Chronic Myeloid Leukemia (CML) Matt Walter Markey Program October 21, 2014

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1 Chronic Myeloid Leukemia (CML) Matt Walter Markey Program October 21, 2014

2 CML: paradigm for cancer treatment Identify primary genetic abnormality Develop an agent that targets that abnormality Monitor for efficacy Overcoming resistance

3 Chronic Myeloid Leukemia Session Overview 1. Matt Walter = Hematopoiesis overview 2. Dan Link = Signaling, animal models 3. Peter Westervelt = Clinical overview * 4. Steve Oh = Imatinib preclinical/clinical 5. Geoff Uy = Imatinib resistance * 6. Rizwan Romee = Bone marrow transplant * 7. Jerry Radich = Guest speaker on CML 8. Last class = round table lunch * Patient interviews

4 Hematopoiesis Stem cell Bone marrow Progenitors Blood Terminally differentiated NEJM (2006) 354;19, p.2034

5 Hematopoietic growth factor (HGF) signaling Receptor Tyrosine Kinase (RTK) (ABL, non Receptor TK) *Frequent targets for mutation in leukemia * * * * * NEJM (2006) 354;19, p.2034

6 Classification of Leukemias Lymphoid Myeloid CLL Chronic Lymphocytic Leukemia ALL Acute Lymphocytic Leukemia CML Chronic Myeloid Leukemia AML Acute Myeloid Leukemia 10/100,000 1/100,000 2/100,000 (~6,000 new/year) Acute = fast clinical progression. Block in differentiation (weeks to months) 5/100,000

7 Chronic Leukemias & MPNs: differentiation occurs Blood Terminally differentiated Essential Thrombocytosis (ET) Chronic Myelo Monocytic Leukemia (CMML) Polycythemia Vera (PV) Chronic Myeloid Leukemia (CML) Chronic Lymphocytic Leukemia (CLL) Myeloproliferative Neoplasms (MPN)

8 Genetics of Myeloproliferative Neoplasms (MPN) Essential Thrombocytosis (ET) (JAK2) Myeloid Neoplasm with Eosinophilia (PDGFRA/B) Polycythemia Vera (PV) (JAK2) Chronic Myeloid Leukemia (CML) (BCR:ABL) Mastocytosis (KIT) Mutation/rearrangement of tyrosine kinase genes

9 Westervelt CML 1.4:1 ( : ), average age 45 Increased blood WBC (>25k/uL) Minimal/no fibrosis or dysplasia Granulocyte maturation basophil Normal peripheral blood CML blood eosinophil Normal BM Peter Maslak, ASH Image Bank 2011;

10 CML chronic Blastic phase (block in differentiation)

11 Westervelt CML Chronic phase Driven by BCR ABL 50% no symptoms, found on routine CBC WBC PB, BM, spleen Fatigue, weight loss, abdominal fullness, bleeding, sweats, large spleen/liver 75% had accelerated phase 3 18 months prior to blastic phase Blast phase Acquire secondary mutations 1/3 = ALL, 2/3 = AML Decreased response to treatment and duration not long lasting

12 BCR:ABL fusion in CML Cytogenetics = t(9;22), Philadelphia chromosome + (Ph+), (100% BCR ABL transcript) BCR ABL transcript +

13 Flemming draws first chromosome Waldeyer coins chromosome Painter reports 48 chromosomes = normal Tjio reports 46 chromosomes = normal syndromes Down +21 Turner XO Klinefelter XXY 1890 von Hansemann observes mitotic aberrations in tumor cells dark ages Technical problems 1. better cultures 2. hypotonic solution 3. colchicine 1960 Nowell & Hungerford = Ph chromosome & CML The findings suggest a causal relationship between the chromosomal abnormality observed and chronic granulocytic leukemia.

