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Flow cytometric signal typing for therapy response prediction in pediatric myeloid leukemia

Flow cytometric signal typing for therapy response prediction in pediatric myeloid leukemia Michael N. Dworzak Veronika Sexl. Linking Research and Patients' Needs. Childhood cancers – Cure rates still poor in acute myeloid leukemia (AML). AML: New therapeutic options by

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Flow cytometric signal typing for therapy response prediction in pediatric myeloid leukemia

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  1. Flow cytometric signal typing for therapy response prediction in pediatric myeloid leukemia Michael N. Dworzak Veronika Sexl Linking Research and Patients' Needs

  2. Childhood cancers – Cure rates still poor in acute myeloid leukemia (AML)

  3. AML: New therapeuticoptions by blockingintracellular signal transduction external stimuli SCF FL IL3 GCSF GMCSF TPO SDF-1 IL6 VEGF PDGF TGFβ IFNγ mutation RTKs: FLT3, KIT, PDGFR, VEGFR Cytokine-Receptors survival apoptosis AKT PI3K Ras mTOR JAK2,3 SRC Rac Raf Bad Bcl-XL RS6K JNK (p38) MEK S6 protein- synthesis ERK NFκB STAT1,3,5 Bad Bcl-XL c-Myc,Elk,CREB,c-Jun,FKHR,… proliferation differentiation

  4. AML: New therapeuticoptions by blockingintracellular signal transduction external stimuli SCF FL IL3 GCSF GMCSF TPO SDF-1 IL6 VEGF PDGF TGFβ IFNγ mutation RTKs: FLT3, KIT, PDGFR, VEGFR Cytokine-Receptors AKT PI3K Ras mutation mTOR JAK2,3 SRC Rac ?? Raf Bad Bcl-XL RS6K JNK (p38) MEK S6 ERK NFκB STAT1,3,5 Bad Bcl-XL c-Myc,Elk,CREB,c-Jun,FKHR,…

  5. Study premises and aims Work package 2 “Flow cytometric assay development” Aim: Development and validation of a flow cytometric assay allowing for interrogation of the activation state of multiple signal pathways potentially relevant for the pathobiology of acute myeloid leukemia in children. Work package 3 “Clinically relevant leukemic cell analysis” Aim: Assess and correlate the signaling profiles of pediatric AML samples with morphological, genetic, as well as clinical parameters, aiming at describing AML-subtype- or patient-specific patterns potentially relevant for the choice of signal transduction inhibitors. Work package 4 “Transplantation of human AML and CML cells into NSG mice” Aim: Amplification of limiting human patient samples of AML and CML through the transplantation of human cells into NSG for assay development. Testing long term effects of inhibitor treatment in vivo. Leukemia arises in NSG animals only when viable stem cellsare injected.

  6. Divergent signalpathwayactivationafter different cytokinestimuli BASAL GCSF GMCSF FL SCF SDF1 IL3 TPO COCK IFNg 7400signalactivationread-outs from N=74 pediatric AML cases

  7. MLL-normal FAB myeloid MLL-rearranged FAB monoblastic HCA-analysis

  8. Patient classificationandstratificationupon patient-specificsignal-activation bio-signatures

  9. “hot” cohort relapsed 44% (11/25) “cold” cohort relapsed 15% (8/52)

  10. additional effects over constitutive • Signal activationsignatures • in pediatric AML: • patient-specific „finger-prints“ • differentiation-associated (FAB types) • genotype-related • outcome-associated

  11. AML: New therapeuticoptions by blockingintracellular signal transduction Cytokine-R RTKs, eg. FLT3, KIT, PDGFRb, VEGFR LY294002 AKT mutation Everolimus PI3K Ras mTOR JAK2,3 SRC Rac Sunitinib Sorafenib PKC412 Raf Bad Bcl-XL RS6K JNK (p38) MEK Bortezomib S6 ERK NFκB STAT1,3,5 AG490 Bad Bcl-XL c-Myc,Elk,CREB,c-Jun,FKHR,… Dasatinib Sorafenib PD98059

  12. 2. Signal inhibition-assay in pediatric AML correlates well with in-vitro drug activity - cell death 1. 3.

  13. In vitro drug activity in pediatric AML correlates well with outcome in-vivo in the xenograft model BM or spleen of 1st TP Incubation: 24h supplemented DMSO Sorafenib PKC412 Rapamycin Ruxolitinib Preliminary data: One representative experiment out of 2

  14. Signals in FLOW in pediatric AML – the international network M. van Heuvel, M. Zwaan Erasmus MU Rotterdam mutational screening cross-platform validation D. Reinhardt, MH Hannover AML-BFM study headquarter sample recruitment clinical data repository genetic data repository mutational screening research data website M. Dworzak, STAK Vienna AML-BFM-A study headquarter signal transduction profiling pharmacodynamic monitoring M. Dworzak, CCRI Vienna xenograft models V. Sexl, Veterinary University Vienna

  15. Output and Impact New options

  16. Output and Impact Publications (preliminary) Enhanced Ratio of Activated STAT5/STAT3 after G‑CSF Stimulation in vitro is Associated with Favorable Prognosis in Pediatric Acute Myeloid Leukemia Maibach S, Herbst C, Zimmermann M, Reinhardt K, Böhmer K, Dworzak M, Creutzig U, Reinhardt D, Ehlers S submittedLeukemia 2013

  17. Output and Impact Clinical application

  18. Leukemia – identification of vulnerable nodes Andrea Hölbl-Kovacic Veronika Sexl Michael Dworzak

  19. Leukemia – Aims of the Study To find Achilles` heelswithinleukemiccellsvia identificationof essential signaltransductionpathways

