4 ± .05% to 29 ± 16% (P < .05). Memory Tregs were the prevalent population, whereas naive Tregs almost disappeared at the end of the culture. mRNA analysis displayed significant increases in CD39, IL-10, granzyme B, and IL-35 levels at the end of culture period (P < .05). Conversely, IFNγ expression decreased significantly by day +14. Expanded Tregs were sorted according to TIM3, CD39, and CD62L expression levels (purity >95%). When sorted populations were analyzed, TIM3 cells showed significant increases in IL-10 and granzyme B (P < .01) .When expanded Tregs were infused in an NSG murine model, mice that received Tcons only died of GVHD, whereas mice that received both Tcons and Tregs survived without GVHD. GMP grade expanded cells that display phenotypic and functional Treg characteristics can be obtained using a fully automated system. Treg suppression is mediated by multiple overlapping mechanisms (eg, CTLA-4, CD39, IL-10, IL-35, TGF-β, granzyme B). TIM3 cells emerge as a potentially highly suppressive population. © 2020 American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc.
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http://dx.doi.org/10.1016/j.bbmt.2020.08.034DOI Listing
December 2020

COVID-19 severity and mortality in patients with chronic lymphocytic leukemia: a joint study by ERIC, the European Research Initiative on CLL, and CLL Campus.

Leukemia 2020 09 9;34(9):2354-2363. Epub 2020 Jul 9.

Università Vita-Salute San Raffaele and IRCC Ospedale San Raffaele, Milan, Italy.

Chronic lymphocytic leukemia (CLL) is a disease of the elderly, characterized by immunodeficiency. Hence, patients with CLL might be considered more susceptible to severe complications from COVID-19. We undertook this retrospective international multicenter study to characterize the course of COVID-19 in patients with CLL and identify potential predictors of outcome. Of 190 patients with CLL and confirmed COVID-19 diagnosed between 28/03/2020 and 22/05/2020, 151 (79%) presented with severe COVID-19 (need of oxygen and/or intensive care admission). Severe COVID-19 was associated with more advanced age (≥65 years) (odds ratio 3.72 [95% CI 1.79-7.71]). Only 60 patients (39.7%) with severe COVID-19 were receiving or had recent (≤12 months) treatment for CLL at the time of COVID-19 versus 30/39 (76.9%) patients with mild disease. Hospitalization rate for severe COVID-19 was lower (p < 0.05) for patients on ibrutinib versus those on other regimens or off treatment. Of 151 patients with severe disease, 55 (36.4%) succumbed versus only 1/38 (2.6%) with mild disease; age and comorbidities did not impact on mortality. In CLL, (1) COVID-19 severity increases with age; (2) antileukemic treatment (particularly BTK inhibitors) appears to exert a protective effect; (3) age and comorbidities did not impact on mortality, alluding to a relevant role of CLL and immunodeficiency.
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http://dx.doi.org/10.1038/s41375-020-0959-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7347048PMC
September 2020

NPM1-mutated acute myeloid leukemia: from bench to bedside.

Blood 2020 Oct;136(15):1707-1721

Institute of Hematology, Centro Ricerche Emato-Oncologiche, Ospedale S. Maria della Misericordia, University of Perugia, Perugia, Italy.

The nucleophosmin (NPM1) gene encodes for a multifunctional protein with prominent nucleolar localization that shuttles between nucleus and cytoplasm. NPM1 mutations represent the most common genetic lesion in adult acute myeloid leukemia (AML; about one third of cases), and they act deterministically to cause the aberrant cytoplasmic delocalization of NPM1 mutants. Because of its unique features, NPM1-mutated AML is recognized as a distinct entity in the 2017 World Health Organization (WHO) classification of hematopoietic neoplasms. Here, we focus on recently identified functions of wild-type NPM1 in the nucleolus and address new biological and clinical issues related to NPM1-mutated AML. The relevance of the cooperation between NPM1 and other mutations in driving AML with different outcomes is presented. We also discuss the importance of eradicating NPM1-mutated clones to achieve AML cure and the impact of preleukemic clonal hematopoiesis persistence in predisposing to second AML. The contribution of HOX genes' expression to the development of NPM1-mutated AML is also highlighted. Clinically, yet unsolved diagnostic issues in the 2017 WHO classification of myeloid neoplasms and the importance of NPM1 mutations in defining the framework of European LeukemiaNet genetic-based risk stratification are discussed. Finally, we address the value and limits of NPM1-based measurable residual disease assessment for treatment guidance and present the results of promising preclinical studies with XPO1 and menin-MLL inhibitors.
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http://dx.doi.org/10.1182/blood.2019004226DOI Listing
October 2020

Blockade of Oncogenic NOTCH1 with the SERCA Inhibitor CAD204520 in T Cell Acute Lymphoblastic Leukemia.

