Research

The hematology oncology lab is conducting research focused on the mechanisms regulating normal blood cell development (hematopoiesis) and malignant transformation to tumor cells.  By making use of genetic, biochemical, cellular and animal models, as well as clinical specimens, we pursue the ultimate goal to unravel the causes of malignant blood tumors and to explain basic mechanisms governing normal hematopoiesis. Our interests are centered on survival mechanisms in normal and tumoral blood cells and on signal transduction pathways important for the response to endogenous and exogenous cellular stresses.  In particular, over the past few years we have set a research program aimed at clarifying the role of Ser Thr kinasaes in the survival of hematopoietic stem cells and mature blood cells and in the growth of B lymphocyte derived malignancies, such as multiple myeloma and non Hodgkin's lymphomas. Our research also is trying to uncover novel regulatory pathways of stem cell-regulating signaling pathwas, in particular the Wnt/beta-catenin and the Hedgehog cascades as well as to clarify other mechanisms of activation and regulation of stress and survival managing signals like the ones mediated by the NF-kB and STAT3 transcription factors.

 We believe that investigating at the same time the normal physiology of hematopoiesis and malignant blood cell biology could represent a powerful strategy that add more value to the research in this area of biomedicine.

Main topics currently under study in the lab are:

1. Role of protein kinases CK1alpha and CK2beta in normal blood cells development.
2. Role of protein kinases CK1alpha and CK2beta in B lymphopoiesis.
3. Mechanims of multiple myeloma plasma cell growth and survival.
4. Stress response pathways in non Hodgkin's lymphomas and multiple myeloma.

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Mechanisms of Growth and Differentiation of Normal and Malignant Hematopoietic Cells.

Hematopoietic stem (HSC) and progenitor cells constitute the reservoir of all the blood cell lineages during the organism lifespan.  These cells are endowed with the capability of self-renewing or commit towards the differentiation of erythroid, megakaryocytic, granulo-monocytic and lymphoid cells.  Homeostatic processes that balance the HSC self-renewing activity, the output towards lineage committment, progenitor cell proliferation and the attainment of the terminally differentiated lineage-specific functions are involved to finely tune these processes.  Malignant transformation can occur at any level during the hematopoietic cascade.  If the oncogenic event hits early, acute leukemias, both lymphoblastic (from lymphoid progenitors) or myeloblastic (from myeloid progenitors), or chronic myeloproliferative neoplasms can develop.  If it hits at later stages, mature blood cell tumors can develop, mostly of lymphoid origin (non Hodgkin lymphomas, chronic lymphocytic leukemia, multiple myeloma and other plasma cell dyscrasias). 

A long-standing interest of our research has been the identification of novel regulators of both normal and malignant hematopoieis, in particular in acute promyelocytic leukemia and in non-Hodgkin lymphomas. In keeping with pursuing these goals, our current research efforts are aimed at investigating signalling regulators (in particular protein kinases) in hematopoiesis and leukemogenesis.  We are also exploring the role of protein kinases and stress signals in normal B cell development and in B-cell malignancies.

