1. New molecular therapeutical targets - in leukemia T-ALL signals: translating posttranslational alterations into therapy? João T. Barata Instituto de Medicina Molecular, Lisboa, Portugal

2. from Nature Reviews Cancer

3. Extracellular Factors Intracellular Alterations Cancer Progression

4. Extracellular Factors Intracellular Alterations Signal Transduction Pathways Cancer Progression

5. Extracellular Factors Intracellular Alterations Signal Transduction Pathways T-ALL Progression

6. Acute Lymphoblastic Leukemia (ALL) The most frequent childhood malignancy Malignant clones originate from T or B cell progenitors arrested at different stages of lymphoid development T-Cell Acute Lymphoblastic Leukemia (T-ALL) Worse prognosis than B-lineage ALL patients The use multi-agent chemotherapy has significantly improved treatment outcome Roughly 20% of the patients relapse and have a dismal prognosis Novel, more selective, less detrimental therapies are required

7. T-Cell Acute Lymphoblastic Leukemia (T-ALL) Roughly 20% of the patients relapse and have a dismal prognosis It is estimated that a significant fraction of the patients are unnecessarily treated with intensive regimens Long-term severe complications Novel, more selective, less detrimental therapies are required

8. Extracellular Factors Intracellular Alterations Signal Transduction Pathways T-ALL Progression

9. Extracellular Factors Intracellular Alterations Signal Transduction Pathways T-ALL Progression

10. PI3K/Akt(PKB) pathway – hyperactivated in human cancer PI3K PIP2PIP3 PTEN Akt/ PKB ProliferationViabilityMigrationGrowth PTEN – frequently inactivated in human cancer:  genetic lesions / epigenetic alterations  diminished/absent mRNA and protein

11. Is PI3K/Akt(PKB) pathway constitutively hyperactivated in primary T-ALL cells?

12. PI3K/Akt signaling pathway constitutive hyperactivation in primary T-ALL cells Most primary T-ALLs (87.5% - 21/24) display PI3K/Akt pathway hyperactivation Silva et al, JCI 2008, 118:3762

13.  Which mechanisms lead to PI3K/Akt pathway hyperactivation in T-ALL? PI3K PIP2PIP3 PTEN Akt/ PKB

14. 80% of the cases express PTEN. PTEN mutations and protein expression alterations

15. Increased PTEN expression associates with decreased activity and increased phosphorylation

16. PTEN P P P InactiveActive Degradation Phosphatase CK2 Stabilization Vazquez et al, MCB 2000, 20:5010; Torres & Pulido 2001, JBC 276:993; Miller et al, FEBS Lett 528:145

17. CK2 in cancer CK2 overexpression is often observed in human solid tumors (e.g. lung, breast and renal cell carcinomas) (Daya-Makin et al., 1994; Landesman-Bollag et al., 2001; Scaglioni et al., 2006; Stalter et al., 1994) Essential for multiple myeloma cell survival (Piazza et al., 2006) Transgenic mice with targeted expression of CK2 in T cells develop lymphomas (Seldin and Leder, 1995)

18. PTEN expression and activity are regulated by CK2 in PTEN+ T-ALL cells

19.  Are primary T-ALL cells dependent on these mechanisms for their survival?

20. Inhibition of PI3K or CK2 promotes cell death selectively in T-ALL cells

21. T-ALL PI3K inhibition (with LY294002) promotes cell death selectively in T-ALL cells with hyperactivation of PI3K/Akt pathway...

22. ... independently of PTEN expression

23. CK2 inhibition (with TBB or DRB) induces cell death selectively in T-ALL cells especially in those that express PTEN protein

24. Silva et al, JCI 2008, 118:3762

25. Extracellular Factors Intracellular Alterations Signal Transduction Pathways T-ALL Progression

26. IL-7 ViabilityCell Cycle p27 kip1 Bcl-2pRb hyper-P PI3K / Akt (PKB) Cell Growth Glut1 Glucose uptake Mitochondrial homeostasis TfR T-ALL 72% patients Barata et al. Blood 2001, 98:1524; Haematologica 2004, 89:1459; Blood 2004, 103:1891; J Exp Med 2004, 200:659

27. Does IL-7 contribute to leukemia progression in vivo?

28. IL-7 / IL-7R in T-cell leukemia Notch upregulates IL-7R  expression in T-ALL (Gonzales-Garcia et al, JEM 2009, 206:779) IL-7 transgenic mice develop T and B cell lymphomas (Rich et al, JEM 1993, 177:305) AKR/J mice with IL-7R  overexpression in thymic progenitors develop spontaneous lymphomas (Laouar et al, Blood 2004, 103:1985)

29. Transgenic IL-7R expression promotes leukemogenesis and tumor-associated death F5 Tet-IL-7R fed dox (n=10) F5 Rag1 -/- IL-7R -/- rtTA huCD2 fed dox (n=4) F5 Tet-IL-7R no dox (n=3) Age (days) Survival Thymic whole cells extracts P-AKT (S473) PTEN p27 KIP1 Actin ControlsTumours Silva et al, unpublished – collaboration with Ben Seddon’s lab

30. Rag2 -/- Il2rg -/- (IL-7 WT) Rag2 -/- Il2rg -/- Il7 -/- (IL-7 KO) vs mIL-7 Human T-ALL cells Human T-ALL cells

31. IL-7 accelerates leukemia expansion in mice xenotransplanted with T-ALL cell lines HPB-ALL-luc-eGFP Silva et al, unpublished

32. IL-7 accelerates leukemia expansion and leukemia-related death in mice xenotransplanted with T-ALL cell lines TAIL7

33. IL-7 accelerates leukemia expansion in mice xenotransplanted with human primary T-ALL cells

34. IL-7 promotes p27 Kip1 downregulation, Bcl-2 upregulation, proliferation and viability of xenotransplanted human primary T-ALL cells Silva et al, unpublished

35. Ex vivo evidence for leukemia cell responsiveness to IL-7 in T-ALL patients IL-7IL7RA Park et al, Immunity 2004, 21:289 IL-7- IL-7RIL-7 (consumption) Guimond et al, Nat Immunol 2009, 10:149

36. IL-7 contributes to human T-ALL in vivo

37. Extracellular Factors Intracellular Lesions Signal Transduction Pathways T-ALL Progression

38. IL-7 CK2 PI3K/Akt T-ALL Progression  -IL-7;  -IL-7R  Abs; Pharmacological inhibitors PI3K/Akt inhibitors CK2 inhibitors (CX-4945)

39. Cancer Biology Unit (IMM) Ana Silva Bruno Cardoso Leila Martins Centro Infantil Boldrini, SP, Brazil J. Andrés Yunes Patricia Jotta Angelo Laranjeira University of Dundee, UK Nick Leslie National Institute for Medical Research, London, UK Ben Seddon Hospital de Santa Cruz, Portugal Miguel Abecasis Fundação para a Ciência e a Tecnologia Fundação Calouste Gulbenkian Associação Portuguesa Contra a Leucemia Children with Leukaemia, UK LPC/NRS – Terry Fox