What are the molecular and genetic aspects involved in the malignant transformation of the leukemic cells?
Could they be used as target for the development of new therapies?
Normal hematopoietic cell biology is strictly driven by complex interactions with microenvironment, involving signaling molecules that finely tune their activation, survival, proliferation and differentiation. The knowledge on anomalous expression or activity of signaling molecules in the leukemic counterparts still requires transformation, in order to better comprehend malignant hematopoietic cell conversion, invasiveness, migration, trafficking and homing, and to identify new targets for innovative therapies. Our Unit faces these issues studying two different kinds of chronic leukemia: the Chronic Lymphocytic Leukemia (CLL), characterized by leukemic B cells, and the Lymphoproliferative Disorders of Large Granular Lymphocytes (LGL Leukemias), elicited by malignant T and NK lymphocytes.
INVESTIGATION OF REGULATORY MECHANISMS INVOLVED IN THE PATHOGENESIS OF CHRONIC LEUKEMIAS TO DEFINE NEW TARGETS FOR THERAPY
Normal hematopoietic cell biology is continuously and strictly driven by multifaceted interactions with microenvironment, involving signaling molecules that finely tune their activation, survival, proliferation and differentiation. Although many studies are currently available on normal hematopoietic cell physiology, the knowledge on anomalous expression or activity of signaling molecules in the malignant counterparts still requires further investigation. The research efforts undertaken in our Unit are aimed to dissect molecular pathways involved in: i) mechanisms accounting for growth and survival of malignant hematopoietic cells and their interactions with the microenvironment; ii) normal and malignant hematopoietic cell invasiveness, migration, trafficking and homing; iii) in vitro responsiveness to both traditional and innovative drugs to suggest new therapeutic strategies. All these points are developed in two different kinds of chronic leukemia:
1. B-cell Chronic Lymphocytic Leukemia (CLL)
2. Lymphoproliferative Disorders of Large Granular Lymphocytes (LGL Leukemias)
1. Investigation of regulatory mechanisms involved in the pathogenesis of CLL
CLL is characterized by the accumulation of mature clonal CD19+/CD5+/CD23+ B lymphocytes in peripheral blood, bone marrow, and lymphoid tissues. Structural and functional features of the B-cell receptor (BCR) and deregulation of the BCR-dependent signaling are associated with the pathogenesis, progression and prognosis of this disease (Burger JA, Trends Immunol. 2013). We previously demonstrated that the Src-kinase Lyn, the switch molecule that couples the BCR to downstream signaling, is crucially involved in the regulation of CLL cells survival (Contri A, J Clin Invest. 2005 and Trentin L, Blood 2008) thus highlighting the importance to better characterize both signals coming from Lyn and those regulating this kinase.
1.1 Cortactin involvement in migration/invasion of CLL leukemic cells
The Lyn substrate Cortactin is known to play an actin cytoskeletal regulatory role involved in both cell migration and cancer progression. Phosphorylated Cortactin at Tyr421 is required for tumor cell motility and invasion. Basing on our previous work on Cortactin (Gattazzo C, Haematologica 2014) we are now aimed at better defining the functional role of Cortactin in CLL. We demonstrated that Cortactin is expressed at different degree in CLL patients (Figure 1) and this expression correlates with the release of MMP-9 and with the motility of neoplastic cells by a direct interaction. Furthermore, the incubation of CLL cells with the Src-kinase inhibitor PP2 decreased phosphorylation of Cortactin at Tyr421 thus reducing the release of MMP-9 in culture medium. This phenomenon was also observed following CXCL12 triggering. These data have recently been published (Martini V, Br J Haematol. 2017). With these and other experiments we are aimed at outline and deepen the role of Cortactin in CLL homing and aggressiveness.
