Transcript Slide 1
Chronic Myelogenous Leukemia (CML) - History • 1845- The first documented clinical description. • 1960- The discovery of Philadelphia chromosome in CML cells • 1970- The isolation of Abelson murine leukemia virus (A-MuLV). • 1973- The discovery that Philadelphia chromosome results from a reciprocal translocation of chromosomes 9 and 22. • 1980-1983 The cloning of v-Abl oncogene in A-MuLV, cloning the c-Abl proto-oncogene, locating c-Abl to chromosome 9, and demonstrating that Abl encodes a protein tyrosine kinase. • 1986- The cloning of BCR-ABL cDNA from CML cells. • 1987-present: Understanding how BCR-ABL causes CML. • 1996- The demonstration that imatinib (Gleevec) inhibits BCR-ABL tyrosine kinase and CML growth in vitro. • 2001- FDA approval of Gleevec for CML therapy. • 2002-present : Gleevec resistance, CML stem cells 9p Normal Chromosomes Reciprocal Translocation 22p Bcr Bcr-Abl 22q Ph1 Chromosome 9q c-Abl Myristoyl group Gag GAG SH3 V-Abl SH2 Y-kinase SH2 Y-kinase C-Abl • V-Abl, but not c-Abl, Transforms Cells. • V-Abl, but not c-Abl, is Tyr-Phosphorylated. • ∆SH3-c-Abl becomes Tyr-Phosphorylated, and can Transform Cells Circa ~1990 v-Abl BCR-ABL Gag NLSs BCR Abl(1a) Abl(1b) SH3 SH2 245 412 Y Y Kinase NES 1 2 3 Domain DNA Binding CAP Proline-rich motifs Myristoyl group Actin Binding Mol Cell Biol. 1993 Dec;13(12):7587-95. (see also McWhirter and Wang, 1991) A coiled-coil oligomerization domain of Bcr is essential for the transforming function of Bcr-Abl oncoproteins. McWhirter JR, Galasso DL, Wang JY. Department of Biology, University of California, San Diego, La Jolla 92093-0116. In Philadelphia chromosome-positive human leukemias, the c-abl proto-oncogene on chromosome 9 becomes fused to the bcr gene on chromosome 22, and chimeric Bcr-Abl proteins are produced. The fused Bcr sequences activate the tyrosine kinase, actin-binding, and transforming functions of Abl. Activation of the Abl transforming function has been shown to require two distinct domains of Bcr: domain 1 (Bcr amino acids 1 to 63) and domain 2 (Bcr amino acids 176 to 242). The amino acid sequence of domain 1 indicates that it may be a coiled-coil oligomerization domain. We show here that domain 1 of Bcr forms a homotetramer. Tetramerization of Bcr-Abl through Bcr domain 1 correlates with activation of the tyrosine kinase and F-actin-binding functions of Abl. Disruption of the coiled coil by insertional mutagenesis inactivates the oligomerization function as well as the ability of Bcr-Abl to transform Rat-1 fibroblasts or to abrogate interleukin-3 dependence in lymphoid cells. These results strongly suggest that Bcr-Abl oligomers are the active entities in transformation. N BCR Kinase Function Abl tyrosine kinase SH3 PXXP ATP SH2 Peptide YpXXP YXXP ABL Location Cues C Three Nuclear Localization Signals (NLS). One Nuclear Export Signal (NES). DNA-binding (bubble DNA). Actin-binding (G and F). ABL N-terminal Structure Nagar, B., et al. (2003) Cell 112: 859 Cryrstal Structure of the N-terminal Region of Abl v-Abl BCR-ABL Gag BCR Abl(1a) Abl(1b) SH3 245 412 Y Y SH2 Kinase NLSs NES 1 2 3 Domain DNA Binding Actin