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STEFANIA FIORCARI

COLLABORATORE DI RICERCA
Dipartimento di Scienze Mediche e Chirurgiche Materno-Infantili e dell'Adulto

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Pubblicazioni

2022 - Indoleamine 2, 3-Dioxygenase 1 Mediates Survival Signals in Chronic Lymphocytic Leukemia via Kynurenine/Aryl Hydrocarbon Receptor-Mediated MCL1 Modulation [Articolo su rivista]
Atene, C. G.; Fiorcari, S.; Mesini, N.; Alboni, S.; Martinelli, S.; Maccaferri, M.; Leonardi, G.; Potenza, L.; Luppi, M.; Maffei, R.; Marasca, R.
abstract

The indoleamine 2,3-dioxygenase 1 (IDO1) metabolic circuitry, comprising the first tryptophan (Trp) catabolite L-kynurenine (Kyn) and the aryl hydrocarbon receptor (AHR), has emerged as a mechanism of cancer immune evasion. Here, we investigated the functional role of the IDO1/Kyn/AHR axis in chronic lymphocytic leukemia (CLL). Our data show that CLL cells expressed an active form of the IDO1 enzyme and microenvironmental stimuli can positively modulate its expression. Interferon (IFN)-γ induces IDO1 expression through the Jak/STAT1 pathway and mediates Kyn production concomitantly with Trp consumption in CLL-conditioned media, while INCB018424 (ruxolitinib), a JAK1/2 inhibitor, impaired both effects. To characterize the involvement of IDO1 in leukemic cell maintenance, we overexpressed IDO1 by vector transfection measuring enhanced resistance to spontaneous apoptosis. IDO1 pro-survival influence was confirmed by treating CLL cells with Kyn, which mediated the increase of induced myeloid leukemia cell differentiation protein (MCL1). Conversely, AHR silencing or its blockade via CH-223191 improved the apoptosis of leukemic clones and mitigated MCL1 expression. Moreover, Kyn-treated CLL cells are less affected by the pro-apoptotic effect of ABT-199 (venetoclax), while CH-223191 showed synergistic/additive cytotoxicity with this drug. Lastly, targeting directly MCL1 in CLL cells with AMG-176, we abrogate the pro-survival effect of Kyn. In conclusion, our data identify IDO1/Kyn/AHR signaling as a new therapeutic target for CLL, describing for the first time its role in CLL pathobiology.

2020 - BTK Inhibition Impairs the Innate Response Against Fungal Infection in Patients With Chronic Lymphocytic Leukemia [Articolo su rivista]
Fiorcari, S.; Maffei, R.; Vallerini, D.; Scarfo, L.; Barozzi, P.; Maccaferri, M.; Potenza, L.; Ghia, P.; Luppi, M.; Marasca, R.
abstract

Infections represent a cause of morbidity and mortality in patients affected by chronic lymphocytic leukemia (CLL). Introduction of new drugs in CLL clinical practice has showed impressive efficacy, in particular those targeting BTK. Among the consistent clinical data, an increasing number of reports describing the occurrence of unexpected opportunistic fungal infections has been reported during treatment with ibrutinib in the first 6 months of treatment. The reason underlying manifestations of invasive fungal infections in patients treated with ibrutinib is still under investigation. Our study aimed to understand the impact of BTK inhibition on immune response to fungal infection mediated by macrophages and CD14+ monocytic population obtained from CLL patients. Exposure to ibrutinib and acalabrutinib reduced signaling pathways activated by Aspergillus fumigatus determining an exacerbation of an immunosuppressive signature, a reduction of phagocytosis and a significant deficit in the secretion of inflammatory cytokines either in macrophages and monocytes isolated from CLL patients and healthy donors. These effects lead to a failure in completely counteracting conidia germination. In addition we investigated the biological effects of ibrutinib on monocyte counterpart in patients who were undergoing therapy. A significant impairment in cytokine secretion and a deficit of phagocytosis in circulating monocytes were detected after 3 months of treatment. Thus, our results uncover modifications in the innate response in CLL patients induced by ibrutinib that may impair the immunological response to fungal infection. BTK inhibition affects a productive immune response of CLL-associated macrophages (NLC) during Aspergillus fumigatus infection. Reduction of TNF-α secretion and phagocytosis are detected in monocytes isolated from CLL patients during ibrutinib therapy.

2020 - Immunomodulatory effect of ibrutinib: Reducing the barrier against fungal infections [Articolo su rivista]
Maffei, R.; Maccaferri, M.; Arletti, L.; Fiorcari, S.; Benatti, S.; Potenza, L.; Luppi, M.; Marasca, Roberto
abstract

The Bruton tyrosine kinase (BTK) inhibitor ibrutinib is increasingly used in the treatment of chronic lymphocytic leukemia (CLL). Moreover, very promising results have been reported in other B-cell malignancies, including primary central nervous system lymphoma (PCNSL). Although well-tolerated in the majority of patients, ibrutinib demonstrates in some cases troublesome toxicities, including invasive fungal infections (IFIs). In the present review, we summarize clinical manifestations of IFIs in patients treated with ibrutinib, generally characterized by an early onset, mild clinical manifestations, asymptomatic/low symptomatic pulmonary localization and high incidence of central nervous system (CNS) involvement. IFI risk appears particularly increased in patients receiving ibrutinib associated with other immune modulator agents, especially with steroids or immune-chemotherapy. Moreover, the immunomodulatory effect of ibrutinib is described, pointing the attention on the involvement of specific molecules targeted by ibrutinib in innate and adaptive response to fungal infection. Overall, the findings indicate the ibrutinib may rapidly impair innate immune cell functions, while concomitantly restoring an effective protective potential of adaptive immune compartment. A correct awareness, especially when other predisposing factors are present, is warranted about the potential risk of IFIs in ibrutinib-treated patients.

