Hematological malignancy unit

Past and current research projects are focused on two major areas: the first is the understanding the events regulating the neoplastic traffic and growth in patients with leukemias, lymphomas, and myelomas. In particular, in the last years, the following research topics were investigated.

Signal transduction in B-cell lymphocytic leukemia

Figure 1. Subcellular localization of HS1 analyzed by confocal microscopy. HS1 is uniformly distribuited in the cytosol of normal B cells, while it shows a nuclear spotting distribution in B-CLL cells. Figure 2. The chemokine/cytokine network underlying the pathogenesis of IPF

Figure 1. Subcellular localization of HS1 analyzed by confocal microscopy. HS1 is uniformly distribuited in the cytosol of normal B cells, while it shows a nuclear spotting distribution in B-CLL cells. Figure 2. The chemokine/cytokine network underlying the pathogenesis of IPF
[click image to enlarge]

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of clonal CD5+ B lymphocytes. Src kinase Lyn, the switch molecule that couples the B cell receptor to downstream signaling, is over-expressed, anomalously present in the cytosol and displays a high constitutive activity, in B-CLL cells as compared to normal B lymphocytes. Furthermore, these aberrant properties of Lyn are related to defective apoptosis in B-CLL cells. Our attention recently focused on the cytosolic form of Lyn. We found that Lyn shows an active conformation as integral component of an aberrant cytosolic 600 kDa multiprotein complex in B-CLL cells, associated with several proteins, such as Hsp90 through its catalytic domain, and HS1 and SHP-1L through its SH3 domain. In particular, Hsp90 appears tightly bound to cytosolic Lyn, thus stabilizing the aberrant complex and converting individual transient interactions into stable ones. We also demonstrated that treatment of B-CLL cells with geldanamycin, an Hsp90 inhibitor already reported to induce cell death, is capable of dissociating the CL complex in the early phases of apoptosis and thus inactivating CL itself. These data identify the CL complex as a potential target for therapy in B-CLL. (Trentin L et al., Geldanamycin-induced Lyn dissociation from aberrant hsp90-stabilized cytosolic complex is an early event in apoptotic mechanisms in B-chronic lymphocytic leukemia, Blood 2008, in press).

Neoplastic growth and signal transduction in Multiple Myeloma

The serine-threonine GSK3 displays a crucial role in different cancer-pathogenetic pathways, including the PI3K/AKT, Wnt β-catenin and NF-κB signaling cascades, either promoting or counteracting cell survival. We investigated the role of GSK3 in multiple myeloma (MM) cell growth. GSK3α and β total and phosphorylated protein levels were found differentially expressed in malignant plasma cells as compared to normal resting B-lymphocytes and to normal in vitro generated plasmablasts. Intriguingly, in freshly isolated malignant plasmacells from patients, most of GSK3 was found colocalized with Wnt receptor LRP6 and casein kinase I next to the cell membrane as compared to normal plasmacells or B-cells from other malignancies, where it was distributed in the cytosol and in the nucleus, thus suggesting a peculiar role of this kinase in these cells. Upon stimulation of MM cells with IL-6 and IGF-I, GSK3 enzymatic activity was hampered, while stimulation with TNFα did not affect GSK3 function nor the early events in NF-κB activation. Basal and IL-6 and IGF-I driven proliferation of MM cells was slightly impaired by GSK3 blockade. GSK3α inhibition with specific compounds (SB216763 and SB415286) lead a significant rescue from cell death of growth factor-deprived MM cells while resulted in reduced cell proliferation and apoptosis of MM cells grown with serum or growth factors. When GSK3 inhibitors were added to MM cell cultured with bone marrow stromal cells (BMSC), MM cells survival increased and NF-κB and β-catenin-mediated gene transcription (of IAPs and cyclinD1, respectively) was deregulated. GSK3 activity inhibition did not modify the rate of proteasome inhibitor-induced cell death in co-colture experiments with BMSC, suggesting that the sensitivity to bortezomib-induced MM cell apoptosis is independent on GSK3.
Altogether, our data indicate that: i) GSK3 localizes on the cell membrane in primary MM cells; ii) GSK3 is differently regulated downstream from growth factors or TNFα-induced signaling pathways in MM cells; and iii) a critical role of this kinase in regulating the MM microenvironment. (Piazza FA et al., Role of GSK3 in Multiple Myeloma Cell Growth and Survival, ASH Annual Meeting 2007, Blood 110: Abs 2509)

Signal transduction pathways in survival and differentiation block of acute myeloid leukemias cells