Role of Protein Kinases CK1 and CK2 in Hematopoietic Stem Cell Growth

HSC self-renewal and differentiation are tightly regulated by intrinsic and extrinsic signals. The Wnt/bcatenin, Hedgehog (Hh) and PI3K/AKT signaling pathways play a critical role in HSC fate decision and biochemical and functional genetic studies have demonstrated their essential function in blood cell development.  Protein kinases CK1a and CK2 are acidophilic serine-threonine kinases, which regulate fundamental cellular processes.  These two kinases, despite structurally unrelated, share the property of impinging on a number of hematopoiesis- associated signal transduction pathways; in particular, CK1a and CK2 regulate at different levels the Wnt/b-catenin, Hh and PI3K/AKT signaling either by direct phosphorylation of pathway members or through indirect modulation of other regulatory proteins. CK1a regulates Wnt signalling by phosphorylating b-catenin on Ser⁴⁵ and priming it for subsequent phosphorylation by GSK3b on Ser³³, Ser³⁷ and Thr⁴¹ in the b-catenin destruction complex.  CK1a also regulates Hh signalling by phosphorylating Cubitus Interruptus/Gli proteins in concert with the kinases PKA and GSK3b, driving their proteolytic degradation and extinguishing Hh signalling.  CK2 positively regulates Wnt signaling by phosphorylating Dishevelled and bcatenin at Thr³⁹³ and also regulates Hh signalling in a positive manner.  In Drosophila, CK2 phosphorylates Smoothened, favoring the Hh-transduced signal and the downstream molecule Ci (the homolog of mammalian Gli), stabilizing it and overall causing a positive regulation.  Moreover, the antagonistic actions of CK2 and B56-containing PP2A phosphatase might regulate the stability of Gli proteins through phosphorylating/dephosphorylating Daz interacting protein 1 (Dzip1), which has been shown pivotal for Gli proteins degradation.  However, despite these notions on the role of CK1a and CK2 in these HSC-regulating pathways, there are no studies that investigated the function of these two protein kinases in hematopoiesis.  On these grounds, we have started a research program aimed at addressing the specific roles of CK1a and CK2 in normal hematopoietic cell development.  We have set different experimental model systems including analysis of cell lines, normal hematopoietic cells from healthy human subjects and genetically modified mice. In particular, we have generated mice with LoxP sites flanking exon 1 of the CK1a gene that will be used to conditionally knockout CK1a in the hematopoietic and B-cell and T-cell compartments.  We have also generated hematopoietic and B-cell specific knockout mice for the regulatory b subunit of CK2 using CK2b flox (generated at the University of Grenoble, France, by T. Bochou). These mice are currently under examination. 

The Figure shows two mouse fetuses at 15.5 dpc. On the left, the CK2beta floxed/floxed-Vav1-CRE mouse (CK2beta knockout in the hemopoietic compartment) appears pale, hydropic with petechial hemorrhages and a paler liver compared to a control (Vav1-CRE) mouse. Flow cytometry, histopathological, cytological analysis of feta liver cells showed a marked reduction of erythroid cells.

Phenotypic and Biological Characterization of Leukemia Stem Cell

Leukemia stem cells (LSCS) represent the "ignition fuel" of acute and chronic leukemias and are believed to be refractory to cytotoxic treatment. The characterization of their biological properties is instrumental to develop novel "leukemia-eradicating" therapeutic approaches.  We have started a program of phenotypic and biochemical characterization of LSC obtained from leukemia patients referred at the University of Padova Hospital and other centers. We have developed a 5 or 6-color flow cytometry protocol to isolate these cells for downstream assays. We are investigating the expression of protein kinases CK1a and CK2 and Wnt and Hh gene targets in order to correlate these data with clinico-biological variables.  Another goal is to set protocols for the identification of differential phosphoproteome patterns in LSC as compared to HSC in order to identify potential druggable targets among protein kinases.

Regulation of Multiple Myeloma and Non-Hodgkin Lymphoma Cell Growth

Multiple myeloma (MM) is an incurable plasma cell malignancy, which causes a significant morbidity due to organ damage and bone tissue destruction. Non-Hodgkin lymphomas are a heterogeneous group of B-cell or T-cell derived blood malignancies that remain in large part incurable. Exploring the role of potential new regulators of these malignancies growth and survival, our group was the first to demonstrate that protein kinases CK2 and GSK3 play an oncogenic function in MM.  These protein kinases have been demonstrated to act by impinging on pivotal signaling pathways that control malignant clone growth. Our Laboratory is currently investigating the mechanisms through which CK2 and GSK3 control the NF-kB, STAT3 and endoplasmic reticulum (ER) stress/unfolded protein response (UPR) signaling in MM and non-Hodgkin Lymphomas.  We could prove that CK2 is a master regulator of a compensatory UPR response in malignant plasma cells by sustaining the activity of the kinase/endoribonuclease IRE1a and the function of the chaperoning complex composed by Heat shock protein 90 /Cell division cycle 37 (Hsp90/Cdc37).  Another issue of our research deals with the mechanisms linking the DNA damage response, the ER stress response and the activation of signal transducers of a stress response (in particular the roles of the transcription factors XBP1, STAT3 and NF-kB).  In tight collaborations with the Division of Pathology of the University of Padova, we are also investigating the expression of these kinases and of stress-regulating proteins in tissue samples from MM and NHL patients and the potential correlations with clinico-biological features of these diseases. Xenotransplants of human MM and NHL cells in immunodeficient mice will be used to analyze the efficacy of manipulating the activity of CK2 and GSK3 kinases and of ER stress signalling.  Genetic mouse models will also be employed to this purpose.  We are generating mice conditionally deleted for the Hsp90 Cdc37 co-chaperone in the hemopoietic compartment.