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GIULIA DEBBIA

DIPENDENTE AZIENDA POLICLINICO
Dipartimento di Scienze Mediche e Chirurgiche Materno-Infantili e dell'Adulto

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Pubblicazioni

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.

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.

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 - 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.

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.