Cell Chem Biol 2020 Jun 7;27(6):678-697.e13. Epub 2020 May 7.

University of Parma, Department of Medicine and Surgery, Parma 43126, Italy. Electronic address:

The identification of SERCA (sarco/endoplasmic reticulum calcium ATPase) as a target for modulating gain-of-function NOTCH1 mutations in Notch-dependent cancers has spurred the development of this compound class for cancer therapeutics. Despite the innate toxicity challenge associated with SERCA inhibition, we identified CAD204520, a small molecule with better drug-like properties and reduced off-target Ca toxicity compared with the SERCA inhibitor thapsigargin. In this work, we describe the properties and complex structure of CAD204520 and show that CAD204520 preferentially targets mutated over wild-type NOTCH1 proteins in T cell acute lymphoblastic leukemia (T-ALL) and mantle cell lymphoma (MCL). Uniquely among SERCA inhibitors, CAD204520 suppresses NOTCH1-mutated leukemic cells in a T-ALL xenografted model without causing cardiac toxicity. This study supports the development of SERCA inhibitors for Notch-dependent cancers and extends their application to cases with isolated mutations in the PEST degradation domain of NOTCH1, such as MCL or chronic lymphocytic leukemia (CLL).
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http://dx.doi.org/10.1016/j.chembiol.2020.04.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7305996PMC
June 2020

Frontline treatment with the combination obinutuzumab ± chlorambucil for chronic lymphocytic leukemia outside clinical trials: Results of a multinational, multicenter study by ERIC and the Israeli CLL study group.

Am J Hematol 2020 06 14;95(6):604-611. Epub 2020 Mar 14.

Division of Experimental Oncology, IRCCS Ospedale San Raffaele, Milan, Italy.

In recent years, considerable progress has been made in frontline therapy for elderly/physically unfit patients with CLL. The combination of obinutuzumab and chlorambucil (O-Clb) has been shown to prolong progression free survival (PFS, median PFS-31.5 months) and overall survival (OS) compared to chlorambucil alone. More recently, obinutuzumab given in combination with either ibrutinib or venetoclax improved PFS but not OS when compared to O-Clb. In this retrospective multinational, multicenter co-operative study, we evaluated the efficacy and safety of frontline treatment with O ± Clb in unfit patients with CLL, in a "real-world" setting. Patients with documented del (17p13.1)/TP53 mutation were excluded. A total of 437 patients (median age, 75.9 years; median CIRS score, 8; median creatinine clearance, 61.1 mL/min) were included. The clinical overall response rate was 80.3% (clinical complete and partial responses in 38.7% and 41.6% of patients, respectively). Median observation time was 14.1 months and estimated median PFS was 27.6 months (95% CI, 24.2-31.0). In a multivariate analysis, high-risk disease [del (11q22.3) and/or IGHV-unmutated], lymph nodes of diameter > 5 cm, obinutuzumab monotherapy and reduced cumulative dose of obinutuzumab, were all independently associated with shorter PFS. The median OS has not yet been reached and estimated 2-year OS is 88%. In conclusion, in a "real-world" setting, frontline treatment with O-Clb achieves PFS comparable to that reported in clinical trials. Inferior outcomes were noted in patients with del (11q22.3) and/or unmutated IGHV and those treated with obinutuzumab-monotherapy. Thus, O-Clb can be still considered as legitimate frontline therapy for unfit CLL patients with low-risk disease.
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http://dx.doi.org/10.1002/ajh.25766DOI Listing
June 2020

Decreased NOTCH1 Activation Correlates with Response to Ibrutinib in Chronic Lymphocytic Leukemia.

Clin Cancer Res 2019 12 2;25(24):7540-7553. Epub 2019 Oct 2.

Institute of Hematology-Centro di Ricerca Emato-Oncologica (CREO), University of Perugia, Perugia, Italy.