1.2 Role of c-Cbl and CIN85 in the regulation of BCR signalling
In CLL, little is known about the alterations affecting the mechanisms involved in the preservation of Signal Transduction Pathways (STPs) homeostasis that is maintained by different molecules. c-Cbl (c-Casitas B-lineage lymphoma), an E3 ubiquitin ligase, and CIN85 (Cbl-interacting protein of 85kDa), an adapter proteins functioning as docking partners for several signaling proteins, control protein kinase degradation and receptor down-regulation (Dikic I, Cell And Mol Life Sci. 2003). Abnormalities of molecules involved in STPs are connected to CLL pathogenesis and a critical role has already been ascribed, as previously highlighted, to BCR-Lyn axis (Contri A, J Clin Invest. 2005 and Trentin L, Blood 2008). Despite Lyn protein overexpression in CLL, its mRNA level is similar in leukemic vs normal B cells. This suggests that the anomalous Lyn protein expression was not related to differences in gene transcription and/or mRNA stability but to a deregulation in Lyn turnover. In this context, we are aimed at investigating the expression and the role of c-Cbl and CIN85 that in normal B cells are involved in the ubiquitin-dependent Lyn degradation and in the down-regulation of BCR signaling. Preliminary data demonstrated that both CIN85 and c-Cbl are overexpressed and not-interacting with Lyn in CLL B cells. This suggest that they are not involved in Lyn turnover. We are currently investigating the basal level of c-Cbl phosphorylation at Tyr731 in neoplastic B cells and, in the complex, the role of CIN85 and c-Cbl in the development of neoplastic clone.
1.3 Role of JAK/STAT pathway in the survival of CLL cells
Several molecules released by microenvironmetal players signal through JAK(Janus kinases)-STAT(signal transducers and activators of transcription) pathways (Murray PJ, J. Immunol. 2007). We focused on JAK2/STAT3 axis since IL-6, one of the most abundant cytokine released in CLL microenvironment, is the key ligand of this pathway. The deregulation of JAK2/STAT3 axis may lead to aberrant activation of STAT3 and, as a result, to tumor development in hematopoietic cells. We demonstrated that STAT3 is overexpressed and hyper-phosphorylated at Tyr705 in malignant vs normal B cells, thus proving its constitutive activation in CLL. The in vitro incubation of leukemic B cells with specific inhibitors of JAK2 and STAT3 (AG490 and Stattic) induced a dose-dependent apoptosis of CLL cells bypassing the protection provided by microenvironment. We demonstrated that AG490 treatment mediated the activation of SHP-1, leading to inactivation of Lyn protein. Lyn and SHP-1 are both involved in the prolonged lifespan of neoplastic CLL cells. Bypassing the pro-survival stimuli provided by the tumor microenvironment, the ability of AG490 and Stattic to induce apoptosis in leukemic B cells represent a starting point for the development of new therapeutic strategies in CLL.
2. Investigation of regulatory mechanisms involved in the pathogenesis of LGL Leukemias
LGLL is a rare and heterogeneous disorder characterized by the chronic proliferation of clonal Large Granular Lymphocytes (LGL) belonging to T (CD8 or CD4 alpha/betaTCR and gamma/deltaTCR) or NK lineage, accounting for different LGLL subtypes. Somatic mutations, particularly involving STAT genes, mark and contribute to the disease course as the development of neutropenia (Lamy T, Blood, 2017; Teramo A, Oncotarget, 2017). This genetic abnormality combined with the pressure provided by relevant cytokines (notably IL-15 and IL-6; Teramo A, Blood, 2013) might favor the evolution of an indolent condition to an aggressive symptomatic disease through still unrecognized cell pathways (Figure 2). A specific therapy for the disease does not exist, immunosuppressive drugs up to date representing the conventional, although poor effective, treatment for LGLL.
2.1 Biological characterization of the different subtypes of LGL Leukemia
To identify the key mechanisms underlying each LGLL subtype the first step is to define the peculiarities characterizing different subsets of LGL leukemia by analyzing the following LGL leukemic subsets: CD8+, CD4+, and γδ T-LGLL and CLPD-NK.
In this context, the Unit is currently investigating:
- Molecular dissection of CD8+ and CD4+ T-LGLL;
- Clonotype and mutational pattern analysis in γδ T-LGLL;
- Insights into KIR role on pathogenesis of NK-CLPD;
- Identification and functional prioritization of genomic variants, genes, pathways and functions hit by mutations in LGLL subsets.