14 Chromosome Gene Drug Baltimore, v Abl Nowell & Hungerford = Ph chromosome & CML Rowley reports Ph chr. = t(9;22) Heisterkamp, Stephenson, Groffen, Grosveld clone genes at breakpoints (BCR, ABL) Druker uses Gleevec to treat CML

15 v ABL oncogene Lymphosarcoma: virus induced thymic independent disease in mice. Abelson HT, Rabstein LS. Cancer Res Aug;30(8): v Abl Cell Dec;18(4): Cell Dec;22(3):777 85

16 (coil coil) p190 = 20 30% ALL (most B cell) p210 (p230) = 99% CML ABL = Loss of N terminal Cap region of ABL, normally inhibits kinase activity BCR = Gain coil coil of BCR allows dimerization of BCR ABL Results in constitutive activation of the ABL tyrosine kinase. Effects on cellular proliferation, survival and motility Dan Link Heisterkamp, Oncogene, 2002 ;21(56):

17 What cell is transformed in CML? Stem cell Bone marrow Progenitors Blood Terminally differentiated Link NEJM (2006) 354;19, p.2034

18 Colony forming Cell Assay (progenitors) Bone Marrow Cells Culture with specific growth factors for 7-14 days Semi-solid media (methylcellulose) CFU-GM (GM-CSF) BFU-E (Epo)

19 Methylcellulose cultures

20 Hematopoietic Stem Cell Assays HSC Definition: capable of long term (6 months) repopulation of ALL hematopoietic lineages Bone marrow transplantation (gold standard) Xenotransplantation models to assay human HSC Long term culture initiating cell (LTC IC) assay

21 Murine or Xenotransplantation radiation Human hematopoietic cells NOD/SCID mice NOD/SCID gamma (NSG) (immunodeficient) Analyze after 3-6 months for blood cells Mouse hematopoietic donor cells (CD45.2) congenic recipient mice (CD45.1) Analyze after 3-6 months for blood cells

22 Better assays needed to study hematopoiesis Methylcellulose and transplant assays are still the gold standard, but is there a faster, cheaper way to study these cells? How can you prospectively identify the cells and isolate them to study?

23 Flow cytometry

24 Murine or Xenotransplantation Human hematopoietic cells Mouse hematopoietic cells radiation NOD/SCID mice (immunodeficient) CD34+ xenotransplant assays = 1:5000 HSCs CD34+/CD38- = 1:617 HSCs congenic mice (CD45.1/CD45.2) Kit+/Lineage-/Sca+ (KLS) cells = 1:10 HSCs CD150+/CD48- (SLAM) cells = 1:2 HSCs Analyze after 3-6 months for blood cells

25 GOALS: 1) prospectively identify cells using flow cytometry (Weissman group = 1 st mouse, 2 nd human hematopoiesis). 2) Define what cell is transformed in CML (chronic phase and blast crisis). Stem cell Bone marrow Progenitors Blood Terminally differentiated

26 929 citations

27 CMP MEP GMP 288 single cells of each type CMP GMP No Epo+Tpo MEP

28 200 cells each CMP Cytospin of 5 pooled colonies MEP GMP

29 1000 cells on stroma Lineage commitment at CMP single cell level: GMP CMP MEP Single cell sorted

30 Regulation of Hematopoiesis Transcription factors Regulate RNA expression of target genes Critical determinants of lineage commitment and terminal differentiation Frequent targets for mutation in leukemia

33 HUMAN PROGENITOR ASSAY PNAS (2002)99;18, p.11872

34 What cell is transformed in CML? Stem cell Bone marrow Progenitors Blood Terminally differentiated Link NEJM (2006) 354;19, p.2034

35 Oh Uy Romee Radich CML therapy Lancet (2007)370, p.342

36 Westervelt Oh Uy Radich Targeted therapy (chronic phase) Imatinib = discovered in high throughput screen of kinase inhibitors. NEJM Oct 9;349(15):

37 Issues Cure vs. control of chronic phase CML Resistance in chronic phase CML Treatment of blastic phase CML

Leukemias and Lymphomas: A primer

Leukemias and Lymphomas: A primer Normal blood contains circulating white blood cells, red blood cells and platelets 700 red cells (oxygen) 1 white cell Neutrophils (60%) bacterial infection Lymphocytes