Advances 2010 Ulrich Steidl

1. Targeting Cancer Stem Cells in Acute Myeloid Leukemia Ulrich G. Steidl, M.D., Ph.D. Assistant Professor Department of Cell Biology AECC Advances Meeting May 5, 2010

2. Stem Cell Stem or Progenitor Cell Leukemia Stem Cell (LSC) Early Events Additional Transforming Events Leukemic Bulk Population (Leukemic Blasts) The Leukemia-Initiating Cell (Leukemia Stem Cell) Model Self-renewal Malignant Self-renewal

3. Stem Cell Stem or Progenitor Cell Leukemia Stem Cell (LSC) Early Events Additional Transforming Events Leukemic Bulk Population (Leukemic Blasts) Self-renewal Malignant Self-renewal • Research efforts, but also diagnosis and therapy have been focusing on characterization, detection and eradication of the leukemic blast population • Therapy response is evaluated based on blast percentages in the blood and marrow (“complete remission”) The Leukemia-Initiating Cell (Leukemia Stem Cell) Model

4. Stem Cell Stem or Progenitor Cell Leukemia Stem Cell (LSC) Early Events Additional Transforming Events Leukemic Bulk Population (Leukemic Blasts) Self-renewal Malignant Self-renewal • What are the molecular mechanisms driving LSC function ? The Leukemia-Initiating Cell (Leukemia Stem Cell) Model

6. 0 1 2 3 4 5 6 7 8 9 Time (Months) wt/wt Controls PU.1 knockdown Survival (%) Preleukemic Phase Leukemic Phase 80% Reduction of PU.1 Induces Acute Myeloid Leukemia AML normal Splenomegaly Myeloid blasts Rosenbauer et al., Nat Genet 2004 +/+ (n=40) kd/kd (n=45)

7. Green : Genes downregulated in PU.1 kd HSC Pathway Assist 5.2 Software - Simplified Schematics Downregulated genes in PU.1 knockdown stem cells C-Jun JunB

8. Free Probe* Probe* Self compete PU.1 PU.1 mut. PU.1 Ab Elf1 Ab PU.1 binds the JUNB promoter PU.1 complex PU.1 knockdown leukemia is mediated by downregulation of the AP-1 transcription factor JunB Restoration of JunB expression rescues leukemia in a murine transplantation assay (NOD-SCID mice) Steidl et al., Nat Genet 2006 100 80 60 40 20 0 0 50 100 150 Time (days) Survival (%) LV JunB LV PU.1 LV c-Jun LV empty

10. HSC CLP CMP GMP MEP Blasts (= leukemic bulk population) <5% in normal BM 20-95% in AML French-American-British (FAB) Classification M0 - undifferentiated AML M1/M2 - myeloblastic leukemia (without/with maturation) M3 - promyelocytic leukemia M4 - myelomonocytic leukemia M5 - monocytic leukemia M6 - erythroleukemia M7 - megakaryoblastic leukemia Leukemia Stem Cell (LSC) Self Renewal Classification of AML based on blast morphology M6 M7 M0 M1 M2 M5 M4 M3 ? ? ? ? normal

11. Lin CD38 CD34 CD45RA CD90 CD123 CMP GMP MEP LT-HSC ST-HSC SSC Events Multi-Parameter FACS of Hematopoietic Stem and Progenitor Cells in AML Used in AML: Nat Genet. 2006 J Clin Invest. 2007 Genes Dev. 2008 Originally developed for normal BM cells by Manz et al. PNAS 2002

12. Genome-wide transcriptional analysis of the hierarchy of LT-HSC, ST-HSC, and GMP from patients with AML, and healthy controls (ECOG-supported Pilot Study) Outline: 1. Fractionation of Lin-CD34+CD38-Thy1+, Lin-CD34+CD38-Thy1-, and Lin-CD34+CD38+CD45RA+CD123+ BM cells 2. RNA isolation and linear amplification (SPIA) 3. Genome-wide expression analysis (Affymetrix arrays) 4. Group comparison AML vs. Normal, as well as intra-individual comparison during development 5. Target identification and functional validation