2020 - Selective inhibition of PI3Kγ affects survival and proliferation of chronic lymphocytic leukemia B cells [Articolo su rivista]
Maffei, R.; Benatti, S.; Atene, C. G.; Debbia, G.; Zucchini, P.; Potenza, L.; Luppi, M.; Fiorcari, S.; Marasca, R.
abstract

the catalytic p110gamma of PI3K is implicated in the survival and proliferation of CLL cells. Our findings support the idea that CLL cells are peculiar in the attitude to sense microenvironmental signals throughout the engagement of multiple PI3Ks. PI3Kgamma inhibition by IPI-549 may directly promote CLL apoptosis, but may also interfere with signals derived from several accessory cells of stromal and immune system. Together with the reported ability of PI3Kgamma inhibition in prevention of CLL migration and adhesion, our data provide knowledge to justify further clinical development of PI3Kc inhibition in CLL cells.

2019 - Overexpression of CD49d in trisomy 12 chronic lymphocytic leukemia patients is mediated by IRF4 through induction of IKAROS [Articolo su rivista]
Fiorcari, Stefania; Benatti, Stefania; Zucchetto, Antonella; Zucchini, Patrizia; Gattei, Valter; Luppi, Mario; Marasca, Roberto; Maffei, Rossana
abstract

2018 - Angiopoietin-2 acts as a survival factor for chronic lymphocytic leukemia B cells throughout Tie-2 receptor engagement [Articolo su rivista]
Maffei, Rossana; Fiorcari, Stefania; Martinelli, Silvia; Guarnotta, Carla; Benatti, Stefania; Belmonte, Beatrice; Potenza, Leonardo; Luppi, Mario; Marasca, Roberto
abstract

we demonstrated that: (i) CLL cells expressed Tie-2 receptor both in peripheral blood and in lymph nodes; (ii) Ang2 may interact with Tie-2 in CLL mediating a survival signal throughout PI3K-AKT signalling, and (iii) the interruption of Ang2/Tie-2 signalling may be effective in CLL.

2018 - Casein kinase 1: A new tale of chronic lymphocytic leukemia (CLL) microenvironment [Articolo su rivista]
Fiorcari, S.; Maffei, R.; Marasca, R.
abstract

2018 - Casein kinase 1: A new tale of chronic lymphocytic leukemia (CLL) microenvironment [Articolo su rivista]
Fiorcari, S.; Maffei, R.; Marasca, R.
abstract

2018 - Idelalisib impairs T-cell-mediated immunity in chronic lymphocytic leukemia [Articolo su rivista]
Martinelli, Silvia; Maffei, Rossana; Fiorcari, Stefania; Quadrelli, Chiara; Zucchini, Patrizia; Benatti, Stefania; Potenza, Leonardo; Luppi, Mario; Marasca, Roberto
abstract

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2018 - Increased SHISA3 expression characterizes chronic lymphocytic leukemia patients sensitive to lenalidomide [Articolo su rivista]
Maffei, Rossana; Fiorcari, Stefania; Martinelli, Silvia; Benatti, Stefania; Bulgarelli, Jenny; Rizzotto, Lara; Debbia, Giulia; Santachiara, Rita; Rigolin, Gian Matteo; Forconi, Francesco; Rossi, Davide; Laurenti, Luca; Palumbo, Giuseppe A.; Vallisa, Daniele; Cuneo, Antonio; Gaidano, Gianluca; Luppi, Mario; Marasca, Roberto
abstract

Lenalidomide is a therapeutically effective drug in chronic lymphocytic leukemia (CLL). Twenty-seven CLL patients were treated with lenalidomide in a phase II clinical trial. Ten patients were grouped as responders (R) and 6 as nonresponders (NR). We evaluated T lymphocytes, NK, monocytes and dendritic cells at baseline and after treatment. A gene expression analysis was performed on 16 CLL samples collected before treatment. The levels of immune cells or immune-related cytokines are not different between R and NR patients. However, CLL patients sensitive to lenalidomide clearly show a peculiar gene expression profile in leukemic cells. The most up-regulated gene (fold change =  +23 in R vs. NR) is Wnt inhibitor SHISA homolog 3 (SHISA3). SHISA3highCLL are characterized by a restrained activation of Wnt signaling and sensibility to lenalidomide-induced apoptosis. In conclusion, SHISA3 is a candidate gene for the identification of CLL patients who will benefit of lenalidomide treatment as single agent.

2017 - Macitentan, a double antagonist of endothelin receptors, efficiently impairs migration and microenvironmental survival signals in chronic lymphocytic leukemia [Articolo su rivista]
Maffei, Rossana; Fiorcari, Stefania; Vaisitti, Tiziana; Martinelli, Silvia; Benatti, Stefania; Debbia, Giulia; Rossi, Davide; Zucchini, Patrizia; Potenza, Leonardo; Luppi, Mario; Gaidano, Gianluca; Deaglio, Silvia; Marasca, Roberto
abstract

The crosstalk between chronic lymphocytic leukemia (CLL) cells and tumor microenvironment is essential for leukemic clone maintenance, supporting CLL cells survival, proliferation and protection from drug-induced apoptosis. Over the past years, the role of several soluble factors involved in these processes has been studied. CLL cells express higher levels of endothelin 1 (ET-1) and ETA receptor as compared to normal B cells. Upon ET-1 stimulation, CLL cells improve their survival and proliferation and reduce their sensitivity to the phosphoinositide-3-kinase d inhibitor idelalisib and to fludarabine. Here, we demonstrate that CLL cells express not only ETA receptor but also ETB receptor. ET-1 acts as a homing factor supporting CLL cells migration and adhesion to microenvironmental cells. In addition, ET-1 stimulates a pro-angiogenic profile of CLL cells increasing VEGF expression through hypoxia-inducible factor-1 (HIF-1α) accumulation in CLL cells. Macitentan, a specific dual inhibitor of ETAand ETBreceptors, targets CLL cells affecting leukemic cells migration and adhesion and overcoming the pro-survival and proliferation signals mediated by microenvironment. Furthermore, macitentan cooperates with ibrutinib inhibiting the BCR pathway and with ABT-199 disrupting BCL2 pathway. Our data describe the biological effects of a new drug, macitentan, able to counteract essential processes in CLL pathobiology as survival, migration, trafficking and drug resistance. These findings envision the possibility to interfere with ET receptors activity using macitentan as a possible novel therapeutic strategy for CLL patients.