Acute myeloid leukemia (AML) arises from an hematopoietic stem cell disorder characterized by both differentiation failure and overproliferation. Recent studies support CK2 as a potential target for cancer therapy. CK2 is essential for cell survival, and considerable evidence suggests that it can also exert potent suppression of apoptosis in cells. This project is aimed to establish a role for CK2 in AML pathogenesis. Acute Promyelocytic Leukemia, (APL, M3 FAB-subtype) is characterized by the chromosomal translocation t(15;17) and the expression of the oncogenic fusion protein PML-RARa (promyelocytic leukemia gene-retinoic acid receptor α); this AML subtype is strikingly responsive to pharmacological doses of retinoic acid (RA), which can overcome the proliferative and anti-differentiation effects of PML-RARa, thus representing a mainstay of the induction and maintenance therapy of this leukemia. Despite the progress achieved in the last decade in the understanding of the RA-dependent molecular pathways, the discrete regulation of RA receptors function in APL still remains unclear. CK2 is a tetrameric (2 α catalytic and 2 β regulatory subunits) protein kinase overexpressed in a large array of tumors, which has been found to phosphorylate and regulate the activity of key myeloid transcription factors and proteins involved in chromatin remodelling. However, CK2 ability to modulate the myeloid developmental program and the RA-dependent cascade has never been investigated. We found that CK2 is highly expressed and active in APL cells. Its localization was usually detected in the cytoplasm, with a scant speckled nuclear pattern; upon RA stimulation most of the protein relocalized in perinuclear areas in the cytosol. Moreover, while the protein levels of the β regulatory subunit progressively fell, the levels and activity of the α catalytic subunit, after RA treatment of APL cells, was found consistently elevated.
To understand whether CK2 plays a role in RA-induced myeloid differentiation, we employed specific CK2 inhibitors (tetrabromobenzotriazole (TBB)-derivatives K27 and K17) and RNA interference to hamper its function. NB4 and primary APL cells displayed an inhibited terminal differentiation upon RA if CK2 activity was blocked with either one of these means, as evaluated by i) the persistence of an immature morphology, ii) the absence of expression of CD11b and CD11c and iii) the greatly reduced production of superoxide anion at the NBT-reduction assay. All these effects were not caused by an increased apoptosis of APL cells, nor by an inhibition of the reshaping of PML nuclear bodies. Instead, CK2 inhibition affected the RA-dependent G1 arrest of APL cells, a prerequisite for the terminal differentiation program to take place. We have also found that RARα-dependent transcription was greatly impaired by CK2 inhibition, and we have evidence of a physical interaction between these two molecules. Last, to clarify the molecular basis of this unpredicted role of CK2 in myeloid differentiation, we have undertaken a proteomic/phosphoproteomic analysis in response to RA. Surprisingly, CK2 inhibition prevented or attenuated a significant amount of the major phosphorylation changes occurring in response to RA. This study emphasizes the role of CK2 in the differentiation induced by RA in AML. Further studies are needed to identify the CK2 targets involved the RA action. (Gurrieri C et al., Role of Protein Kinase CK2 in the Retinoic Acid-Induced Differentiation of Acute Promyelocytic Leukemia Cells, ASH Annual Meeting Abstracts 2007, Blood 110: abs 879).

Lymphoproliferative Disease of Granular Lymphocytes

Recently we investigated the presence of a putative genetic background predisposing to development of the natural killer (NK)-type Lymphoproliferative Disease of Granular Lymphocytes (LDGL). Using polymerase chain reaction (PCR)-based sequence-specific primers, the killer immunoglobulin-like receptor (KIR) genotypes of 35 patients with NK-type LDGL and of 50 normal subjects were investigated to evaluate whether genes coding for activating KIRs were more frequently detected in patients with NK-LDGL. Genotype frequency indicated that the most frequently found gene content was eight genes in controls and 14 in patients (p<0.05). The KIR genotype analysis revealed that patient and, surprisingly, control KIR genotypes preferentially consisted of type B haplotypes characterized by the presence of multiple-activating KIRs. Evidence was also provided that the same KIR genotype was shared by a variable number of patients. Interestingly, the recurrent genotypes observed in the patient group were not found in controls. Concerning inhibitory genes, KIR2DL5a and 2DL5b were more frequently detected in patients than in controls (p<0.01), likely representing a discrete feature of the genetic repertoire of the patients. KIR gene repertoire analysis in patients suggests that the susceptibility to NK-LDGL might be related to the presence of activating KIR genes and supports the concept that these receptors may be involved in the priming of granular lymphocytes (GL) proliferation. Population analysis might disclose a genetic background predisposing to this disease. (Scquizzato E et al., Genotypic evaluation of killer immunoglobulin-like receptors in NK-type lymphoproliferative disease of granular lymphocytes, Leukemia 2007, 21: 1060-9). With these results as a background, in 13 patients with NK-type LDGL and in 10 healthy controls we investigated the mRNA levels of the KIR3DL1 inhibitory and the related KIR3DS1 activating receptors. These genes are usually present and expressed in more than 75% of cases in Caucasian population. Since DNA methylation is the main mechanism involved in the regulation of KIR gene expression, we also analyzed the methylation pattern of CpG islands in the promoter of these genes and the expression levels of DNMTs, responsible of DNA methylation. In spite of 100% expression of KIR3DS1, the complete lack of KIR3DL1 expression was demonstrated in most patients (p<0.01). Moreover, the results of methylation patterns identified a higher methylation status in patients as compared to controls (p<0.01), even if the expression level of DNMTs was not different. In conclusion, we show for the first time a consistent downregulation of inhibitory signal associated to a significantly increased DNA metylation in the KIR3D1 promoter of NK-type LDGL patients, providing evidence that together with the increased expression of activating receptors, also the decrease of the inhibitory signals could account for the NK cell proliferation. (Gattazzo C et al., Inhibitory KIR3DL1 signal down regulation in patients with NK-type lymphoproliferative disease of granular lymphocytes, manuscript submitted).

