Phd RESEARCH PROJECT

LIPID SIGNALING AND MYELOID DIFFERENTIATION: ROLE OF PI-PLC BETA 1

STATE OF THE ART (STATO DELL’ARTE)

Phosphoinositides (PIs) are a small family of phospholipids that control and modulate different cellular processes. They are involved in both cytoplasmatic and nuclear signalling pathways, regulating membrane trafficking, cellular differentiation, proliferation and apoptosis.1,2 Nuclear phosphoinositides are located in the nuclear speckles and work as co-factors in transcription regulation, DNA repair and RNA expression.3These two different locations, cytoplasmatic and nuclear, are completely independent.4Different kinases, phosphatases and lipases, act on PI pathways in order to release second messengers that take part in several pathways. Among the enzymes involved in the nuclear lipid signal transduction, Phospholipase Cβ1 (PLCβ1) has an important and pivotal role.5 6 Beyond the classic cytoplasmatic PI cycle, PLCβ1 regulates the nuclear PI cycle, playing an important role in the control of cell cycle, proliferation and differentiation.4,7PLCβ1 gene is located on the short arm of chromosome 20 (20p12)8 and produces two splicing variants: PLCβ1a (150 kDa) and PLCβ1b (140 kDa). They differentiate one another for the C-terminal sequence and their localization. In fact, both of them have a Nuclear Localization Sequence (NLS), but PLCβ1a has also a Nuclear Export Sequence (NES), and for this reason it localizes also in the cytoplasm. On the other hand, PLCβ1b is mainly located inside the nucleus.9,10 Recently, the evidence of a clinical correlation between the presence of a PLCβ1 monoallelic gene deletion and the progression of Myelodysplastic Syndromes (MDS) to Acute Myeloid Leukemia (AML) opened new perspectives of research and treatments.11,12,13 The MDS are a heterogeneous group of bone marrow disorders characterized by alterations of the hematopoietic stem cells that lead to anemia, neutropenia, bleeding problems and infections.14,15 MDS are not very common syndromes, with an overall incidence of 4-5 cases/100.000/year, and 20-40 cases/100.000/year in over 70 year-old patients.16 The FAB (French-American-British) and IPSS (International Prognostic Scoring Systems) classifications17,18, mainly based on the blast number and karyotype, are used to divide the patients in two major groups: high and low risk of AML progression. MDS patients should undergo allogenic hematopoietic stem cell transplantation (allo-HSCT), but for those who are not candidates, hypomethylating agents (HMAs), such Azacitidine (AZA), are now the first therapeutic choice. AZA is both a hypomethylating and a direct cytotoxic agent for abnormal hematopoietic cells. Moreover, these drugs have also been tested before and after allo-HSCT, but we are not still completely aware of their role.23 Patients with MDS need a lot of time to be able to benefit of the effect of AZA, and sometimes they can lose their positive response to the treatment after a certain time.20, 26 For these reasons, molecular markers able to predict the clinical outcome and responsiveness to HMAs are needed, in order to reach a better, safer and more specific use of AZA or other HMAs. 23 Patients affected by MDS with a high risk of AML evolution have a reduction in the expression of the nuclear PLCβ1 variant, that can also be related to the presence of a mono-allelic deletion of the gene.14 ,18 As PLCβ1b can physiologically regulate the progression trough the G1 phase of the cell cycle19,12, the drastic reduction of nuclear PLCβ1 expression could alter the normal cell cycle in patients with MDS.

Nuclear PLCβ1 in MDS is also epigenetically relevant. In fact, several studies have shown that PLCβ1 is a molecular target for AZA.20,21,22 As a matter of fact, high risk patients that respond to the drug have shown an early increase of nuclear PLCβ1 expression, an induction of normal myeloid differentiation, and a better prognosis21,23. At the same time, the demethylation of the PLCβ1 gene promoter also decreases the activation of phosphorylated AKT, which reduces the cellular apoptosis and increase proliferation. Therefore, also the decrease of AKT activation may improve the patients' prognosis.22,23,21For all these reasons, PLCβ1 could be both a prognosis stratification marker and a treatment predictive outcome marker in patients with MDS, but we still do not know many of the molecular processes and effects.

PROJECT DESCIPTION (DESCRIZIONE DEL PROGETTO)

Hypothesis: if PLCβ1 does have a central role in myeloid differentiation, we could study its modulation during the differentiation induced by hypomethylating agents (HMAs) as Azacytidine24 and Phorbol 12-myristate 13-acetate (PMA), a specific activator of PKC, hence of NF-κB25. The focus will be on PLCβ1 different localization. Indeed, PLCβ1 could interact with phosphokinases, lipase and cyclins at a nuclear level, thus specifically affecting cell cycle and differentiation.
Goal: the aim of this project is to analyse and better understand the role of nuclear PI-PLCβ1 in myeloid differentiation. This could identify patients with a higher probability to respond to HMAs with the possible result of minimizing the therapeutic intrinsic risks and, at the same time, optimizing the clinical outcomes. Eventually, we could try to create the bases for individualized therapeutic strategies, considering the PLCβ1 genetic and epigenetic features, in order to reach the best results with the minimum risks and costs.