2017 - The expression of endothelin-1 in chronic lymphocytic leukemia is controlled by epigenetic mechanisms and extracellular stimuli. [Articolo su rivista]
Martinelli, Silvia; Maffei, Rossana; Fiorcari, Stefania; Quadrelli, Chiara; Zucchini, Patrizia; Benatti, Stefania; Potenza, Leonardo; Luppi, Mario; Marasca, Roberto
abstract

Endothelin-1 (ET-1) is a hormone peptide widely expressed and is involved in several biological processes, important not only for normal cell function but also for tumor development, including cell proliferation, invasion, metastasis, angiogenesis and osteogenesis. In accordance, ET-1 was already shown to contribute to the growth and progression of many different solid cancers. We recently demonstrated that ET-1 has a role in the pathogenesis of chronic lymphocytic leukemia (CLL) where it is abnormally expressed. In the context of this malignancy, ET-1 is able to mediate survival, drug-resistance and growth signals in leukemic cells. Previous studies, not conducted in CLL, have shown that ET-1 regulatory mechanisms are numerous and cell specific. Here, we valued the expression of ET-1 in CLL, in relation to DNA methylation but also in response to stimulation of some important pathways for the dialogue between CLL and microenvironment. We found that a high methylation of ET-1 first intron affects the basal expression of ET-1 in CLL. Moreover, we showed that the activation of CD40 or Toll-like receptor (TLR) by extracellular stimuli produces an augment of ET-1 level in CLL cells. Finally, we demonstrated the fundamental role of NF-kB signalling pathway in promoting and maintaining ET-1 expression in CLL cells, both in basal conditions and after CD40 activation.

2016 - Ibrutinib modifies the function of monocyte/macrophage population in chronic lymphocytic leukemia [Articolo su rivista]
Fiorcari, Stefania; Maffei, Rossana; Audrito, Valentina; Martinelli, Silvia; Hacken, Elisa Ten; Zucchini, Patrizia; Grisendi, Giulia; Potenza, Leonardo; Luppi, Mario; Burger, Jan A; Deaglio, Silvia; Marasca, Roberto
abstract

In lymphoid organs, nurse-like cells (NLCs) show properties of tumor-associated macrophages, playing a crucial role in chronic lymphocytic leukemia (CLL) cell survival. Ibrutinib, a potent inhibitor of Bruton's tyrosine kinase (BTK), is able to counteract pro-survival signals in CLL cells. Since the effects on CLL cells have been studied in the last years, less is known about the influence of ibrutinib on NLCs properties. We sought to determine how ibrutinib modifies NLCs functions focusing on the balance between immunosuppressive and inflammatory features. Our data show that ibrutinib targets BTK expressed by NLCs modifying their phenotype and function. Treatment with ibrutinib reduces the phagocytic ability and increases the immunosuppressive profile of NLCs exacerbating the expression of M2 markers. Accordingly, ibrutinib hampers LPS-mediated signaling, decreasing STAT1 phosphorylation, while allows IL-4-mediated STAT6 phosphorylation. In addition, NLCs treated with ibrutinib are able to protect CLL cells from drug-induced apoptosis partially through the secretion of IL-10. Results from patient samples obtained prior and after 1 month of treatment with ibrutinib show an accentuation of CD206, CD11b and Tie2 in the monocytic population in the peripheral blood. Our study provides new insights into the immunomodulatory action of ibrutinib on monocyte/macrophage population in CLL.

2016 - Lenalidomide in chronic lymphocytic leukemia: The present and future in the era of tyrosine kinase inhibitors [Articolo su rivista]
Maffei, Rossana; Colaci, Elisabetta; Fiorcari, Stefania; Martinelli, Silvia; Potenza, Leonardo; Luppi, Mario; Marasca, Roberto
abstract

Lenalidomide is an immunomodulatory agent (IMiD) clinically active in chronic lymphocytic leukemia (CLL), both in heavily pre-treated patients and upfront. Lenalidomide has a unique mechanism of action in CLL. Its efficacy relies on a multifactorial mode-of-action (MOA), comprising a plethora of immunomodulatory actions, the disruption of mutualistic interactions inside CLL microenvironment and direct effects against leukemic cells. In the last few years, a number of new and highly effective drugs appeared in the scenario of CLL therapeutic options, i.e. tyrosine kinase inhibitors (TKIs), showing a good safety profile and impressive clinical response, also in high-risk patients. In this review, we describe the data from clinical studies about lenalidomide efficacy in CLL and we critically dissect the different mechanisms of action of this drug. We point the attention on open issues, including drug dosage and administration schedule, prediction of clinical response to lenalidomide, and combination therapeutic strategies. This overview would be useful to envision a possible role of lenalidomide in the treatment flow-chart of CLL, exploiting its peculiar MOA and also exploring the possible synergetic effect with new drugs.