Purpose: Ibrutinib, a Bruton tyrosine kinase inhibitor (BTKi), has improved the outcomes of chronic lymphocytic leukemia (CLL), but primary resistance or relapse are issues of increasing significance. While the predominant mechanism of action of BTKi is the B-cell receptor (BCR) blockade, many off-target effects are unknown. We investigated potential interactions between BCR pathway and NOTCH1 activity in ibrutinib-treated CLL to identify new mechanisms of therapy resistance and markers to monitor disease response.

Experimental Design: NOTCH activations was evaluated either and in CLL samples after ibrutinib treatment by Western blotting. Confocal proximity ligation assay (PLA) experiments and analyses of down-targets of NOTCH1 by qRT-PCR were used to investigate the cross-talk between BTK and NOTCH1.

Results: ibrutinib treatment of CLL significantly reduced activated NOTCH1/2 and induced dephosphorylation of eIF4E, a NOTCH target in CLL. BCR stimulation increased the expression of activated NOTCH1 that accumulated in the nucleus leading to HES1, DTX1, and c-MYC transcription. Results of PLA experiments revealed the presence of NOTCH1-ICD/BTK complexes, whose number was reduced after ibrutinib treatment. In ibrutinib-treated CLL patients, leukemic cells showed NOTCH1 activity downregulation that deepened over time. The NOTCH1 signaling was restored at relapse and remained activated in ibrutinib-resistant CLL cells.

Conclusions: We demonstrated a strong clinical activity of ibrutinib in a real-life context. The ibrutinib clinical efficacy was associated with NOTCH1 activity downregulation that deepened over time. Our data point to NOTCH1 as a new molecular partner in BCR signaling with potential to further improve CLL-targeted treatments.
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http://dx.doi.org/10.1158/1078-0432.CCR-19-1009DOI Listing
December 2019

Germline NPM1 mutations lead to altered rRNA 2'-O-methylation and cause dyskeratosis congenita.

Nat Genet 2019 10 30;51(10):1518-1529. Epub 2019 Sep 30.

Cancer Research Institute, Beth Israel Deaconess Cancer Center, Department of Medicine and Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.

RNA modifications are emerging as key determinants of gene expression. However, compelling genetic demonstrations of their relevance to human disease are lacking. Here, we link ribosomal RNA 2'-O-methylation (2'-O-Me) to the etiology of dyskeratosis congenita. We identify nucleophosmin (NPM1) as an essential regulator of 2'-O-Me on rRNA by directly binding C/D box small nucleolar RNAs, thereby modulating translation. We demonstrate the importance of 2'-O-Me-regulated translation for cellular growth, differentiation and hematopoietic stem cell maintenance, and show that Npm1 inactivation in adult hematopoietic stem cells results in bone marrow failure. We identify NPM1 germline mutations in patients with dyskeratosis congenita presenting with bone marrow failure and demonstrate that they are deficient in small nucleolar RNA binding. Mice harboring a dyskeratosis congenita germline Npm1 mutation recapitulate both hematological and nonhematological features of dyskeratosis congenita. Thus, our findings indicate that impaired 2'-O-Me can be etiological to human disease.
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http://dx.doi.org/10.1038/s41588-019-0502-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6858547PMC
October 2019

IL-4-dependent Jagged1 expression/processing is associated with survival of chronic lymphocytic leukemia cells but not with Notch activation.

Cell Death Dis 2018 11 26;9(12):1160. Epub 2018 Nov 26.

Department of Experimental Medicine, Biosciences and Medical Embryology Section, University of Perugia, Perugia, Italy.

