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Id proteins as determinants of drug sensitivity in B chronic lymphocytic leukaemia

Id proteins as determinants of drug sensitivity in B chronic lymphocytic leukaemia Sarah Weiler 1 , Debo Ademokun 2 , Andrew Poynter 3 and John Norton 1 1 Department of Biological Sciences, University of Essex, 2 Departments of Haematology and 3 Radiotherapy, Ipswich Hospital NHS Trust.

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Id proteins as determinants of drug sensitivity in B chronic lymphocytic leukaemia

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  1. Id proteins as determinants of drug sensitivity in B chronic lymphocytic leukaemia Sarah Weiler1, Debo Ademokun2, Andrew Poynter3 and John Norton1 1Department of Biological Sciences, University of Essex, 2Departments of Haematology and 3Radiotherapy, Ipswich Hospital NHS Trust

  2. Id proteins as dominant negative antagonists of bHLH transcription factors

  3. Cellular processes involving ID proteins • lineage commitment • differentiation • embryogenesis/organogenesis • cell cycle regulation • cell growth • apoptosis • tumorigenesis • angiogenesis

  4. The global interactome of ID proteins

  5. Id gene expression in various tumours compared to normal tissue Fold down-regulated Fold up-regulated

  6. ID proteins are involved in drug-sensitivity in various tumours

  7. B-cell chronic lymphocytic leukaemia (BCLL) • Most common type of leukaemia in western world • Accumulation of mature clonal B-cells expressing CD5 and CD19 • Defective apoptosis not increased proliferation • Increased resistance to chemotherapy (effectively incurable) • Highly variable therapeutic response among patients

  8. BCLL treatment Current prognostic markers ● IgHV mutational status ● ZAP70 + CD38

  9. Aims of the study • investigate role of ID proteins in drug-induced apoptosis in BCLL • determine pathways and molecular mechanisms • determine whether ID proteins can be used as molecular markers to predict therapeutic response in BCLL

  10. Dynamics of in vivo Id gene expression in BCLL patients following treatment with fludarabine ID1 ID2 ID3 GEO datasets analysed from Rosenwald et al (2004) Blood 104: 1428-1434

  11. Identification of genes up-regulated by fludarabine in BCLL E: 0- 6hr L: 3-5 days Most of the significant fludarabine-induced changes involve up-regulation of gene expression The expression of very few genes (<100) is significantly changed by fludarabine (P = ≤ 0.01) Occurrences Feature P

  12. Expression profile of genes that are most significantly up-regulated by fludarabine in BCLL ID2 is third-most fludarabine-responsive gene within a gene signature defining a classic p53-dependent cell death pathway

  13. GEO microarray datasets Co-expression analysis of ID genes inBCLL Gene Network Central (SABiosciences) Primary interactions network of co-expressed genes CYTOSCAPE Construction of interactive gene/protein interaction networks Aracne (transcription factor binding) “Reverse-engineering” of regulatory networks of ID proteins export export

  14. Protein and gene interaction network connecting ID2 to apoptosis in BCLL

  15. Analysis of apoptotic response of leukaemic B cells after introduction of exogenous genes Live cells – large faint nuclei Apoptotic cells – condensed bright nuclei

  16. Anti-apoptotic genes (Bcl2 and Bclxl) protect leukeamic B cells from cell death induced by oxidative stress % apoptosis in TEGFP positive population Bcl2: P <0.0001 Bcl-Xl: P <0.0001 [H2O2] (µM)

  17. Apoptotic effects of gain- and loss-of-function of Id proteins in transfected human fibroblast cells

  18. Effect of gain- and loss-of-function of Id proteins on apoptosis induced by oxidative stress % apoptosis Bcl2: P <0.0001 ID1: P =0.0017 ID3: P <0.0001 aHLH: P =0.0026 [H2O2] (µM)

  19. Summary • ID expression is deregulated in BCLL • BCLL microarray datasets show ID gene expression changes in response to various chemotherapeutic drugs • Fludarabine consistently up-regulates ID2 • The response of ID1 and ID3 is heterogenous in different patients • Regulatory pathways link ID genes to cell death/survival mechanisms in BCLL • Bcl2 and Bcl-XL protect from oxidative stress-induced apoptosis in B-cell lines • In human adherent fibroblasts, ID1 sensitizes cells to apoptosis induced by oxidative stress while ID3 protects cells • Functional ablation of all four ID proteins rescues cells from oxidative stress-induced cell death Acknowledgments This project is generously supported by a grant from the Ipswich Hospital Haematology and Oncology Research Fund

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