2016 - The histone deacetylase inhibitor romidepsin synergizes with lenalidomide and enhances tumor cell death in T-cell lymphoma cell lines [Articolo su rivista]
Cosenza, Maria; Civallero, Monica; Fiorcari, Stefania; Pozzi, Samantha; Marcheselli, Luigi; Bari, Alessia; Ferri, Paola; Sacchi, Stefano
abstract

We investigated the cytotoxic interactions of romidepsin, a histone deacetylase inhibitor, and lenalidomide, an immunomodulatory agent, in a T-cell lymphoma preclinical model. Hut-78 and Karpas-299 cells were treated with romidepsin and lenalidomide alone and in combination. The interaction between romidepsin and lenalidomide was evaluated by the Chou–Talalay method, and cell viability and clonogenicity were also evaluated. Apoptosis, reactive oxygen species (ROS) levels, and cell cycle distribution were determined by flow cytometry. ER stress, caspase activation, and the AKT, MAPK/ERK, and STAT-3 pathways were analyzed by Western blot. Combination treatment with romidepsin and lenalidomide had a synergistic effect in Hut-78 cells and an additive effect in Karpas-299 cells at 24 hours and did not decrease the viability of normal peripheral blood mononuclear cells. This drug combination induced apoptosis, increased ROS production, and activated caspase-8, −9, −3 and PARP. Apoptosis was associated with increased hallmarks of ER stress and activation of UPR sensors and was mediated by dephosphorylation of the AKT, MAPK/ERK, and STAT3 pathways.The combination of romidepsin and lenalidomide shows promise as a possible treatment for T-cell lymphoma. This work provides a basis for further studies.

2015 - Lenalidomide interferes with tumor-promoting properties of nurse-like cells in chronic lymphocytic leukemia [Articolo su rivista]
Fiorcari, Stefania; Martinelli, Silvia; Bulgarelli, Jenny; Audrito, V; Zucchini, Patrizia; Colaci, Elisabetta; Potenza, Leonardo; Narni, Franco; Luppi, Mario; Deaglio, S; Marasca, Roberto; Maffei, Rossana
abstract

Lenalidomide is an immunomodulatory agent clinically active in chronic lymphocytic leukemia patients. The specific mechanism of action is still undefined, but includes modulation of the microenvironment. In chronic lymphocytic leukemia patients, nurse-like cells differentiate from CD14(+) mononuclear cells and protect chronic lymphocytic leukemia cells from apoptosis. Nurse-like cells resemble M2 macrophages with potent immunosuppressive functions. Here, we examined the effect of lenalidomide on the monocyte/macrophage population in chronic lymphocytic leukemia patients. We found that lenalidomide induces high actin polymerization on CD14(+) monocytes through activation of small GTPases, RhoA, Rac1 and Rap1 that correlated with increased adhesion and impaired monocyte migration in response to CCL2, CCL3 and CXCL12. We observed that lenalidomide increases the number of nurse-like cells that lost the ability to nurture chronic lymphocytic leukemia cells, acquired properties of phagocytosis and promoted T-cell proliferation. Gene expression signature, induced by lenalidomide in nurse-like cells, indicated a reduction of pivotal pro-survival signals for chronic lymphocytic leukemia, such as CCL2, IGF1, CXCL12, HGF1, and supported a modulation towards M1 phenotype with high IL2 and low IL10, IL8 and CD163. Our data provide new insights into the mechanism of action of lenalidomide that mediates a pro-inflammatory switch of nurse-like cells affecting the protective microenvironment generated by chronic lymphocytic leukemia into tissues.

2015 - Mesenchymal progenitors expressing TRAIL induce apoptosis in sarcomas [Articolo su rivista]
Grisendi, Giulia; Spano, Maria Carlotta; D'Souza, Naomi; Rasini, Valeria; Veronesi, Elena; Prapa, Malvina; Petrachi, Tiziana; Piccinno, MARIA SERENA; Rossignoli, Filippo; Burns, Jorge Phillip Joaquin Sans; Fiorcari, Stefania; Granchi, Donatella; Baldini, Nicola; Horwitz, EDWIN MARK; Guarneri, Valentina; Conte, Pierfranco; Paolucci, Paolo; Dominici, Massimo
abstract

Sarcomas are frequent tumors in children and young adults that, despite a relative chemo-sensitivity, show high relapse rates with up to 80% of metastatic patients dying in 5 years from diagnosis. The real ontogeny of sarcomas is still debated and evidences suggest they may derive from precursors identified within mesenchymal stromal/stem cells (MSC) fractions. Recent studies on sarcoma microenvironment additionally indicated that MSC could take active part in generation of a supportive stroma. Based on this knowledge, we conceived to use modified MSC to deliver tumor necrosis factor related apoptosis inducing ligand (TRAIL) targeting different sarcoma histotypes. Gene modified MSC expressing TRAIL were co-cultured with different osteosarcoma, rhabdomyosarcoma and Ewing's Sarcoma (ES) cell lines assessing viability and caspase-8 activation. An in vivo model focused on ES was then implemented considering the impact of MSC-TRAIL on tumor size, apoptosis and angiogenesis. MSC expressing TRAIL induced significantly high apoptosis in all tested lines. Sarcoma death was specifically associated with caspase-8 activation starting from 8 hours of co-culture with MSC-TRAIL. When injected into pre-established ES xenotransplants, MSC-TRAIL persisted within its stroma, causing significant tumor apoptosis versus control groups. Additional histological and in vitro studies reveal that MSC-TRAIL could also exert potent anti-angiogenic functions. Our results suggest that MSC as TRAIL vehicles could open novel therapeutic opportunities for sarcoma by multiple mechanisms.

2015 - Targeting neoplastic B cells and harnessing microenvironment: the “double face” of ibrutinib and idelalisib [Articolo su rivista]
Maffei, Rossana; Fiorcari, Stefania; Martinelli, Silvia; Potenza, Leonardo; Luppi, Mario; Marasca, Roberto
abstract

Relapsed or refractory diffuse large B-cell lymphoma (DLBCL) not suitable for high dose chemotherapy with autologous stem cell transplantation (ASCT) has a dismal prognosis and no standard therapy. We designed an Italian multicenter retrospective study aimed at evaluating the safety and efficacy of rituximab plus bendamustine (R–B) as salvage treatment in patients not eligible for ASCT because of age and/or comorbidity or in patients with post-ASCT recurrence. Fifty-five patients with a median age of 76 years were included. The overall response rate was 50%, including 28% complete remission and 22% partial remission. The median overall survival (OS) was 10.8 months. The median progression free survival (PFS) was 8.8 months. Eleven patients are still alive and in complete remission at last follow-up (12–71 months). Toxicity was moderate, mainly grades 1 and 2. R–B showed promising efficacy results with an acceptable toxicity profile and should be further investigated, possibly in combination with novel drugs.