As previously reported, chronic lymphocytic leukemia (CLL) cells show constitutive Notch1/2 activation and express the Notchligand Jagged1. Despite increasing knowledge of the impact of Notch alterations on CLL biology and pathogenesis, the role of Jagged1 expressed in CLL cells remains undefined. In other cell types, it has been shown that after Notch engagement, Jagged1 not only activates Notch in signal-receiving cell, but also undergoes proteolytic activation in signal-sending cell, triggering a signaling with biological effects. We investigated whether Jagged1 expressed in CLL cells undergoes proteolytic processing and/or is able to induce Notch activation through autocrine/paracrine loops, focusing on the effect that CLL prosurvival factor IL-4 could exert on the Notch-Jagged1 system in these cells. We found that Jagged1 was constitutively processed in CLL cells and generated an intracellular fragment that translocated into the nucleus, and an extracellular fragment released into the culture supernatant. IL-4 enhanced expression of Jagged1 and its intracellular fragments, as well as Notch1/2 activation. The IL-4-induced increase in Notch1/2 activation was independent of the concomitant upregulated Jagged1 levels. Indeed, blocking Notch-Jagged1 interactions among CLL cells with Jagged1 neutralizing antibodies did not affect the expression of the Notch target Hes1. Notably, anti-Jagged1 antibodies partially prevented the IL-4-induced increase in Jagged1 processing and cell viability, suggesting that Jagged1 processing is one of the events contributing to IL-4-induced CLL cell survival. Consistent with this, Jagged1 silencing by small interfering RNA partially counteracted the capacity of IL-4 to promote CLL cell survival. Investigating the pathways whereby IL-4 promoted Notch1/2 activation in CLL cells independent of Jagged1, we found that PI3Kδ/AKT and PKCδ were involved in upregulating Notch1 and Notch2 proteins, respectively. Overall, this study provides new insights into the Notch-ligand system in CLL cells and suggests that targeting this system may be exploited as a novel/additional therapy approach for CLL.
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http://dx.doi.org/10.1038/s41419-018-1185-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6255763PMC
November 2018

Uncommon lymphoplasmacytic lymphoma with IgA paraproteinemia: a challenging clinical diagnosis solved by MYD88 mutation analysis.

Ann Hematol 2019 Jun 13;98(6):1507-1508. Epub 2018 Nov 13.

Hematology and Clinical Immunology, University of Perugia, Perugia, Italy.

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http://dx.doi.org/10.1007/s00277-018-3545-9DOI Listing
June 2019

Mutant NPM1 Maintains the Leukemic State through HOX Expression.

Cancer Cell 2018 09;34(3):499-512.e9

Stem Cell and Regenerative Medicine, Baylor College of Medicine, Houston, TX 77030, USA; Center for Cell and Gene Therapy, Texas Children's Hospital and Houston Methodist Hospital, Baylor College of Medicine, Houston, TX 77030, USA; Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA; Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030, USA; Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02215, USA. Electronic address:

NPM1 is the most frequently mutated gene in cytogenetically normal acute myeloid leukemia (AML). In AML cells, NPM1 mutations result in abnormal cytoplasmic localization of the mutant protein (NPM1c); however, it is unknown whether NPM1c is required to maintain the leukemic state. Here, we show that loss of NPM1c from the cytoplasm, either through nuclear relocalization or targeted degradation, results in immediate downregulation of homeobox (HOX) genes followed by differentiation. Finally, we show that XPO1 inhibition relocalizes NPM1c to the nucleus, promotes differentiation of AML cells, and prolongs survival of Npm1-mutated leukemic mice. We describe an exquisite dependency of NPM1-mutant AML cells on NPM1c, providing the rationale for the use of nuclear export inhibitors in AML with mutated NPM1.
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http://dx.doi.org/10.1016/j.ccell.2018.08.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6159911PMC
September 2018

NOTCH and Graft-Versus-Host Disease.

Front Immunol 2018 10;9:1825. Epub 2018 Aug 10.

Institute of Hematology-Centro di Ricerche Emato-Oncologiche (CREO), University of Perugia, Perugia, Italy.

In allogeneic hematopoietic stem cell transplantation, which is the major curative therapy for hematological malignancies, T cells play a key role in the development of graft-versus-host disease (GvHD). NOTCH pathway is a conserved signal transduction system that regulates T cell development and differentiation. The present review analyses the role of the NOTCH signaling as a new regulator of acute GvHD. NOTCH signaling could also represent a new therapeutic target for GvHD.
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http://dx.doi.org/10.3389/fimmu.2018.01825DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6096230PMC
September 2019

NOTCH1 Aberrations in Chronic Lymphocytic Leukemia.

Front Oncol 2018 27;8:229. Epub 2018 Jun 27.

Institute of Hematology-Centro di Ricerche Emato-Oncologiche (CREO), University of Perugia, Perugia, Italy.