  20. A candidate approach: The JAK/STAT Signalling Pathway oncoproteins Tumor promoters: JAK2, STAT3, STAT5 Tumor suppressors: STAT1

  21. Key findings of the STAT5 Study B-ALL maintenance LeukemicStem Cells STAT5 Imatinib-responsiveness Mechanism: Serinephosphorylationof Stat5 iscritical

  22. STAT5 is critical for B-ALL maintenance Stat5 present Stat5 absent 44 0.2 bone marrow B220 CD19 delete Stat5 in leukemic cells Hoelbl, Schuster et al, EMBO Mol Med 2010

  23. STAT5 is critical for leukemic stem cells leukemicmouse bone marrow SSC c-kit GFP Sca-1 STAT5 deletion ex vivo 0 SSC c-kit GFP Sca-1 2nd transplant Hoelbl, Schuster et al, EMBO Mol Med 2010

  24. Imatinib treatment selects STAT5highcells daysof treatment: Days p + imatinib Warsch et al, Blood 2011

  25. Serine phosphorylation of STAT5 is critical for leukemia STAT5 serine phosphorylation – a potential therapeutic target Friedbichler et al, Blood 2011

  26. Another Candidate Approach: Switching gears from JAK/STAT signalling to cell cycle regulation The cell-cycle dependent kinase 6 (CDK6) comes into a privileged role in angiogenesis… Friedbichler et al, Blood 2011

  27. CDK6 regulates angiogenesis in lymphoma * 100 0.50 Cdk6+/+ Cdk6-/- 80 0.40 60 tumor weight (g) 0.30 % survival 40 0.20 20 0.10 Nu/Nu 0 0 10 20 0.00 time (days) Cdk6+/+ Cdk6-/- NPM-ALK+tumors B-ALL tumors Cdk6+/+ Cdk6-/- Cdk6+/+ Cdk6-/- Kollmann et al, Cancer Cell 2013

  28. Output and Impact Follow-up publication NPM-ALK+tumors + imatinib Imatinib treatment of a human ALCL patient Laimer, Dolznig, Kollmann, Vesely et al, Nature Medicine 2012 Lab of Lukas Kenner, LBI-CR

  29. Output and Impact Follow-up projects STAT5 serinephosphorylation in leukemia (FWF-Grant to A.H.) CDK6 in stem-cell-derivedleukemia (FWF-Grant to V.S.)

  30. Output and Impact - Publications Hoelbl A, Schuster C, Kovacic B, Hoelzl M, Fajmann S, Grebien F, Warsch W, Stengl G, Hennighausen L, Beug H, Moriggl R, Sexl V. Stat5 is indispensable for the maintenance of bcr/abl positive leukemia (2010). EMBO Mol Med; 2: 98-110. Friedbichler K, Kerenyi MA, Kovacic B, Li G, Hoelbl A, Yahiaoui S, Sexl V, Müllner E, Fajmann S, Cerny-Reiterer S, Valent P, Beug H, Gouilleux F, Bunting KD, Moriggl R. Stat5a serine 725 and 779 phosphorylation is a prerequisite for hematopoietic transformation (2010). Blood. 116 1548. Warsch W, Kollmann K, Eckelhart E, Fajmann S, Cerny-Reiterer S, Hoelbl A, Gleixner K, Dworzak M, Mayerhofer M, Hoermann G, Hermann H, Sillaber C, Egger G, Valent P, Moriggl R, Sexl V. High Stat5 levels mediate imatinib –resistance and indicate disease progression in chronic myeloid leukemia (2011). Blood. 117:3409 Hantschel O, Warsch W, Eckelhart E, Grebien F, Superti-Furga G, Sexl V. Bcr/Abl directly activates Stat5 independent of Jak2. (2012) Nat. Chem Biol 8(3):285-93 Kovacic, B; Hoelbl, A; Litos, G; Alacakaptan, M; Schuster, C; Fischhuber, K; Kerenyi, M; Stengl, G; Moriggl, R; Sexl, V; Beug, H: Diverging fates of cells of origin in acute and chronic leukemia. EMBO Mol Med, Apr;4(4):283-97 Kollmann K, Heller G, Schneckenleithner C, Warsch W, Scheicher R, Ott RG, Schäfer M, Fajmann S, Schlederer M, Schiefer AI, Reichart U, Mayerhofer M, Hoeller C, Zöchbauer-Müller S, Kerjaschki D, Bock C, Kenner L, Hoefler G, Freissmuth M, Green AR, Moriggl R, Busslinger M, Malumbres M, Sexl V. A Kinase-Independent Function of CDK6 Links the Cell Cycle to Tumor Angiogenesis. Cancer Cell. 2013 Aug 12;24(2):167-81

  31. Output and Impact Career Development • Veronika Sexl: • 2010: fullprofessorshipattheVetmedUni Vienna (VUV); headoftheinstitute • 2013: memberofthe EHA Fellowship Grant Committee • 2013: Supervisory Board Member ofthe Medical University Vienna (MUV) • Andrea Hölbl-Kovacic: • 2009: University Assistantatthe MUV • 2011: FWF-Project Leader „STAT5 SerinePhosphorylation in Leukemia“ • 2011: Award „Forschungspreis der Stadt Wien für innovative Krebsforschung“ • 2012: University AssistantattheVUV • Wolfgang Warsch: • 2012: Award „Forschungspreis der Stadt Wien für innovative Krebsforschung“ • 2012: PostDocatthe „Cambridge Institute of Medical Research“, • Lab of Tony Green

  32. Thanks to… Christine Schneckenleithner Wolfgang Warsch Sabine Fajmann Michaela Prchal Michael Dworzak and Angela Schumich …you for your attention

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