Which are the mechanisms regulating malignant B lymphocyte growth, fitness and resilience to cytotoxic agents?

Non-Hodgkin lymphomas (NHL) and multiple myeloma (MM) are B-cell derived tumours and represent the vast majority of hematologic cancers, in many instances still incurable. We aim at defining the determinants of intrinsic and extrinsic growth signals for NHL and MM cells. Our laboratory is particularly interested in identifying novel regulators of cellular fitness and stress resistance to be targeted in novel therapeutic approaches for NHL and MM.

Background
Research
Group Members
Key Publications
NHL and MM cell growth and proliferation implies a continuous exposition to different kinds of stresses, as for instance the oxidative, replicative, proteotoxic and DNA damage-related harmful burdens. The precise mechanisms regulating NHL and MM cell capability of coping with such damaging noxae are largely unknown. Since B cells have physiological mechanisms regulating immunoglobulin gene rearrangements (class switch recombination and somatic hypermutation) handling DNA double strand breaks and, at the plasmacellular differentiation stage, coping with the protetoxic and oxidative noxious load, one possible way the malignant B cell counterparts may survive to different stresses is through an over-exploitation of these pathways. Therefore - while essential for malignant B cell survival – these stress-managing cascades also represent exquisite points of therapeutic vulnerability. By using multifaceted experimental approaches, utilizing NHL and MM cell lines, primary cells from patients, tissue sections, mouse models, genomic, transcriptomic and proteomic tools, we demonstrated that the family of serine-threonine kinases CK1 and CK2 are important modulators of the activation of the NF-B, PI3K/AKT and JAK/STAT signaling pathways in NHL and MM and are fundamental to sustain the response to endoplasmic reticulum (ER)/proteotoxic stress. Our loss-of-function models of CK1 and CK2 in mouse B lymphocytes demonstrate the essential role of these kinases and are fundamental tools to study their function in B cell physiology and pathobiology. We aim to explore 1) the physiological role of CK1 and CK2 in hemo-lymphopoiesis; 2) the exact function of CK1 and CK2 in the transmission of signals from the B-cell receptor to the nucleus in malignant lymphoma; 3) the role of CK1 in autophagy in NHL and MM; 4) the role of CK2 in the ER stress and in the DNA damage response in NHL and MM.

What are the physiological roles of protein kinases CK1 and CK2 in hemolymphopoiesis?
Hematopoietic stem cell (HSC) self-renewal and differentiation in myeloid and lymphoid cells are processes tightly regulated by intrinsic and extrinsic signals. The Wnt/β-catenin, Hedgehog (Hh) and PI3K/AKT signaling pathways play a crucial role in HSC fate decision and biochemical and functional genetic studies have demonstrated their essential function in blood cell development. Protein kinases CK1α and CK2 are acidophilic serine-threonine kinases, which, despite structurally unrelated, share the property of impinging on the Wnt/β-catenin, Hh and PI3K/AKT signaling. CK1α regulates Wnt signalling by phosphorylating β-catenin on Ser45 and priming it for subsequent phosphorylation by GSK3β on Ser33, Ser37 and Thr41 in the β-catenin destruction complex. CK1α also regulates Hh signalling by phosphorylating Cubitus Interruptus/Gli proteins in concert with the kinases PKA and GSK3β, driving their proteolytic degradation and extinguishing Hh signalling. CK2 positively regulates Wnt signaling by phosphorylating Dishevelled and catenin 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 (Figure 1). However, there are no studies that investigated the function of CK1α and CK2 in hematopoiesis. On these grounds, we have started a research program aimed at addressing the specific roles of CK1 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 CK1α gene that will be used to conditionally knockout CK1α in the hematopoietic and B-cell and T-cell compartments. We have also generated hematopoietic and B-cell specific knockout mice for the regulatory β subunit of CK2 using CK2β flox. These mice are currently under examination.