Binding CAP Proline-rich motifs Myristoyl group PD166326 SH3 N-lobe Y Y CAP SH2 C-lobe Myristate [Nagar et al, Cell, 2003] ABL Inhibitors • Welch, P.J. & Wang, J.Y.J. (1993) RB binds the ATP-binding lobe of Abl kinase domain and inhibits its kinase activity. Cell 75, 779-90. • Wen, S.T. & Van Etten, R.A. (1996) PAG/PrdxI binds Abl SH3 domain and inhibits its kinase activity. Genes Dev 11, 2456-67. • Woodring, P.J., Hunterm T. & Wang, J. Y. J. (2001) F-actin binds the C-terminus of Abl and inhibits its kinase activity. J. Biol. Cell. 276: 27104-27110. A Model for the Regulation of Abl Kinase: Auto-inhibition and Co-inhibition Catalytically inactive Equilibrium of Low to medium activities Coinhibitor 1 Abl Abl Autoinhibited High Activity (Transforming) P Abl P Co-inhibitors Abl Activated Abl Coinhibitor 2 Uninhibited Coinhibited Wang, J.Y.J. NCB, 2004 Partitioning of Latent Abl Kinase into Implications: Distinct Signaling Complexes by its Co-Inhibitors RB-Abl complex: Abl Transduces Signals that Disrupt RB-Abl Interaction: e.g., RB phosphorylation, RB degradation. F-actin-Abl complex: Abl Transduces Signals that Disrupt F-actin-Abl Interaction: e.g., Cell adhesion. Prdx-Abl complex: Abl Transduces Signals that Disrupt Prdx-Abl interaction: oxidative stress? Master Protein Kinase Slave Protein Kinase Signal (ligand, second messenger) Signals (various) Kinase Kinase Protein Substrates Substrates BCR-ABL phosphorylates proteins that are substrates or non-substrates of Abl tyrosine kinase. • BCR-ABL contains BCR-sequences. • BCR-ABL does not enter the nucleus. Biological Activity Reported for BCR-ABL • Many! • Abrogates the cytokine requirement for cell survival.. •Abrogates the adhesion requirement for cell proliferation. • Does not abrogate the serum requirement for growth. • Stimulates random motility. • Phosphorylates different proteins in different cell lines. N BCR Kinase Function Abl tyrosine kinase SH3 PXXP ATP SH2 Peptide YpXXP YXXP ABL Location Cues C Three Nuclear Localization Signals (NLS). One Nuclear Export Signal (NES). DNA-binding (bubble DNA). Actin-binding (G and F). Imatinib mesylate is the active component of Gleevec C29H31N7O.CH4SO3, relative molecular mass is 589.7. Cohen, M. H. et al. Clin Cancer Res 2002;8:935-942 Copyright ©2002 American Association for Cancer Research Imatinib-resistant BCR-ABL kinase domain mutations N Shah, C Sawyers et al. CANCER CELL 2, 2002 Imatinib-resistant mutations at ABL kinase domain ❶ F317L ❷ T315I ❸ F359V ❹ ❺ ❻ ❼ ❽ 9 10 11 12 13 N Shah, C Sawyers, et al. Cancer Cell 2, 2002 M244V G250E Q252H/R Y253F/H E255K M351T E355G V379I L387M H396R Mathematical models suggest CML stem cells to be refractory to Imatinib F Michor, et al. Nature 435, 2005 I Roeder, et al. Nature Med 12, 2006 QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. Stem cells, cancer, and cancer stem cells Tannishtha Reya, Sean J. Morrison, Michael F. Clarke and Irving L. Weissman. Nature 414, 105-111 (1 November 2001) The current model of hematopoiesis CML Stem Cells * CML is a disease of the hematopoietic stem cells (HSC) • During Chronic Phase (CP), Ph1-chromosome can be found in mature