Chronic lymphocytic leukemia (CLL) is an incurable B-cell neoplasm characterized by highly variable clinical outcomes. In recent years, genomic and molecular studies revealed a remarkable heterogeneity in CLL, which mirrored the clinical diversity of this disease. These studies profoundly enhanced our understanding of leukemia cell biology and led to the identification of new biomarkers with potential prognostic and therapeutic significance. Accumulating evidence indicates a key role of deregulated NOTCH1 signaling and mutations in CLL. This review highlights recent discoveries that improve our understanding of the pathophysiological NOTCH1 signaling in CLL and the clinical impact of mutations in retrospective and prospective trials. In addition, we discuss the rationale for a therapeutic strategy aiming at inhibiting NOTCH1 signaling in CLL, along with an overview on the currently available NOTCH1-directed approaches.
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http://dx.doi.org/10.3389/fonc.2018.00229DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6030253PMC
June 2018

NOTCH1 Is Aberrantly Activated in Chronic Lymphocytic Leukemia Hematopoietic Stem Cells.

Front Oncol 2018 20;8:105. Epub 2018 Apr 20.

Institute of Hematology-Centro di Ricerche Emato-Oncologiche (CREO), University of Perugia, Perugia, Italy.

To investigate chronic lymphocytic leukemia (CLL)-initiating cells, we assessed mutation/expression in hematopoietic stem cells (HSCs). In mutated CLL, we detected subclonal mutations in 57% CD34+/CD38- HSCs. mutation was present in 66% CD34+/CD38+ progenitor cells displaying an increased mutational burden compared to HSCs. Flow cytometric analysis revealed significantly higher NOTCH1 activation in CD34+/CD38- and CD34+/CD38+ cells from CLL patients, regardless mutation compared to healthy donors. Activated NOTCH1 resulted in overexpression of the NOTCH1 target c-MYC. We conclude that activated NOTCH1 is an early event in CLL that may contribute to aberrant HSCs in this disease.
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http://dx.doi.org/10.3389/fonc.2018.00105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5919960PMC
April 2018

Bepridil exhibits anti-leukemic activity associated with NOTCH1 pathway inhibition in chronic lymphocytic leukemia.

Int J Cancer 2018 08 23;143(4):958-970. Epub 2018 Mar 23.

Institute of Hematology-Centro di Ricerche Emato-Oncologiche (CREO), University of Perugia, Perugia, Italy.

Dysregulated NOTCH1 signaling, by either gene mutations or microenvironment interactions, has been increasingly linked to chronic lymphocytic leukemia (CLL). Thus, inhibiting NOTCH1 activity represents a potential therapeutic opportunity for this disease. Using gene expression-based screening, we identified the calcium channel modulator bepridil as a new NOTCH1 pathway inhibitor. In primary CLL cells, bepridil induced selective apoptosis even in the presence of the protective stroma. Cytotoxic effects of bepridil were independent of NOTCH1 mutation and other prognostic markers. The antitumor efficacy of bepridil was associated with inhibition of NOTCH1 activity through a decrement in trans-membrane and activated NOTCH1 protein levels with unchanged NOTCH2 protein levels. In a CLL xenotransplant model, bepridil significantly reduced the percentage of leukemic cells infiltrating the spleen via enhanced apoptosis and decreased NOTCH1 activation. In conclusion, we report in vitro and in vivo anti-leukemic activity of bepridil associated with inhibition of the NOTCH1 pathway in CLL. These data provide a rationale for the clinical development of bepridil as anti-NOTCH1 targeted therapy for CLL patients.
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http://dx.doi.org/10.1002/ijc.31355DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6055653PMC
August 2018

Cytogenetic/mutation profile of chronic lymphocytic leukemia/malignant melanoma collision tumors of the skin.

Mol Cytogenet 2018 16;11. Epub 2018 Jan 16.

1Molecular Medicine Laboratory, Hematology and Bone Marrow Transplantation Unit, University of Perugia, Hospital S. Maria della Misericordia, Piazzale Menghini n.9, 06132 Perugia, Italy.

Background: Collision tumors are rare entities that consist of two histologically distinct tumor types arising in the same anatomic site. An association between chronic lymphocytic leukemia (CLL) and malignant melanoma (MM) has been already described. Up to now, they have been documented only at positive regional lymph nodes while we focused on collision tumor in a skin lesion.