How are regulated the growth, stress resistance and fitness of NHL and MM cells?
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, we were the first to demonstrate that protein kinases CK2 and GSK3 play an oncogenic function in MM. We showed that CK2 regulates the STAT3 and NF-B activation downstream from growth factors and cytokines whereas GSK3 modulates catenin and AKT levels in MM cells (Piazza F et al. Blood 2006, Piazza F et al. BMC Cancer 2010). Since we found that CK2 regulates the ER-stress/UPR in MM cells, we are currently investigating the mechanisms through which CK2 and the related kinases GSK3 and CK1 may control the NF-B, 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 IRE1α and the function of the chaperoning complex composed by Heat shock protein 90 /Cell division cycle 37 (Hsp90/Cdc37) (Manni S et al. Clin Can Res 2012). Along this line, we are interested in checking whether mechanisms linking the DNA damage response, the ER stress response and the activation of signal transducers of a stress response could be regulated by the transcription factors XBP1, STAT3 and NF-B in NHL and MM In tight collaborations with the Division of Pathology of the University of Padova, we are also investigating the expression of these kinases, transcription factors and stress-regulating proteins in tissue samples from MM and NHL patients and the potential correlations with clinico-biological features of these diseases (Pizzi M, Piazza F et al. Oncotarget 2015). Moreover, we plan to set up a phospho-proteomic and proteomic program aimed at analysing the pattern in NHL and MM cells under basal and different stress conditions and manipulation of stress-related kinases.

What are the roles of CK1 and CK2 in B-cell receptor signalling?
Both “tonic” antigen (Ag)-independent, PI3K-dependent and “chronic active” NF-B-addicted B-cell receptor (BCR) signaling are crucial for the growth of most of non-Hodgkin lymphomas (NHL). The serine-threonine kinases CK1 and CK2 stimulate the PI3K/Akt/mTOR pathway and - downstream BCR - might interact with the Ikk-NF-B-activating Card11-Bcl10-MALT1 (CBM1) complex. We recently demonstrated high CK2 expression and activity in germinal center (GC)-derived NHL (Pizzi M, Piazza F et al. Oncotarget 2015) and preliminary data in knockout mice we have generated indicate that CK1 and CK2 are pivotal for early and late B cell development and may significantly impact on “tonic” as well as “active” BCR signalling (Zaffino F et al Blood 2014).
We are interested to: a) analyze the in vivo role of CK1 and CK2in normal and NHL B cells by using loss of function mouse and cellular models. b) disentangle the BCR signaling nodes where CK1 and CK2 are likely to be crucial: the PI3K/AKT/FOXOs and the CARD11/IKK/NF-B pathways and the CK2-regulated Hsp90/Cdc37 chaperoning function on signaling kinases. c) identify novel CK1 and CK2 target molecules in normal and NHL B lymphocytes by high throughput phosphoproteomics. d) test in mouse NHL cell xenograft models CK1 and CK2 inhibitors in conjunction with novel anti-lymphoma agents.

What is the role of CK1 and CK2 in autophagy and proteotoxic stress in NHL and MM cells?
Autophagy plays a fundamental role in B cell physiology and during malignant transformation. In MM, autophagy must be tightly regulated to keep cellular homeostasis (Pengo N et al. 2013; Oliva L and Cenci S, 2014). CK1 is downstream from RAS-induced autophagy maintaining the rate of the process under control, while CK2 is a crucial kinase in stimulating the activity of the phagosome cargo protein p62/SQSTM1. We are interested in assessing the pathophysiological roles of these two kinases in autophagy in NHL and MM. We have shown that CK1 regulates autophagy in MM since its inactivation triggers the autophagic flux (Carrino M et al, 2017). Protein kinases members of the CK1 family are under analysis in the autophagic pathways in NHL and MM. The regulation of autophagosome and correct cargoing activity by p62/SQSTM1 by CK2 is analysed in B cell tumors. The effects of the inactivation of CK1 and CK2 on the cytotoxicity on the protestasis exerted by proteasome inhibitors and immunomodulatory drugs (lenalidomide, thalidomide) and are investigated.

