Molecular Characterization of Leukemia Stem Cell Development Scott A. Armstrong MD, Ph.D.
Normal and Leukemic Hierarchies NORMAL HSC (SRC) Myeloid progenitor LTC-IC CFU AML LSC (SL-IC) Leukemic LTC-IC AML CFU Blast cells leukemic clone is maintained by rare SL-IC (CD34+/CD38-): ability to differentiate into leukemic blasts high self-renewal (serial transplantation experiments) Lapidot, Nature 1994 Bonnet, Nature Med 1997
Terminology: 1. Cell of origin: The cell from which the cancer arises. 2. Cancer stem cell: A cell in the fully developed cancer that has properties associated with normal stem cells. a. Able to give rise to cells that can propagate the disease (self-renewal) and more differentiated cells that can t propagate the disease. b. Proliferate extensively c. Rare (how rare) Does not mean cancerous stem cell
-located at 11q23 MLL (HRX, ALL-1) and Leukemia -consistently rearranged in infant acute leukemia (more than 70%) resulting in AML or B-ALL. -frequently rearranged in secondary acute leukemia (AML) after treatment with topoisomerase II inhibitors -Mll (TrxG) maintains Hox gene expression during development, and is necessary for appropriate hematopoietic development. DNA MT homology PHD(Zn 2+ ) fingers activation domain SET domain AT hooks } breakpoint MLL fusion protein (>30 different fusion partners) t(9;11)-- AML t(4;11)-- ALL
Progression from Committed Progenitor to Leukemia Stem Cell Cozzio et al. Genes & Dev. 2003 Huntly et al. Cancer Cell 2004 Krivtsov et al. Nature 2006
Progression from Committed Progenitor to Leukemia Stem Cell Hoxa9/Meis1 MLL-Fusion MLL-Fusion LSC 1/150 Krivtsov et al., Nature 2006
Progression from Committed Progenitor to Leukemia Stem Cell MLL-Fusion Krivtsov et al., Nature 2006
LSC Have Reactivated a Subset of Stem Cell Genes (self-renewal associated signature) Krivtsov et al., Nature 2006 K-means analysis K=10
MLL-AF9 Leukemia Stem Cells MLL-Fusion
MLL-AF9 Leukemia Stem Cells?HoxA9/Meis1 MLL-Fusion
Single Cell Clonogenic Efficiency 40 28 GMP-MLLAF9 Infection Single-cell sorting 40 22 GMP/KLS selection GFP+ Serial replating 40 GMP-A9M 18 LSK-A9M 40 Immunophenotype Identical Mac1+, Gr1+, ckit variable 15 LSK-MLL-AF9 1 2 3 4 5 6 Transplantation 5x10 5 cells
LSC Immunophenotype 100 GMP-HoxA9+Meis1 % survival 80 60 40 HSC-HoxA9+Meis1 GMP-MLLAF9 identical HoxA9/Meis1 20 HSC-MLL-AF9 0 0 50 100 150 200 250 Days after transplantations LSC = LGMP
Gene expression in multiple LSC populations 170 100 GMP-HoxA9+Meis1 % survival 80 60 40 HSC-HoxA9+Meis1 GMP-MLLAF9 294 20 HSC-MLL-AF9 0 0 50 100 150 200 250 Days after transplantations 534
GMP-A9M cells lose in vivo proliferative capacity SCC Control LSK-HOXA9/M GMP-HOXA9/M (N>3) (N>3) 18hr 0 0.046 0.022 1000 800 8days 1mon 4mon 0 1.6 0.13 0.13 10 0 10 1 10 2 10 3 10 4 GMPA9M BM_GFP 0 24 4 0 49 0 SCC 600 400 200 0 No. of GFP+ cells in BM 980x 480x 30x 200x 8x 0 GFP LSK HOXA9/M GMP-HOXA9/M
c-kit population is associated with leukemia development LSK-HOXA9/M GMP-HOXA9/M Gr-1 c-kit high in vitro 10days 1month c-kit low 4 months Leukemia GFP- c-kit
Assessment of leukemia propagation by c-kit high vs. c-kit low cells c-kit low c-kit high Gr-1 c-kit Limiting dilution transplantation 15 days & 1 month post-injection FACS
Heterogeneity of HoxA9/M driven leukemia cells Gr-1 KLS-HoxA9/M In vitro 10 5 10 4 10 3 10 2 kit low Day10 1mon Survival % 10 5 Kit hi 10 4 Kit hi 10 3 Kit hi 10 2 Kit hi 4mon LSC Days after transplant c-kit
Summary 1: 1. MLL-AF9 can transform HSC or Progenitor cells to induce LSC with a maturing myeloid cell immunophenotype 2. HoxA9+ Meis1 efficiently transforms HSC, but inefficiently transforms progenitors 3. LSC in HoxA9 + Meis1 leukemias have a similar immunophenotype to MLL-AF9 LSC. (c-kit high)
LSC in Myeloid Malignancies Mouse: AML: 1: MLL-AF9 Mid myeloid phenotype (L-GMP) 2: HOXA9 Mid myeloid phenotype (L-GMP) 3: RAR-PML Late myeloid phenotype (past GMP stage) 4: Inv16 Mid myeloid phenotype (CMP like) 5: Pu.1 KO Lin- Kit+ Sca1+ (early myeloid/hsc) MPD: 1. BCR-ABL Lin- Kit+ Sca1+ (early myeloid/hsc) 2. Ras+ MPD? Hard to transplant 3. Jak2+ MPD? Human: AML: Either CD34+ CD38- or CD34+ CD38+ CML:? HSC Blast Crisis CML: Mid myeloid phenotype (L-GMP)