Results: the proposed experiments aim to observe an increase of nuclear PLCβ1 after AZA treatment that is associated with an increased myeloid differentiation. Moreover, we also expect to see a clinical improvement of the patients (reduction of anemia, neutropenia/infections, bleeding and a decreased need for support therapy). If both the molecular and clinical data are encouraging, in the future the molecular gait could precede the clinical one so as to guide the therapeutic strategies.

FELLOW TRAINING PLAN (PIANO FORMAZIONE ASSEGNISTA)

First year: PLCβ1 role in myeloid differentiation

The purpose of the first year will be to clarify the role of nuclear PLCβ1 in the differentiation process and see its correlation with other enzymes involved in the PI nuclear cycle. The work will start on AML cellular lines, in order to observe and quantify their myeloid differentiation under PMA or Azacytidine (AZA) treatments. These two treatments should have effects on the amount, and possibly the localisation, of PLCβ1. This could result in an alteration of PLCβ1-dependent cellular differentiation and cell cycle regulation. In fact, PLCβ1 regulates molecules like Cyclin D3, Cyclin B1 and PKCα during cell cycle, thus regulating the G1 checkpoint and the G2/M transition.27 We will need to use Electroporation techniques in order to overexpress or silence PLCβ1. We will measure the gene expression by Real time-PCR. The investigation will focus on the two different isoforms of PLCβ1: 1a and 1b, but also on other molecules correlated to the PI cycle. Instead, to follow the myeloid differentiation we will measure, by flow cytometry, the level of CD11b and CD14, that are respectively granulocyte and monocyte differentiation markers.

Second year: morphological studies and clinical approach

 If we show that PLCβ1 has a clear role in myeloid differentiation, we should then investigate its morphological role, through different techniques, before and after treatments with AZA. Indeed, the nuclear localization of PLCβ1 could lead to a specific morphological and molecular phenotype. In particular, we would like to know how nuclear PLCβ1 is involved in the myeloid process and quantify its expression during the treatments, by immunocytochemistry and analysing both cytoplasmatic and nuclear fractions.

 During this year we will start also the patients’ enrollment. The collaboration with the Department of Haematology "L. and A. Seràgnoli" of the S.Orsola-Malpighi Hospital of Bologna will be essential. In particular, we will set a prospective study for patients with MDS treated with AZA or supportive therapy, in order to correlate molecular and clinical results.

Our cohort will be made of 30 patients:

20 High-risk MDS patients treated with AZA
10 High-risk MDS patients treated with supportive therapy

Third year: from molecular to clinical data

During the last year of the research project we aim to analyse MDS patients’ peripheral blood and bone marrow. We will study the molecular behaviour of PLCβ1 in the patients’ cells under treatment (AZA) or without it (supportive care only). We will then correlate patients’ clinical outcome with their molecular asset.

BIBLIOGRAPHY (BIBLIOGRAFIA)

1. Cocco L, Martelli AM, Vitale M, et al. Inositides in the nucleus: regulation of nuclear PI-PLCβ1. Adv Enzyme Regul. 2002;42:181-193. doi:10.1016/S0065-2571(01)00030-9.

2. Follo MY, Marmiroli S, Faenza I, et al. Nuclear phospholipase C β1 signaling, epigenetics and treatments in MDS. Adv Biol Regul. 2013;53(1):2-7. doi:10.1016/j.jbior.2012.09.009.

3. Martelli AM, Manzoli L, Cocco L. Nuclear inositides: facts and perspectives. Pharmacol Ther. 2004;101(1):47-64. http://www.ncbi.nlm.nih.gov/pubmed/14729392.

4. Martelli AM, Gilmour RS, Bertagnolo V, Neri LM, Manzoli L, Cocco L. Nuclear localization and signalling activity of phosphoinositidase C beta in Swiss 3T3 cells. Nature. 1992;358(6383):242-245. doi:10.1038/358242a0.

5. Follo MY, Faenza I, Piazzi M, et al. Nuclear PI-PLCβ1: An appraisal on targets and pathology. Adv Biol Regul. 2014;54:2-11. doi:10.1016/j.jbior.2013.11.003.

6. Follo MY, Bosi C, Finelli C, et al. Real-time PCR as a tool for quantitative analysis of PI-PLCbeta1 gene expression in myelodysplastic syndrome. Int J Mol Med. 2006;18(2):267-271.

7. Manzoli L, Billi AM, Rubbini S, et al. Essential role for nuclear phospholipase C beta1 in insulin-like growth factor I-induced mitogenesis. Cancer Res. 1997;57(11):2137-2139.

8. Cocco L, Manzoli L, Palka G, Martelli AM. Nuclear phospholipase C beta1, regulation of the cell cycle and progression of acute myeloid leukemia. Adv Enzyme Regul. 2005;45:126-135. doi:10.1016/j.advenzreg.2005.02.001.

9. Piazzi M, Blalock WL, Bavelloni A, et al. Phosphoinositide-specific phospholipase C β 1b (PI-PLCβ1b) interactome: affinity purification-mass spectrometry analysis of PI-PLCβ1b with nuclear protein. Mol Cell Proteomics. 2013;12(8):2220-2235. doi:10.1074/mcp.M113.029686.