2014 - Endothelin-1 promotes survival and chemoresistance in chronic lymphocytic leukemia B cells through eta receptor [Articolo su rivista]
Maffei, Rossana; Bulgarelli, Jenny; Fiorcari, Stefania; Martinelli, Silvia; Castelli, Ilaria; Valenti, Vanessa; Rossi, Davide; Bonacorsi, Goretta; Zucchini, Patrizia; Potenza, Leonardo; Vallisa, Daniele; Gattei, Valter; Del Poeta, Giovanni; Forconi, Francesco; Gaidano, Gianluca; Narni, Franco; Luppi, Mario; Marasca, Roberto
abstract

The endothelin axis, comprising endothelins (ET-1, ET-2 and ET-3) and their receptors (ET(A)R and ETBR), has emerged as relevant player in tumor growth and metastasis. Here, we investigated the involvement of ET-1/ET(A)R axis in chronic lymphocytic leukemia (CLL). CLL cells expressed higher levels of ET-1 and ETA receptor as compared to normal B cells. ET-1 peptide stimulated phosphoinositide-3-kinase and mitogen-activated protein kinase signaling pathways, improved survival and promoted proliferation of leukemic cells throughout ET(A)R triggering. Moreover, the blockade of ET(A)R by the selective antagonist BQ-123 inhibited the survival advantage acquired by CLL cells in contact with endothelial layers. We also found that blocking ET(A)R via BQ-123 interferes with ERK phosphorylation and CLL pro-survival effect mediated by B-cell receptor (BCR) activation. The pro-apoptotic effect of phosphoinositide-3-kinase δ inhibitor idelalisib and mitogen-activated protein kinase inhibitor PD98059 was decreased by the addition of ET-1 peptide. Then, ET-1 also reduced the cytotoxic effect of fludarabine on CLL cells cultured alone or co-cultured on endothelial layers. ET(A)R blockade by BQ-123 inhibited the ET-1-mediated protection against drug-induced apoptosis. Lastly, higher plasma levels of big ET-1 were detected in patients (n = 151) with unfavourable prognostic factors and shorter time to first treatment. In conclusion, our data describe for the first time a role of ET-1/ET(A)R signaling in CLL pathobiology. ET-1 mediates survival, drug-resistance, and growth signals in CLL cells that can be blocked by ET(A)R inhibition.

2014 - Endothelium-mediated survival of leukemic cells and angiogenesis-related factors are affected by lenalidomide treatment in chronic lymphocytic leukemia [Articolo su rivista]
Maffei, Rossana; Fiorcari, Stefania; Bulgarelli, Jenny; Rizzotto, Lara; Martinelli, Silvia; Rigolin, Gian Matteo; Debbia, Giulia; Castelli, Ilaria; Bonacorsi, Goretta; Santachiara, Rita; Forconi, Francesco; Rossi, Davide; Laurenti, Luca; Palumbo, Giuseppe A.; Vallisa, Daniele; Cuneo, Antonio; Gaidano, Gianluca; Luppi, Mario; Marasca, Roberto
abstract

Lenalidomide is an IMID immunomodulatory agent clinically active in patients with chronic lymphocytic leukemia (CLL). We evaluated the activity of lenalidomide inside an in vitro coculture system of endothelial and CLL cells. Lenalidomide was able to inhibit CLL survival advantage mediated by endothelial contact. Moreover, the marked increase of in vitro angiogenesis determined by CLL-derived conditioned media was reduced by lenalidomide. We also analyzed peripheral blood collected from 27 patients with relapsed or refractory CLL being treated with lenalidomide within a phase II trial. Plasma levels of VEGF and THBS-1 decreased, whereas Ang2 and Ang increased during treatment. Patients who respond to lenalidomide showed a more pronounced decrease of VEGF and bFGF than did patients with stable or progressive disease (p = 0.007 and p = 0.005). Furthermore, lenalidomide reduced circulating endothelial cells and endothelial progenitors by increasing the percentage of apoptotic cells. Conversely, for six matched bone marrow biopsies available before and after treatment, we did not detect any modification in vessel density, suggesting a possible mechanism of vessel normalization rather than regression. In conclusion, our study provides further evidence that the anti-CLL effect of lenalidomide is mediated through the alteration of microenvironmental elements, implying the modulation of several angiogenesis-related factors and disruption of CLL crosstalk with endothelial cells.

2013 - ANGPT2 promoter methylation is strongly associated with gene expression and prognosis in chronic lymphocytic leukemia [Articolo su rivista]
Martinelli, Silvia; Kanduri, M; Maffei, Rossana; Fiorcari, Stefania; Bulgarelli, Jenny; Marasca, Roberto; Rosenquist, R.
abstract

Increasing evidence suggests a key role for angiopoietin-2 (ANGPT2) in influencing the aggressiveness of chronic lymphocytic leukemia (CLL). In the presence of vascular endothelial growth factor (VEGF), ANGPT2 causes vessel destabilization leading to neoangiogenesis. Accordingly, high expression levels of ANGPT2 and high degree of angiogenesis have consistently been associated with poor prognosis in CLL; however, the molecular mechanisms behind the variability in ANGPT2 expression are still to be discovered. Here, for the first time, we investigated the DNA methylation status of the ANGPT2 promoter in a large CLL cohort (n = 88) using pyrosequencing and correlated methylation data with ANGPT2 expression levels, prognostic factors and outcome. Importantly, methylation levels of the ANGPT2 gene correlated inversely with its mRNA expression levels (p<0.001). Moreover, low ANGPT2 methylation status was highly associated with adverse prognostic markers, shorter time to first treatment and overall survival. Finally, treatment with methyl inhibitors induced re-expression of ANGPT2 in two B-cell lymphoma cell lines, underscoring the importance of DNA methylation in regulating transcriptional silencing of this gene. In conclusion, we believe that the known variability in ANGPT2 expression among CLL patients could be explained by differential promoter DNA methylation and that low methylation levels of the ANGPT2 promoter have an adverse prognostic impact in CLL.