Sabrina Manni

Assistant Professor

sabrina.manni@unipd.it

Laura Quotti Tubi

Postdoctoral Fellows

laura.quottitubi@gmail.com

Arash Arjomand

Postdoctoral Fellows

arash_arjomand@hotmail.com

Marilena Carrino

PhD Student

marilena.carrino@studenti.unipd.it

  1. Manni S, Carrino M, Piazza F. Role of protein kinases CK1α and CK2 in multiple myeloma: regulation of pivotal survival and stress-managing pathways. J Hematol Oncol. 2017 Oct 2;10(1):157. doi: 10.1186/s13045-017-0529-5. Review. PubMed PMID: 28969692; PubMed Central PMCID: PMC5625791.
  2. Mandato E, Nunes SC, Zaffino F, Casellato A, Macaccaro P, Tubi LQ, Visentin A,Trentin L, Semenzato G, Piazza F. CX-4945, a selective inhibitor of casein kinase 2, synergizes with B cell receptor signaling inhibitors in inducing diffuse large B cell lymphoma cell death. Curr Cancer Drug Targets. 2017 Apr 26. doi: 10.2174/1568009617666170427110450. [Epub ahead of print] PubMed PMID: 28460620.
  3. Manni S, Carrino M, Manzoni M, Gianesin K, Nunes SC, Costacurta M, Tubi LQ, Macaccaro P, Taiana E, Cabrelle A, Barilà G, Martines A, Zambello R, Bonaldi L, Trentin L, Neri A, Semenzato G, Piazza F. Inactivation of CK1α in multiple myeloma empowers drug cytotoxicity by affecting AKT and β-catenin survival signaling pathways. Oncotarget. 2017 Feb 28;8(9):14604-14619. doi: 10.18632/oncotarget.14654. PubMed PMID: 28099937; PubMed Central PMCID: PMC5362429.
  4. Quotti Tubi L, Canovas Nunes S, Brancalion A, Doriguzzi Breatta E, Manni S, Mandato E, Zaffino F, Macaccaro P, Carrino M, Gianesin K, Trentin L, Binotto G, Zambello R, Semenzato G, Gurrieri C, Piazza F. Protein kinase CK2 regulates AKT, NF-κB and STAT3 activation, stem cell viability and proliferation in acute myeloid leukemia. Leukemia. 2017 Feb;31(2):292-300. doi: 10.1038/leu.2016.209. Epub 2016 Aug 1. PubMed PMID: 27479180.
  5. Mandato E, Manni S, Zaffino F, Semenzato G, Piazza F. Targeting CK2-driven non-oncogene addiction in B-cell tumors. Oncogene. 2016 Nov 24;35(47):6045-6052. doi: 10.1038/onc.2016.86. Epub 2016 Apr 4. Review. PubMed PMID: 27041560.
  6. Manni S, Toscani D, Mandato E, Brancalion A, Quotti Tubi L, Macaccaro P, Cabrelle A, Adami F, Zambello R, Gurrieri C, Semenzato G, Giuliani N, Piazza F. Bone marrow stromal cell-fueled multiple myeloma growth and osteoclastogenesis are sustained by protein kinase CK2. Leukemia. 2014 Oct;28(10):2094-7. doi: 10.1038/leu.2014.178. Epub 2014 Jun 4. PubMed PMID: 24897506.
  7. Manni S, Brancalion A, Mandato E, Quotti Tubi L, Colpo A, Pizzi M, Cappellesso R, Zaffino F, Di Maggio SA, Cabrelle A, Marino F, Zambello R, Trentin L, Adami F, Gurrieri C, Semenzato G, Piazza F (2013) Protein kinase CK2 inhibition down modulates the NF-kB and STAT3 survival pathways, enhances the cellular proteotoxic stress and synergistically boosts the cytotoxic effect of bortezomib on multiple myeloma and mantle cell lymphoma cells. PLOS One 8: e75280.
  8. Manni S, Brancalion A, Quotti Tubi L, Colpo A, Pavan L, Cabrelle A, Ave E, Zaffino F, Di Maira G, Ruzzene M, Adami F, Zambello R, Pitari MR, Tasson P, Pinna LA, Gurrieri C, Semenzato G, Piazza F (2012) Protein kinase CK2 protects multiple myeloma cells from ER stress-induced apoptosis and from the cytotoxic effect of HSP90 inhibition through regulation of the unfolded protein response. Clin Cancer Res 18:1888-900.
  9. Piazza F, Manni S, Ruzzene M, Pinna LA, Gurrieri C, Semenzato G (2012) Protein kinase CK2 in hematologic malignancies: reliance on a pivotal cell survival regulator by oncogenic signaling pathways. Leukemia 26:1174-1179.
  10. Piazza F, Manni, S, Quotti Tubi L, Montini B, Pavan L, Colpo A, Gnoato M, Cabrelle A, Adami F, Zambello R, Trentin L, Gurrieri C, Semenzato G (2010) Glycogen Synthase Kinase-3 regulates multiple myeloma cell growth and bortezomib-induced cell death. BMC Cancer 10:526.

FRANCESCO PIAZZA

  • • MD, University of Padova, Italy (1995)
  • Board in Medical Oncology, University of Padova, Italy (2000)
  • Postdoc: Memorial Sloan-Kettering Cancer Center, New York, USA (1999-2003)
  • Assistant Professor of Hematology, Dept. of Medicine, University of Padua (since 2008)
  • Group leader: Hematological Malignancies, Lymphoma and Myeloma Pathobiology, Venetian Institute of Molecular Medicine, Padua, Italy (since 2013)

Selected Awards

  • 2004: Brian D. Novis Junior Award, International Myeloma Foundation (USA)
  • 2006: “Guido Berlucchi” Award for young oncologists, Italy