blood cells of various myeloid and lymphoid lineage. • Gleevec is effective during CP, but ineffective if disease has progressed to accelerated phase (AP) or Blast Crisis (BC). • CML stem cell identified in myeloid blast crisis patient samples. Jamieson et al, 2004 Increased Nuclear b-catenin in Blast Crisis CML Granulocyte-Macrophage Progenitors n=6 n=3 n=4 Jamieson et al, New Engl J Med 2004;351:657-67 CML-like phenotypes in mHPC/p210-transplanted mice Spleen (8w) mHPC/GFP mHPC/p210 87 (21) mg 782 (84) mg n=6 n=12 Peripheral Blood smear of mHPC/p210 mice (7w) (Wright-Giemsa staining) mHPC/GFP (8w) Bone Marrow Histopathology (H&E staining) at 8 Weeks PostSpleen transplantation Liver mHPC/p210 (8w) Irradiated 20-recipients Schedule of Imatinib-treatment 11 days Day 0 16 27 28 ~ Transplantation Imatinib-treatment 1 x 106 BM cells from leukemic mice (10) AM: 50 mg/kg, p.o. PM: 100 mg/kg, p.o. v.s. Vehicle control Effects of Imatinib WBC (/l) 40000 30000 20000 10000 0 GFP P0.01 Spleen sizes (mg) P0.01 600 400 200 0 Vehicle Imatinib p210 GFP P0.01 Vehicle Imatinib p210 P=0.11 6 80 GMP (%) BM GFP (%) 100 60 40 4 2 20 0 GFP Vehicle Imatinib p210 0 mHPC/GFP Vehicle Imatinib mHPC/p210 Transplantation of Imatinib-treated mouse bone marrow induced leukemia 30-#1 (Day 38, 30) 30-#1 (Day 38, 30) 71.5 % 30-#2 (Day 41, 30) 55.5 % Spleen 587 ( 146) mg n=5 85.3 % 80.8 % GFP GFP Bone Marrow QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. In the Cytoplasm: BCR-ABL inhibits apoptosis. Oncogenic In the Nucleus: (not where it is usually found) BCR-ABL induces apoptosis. Anti-oncogenic Vigneri & Wang, Nat. Med. 2001 Extra Cellular Matrix, PDGF Outside actin dynamics F-actin Abl Abl Cytoplasm Nucleus NES NLS DNA Abl Damage TNF APOPTOSIS Inactive Abl RB Abl Inactive TNF-induced apoptosis of mouse thymocytes requires Abl and is blocked by Rb-MI Chau et al, MCB, 2004 Abl contributes to TNF-induced apoptosis Chau et al, MCB, 2004. LMB Covalent modification of CRM1 by Leptomycin B CRM1=exportin-1 T Kau, P Silver et al. Nature Reviews Cancer 4, 2004 Trapping BCR-ABL in the Nucleus: Leptomycin B NES ** NLS ** *STI571 cytoplasm Nucleus Nuclear BCR-ABL kinase kills: Leptomycin B NES Death cytoplasm Nucleus Imatinib or LMB Extensive Washing Count viable cells every 2 days or Imatinib + LMB 48 hours 16 days Complete Media, NO DRUGS 1x10 1x10 Un-Induced for TonB (+IL3) BCR-ABL Expression 8 non-treated Imatinib 7 LMB Imatinib+LMB 1x10 1x10 1x10 6 5 4 2 4 6 8 10 12 14 days TonB/BCR-ABL (-IL3) 1x10 1x10 1x10 1x10 8 Induced for BCR-ABL Expression non-treated Imatinib LMB 6 4 2 Imatinib+LMB 0 2 4 6 8 10 12 14 16 days TonB/BCR-ABL (+IL3) 1x10 1x10 1x10 1x10 8 Induced for BCR-ABL Expression Imatinib non-treated 6 LMB 4 2 Imatinib+LMB 0 2 4 6 8 10 12 14 days K562 (-IL3) 1x10 8 non-treated Imatinib 1x10 1x10 1x10 6 LMB 4 2 Imatinib+LMB 0 3 5 7 9 11 13 15 days Questions1. How can the mathematical prediction depicted in Fig. 4A (sc panel) of the Michor et al paper be tested in the clinic? 2. What may account for the innate resistance of CML stem cells to imatinib? 3. What may account for the activation of cell death by nuclear BCR-ABL?