2.2 Evaluation of different strategies for LGLL therapy: from the already used immunomodulators to new therapeutic options
Studies on the molecular mechanisms of action (MMOA) of methotrexate (MTX), cyclophosphamide (CTX) or cyclosporine A (CyA) on leukemic LGL are not available, so the biological rationale of each therapeutic approach has not yet been demonstrated. Interestingly, some biological features (e.g. STAT3 mutations at Y640F) seemed to predict a response to MTX, since most patients with this mutation responded to the immunosuppressive agent (Loughran TP, Leukemia, 2015). Furthermore, the overall response rate (ORR) to the first-line therapies with each of this drug ranges from 50 to 60%, with a relevant number of patients refractory to treatments or relapsed in a short time. This scenario justifies the need of new therapeutic options to be added to the immunosuppressive drugs already in use. Our purpose is to explore the MMOA of the currently used drugs in order to identify the biological targets on which they exert their effects and evaluate new potential drugs.
As regards the research on LGLL therapy, our Unit is dealing with:
- MMOA of the immunosuppressive drugs already used in LGLL therapy;
- Proteasome inhibitors as new therapeutic options for LGLL;
- Evaluation of JAK/STAT inhibitors as new therapeutic options for LGLL.
Future research plans
Involvement of HSP70 in neoplastic B cell survival of Chronic Lymphocytic Leukaemia
By meanings of Reverse Phase Protein Array (RPPA) technique, we previously demonstrated that HSP70 was significantly overexpressed in CLL vs normals (Frezzato F, J Leukoc Biol. 2016). Considering the anti-apoptotic and cytoprotective roles of HSP70 in cancer (Mayer MP, Cell Mol Life Sci. 2005), we want to characterize this protein in CLL. Preliminary data by western blotting analysis demonstrated that HSP70 was overexpressed in leukemic vs normal B cells and correlated to poor-prognosis. HSP70 levels significantly decreased in ex vivo leukemic cells of patients responding to in vivo treatment (i.e. Fludarabine or Bendamustine). We also observed that HSP70 is localized in the nucleus of neoplastic B cells at baseline and colocalized with its co-chaperone Bag3 upon heat shock. We also found that HSP70 inhibition with different molecules (Pifthrin-μ, MKT-077 and VER155008) caused apoptosis in leukemic B cells. These early results suggest a pivotal role for HSP70 in the regulation of cell survival of leukemic B cells and hint that this protein might represent a target for the development of new therapeutic strategies. Now, we are aimed at studying the mechanisms by which HSP70 promotes survival in CLL B cells.
Investigation of miRNAs in LGL disorder progression
Several small regulatory RNAs, microRNAs (miRNAs), contribute to normal hematopoietic processes and some miRNAs act both as tumor suppressors and oncogenes in the pathogenesis of hematological disorders. The collaboration of our group with Prof. Flavia Bazzoni (Verona University) has allowed us to find interesting preliminary results on the topic of miRNAs, an issue still unknown in LGL disorders. We found that some miRNAs are exclusively down-modulated in leukemic LGLs as compared to LGLs derived from healthy controls. Interestingly, a discrete number of miRNAs seems to specifically characterize patients’ LGLs carrying STAT3 mutations. These preliminary results prompt us to perform a detailed quantitative and qualitative analysis of the miRNA expression profile in leukemic LGLs to define whether this analysis can contribute to understand the mechanisms leading to the disease progression in these disorders. The identification of the miRNA profile would allow the discovery of new markers of LGL disease progression potentially useful for diagnosis/prognosis and hopefully therapy.