The second area of research of Semenzato's unit deals with the characterization of the mechanisms which regulate immune cell response at pulmonary level during interstitial lung disorders. In particular, in the last years, the following topics have been investigated.

Idiopathic pulmonary fibrosis pathogenesis

Figure 2. The chemokine/cytokine network underlying the pathogenesis of IPF

Figure 2. The chemokine/cytokine network underlying the pathogenesis of IPF
[click image to enlarge]

Idiopathic pulmonary fibrosis (IPF) is a progressive chronic inflammatory lung disease characterized by fibroblasts proliferation and deposition of extracellular matrix. Animal models of bleomycin induced pulmonary fibrosis have provided contributions in dissecting the molecular basis of this disease, focusing on the role of cytokines and chemokines as pathogenetic mediators of lung fibrosis. GSK3 has been recently shown to modulate inflammatory cytokine production. Since bleomycin causes lung injury, characterized by an inflammatory response followed by a fibrotic degeneration, we postulated that blockade of GSK3 activity by a GSK3 inhibitor could affect the inflammatory and pro-fibrotic cytokine network which sustains bleomycin induced lung injury. We showed that inhibition of GSK3 activity significantly prevented bleomycin induced alveolitis and lung fibrosis. In particular, GSK3 blockade affected the chemokine/cytokine inflammatory and profibrotic milieu, by hampering lung macrophages and Th2 cells, suggesting that GSK3 inhibition has a protective effect on lung fibrosis induced by bleomycin and candidate GSK3 as a potential therapeutical target for preventing pulmonary fibrosis (Gurrieri C et al., GSK3 blockade prevents bleomycin induced lung inflammation and fibrosis, Modern Pathology 2007, 20: 322A, abs 1483).

Pathogenesis and mechanisms of Interstitial Lung Disease

Lung inflammatory diseases, known as interstitial lung diseases (ILDs), while heterogeneous, share common pathogenic mechanisms. The alveolar interstitium, i.e. the space between the alveolar epithelium and the capillary endothelium, is the preferential target of inflammatory processes during ILDs, where the damage derives from the accumulation of inflammatory cells in interstitial and alveolar spaces. In sarcoidosis, T cells and alveolar macrophages (Ams) compartmentalize in the pulmonary microenvironment (alveolitis). This project is aimed at obtaining further clues on the mechanisms leading to the development and persistence of pulmonary inflammation and the continuous recruitment of inflammatory and immunocompetent cells at alveolar and interstitial space. Our recent data on inflammatory cells have clearly demonstrated that the cytokine/chemokine pattern varies during the different stages of the ILDs. We have shown previously that the chemokine receptors CXCR3 and CXCR6 are coexpressed by Th1 cells infiltrating the lung and the granuloma of patients with sarcoidosis. Recently, we evaluated the role of CCL20/CCR6 interaction in the pathogenesis of acute and chronic pulmonary sarcoidosis. We demonstrated that Th1 cells isolated from the bronchoalveolar lavage (BAL) of patients with sarcoidosis and T cell alveolitis are equipped with CCR6. Furthermore, CCR6+ T cells coexpressed the chemokine receptors CXCR3 and CXCR6. Immunohistochemical analysis of lung specimens has shown that CCR6+ T cells infiltrate lung interstitium and surround the central core of the granuloma. It is interesting that CCR6 was never detected on the AM surface, and it is observed in the cytoplasm of AMs from patients with sarcoidosis and alveolitis. The CCR6 ligand CCL20 was expressed by macrophages, multinucleated giant cells, and epithelioid cells infiltrating the granuloma. Furthermore, detectable levels of CCL20 protein are seen in the BAL fluid components of patients with active sarcoidosis, and sarcoid AMs release the CCR6 ligand in vitro. From a functional point of view, sarcoid Th1 cells were able to respond to CXCL10, CXCL16, and CCL20 in migratory assays. In vitro kinetic studies demonstrated that CCR6 is induced rapidly by IL-2, IL-18, and IFN-gamma. In conclusion, T cells expressing CCR6, CXCR3, and CXCR6 act coordinately with respective ligands and Th1 inflammatory cytokines in the alveolitic/granuloma phases of the disease. (Facco M et al., Expression and role of CCR6/CCL20 chemokine axis in pulmonary sarcoidosis, J Leukoc Biol 2007, 82: 946-55).

Synoptic CV

Honours

** Selected Publications (VIMM)

** Other Publications (VIMM)

** Additional Publications

Selected Seminars

Contact

Gianpietro Semenzato Venetian Institute of Molecular Medicine Via Orus 2 35129 Padua — Italy

Tel.:  (+39) 049 7923 263 Tel. lab.:  (+39) 049 7923 241 Fax:  (+39) 049 7923 250

Last updated: ---, GS ·