2013 - Clinical heterogeneity of de novo 11q deletion chronic lymphocytic leukaemia: prognostic relevance of extent of 11q deleted nuclei inside leukemic clone. [Articolo su rivista]
Marasca, Roberto; Maffei, Rossana; Martinelli, Silvia; Fiorcari, Stefania; Bulgarelli, Jenny; Debbia, Giulia; Rossi, D; Rossi, Fm; Rigolin, Gm; Martinelli, S; Gattei, V; Del Poeta, G; Laurenti, L; Forconi, F; Montillo, M; Gaidano, G; Luppi, Mario
abstract

Deletion on the long arm of chromosome 11 occurs in 5-20% of chronic lymphocytic leukaemia (CLL) patients. We analysed clinical-biological characteristics of 131 CLL patients carrying 11q deletion documented before therapy (de novo 11q deleted CLL). De novo 11q deleted CLL were characterized by high frequencies of unmutated immunoglobulin variable heavy genes, multiple fluorescence in situ hybridization aberrations and lymph node involvement. Factors significantly associated with shorter time to first treatment (TTFT) were advanced Binet stages, high white blood cell count, increased β(2) -microglobulin levels, 17p in addition, splenomegaly and more extensive lymphadenopathy. We found that patients with <25% 11q deleted nuclei (n = 22) experienced longer TTFT compared with patients with ≥25% 11q deleted nuclei (n = 87; median TTFT, 40 vs. 14 months, p = 0.011) and also showed better response to treatments (complete response, 50% vs. 21%, p = 0.016). The variables identified by multivariate analysis as independently associated with reduced TTFT were advanced Binet stages [hazard ratio (HR) 4.69; p < 0.001] and ≥25% 11q deleted nuclei (HR 4.73; p = 0.004). De novo 11q deleted CLLs exhibit variable clinical outcome. The percentage of deleted nuclei inside leukemic clone should be included in the prognostic definition of therapy-naïve 11q deleted CLL patients.

2013 - Monocytic population in chronic lymphocytic leukemia shows altered composition and deregulation of genes involved in phagocytosis and inflammation [Articolo su rivista]
Maffei, Rossana; Bulgarelli, Jenny; Fiorcari, Stefania; Bertoncelli, L; Martinelli, Silvia; Guarnotta, C; Castelli, Ilaria; Deaglio, S; Debbia, Giulia; DE BIASI, Sara; Bonacorsi, G; Zucchini, Patrizia; Narni, Franco; Tripodo, C; Luppi, Mario; Cossarizza, Andrea; Marasca, Roberto
abstract

Macrophages reside in tissues infiltrated by chronic lymphocytic leukemia B-cells and the extent of infiltration is associated with adverse prognostic factors. Blood monocyte population was studied by flow cytometry and whole-genome microarrays. A mixed lymphocyte reaction was performed to evaluate T cell proliferation in contact with monocytes from patients and normal donors. Migration and gene modulation in normal monocytes treated with leukemia were also evaluated. Chronic lymphocytic leukemia patients showed an increase in the absolute number of monocytes compared to normal controls (792+/-86 cells/mL vs. 485+/-46 cells/mL, p=0.003). Higher number of nonclassical CD14+CD16++ and Tie-2 expressing monocytes (TEMs) was also detected in patients. Furthermore, we performed a gene expression analysis of monocytes in chronic lymphocytic leukemia patients, showing up-regulation of RAP1GAP and down-regulation of tubulins and CDC42EP3, which would be expected to result in impairment in phagocytosis. We also detected gene alterations such as the down-regulation of PTGR2, a reductase able to inactivate the prostaglandin E2, indicating an immunosuppressive activity. Accordingly, T cell proliferation was inhibited in contact with monocytes from patients compared to normal controls. Finally, normal monocytes in vitro increased migration and up-regulated CD16, RAP1GAP, IL-10, IL-8, MMP9 and down-regulated PTGR2 in response to leukemic cells or conditioned media. In conclusion, altered composition and deregulation of genes involved in phagocytosis and inflammation were found in blood monocytes obtained from chronic lymphocytic leukemia patients, suggesting that leukemia-mediated 'education' of immune elements may also include the establishment of a skewed phenotype in monocyte/macrophage population.

2013 - The PI3-kinase delta inhibitor idelalisib (GS-1101) targets integrin-mediated adhesion of chronic lymphocytic leukemia (CLL) cell to endothelial and marrow stromal cells [Articolo su rivista]
Fiorcari, Stefania; Brown, Wells S; Mcintyre, Bradley W; Estrov, Zeev; Maffei, Rossana; O'Brien, Susan; Sivina, Mariela; Hoellenriegel, Julia; Wierda, William G; Keating, Michael J; Ding, Wei; Kay, Neil E; Lannutti, Brian J; Marasca, Roberto; Burger, Jan A.
abstract

CLL cell trafficking between blood and tissue compartments is an integral part of the disease process. Idelalisib, a phosphoinositide 3-kinase delta (PI3Kδ) inhibitor causes rapid lymph node shrinkage, along with an increase in lymphocytosis, prior to inducing objective responses in CLL patients. This characteristic activity presumably is due to CLL cell redistribution from tissues into the blood, but the underlying mechanisms are not fully understood. We therefore analyzed idelalisib effects on CLL cell adhesion to endothelial and bone marrow stromal cells (EC, BMSC). We found that idelalisib inhibited CLL cell adhesion to EC and BMSC under static and shear flow conditions. TNFα-induced VCAM-1 (CD106) expression in supporting layers increased CLL cell adhesion and accentuated the inhibitory effect of idelalisib. Co-culture with EC and BMSC also protected CLL from undergoing apoptosis, and this EC- and BMSC-mediated protection was antagonized by idelalisib. Furthermore, we demonstrate that CLL cell adhesion to EC and VLA-4 (CD49d) resulted in the phosphorylation of Akt, which was sensitive to inhibition by idelalisib. These findings demonstrate that idelalisib interferes with integrin-mediated CLL cell adhesion to EC and BMSC, providing a novel mechanism to explain idelalisib-induced redistribution of CLL cells from tissues into the blood.