Case Presentation: We characterized the genomic profile of a skin CLL/MM collision tumor in a patient with a 9-years story of CLL. Typical high-grade genomic biomarkers featured the CLL: the immunoglobulin heavy variable genes were unmutated; a clonal del(11q), involving and , was present in the peripheral blood (PB) and skin lesion, while a subclonal large del(13q)/D13S319 was detected only in the PB. Interestingly, the del(13q) clone, increased from 10% to 46% from diagnosis to relapse. , , and were wild type. The MM lesion carried a and a promoter mutation.As the family story was consistent with a genetic predisposition to cancer, we performed mutational analysis of genes involved in familial melanoma and CLL, and of and . No germinal mutation known to predispose to CLL, MM, or breast cancer was found. Interestingly, conventional cytogenetic detected a constitutional t(12;17)(p13;p13).

Conclusions: Our data are consistent with distinct genetic landscape of the two tumors which were characterized by specific disease-related abnormalities. CLL cells carried poor prognostic imbalances, i.e. large deletions of the long arm of chromosomes 11 and 13, while in MM cells two functionally linked mutations, i.e. and a promoter occurred. Although, known germline variations predisposing to MM and/or CLL were ruled out, genetic counseling suggested the proband family was at high risk for MM.
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http://dx.doi.org/10.1186/s13039-017-0353-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5771154PMC
January 2018

A scale of "bad" co-mutations in -driven AML.

Blood 2017 10;130(17):1877-1879

UNIVERSITY OF PERUGIA.

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http://dx.doi.org/10.1182/blood-2017-09-804062DOI Listing
October 2017

Wnt/β-Catenin Signaling Induces Integrin α4β1 in T Cells and Promotes a Progressive Neuroinflammatory Disease in Mice.

J Immunol 2017 11 22;199(9):3031-3041. Epub 2017 Sep 22.

Department of Medicine, University of Perugia, Perugia 06132, Italy;

The mechanisms leading to autoimmune and inflammatory diseases in the CNS have not been elucidated. The environmental triggers of the aberrant presence of CD4 T cells in the CNS are not known. In this article, we report that abnormal β-catenin expression in T cells drives a fatal neuroinflammatory disease in mice that is characterized by CNS infiltration of T cells, glial activation, and progressive loss of motor function. We show that enhanced β-catenin expression in T cells leads to aberrant and Th1-biased T cell activation, enhanced expression of integrin α4β1, and infiltration of activated T cells into the spinal cord, without affecting regulatory T cell function. Importantly, expression of β-catenin in mature naive T cells was sufficient to drive integrin α4β1 expression and CNS migration, whereas pharmacologic inhibition of integrin α4β1 reduced the abnormal T cell presence in the CNS of β-catenin-expressing mice. Together, these results implicate deregulation of the Wnt/β-catenin pathway in CNS inflammation and suggest novel therapeutic strategies for neuroinflammatory disorders.
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http://dx.doi.org/10.4049/jimmunol.1700247DOI Listing
November 2017

Clinical-Grade-Expanded Regulatory T Cells Prevent Graft-versus-Host Disease While Allowing a Powerful T Cell-Dependent Graft-versus-Leukemia Effect in Murine Models.

Biol Blood Marrow Transplant 2017 Nov 17;23(11):1847-1851. Epub 2017 Jul 17.

Department of Hematology, Transfusion Medicine and Biotechnologies, Ospedale Civile, Pescara, Italy; Department of Medicine and Aging Sciences, University of Chieti-Pescara, Italy. Electronic address:

We developed a good manufacturing practices-compatible expansion protocol to improve number and purity of regulatory T cells (Tregs) available for clinical trials. Six clinical-grade separation procedures were performed, followed by expansion with high-dose interleukin (IL)-2, anti-CD3/anti-CD28 TCR stimulation, and rapamycin for 19 days achieving a median of 8.5-fold (range, 6.25 to 13.7) expansion. FOXP3 expression was stably maintained over the culture period, while the percentage of CD127 was significantly reduced. The in vitro suppression assay showed a strong Mixed Lymphocytes Reaction inhibition. In vitro amplification did not induce any karyotypic modification. To evaluate the graft-versus-host disease (GVHD)/graft-versus-leukemia (GVL) bifunctional axis, expanded Tregs and conventional T cells (Tcons) were tested in NOD/SCID/IL2Rgnull mice injected with primary acute myeloid leukemia (AML) cells, AML cell line, acute lymphoid leukemia Philadelphia cell line, or Burkitt-like lymphoma cell line. All mice that received leukemia cells together with expanded Tregs and Tcons were rescued from leukemia and survived without GVHD, showing that Treg expansion procedure did not compromise GVHD control and the strong Tcon-mediated GVL activity. This report might represent the basis for treating high-risk leukemia and/or relapsed/refractory leukemia patients with high-dose Treg/Tcons.
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http://dx.doi.org/10.1016/j.bbmt.2017.07.009DOI Listing
November 2017

Ibrutinib treatment of a patient with relapsing chronic lymphocytic leukemia and sustained remission of Richter syndrome.