Vanessa Rebecca Gasparini
- Teramo A, Barilà G, Calabretto G, Ercolin C, Lamy T, Moignet A, Roussel M, Pastoret C, Leoncin M, Gattazzo C, Cabrelle A, Boscaro E, Teolato S, Pagnin E, Berno T, De March E, Facco M, Piazza F, Trentin L, Semenzato G, Zambello R. “STAT3 mutation impacts biological and clinical features of T-LGL leukemia”. Oncotarget. 2017; 8:61876-61889
- Martini V, Gattazzo C, Frezzato F, Trimarco V, Pizzi M, Chiodin G, Severin F, Scomazzon E, Guzzardo V, Saraggi D, Raggi F, Martinello L, Facco M, Visentin A, Piazza F, Brunati AM, Semenzato G, Trentin L. “Cortactin, a Lyn substrate, is a checkpoint molecule at the intersection of BCR and CXCR4 signalling pathway in chronic lymphocytic leukaemia cells”. Br J Haematol. 2017; 178:81-93
- Tibaldi E, Pagano MA, Frezzato F, Trimarco V, Facco M, Zagotto G, Ribaudo G, Pavan V, Bordin L, Visentin A, Zonta F, Semenzato G, Brunati AM, Trentin L. “Targeted activation of the SHP-1/PP2A signalling axis elicits apoptosis of chronic lymphocytic leukemia cells”. Haematologica 2017; 102:1401-1412
- Leanza L, Romio M, Becker KA, Azzolini M, Trentin L, Managò A, Venturini E, Zaccagnino A, Mattarei A, Carraretto L, Urbani A, Kadow S, Biasutto L, Martini V, Severin F, Peruzzo R, Trimarco V, Egberts JH, Hauser C, Visentin A, Semenzato G, Kalthoff H, Zoratti M, Gulbins E, Paradisi C, Szabo I. “Direct Pharmacological Targeting of a Mitochondrial Ion Channel Selectively Kills Tumor Cells In Vivo”. Cancer Cell 2017; 31:516-531
- Visentin A, Imbergamo S, Gurrieri C, Frezzato F, Trimarco V, Martini V, Severin F, Raggi F, Scomazzon E, Facco M, Piazza F, Semenzato G, Trentin L. “Major infections, secondary cancers and autoimmune diseases occur in different clinical subsets of chronic lymphocytic leukaemia patients”. Eur J Cancer 2017; 72:103-111
- Frezzato F, Accordi B, Trimarco V, Gattazzo C, Martini V, Milani G, Bresolin S, Severin F, Visentin A, Basso G, Semenzato G, Trentin L. “Profiling B cell chronic lymphocytic leukemia by reverse phase protein array: Focus on apoptotic proteins”. J Leukoc Biol. 2016; 100:1061-1070
- Cattaneo F, Patrussi L, Capitani N, Frezzato F, D'Elios MM, Trentin L, Semenzato G, Baldari CT. “Expression of the p66Shc protein adaptor is regulated by the activator of transcription STAT4 in normal and chronic lymphocytic leukemia B cells”. Oncotarget. 2016; 7:57086-57098
- Zonta F, Pagano MA, Trentin L, Tibaldi E, Frezzato F, Trimarco V, Facco M, Zagotto G, Pavan V, Ribaudo G, Bordin L, Semenzato G, Brunati AM. “Lyn sustains oncogenic signaling in chronic lymphocytic leukemia by strengthening SET-mediated inhibition of PP2A”. Blood. 2015; 125:3747-3755
- Gattazzo C, Teramo A, Passeri F, De March E, Carraro S, Trimarco V, Frezzato F, Berno T, Barilà G, Martini V, Piazza F, Trentin L, Facco M, Semenzato G, Zambello R. “Detection of monoclonal T populations in patients with KIR-restricted chronic lymphoproliferative disorder of NK cells”. Haematologica. 2014; 99:1826-1833
- Teramo A, Gattazzo C, Passeri F, Lico A, Tasca G, Cabrelle A, Martini V, Frezzato F, Trimarco V, Ave E, Boscaro E, Piazza F, Facco M, Trentin L, Semenzato G, Zambello R. “Intrinsic and extrinsic mechanisms contribute to maintain the JAK/STAT pathway aberrantly activated in T-type large granular lymphocyte leukemia”. Blood. 2013; 121: 3843-3854
- Chairman, Hematological Network of the Veneto Region (REV), Italy (since 2016)
- Full Professor, University of Padua Medical School, Padova, Italy (since 2000)
- Chief, Hematology and Clinical Immunology Section, Department of Medicine, University of Padova, Italy (since 2000)
- Associate Professor, University of Padua Medical School, Padova, Italy (1990–2000)
- Assistant Professor, University of Padua Medical School, Padova, Italy (1981–1990)
- MD; University of Padua Medical School, Padova, Italy (1974)
- 1990 – A. Minich Award from the Venice Academy of Sciences for studies on the pathogenesis of leukemias.
- 1989 – Cournand Honorary Lecturer for original studies on cellular immunology in interstitial lung diseases.
- 1974 – Award for the best doctoral thesis of the year.