The Wnt/ -catenin pathway in hematopoiesis and leukemia Wnt regulates self-renewal of HSC Constitutive activation increased HSC followed by exhaustion, myeloid failure Genetic inhibition (Axin) reduced repopulating β-catenin deletion in during development--reduced repopulation β-catenin deletion in adult HSC does not affect HSC repopulation β-catenin activation is present in AML stem cells/ CML BC stem cells
Active β-catenin in the c-kit high population c-kit low c-kit high c-kit low LSK- HOXA9/M GMP-HOXA9/M Nucleus active β-catenin Merge c-kit high c-kit low The inability of A9M to transform GMP may be due to lack of -catenin activation Wang et al., Science 2010
GMP-HOXA9/M + βcat(s37a) mimics leukemogenic effect of LSK-A9M cat* GMP-HOXA9/M GMP-HOXA9/M +βcat* LSK-HOXA9/M Days after transplant
Inactivation of β-catenin Hoxa9+Meis1a Single cells β-catenin LoxP LoxP 1 2 3 4 5 6 7 15 LSK GMP puro GFP+ Brault et al. Development 2001 +/- Cre LSK-A9/M GMP-A9/M β-cat β-cat actin
Inactivation of β-catenin suppresses LSK-HOXA9/M cell expansion in vivo LSK-HOXA9/M GMP-HOXA9/M βcat WT βcat -/- βcat WT βcat -/- 18hr 0.0092 0.009 0.006 0.004 8days 0.29 0.096 0.18 0.13 1mon 0.69 0.5 0.4 4mon 88 0.11 0 0
Loss of β-catenin suppresses leukemogenesis 1 0 0 80 60 40 20 Catenin lox/lox LSK-HOXA9/M (+Cre) Catenin lox/lox LSK- HOXA9/M WT LSK-HOXA9/M WT LSK-HOXA9/M (+Cre) 0 0 1 0 0 2 0 0 3 0 0 β-cat β-cat
Assessment of -catenin in fully developed leukemia 1 2 3 4 5 6 7 15 βcat loxp/loxp x Mx1-Cre βcat loxp/loxp MxCre + βcat WT MxCre + (control) βcat WT βcat loxp/loxp p=0.008 βcat -/- HoxA9/M LSK 4D 70D AML 2 nd transplantation Poly (I:C) at day 7, 9, 13, & 17
What about MLL-AF9?
β-catenin deficiency influences leukemic transformation but not normal hematopoiesis βcat loxp/loxp MxCre + βcat WT MxCre + poly (I:C) at day 1, 3, 7, 11 17D After pipc treatment LSK LSK (8 week-old) cat -/- cat WT Koch U, et al. Blood. 2008 Jan 1;111(1):160-4. LSK Jeannet G, et al. Blood. 2008 Jan 1;111(1):142-9. poly (I:C) treatment Survival curve
βcat loxp/loxp MxCre+ β-catenin deficiency inhibits MLL-AF9 induced leukemia development βcat WT MxCre+ (control) MLL-AF9 βcat loxp/loxp MxCre+ βcat WT MxCre+ (control) LSK SCC GFP Spleen 120mg 700mg poly (I:C) treatment
Genetic loss of β-cat prolongs survival of mice with MLL-AF9 cells cat -/- PIPC Survival % cat WT no PIPC cat WT PIPC p<0.002 cat loxp/loxp no PIPC Days after transplant
Indomethacin suppresses -catenin expression c-kit low c-kit high Nucleus active β-catenin Merge c-kit high c-kit low Indo c-kit high Indo - + Indo? -catenin c-kit high actin -catenin expression in c-kit high cells is inhibited by indomethacin in vitro
Chemical inhibition of -cat impairs LSC development Gr-1 Survival (%) Control Indomethacin (Indo) Indo treatment in vivo preferentially eliminates the c-kit high population Indo treatment in vivo reduces the frequency c-kit of LSC 10 3 10 2 Indo 10 4 Indo 100 fold 10 2 contr 10 4 contr 10 3 contr 100 80 60 40 20 0 0 20 40 60 80 100 120 Days Days after after transplant transplant
1. Hoxa9+Meis1a promotes Leukemic self-renewal in HSC but can not confer full properties of self-renewal on GMP 2. Cooperation of Hoxa9-Meis1a and Wnt/β-catenin pathways confer self-renewal properties on GMP. 3. Hoxa9+Meis1a expression in HSC generate LSC with a phenotype consistent with maturing myeloid cells 4. Loss of -catenin suppresses leukemia development from HSC or GMP Summary 5. Indomethacin inhibits -catenin expression and targets LSC catenin Determines Developmental Stage Specific Transformation to LSC
Armstrong Lab Zhaohui Feng Kathrin Bernt Amit Sinha Andrei Krivtsov Matthew Stubbs Jenny Wang Florian Heidel Clark Owyang Natalie Punt Aniruddha Deshpande Liying Zhang Krysta Schlis Tobias Neff Won-Il Kim Ellen Basu Lars Bullinger Inga Hofmann- Zhang Nan Zhu Acknowledgments Kung Lab Andrew Kung Sridhar Vempati Tina Davis DFCI/Childrens Stuart Orkin Len Zon Wolfram Goessling Trista North Lewis Silverman Stephen Sallan BWH Jeff Kutok