Tumori 2017 Nov 15;103(Suppl. 1):e37-e40. Epub 2017 Nov 15.

Institute of Hematology, Centro di Ricerche Emato-Oncologiche (CREO), University of Perugia, Perugia - Italy.

Purpose: Richter syndrome (RS) is a rare event in chronic lymphocytic leukemia (CLL) that is influenced by biological factors and prior CLL treatments. Ibrutinib is a Bruton tyrosine kinase inhibitor that has shown remarkable efficacy in CLL; however, little is known about its relationship to RS. We report a case of ibrutinib efficacy against CLL in a patient with prolonged remission of RS.

Methods: The patient was diagnosed with CLL in 2003. Biological findings at onset included absent ZAP70 expression, mutated IGVH, and NOTCH1 mutation. He was treated with FCR with partial response. In 2013, he progressed to RS, not clonally related to the underlying CLL. The patient was treated with anthracycline- and platinum-based regimens, obtaining a complete remission. After 3 years, he presented a CLL progression with worsening lymphocytosis, anemia, thrombocytopenia, increased splenomegaly, and lymphadenopathies. Positron emission tomography-computed tomography scan excluded pathologic uptake. Thus, he was started on ibrutinib.

Results: At 12 months' follow-up, we observed white blood cell normalization, increased hemoglobin and platelet levels, disappearance of lymphadenopathy, and spleen size reduction. Therapy was well-tolerated with no evidence of RS.

Conclusion: This case demonstrates sustained RS remission in a patient with CLL under ibrutinib therapy, thus improving our knowledge on the use of this new drug in CLL and beyond.
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http://dx.doi.org/10.5301/tj.5000667DOI Listing
November 2017

The landscape of BRAF transcript and protein variants in human cancer.

Mol Cancer 2017 04 28;16(1):85. Epub 2017 Apr 28.

Oncogenomics Unit, Core Research Laboratory, Istituto Toscano Tumori (ITT), AOUP, CNR-IFC, Via Moruzzi 1, 56124, Pisa, Italy.

Background: The BRAF protein kinase is widely studied as a cancer driver and therapeutic target. However, the regulation of its expression is not completely understood.

Results: Taking advantage of the RNA-seq data of more than 4800 patients belonging to 9 different cancer types, we show that BRAF mRNA exists as a pool of 3 isoforms (reference BRAF, BRAF-X1, and BRAF-X2) that differ in the last part of their coding sequences, as well as in the length (BRAF-ref: 76 nt; BRAF-X1 and BRAF-X2: up to 7 kb) and in the sequence of their 3'UTRs. The expression levels of BRAF-ref and BRAF-X1/X2 are inversely correlated, while the most prevalent among the three isoforms varies from cancer type to cancer type. In melanoma cells, the X1 isoform is expressed at the highest level in both therapy-naïve cells and cells with acquired resistance to vemurafenib driven by BRAF gene amplification or expression of the Δ[3-10] splicing variant. In addition to the BRAF-ref protein, the BRAF-X1 protein (the full length as well as the Δ[3-10] variant) is also translated. The expression levels of the BRAF-ref and BRAF-X1 proteins are similar, and together they account for BRAF functional activities. In contrast, the endogenous BRAF-X2 protein is hard to detect because the C-terminal domain is selectively recognized by the ubiquitin-proteasome pathway and targeted for degradation.

Conclusions: By shedding light on the repertoire of BRAF mRNA and protein variants, and on the complex regulation of their expression, our work paves the way to a deeper understanding of a crucially important player in human cancer and to a more informed development of new therapeutic strategies.
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http://dx.doi.org/10.1186/s12943-017-0645-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5410044PMC
April 2017

A mast cell-ILC2-Th9 pathway promotes lung inflammation in cystic fibrosis.