2012 - Physical contact with endothelial cells through β1- and β2- integrins rescues chronic lymphocytic leukemia from spontaneous and drug-induced apoptosis and induces a peculiar gene expression profile on leukemic cells. [Articolo su rivista]
Maffei, Rossana; Fiorcari, Stefania; Bulgarelli, Jenny; Martinelli, Silvia; Castelli, Ilaria; Deaglio, S; Debbia, Giulia; Fontana, M; Coluccio, Valeria; Bonacorsi, G; Zucchini, Patrizia; Narni, Franco; Torelli, Giuseppe; Luppi, Mario; Marasca, Roberto
abstract

Background: Chronic lymphocytic leukemia B-cells display prolonged survival in vivo, but when cultured in vitro rapidly undergo spontaneous apoptosis. We hypothesize that interaction with endothelial cells in infiltrated tissues and during recirculation may have a pathogenetic role in chronic lymphocytic leukemia.Design and Methods: We evaluated apoptosis of leukemic cells after co-culture on HUVEC monolayer with addition of Fludarabine and blocking adhesion antibodies. Then, we compared microarray-based expression profiles between leukemic cells at baseline and after co-culture.ùResults: We found that endothelial layer protected leukemic cells from apoptosis inducing a 2-fold mean decrement in apoptotic cells after 2 days co-culture. Moreover, endothelial layer decreased sensitivity of chronic lymphocytic leukemia B-cells to Fludarabine-induced apoptosis. Physical contact with endothelium mediated by both β1- and β2- integrins is essential for survival advantage. In particular, blocking CD106 on endothelial cells or CD18 on leukemic B-cells determined the almost complete abrogation of survival advantage (&gt;70% inhibition of viability). Conversely, a reduction of apoptosis was also measured in leukemic cells cultured in conditioned medium collected after 2 days of co-culture, implying that survival is partially mediated by soluble factors. Overall, the contact with endothelial cells modulated 1,944 genes on chronic lymphocytic leukemia B-cells, establishing a peculiar gene expression profile: up-regulation of angiogenesis-related genes, increase of genes involved in TGFβ and Wnt signalling pathways, secretion of cytokines recruiting stromal cells and macrophages and increase in anti-apoptotic molecules such as Bcl2 and Survivin. Conclusion: Our study supports the notion that endothelial cells are major players in chronic lymphocytic leukemia microenvironment. Adhesion to endothelium strongly sustains survival, protects from drug-induced apoptosis and widely modifies gene expression profile of leukemic cells.

2011 - INTERACTION BETWEEN ENDOTHELIUM AND CHRONIC LYMPHOCYTIC LEUKEMIA B-CELLS RESCUES FROM APOPTOSIS AND MODULATES GENE EXPRESSION PROFILE OF LEUKEMIC CELLS [Abstract in Rivista]
Maffei, Rossana; Fiorcari, Stefania; Martinelli, Silvia; Bulgarelli, Jenny; Debbia, Giulia; Fontana, M; Faglioni, Laura; Bigliardi, Sara; Zucchini, Patrizia; Narni, Franco; Torelli, Giuseppe; Luppi, Mario; Marasca, Roberto
abstract

Background. Despite an apparent long life in vivo, CLL cells die rap- idly in vitro. This observation suggests that the apoptotic resistance is not intrinsic to leukemia B cells but extrinsic factors are necessary for CLL prolonged survival. Aims. we investigated the interactions be- tween endothelial cells and CLL cells, highlighting molecular net- works involved in this cellular crosstalk. Methods. we co-cultured CLL cells on HUVEC endothelial monolayer (HC) or in medium alone (CLL only). Then, we detected CLL viability by flow cytometry and we performed whole-genome high density microarrays. Results. we found that endothelial cells protected CLL from spontaneous apop- tosis. After 48h, increased number of alive CLL cells was present in HC condition (59.7 ± 4.2%) compared to CLL alone (22.9 ± 5.1%) (p<0.0001). Moreover, we found that spontaneous in vitro apoptosis was higher in unmutated IGHV CLL (UM-CLL) compared to mutated ones (M-CLL). In HC condition, similar survival was detected be- tween M-CLL and UM-CLL, implying a 2.2-fold increase in relative viability in M-CLL and a 6.1-fold increase in UM-CLL. Moreover, the endothelial cell layer decreased the in vitro sensitivity of CLL cells to Fludarabine-induced apoptotic cell death. The mean viability of CLL cells treated with 10 µM Fludarabine was 19.8% (±4.4%) after 48 hours and 3.8% (±1.3%) after 72 hours. In HC with Fludarabine ad- dition, the mean viability of CLL cells was 37.8% (±9.1%) after 48 hours and 14.3% (±3.2%) after 72 hours. Then, we compared gene expression profiles (GEP) between CLL cultured in contact with EC layer and CLL at baseline to unravel the transcriptional modifications induced by EC cells. Overall 1944 genes were found to be modulated (FC≥2, p<0.05). CLL cells in HC condition showed a 22.6-fold in- crease of CCL2, able to recruit tumor-activated monocytes (p=0.0032) and a 6.5-fold increase of PDGFC, chemoattractant for mesenchymal stromal cells (p=0.0051). Other soluble factors up-reg- ulated by EC/CLL contact were VEGFC (FC=9.4, p=0.0061), ANGTL4 (FC=8.6, p=0.015), EDN1 (FC=9.2, p=0.0061), AMOTL2 (FC=4.3, p=0.019) and THBS1 (FC=45.1, p=0.0004) as well as the metalloproteases MMP2 (FC=8.3, p=0.02) and MMP4 (FC=3.0, p=0.039). The GEP data were confirmed by evaluating the secreted levels of soluble factors in conditioned medium collected after 48h- HC culture. In addition, CLL cells on endothelial layer maintained or increased the expression levels of anti-apoptotic factors Bcl-2, Bcl2A1, BIRC3/c-IAP2 and BIRC5/Survivin compared to CLL cells at baseline. Of interest, the Ang2 tyrosine kinase receptor Tie2 mRNA was found to be increased in CLL cells in co-culture (FC=10.7, p=0.017). We confirmed GEP data by flow cytometry finding a 2-fold and a 4.3-fold increase of percentage of Tie2+CLL cells at 48h and 72h in HC. Conclusion. our results demonstrate a role of endothelial cells in CLL survival advantage and Fludarabine-resistance. The inti- mate contacts with EC seem to determine a microenvironmental- driven angiogenic switch of CLL phenotype, improve the secretion of cytokines involved in regulation of microenvironmental elements such as stromal cells and macrophages and increase the expression of anti-apoptotic molecules.

