Signaling monitoring and flow cytometry

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1 Signaling monitoring and flow cytometry Coultry Club, FIAP Paris April 8, 2014 Dr Jacques Nunès Lymphocyte Activation Group 1

2 PLAN I Signalisation intracellulaire & «signal monitoring» II Méthodologie et outils III Résultats IV - Perspectives 2

3 PLAN I Signalisation intracellulaire & «signal monitoring» II Méthodologie et outils III Résultats IV - Perspectives 3

4 Navigating the cell signaling maze guided by Ariadne's thread Acuto O. et al. Nat. Rev. Immunol Minoan Palace in Heraklion, Crete 4

5 Cell signaling modules and their connections Jacques A. Nunès, Guylène Firaguay and Emilie Coppin. Analysis of cellular signaling events by flow cytometry. Flow Cytometry - Recent Perspectives, 2012, ISBN

6 Cell signaling modules and their connections Jacques A. Nunès, Guylène Firaguay and Emilie Coppin. Analysis of cellular signaling events by flow cytometry. Flow Cytometry - Recent Perspectives, 2012, ISBN

7 PI3K / Akt hyperactivation in cancer cells Vivanco I and Sawyers CL & Engelman JA

8 The PI3K/Akt signaling in acute myeloid leukemia Survival, proliferation and drug resistance of AML cells unresolved issues Relevance of PI3K/Akt/mTOR pathway up-regulation in AML patients and its druggability No predictive markers for AML patients with a high probability of responding to PI3K/Akt/mTOR inhibition Martelli AM et al. Expert Opin. Investig. Drugs 2009; 18(9):

9 PLAN I Signalisation intracellulaire & «signal monitoring» II Méthodologie et outils III Résultats IV - Perspectives 9

10 The PI3K / Akt pathway in human cancer PIP 2 PTEN PI3 - kinase PIP 3 PH Akt P P Thr308 Ser473 PDK1 mtor/ Rictor/G L Rapamycin RAD001 Cellular targets : mtor, MDM2,TSC2, GSK3, FKHR, NF- B, BAD... Transformation, cell cycle, DNA repair, apoptosis 10

11 Western-blot versus phosphoflow analysis (FCM) approach Guylène Firaguay human primary T cells - + CD3 + CD28 NT CD3 + CD28 pser473 Akt WB Akt WB pser473 Akt -PE 11

12 Western-blot versus phosphoflow analysis (FCM) approach Guylène Firaguay 0 1 human primary T cells - + CD3 + CD28 NT CD3 + CD28 pser473 Akt WB Akt WB pser473 Akt -PE 12

13 Detecting intracellular signaling events as a barcode signal activation blot overview digital view Thanks to Ron Germain

14 PLAN I Signalisation intracellulaire & «signal monitoring» II Méthodologie et outils III Résultats IV - Perspectives 14

15 Detecting intracellular signaling events by FCM Differential role for CD277 as a co-regulator of the immune signal in T and NK cells. Messal N, Mamessier E, Sylvain A, Celis-Gutierrez J, Thibult ML, Chetaille B, Firaguay G, Pastor S, Guillaume Y, Wang Q, Hirsch I, Nunès JA, Olive D. Eur J Immunol Dec;41(12): human T cells pakt perk1/2 NT CD3+CD28 + IgG1 (17 g/ml) CD3+CD28 + CD277 mab (17 g/ml) 15

16 Analysis of signaling events by dynamic phosphoflow cytometry Pervanadate (pv) treatment Kinase inhibitor (KI) treatment Proportion of cells pv pv KI KI Mean Fluorescence Intensity of phosphoprotein Mean Fluorescence Intensity of phosphoprotein Kinase OFF, large shift in Phosphoprotein intensity Kinase ON, very little change in Phosphoprotein intensity Kinase ON, large shift in Phosphoprotein intensity Kinase OFF, very little change in Phosphoprotein intensity Guylène Firaguay and Jacques A. Nunès. Sci. Signal., 2009 Volume 2 (86) pl3. 16

17 Analysis of signaling events by dynamic phosphoflow cytometry U937 myeloïd cell line lymphocytes pakt (Ser473) pakt (Thr308) pcrkl (Tyr207) perk-1/2 pjnk ps6 ribosomal OFF ON 17

18 Acute myeloid leukemia with normal karyotype (NC-AML) patients «Approximately 50% of adult AMLs have normal cytogenetics (NC-AMLs), and represent a heterogeneous and poorly understood group.» Norbert Vey et al., Oncogene 2004 Molecular determinants of prognosis in NC-AMLs Identification of new groups using dynamic phosphoflow analysis 18

19 Calculation of a PI3K inhibitor sensivity factor (SF) SF = MFI UT / MFI WMN pv U937 myeloïd cell line lymphocytes pakt (Ser473) perk-1/2 ps6 ribosomal 19

20 Dynamic phosphoflow analysis 1- Signaling signature (coupling with gene profiling data) 2- Innovative proposal for new therapeutic protocols targeting intracellular signaling pathways signaling pathway inhibitors - (e.g. wortmannin, a PI3K inhibitor) No predictive markers for AML patients with a high probability of responding to PI3K/Akt/mTOR inhibition 20

21 Dynamic phosphoflow analysis 1- Signaling signature (with gene expression profiling data) Identification of new potential biomarkers in NC-AMLs: OGFRL1, PLSCR1 2- Innovative proposal for new therapeutic protocols targeting intracellular signaling pathways Signaling Inhibitor Sensitivity Factor 21

22 Cell signaling and integrative biology 22

23 Polychromatic phosphoflow analysis Jean-Michel Othoniel, Le Confident Square Doyen Jean Lépine, Nice Jacques A. Nunès, Guylène Firaguay and Emilie Coppin. Analysis of cellular signaling events by flow cytometry. Flow Cytometry - Recent Perspectives, 2012, ISBN

24 Cell signaling modules and their connections 24

25 Cell signaling modules and their connections 25

26 Monitoring of cell signaling pathways by FCM Jacques A. Nunès, Guylène Firaguay and Emilie Coppin. Analysis of cellular signaling events by flow cytometry. Flow Cytometry - Recent Perspectives, 2012, ISBN

27 Monitoring of cell signaling pathways by FCM PerFix EXPOSE kit Cell stimulation on human peripheral blood Cell surface and intracellular staining in one step (CD14 - CD45 - perk - pakt - pstat5) NT GM-CSF LPS pv Emilie Coppin No stimulation- GM CSF 20 ng/ml 10min - LPS 20 ng/ml 7min - pervanadate 50μM 5min 27

28 PLAN I Signalisation intracellulaire & «signal monitoring» II Méthodologie et outils III Résultats IV - Perspectives 28

29 Monitoring of cell signaling pathways by mass cytometry. towards 34 parameters Single-cell mass cytometry of differential immune and drug responses across a human hematopoietic continuum. Bendall SC et al. Science (6030): DVS Sciences and CyTOF are trademarks or registered trademarks of DVS Sciences, Inc. 29

30 Lymphocyte Activation group Valentin Giroux Cédric Favre Emilie Coppin Guylène Firaguay Daniel Birnbaum & Co Emilie Mamessier, Daniel Olive Norbert Vey Cédric Favre Caroline Scifo Fabrice Malergue Calanque de Sugiton, Marseille 30