Nat Commun 2017 01 16;8:14017. Epub 2017 Jan 16.

Department of Experimental Medicine, University of Perugia, 06132 Perugia, Italy.

T helper 9 (Th9) cells contribute to lung inflammation and allergy as sources of interleukin-9 (IL-9). However, the mechanisms by which IL-9/Th9 mediate immunopathology in the lung are unknown. Here we report an IL-9-driven positive feedback loop that reinforces allergic inflammation. We show that IL-9 increases IL-2 production by mast cells, which leads to expansion of CD25 type 2 innate lymphoid cells (ILC2) and subsequent activation of Th9 cells. Blocking IL-9 or inhibiting CD117 (c-Kit) signalling counteracts the pathogenic effect of the described IL-9-mast cell-IL-2 signalling axis. Overproduction of IL-9 is observed in expectorates from cystic fibrosis (CF) patients, and a sex-specific variant of IL-9 is predictive of allergic reactions in female patients. Our results suggest that blocking IL-9 may be a therapeutic strategy to ameliorate inflammation associated with microbial colonization in the lung, and offers a plausible explanation for gender differences in clinical outcomes of patients with CF.
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http://dx.doi.org/10.1038/ncomms14017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5241810PMC
January 2017

Lack of glucocorticoid-induced leucine zipper (GILZ) deregulates B-cell survival and results in B-cell lymphocytosis in mice.

Blood 2015 Oct 14;126(15):1790-801. Epub 2015 Aug 14.

Department of Medicine, Section of Pharmacology and.

Glucocorticoids (GC) are widely used as antiinflammatory/immunosuppressive drugs and antitumor agents in several types of lymphoma and leukemia. Therapeutic doses of GC induce growth-suppressive and cytotoxic effects on various leukocytes including B cells. Molecular mechanisms of GC action include induction of GC target genes. Glucocorticoid-induced leucine zipper (GILZ) is a rapidly, potently, and invariably GC-induced gene. It mediates a number of GC effects, such as control of cell proliferation, differentiation, and apoptosis. Here we show that deletion of GILZ in mice leads to an accumulation of B lymphocytes in the bone marrow, blood, and lymphoid tissues. Gilz knockout (KO) mice develop a progressive nonlethal B lymphocytosis, with expansion of B220(+) cells in the bone marrow and in the periphery, dependent on increased B-cell survival. Decreased B-cell apoptosis in mice lacking GILZ correlates with increased NF-κB transcriptional activity and Bcl-2 expression. B cell-specific gilz KO mice confirmed that the effect of GILZ deletion is B-cell self-intrinsic. These results establish GILZ as an important regulator of B-cell survival and suggest that the deregulation of GILZ expression could be implicated in the pathogenesis of B-cell disorders.
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http://dx.doi.org/10.1182/blood-2015-03-631580DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4705834PMC
October 2015

Notch signaling sustains the expression of Mcl-1 and the activity of eIF4E to promote cell survival in CLL.

Oncotarget 2015 Jun;6(18):16559-72

Department of Experimental Medicine, Biosciences and Medical Embryology Section, University of Perugia, Perugia, Italy.

In chronic lymphocytic leukemia (CLL), Notch1 and Notch2 signaling is constitutively activated and contributes to apoptosis resistance. We show that genetic inhibition of either Notch1 or Notch2, through small-interfering RNA, increases apoptosis of CLL cells and is associated with decreased levels of the anti-apoptotic protein Mcl-1. Thus, Notch signaling promotes CLL cell survival at least in part by sustaining Mcl-1 expression. In CLL cells, an enhanced Notch activation also contributes to the increase in Mcl-1 expression and cell survival induced by IL-4.Mcl-1 downregulation by Notch targeting is not due to reduced transcription or degradation by caspases, but in part, to increased degradation by the proteasome. Mcl-1 downregulation by Notch targeting is also accompanied by reduced phosphorylation of eukaryotic translation initiation factor 4E (eIF4E), suggesting that this protein is another target of Notch signaling in CLL cells.Overall, we show that Notch signaling sustains CLL cell survival by promoting Mcl-1 expression and eIF4E activity, and given the oncogenic role of these factors, we underscore the therapeutic potential of Notch inhibition in CLL.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4599289PMC
http://dx.doi.org/10.18632/oncotarget.4116DOI Listing
June 2015