13. Approach is feasible: ~350 genes differentially expressed (p<0.05; fold>1.5) Hierarchical Clustering distinguishes Healthy Control and AML LT- and ST-HSC Healthy control AML

14. ST-HSC GMP LT-HSC Principal Component Analysis of Stem and Progenitor Populations from Patients with AML

15. Venn Diagram of differential gene expression AML vs. Normal in different stem and progenitor subsets -> Different genes are affected in different stem and progenitors  (LT-HSC)  (ST-HSC)  (GMP)

18. Is Eltrombopag an option in thrombocytopenic patients with AML or MDS ? Thrombocytopenia present in 67% of patients with MDS at initial diagnosis Thrombocytopenic hemorrhage cause of death in 16-30% of patients with MDS Kantarjian et al., Cancer 2007 Thrombocytopenia is caused by the disease itself, as well as (cytotoxic) therapy At least two major questions: 1. Does Eltrombopag potentially stimulate malignant cells, including leukemia stem cells, in AML or MDS ? 2. Is Eltrombopag capable of increasing megakaryopoiesis in MDS/AML, similar to its effect in chronic ITP ? Preclinical study utilizing assay systems for evaluation of LSC

19. Eltrombopag does not affect malignant self-renewal in ex vivo cultures of MDS/AML-derived BM-MNC Number of patients re-plating Primary Plating Serial Replating 1 st 2 nd 3 rd 4 th Progenitor frequency Colony-forming capacity Maintenance of colony-forming capacity Long-term self-renewal Serial Replating Assays NORMAL MDS/AML

20. Eltrombopag does not increase in vivo engraftment of leukemia stem cells in a xenotransplantation model NSG mice (NOD-SCID IL2R  null) MDS/AML cells hCD45 Engraftment (4 weeks) Analysis Eltrombopag treatment for 12 weeks 1 mg p.o. /d Average serum levels: 2290 ng/mL (SD: 769 ng/mL) Human donor cell chimerism in mice treated for 12 weeks (n=20 mice) Will et al., Blood 2009

21. 0 0.1 1 3 10 Eltrombopag Concentration [  g/ml] 150 120 90 60 30 0 Mk-colony numbers [% Tpo control] Eltrombopag stimulates early megakaryopoietic progenitor cells Total Mk-colony numbers [%] 120 80 40 0 0 0.1 1 3 10 TPO Eltrombopag Conc. [  g/ml] 100ng/ml Total Mk-colony numbers [% Tpo control] 0 0.1 1 3 10 100ng/ml TPO “ mixed” colony from Mk/E- progenitor colonies from immature MK-progenitor colony from mature MK-progenitor

22. Conclusions 1. Utilizing a variety of pre-clinical assays (incl. a xenograft model), we found NO evidence that Eltrombopag stimulates malignant blasts or enhances self-renewal capacity of leukemia stem cells in MDS / AML 2. Eltrombopag is capable of increasing megakaryopoiesis in samples from MDS / AML patients 3. The data demonstrate the usefulness of our pre-clinical stem cell assay systems for drug evaluation, and provide a rationale for the clinical testing of Eltrombopag for treatment of thrombocytopenia in patients with MDS / AML -> Single agent clinical study (Amit Verma, Samir Parekh) -> Testing in combination with other (anti-leukemic) drugs

23. Daniel G. Tenen CSI Singapore/ Harvard Medical School University of California, San Francisco Emmanuelle Passegu é Collaborators @ Einstein Amit Verma Samir Parekh Cristina Montagna Art Skoultchi John Greally Eric Bouhassira Einstein Genomics Core David Reynolds Members of the Steidl Lab Britta Will Masahiro Kawahara Laura Barreyro Ashley Pandolfi Cynthia Okoye Julia Luciano Jillian Mayer Tihomira Todorova NIH, NYSTEM, Leukemia Research Foundation, Gabrielle’s Angel Foundation, GlaxoSmithKline Guillermo Simkin Einstein Human Stem Cell FACS Facility Eastern Cooperative Oncology Group Elisabeth Paietta Memorial Sloan Kettering Cancer Center Ross Levine Weill Cornell Medical College Ari Melnick Chris Mason