2010 - Angiopoietin-2 plasma dosage predicts time to first treatment and overall survival in chronic lymphocytic leukemia. [Articolo su rivista]
Maffei, Rossana; Martinelli, Silvia; Santachiara, R; Rossi, D; Guarnotta, C; Sozzi, E; Zucchetto, A; Rigolin, Gm; Fiorcari, Stefania; Castelli, Ilaria; Fontana, M; Coluccio, Valeria; Leonardi, G; Zucchini, P; Tripodo, C; Cuneo, A; Gattei, V; Del Poeta, G; Forconi, F; Gaidano, G; Torelli, Giuseppe; Marasca, Roberto
abstract

The clinical relevance of angiopoietin-2 (Ang2) in chronic lymphocytic leukemia (CLL) was previously suggested by the association between high Ang2, and shorter progression-free survival reported in small series of patients. Here, we evaluated Ang2 glycoprotein levels in plasma samples collected from a multicentric cohort of CLL patients (n = 316) using an enzyme-linked immunosorbent assay method, and we investigated its prognostic role in relation to time to first treatment (TTFT) and overall survival. Based on a cutoff equal to 2459 pg/mL, we divided our cohort in 2 subsets (high and low Ang2) composing 100 (31.6%) and 216 (68.4%) patients, respectively. High Ang2 was predictive of reduced TTFT (P &lt; .001) and overall survival (P = .002). Multivariate analysis confirmed that high Ang2 was an independent prognosticator for TTFT (hazard ratio = 1.739; 95% confidence interval, 1.059-2.857; P = .029). Significant associations were found between high Ang2 and advanced Binet stages (P &lt; .001), high beta(2)-microglobulin (P &lt; .001), unmutated variable region of immunoglobulin heavy chain gene status (P &lt; .001), high CD38 and zeta-chain-associated protein kinase 70 expression (P &lt; .001 and P = .003), and intermediate/high cytogenetic risk (P = .005). Moreover, Ang2 added prognostic power to other conventional prognosticators and helped to refine prognosis among CLL subsets with both high and low vascular endothelial growth factor plasma levels. Ang2 plasma level may be a useful independent prognosticator for CLL.

2010 - Increased angiogenesis induced by chronic lymphocytic leukemia B cells is mediated by leukemia-derived Ang2 and VEGF. [Articolo su rivista]
Maffei, Rossana; Martinelli, Silvia; Castelli, Ilaria; Santachiara, R; Zucchini, Patrizia; Fontana, M; Fiorcari, Stefania; Bonacorsi, G; Ilariucci, F; Torelli, Giuseppe; Marasca, Roberto
abstract

Emerging evidence suggests that angiogenic signalling pathways play important role in the patho-biology of chronic lymphocytic leukemia (CLL). Our goal was to investigate: (i) the spontaneous and hypoxia-induced production of pro-angiogenic factors, VEGF and Ang2, by Real-time PCR and ELISA, (ii) the degree of vascularization in CLL-infiltrated bone marrow (BM) compartment by CD34 immunohistochemical staining of microvessels and (iii) the direct angiogenic effect of CLL-derived VEGF and Ang2 by function-blocking experiments in Matrigel assays. The results demonstrated that CLL cells spontaneously express both VEGF and Ang2 and are able to secrete these factors in surrounding microenvironment. Full-length Ang2 mRNA and truncated form Ang2443 were detectable. Moreover, CLL cells were shown to enhance secretion of both VEGF and Ang2 proteins when subjected to hypoxic condition. Furthermore, increased in vivo and in vitro angiogenesis was induced by CLL cells. Enhanced BM vascularity correlated with Ig-unmutated CLL subset and increased expression of Ang2. Then, we demonstrated that supernatants obtained from CLL cells significantly increase the HUVEC tube formation in Matrigel assays and that this enhanced angiogenic capacity is mediated by both CLL-derived VEGF and Ang2. Taken together, these results suggest that several simultaneous mechanisms may be involved in the CLL capacity to induce the disruption of pre-existing vessel structures to give rise to tumor neoangiogenesis. The preliminary studies in solid tumors, showing that the disruption of Ang2 function can inhibit tumor vessel density and growth, are encouraging and suggest the possibility of new future therapeutic options targeting CLL microenvironment.