10. Martelli AM, Fiume R, Faenza I, et al. Nuclear phosphoinositide specific phospholipase C (PI-PLC)-beta 1: a central intermediary in nuclear lipid-dependent signal transduction. Histol Histopathol. 2005;20(4):1251-1260. http://www.ncbi.nlm.nih.gov/pubmed/16136505. Accessed April 21, 2016.

11. Lo Vasco VR, Calabrese G, Manzoli L, et al. Inositide-specific phospholipase c beta1 gene deletion in the progression of myelodysplastic syndrome to acute myeloid leukemia. Leukemia. 2004;18(6):1122-1126. doi:10.1038/sj.leu.2403368.

12. Follo MY, Finelli C, Clissa C, et al. Phosphoinositide-Phospholipase C 1 Mono-Allelic Deletion Is Associated With Myelodysplastic Syndromes Evolution Into Acute Myeloid Leukemia. J Clin Oncol. 2008;27(5):782-790. doi:10.1200/JCO.2008.19.3748.

13. Itzykson R, Gardin C, Fenaux P. Meeting report: myelodysplastic syndromes at ASH 2007. Leukemia. 2008;22(5):893-897. doi:10.1038/leu.2008.45.

14. Cocco L, Follo MY, Faenza I, et al. Nuclear inositide signaling: an appraisal of phospholipase C beta 1 behavior in myelodysplastic and leukemia cells. Adv Enzyme Regul. 2007;47:2-9. doi:10.1016/j.advenzreg.2006.12.003.

15. Poli A, Billi AM, Mongiorgi S, et al. Nuclear Phosphatidylinositol Signaling: Focus on Phosphatidylinositol Phosphate Kinases and Phospholipases C. J Cell Physiol. 2016;231(8):1645-1655. doi:10.1002/jcp.25273.

16. Herold G. HEROLD’s Internal Medicine (Second Edition) - Vol. 1, Volume 1. Lulu.com; 2014.

17. Nosslinger T. Myelodysplastic syndromes, from French-American-British to World Health Organization: comparison of classifications on 431 unselected patients from a single institution. Blood. 2001;98(10):2935-2941. doi:10.1182/blood.V98.10.2935.

18. Cocco L, Follo MY, Manzoli L, Suh P-G. Phosphoinositide-specific phospholipase C in health and disease. J Lipid Res. 2015;56(10):1853-1860. doi:10.1194/jlr.R057984.

19. Faenza I, Bregoli L, Ramazzotti G, et al. Nuclear phospholipase C beta1 and cellular differentiation. Front Biosci. 2008;13:2452-2463.

20. Follo MY, Finelli C, Bosi C, et al. PI-PLCbeta-1 and activated Akt levels are linked to azacitidine responsiveness in high-risk myelodysplastic syndromes. Leukemia. 2008;22(1):198-200. doi:10.1038/sj.leu.2404855.

21. Cocco L, Finelli C, Mongiorgi S, et al. An increased expression of PI-PLC 1 is associated with myeloid differentiation and a longer response to azacitidine in myelodysplastic syndromes. J Leukoc Biol. 2015;98(5):769-780. doi:10.1189/jlb.2MA1114-541R.

22. Follo MY, Finelli C, Mongiorgi S, et al. Reduction of phosphoinositide-phospholipase C beta1 methylation predicts the responsiveness to azacitidine in high-risk MDS. Proc Natl Acad Sci U S A. 2009;106(39):16811-16816. doi:10.1073/pnas.0907109106.

23. Finelli C, Follo M, Stanzani M, et al. Clinical impact of hypomethylating agents in the treatment of myelodysplastic syndromes. Curr Pharm Des. March 2016.

24. Zeidan AM, Kharfan-Dabaja MA, Komrokji RS. Beyond hypomethylating agents failure in patients with myelodysplastic syndromes. Curr Opin Hematol. 2014;21(2):123-130. doi:10.1097/MOH.0000000000000016.

25. Tímár J, Diczházi C, Bartha, Ladányi A, Tarcsafalvi A, Lapis K. PMA induces shift from chondroitin to heparan sulphate on proteoglycans correlating with fibronectin adhesion of MDS human leukemia cells. Anticancer Res. 14(3A):1227-1231.

26. Nazha A, Sekeres MA, Garcia-Manero G, et al. Outcomes of patients with myelodysplastic syndromes who achieve stable disease after treatment with hypomethylating agents. Leuk Res. 2016;41:43-47. doi:10.1016/j.leukres.2015.12.007.

27. Poli A, Faenza I, Chiarini F, Matteucci A, McCubrey JA, Cocco L. K562 cell proliferation is modulated by PLCβ1 through a PKCα-mediated pathway. Cell Cycle. 2013;12(11):1713-1721. doi:10.4161/cc.24806.

28. Fenaux P, Adès L. How we treat lower-risk myelodysplastic syndromes. Blood. 2013;121(21):4280-4286. doi:10.1182/blood-2013-02-453068.