Growth Control in Multiple Myeloma

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SALL4 in Normal and Leukemic Stem Cells (PROGRESS
REPORT June 2005 May 2006) Yupo Ma, MD,
PhD Chief of Hematopathology Nevada Cancer
Institute
Networking Scientists and Resources to
Strengthen Biomedical Research in Nevada
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MDS
Myelodysplastic syndromes (MDS) are a group of
diseases that involve dysfunction of the bone
marrow. In all forms of MDS, the bone marrow stem
cell functions are disrupted This leads to a
decrease in production of normal red blood cells,
white blood cells, and platelets. Fourty percent
of people with MDS develop acute myeloid leukemia
(AML). Many blood disease experts consider MDS to
be a type of cancer (preleukemia). 14, 000 new
cases per year and incidence of MDS continues to
increase as our population ages Currently, there
is no good treatment for MDS
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Acute Leukemia
In leukemia, bone marrow produces a large number
of abnormal white blood cells. 31,000 new cases
of leukemia are diagnosed in the United States
each year
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The important issues?

• The order and timing of oncogenic events
  involving MDS and MDS transformation are unknown
• The molecular pathways of MDS progression and AML
  transformation are not fully understood.
• This is a major shortfall in our current
  understanding of these diseases

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We hypothesize that constitutive expression of
SALL4B plays a key role in the development of MDS
and drives the progression of MDS to AML.
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Leukemic Stem Cells (LSCs)

• It has recently been hypothesized that
  Leukemogenesis and other types of malignancies
  arise from neoplastic stem cells.
• Our current therapies succeed at eliminating
  bulky disease and rapidly proliferating cells,
  but often miss a tumor reservoir (tumor stem
  cells) that leads to disease recurrence and
  metastasis.

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Leukemia stem cells (LSCs) exhibit functions
similar to those of normal hematopoietic stem
cells (HSCs)
LSC
LSCs may also arise from differentiated
progenitor cells that have reacquired the
capacity for self-renewal.
HSChematopoietc stem cells CMPcommon
multipotent progenitor
cells GMPgranoloctye macrphage progenitor
cells
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SALL4

• Newly identified Zinc finger transcription factor
• Mutations associated with Okihiro syndrome,
  acro-renal-ocular syndrome and IVIC syndrome (an
  acronym for Instituto Venezolano de
  Investigaciones Cientificas)
• -Defects in multiple organ systems and
  leukocytosis

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Summary results presented in the INBRE proposal

• We demonstrated that SALL4 failed to be turned
  off in human primary AML (N96) using
  immunohistochemistry and real-time Q-PCR.
• SALL4B transgenic mice developed myelodysplastic
  symptoms (MDS) and subsequently AML that was
  transplantable.
• Increased apoptosis associated with
  dysmyelopoiesis was evident in SALL4B transgenic
  mouse marrows and colony formation assays (CFU)
  consistent with features of human MDS.
• Both isoforms (SALL4A and SALL4B) were able to
  bind to ß-catenin and synergistically enhanced
  the Wnt/ß-catenin signaling pathway, an important
  pathway involving self-renewal of HSC.

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Publications and Grant Submission

• Publications In Past Fiscal Year (July 2005 to
  May 2006)
• A. Publications
• 1. L Chai, Yang J, Di C, Cui W, Lai R, and Ma
  Y. Transcriptional activation of the SALL1 by the
  human SIX1 domain. 2006 ( JBC, In Press)
• 2. Ma Y, Wei C, Yang J, Qu J, Di C , Amin HM,
  Lai R, Ritz J, Krause DS, and
• L Chai. SALL4, a novel oncogene, is
  constitutively expressed in acute myeloid
  leukemia (AML) and is sufficient to induce AML in
  transgenic mice. 2006 (Blood, In Press)
• Lai R, Lefresne SV, Franko B, Hui D, Hanson J,
  Mirza I, Mansoor A, Amin HM, Ma Y.
  Immunohlonulin Vh somatic hypermutation in mantle
  cell lymphoma-mutated genotype correlates with
  better clinical outcome 2006 (submitted to Blood)
• 4. Wei C, . Ma Y, Nikki Kong N, Yang J, Amin
  HM, Lai R and L Chai. Expression analysis of a
  novel oncogene, SALL4, in lymphoma, multiple
  myeloma, and acute lymphoblastic leukemia. 2006
  ( submitted to American of Surgical Pathology)
• B. Abstracts
• Chai L, Cui W, Yang J, Di C, Amin H, and Ma Y.
  SALL4, a novel oncogene induces myelodysplastic
  syndrome and acute myeloid leukemia via
  Wnt/ß-catenin pathway. Blood Nov. 2005 106
  397a.
• Rai R, Lefresne SV, Franko BC, Shi X, Hui D,
  Mansoor A, Amin HM, Ma Y. Somatic Hypermatation
  of the Immunoglobulin Heavy Chain (IgH) Gene in
  Mantle Cell Lymphoma. Laboratory Investigation,
  2006, 86235A.
• NIH grant submission
• an NIH R01 application in June, 2006,
  entitled a mouse model of myelodysplastic
  syndrome progression and leukemic stem cells.

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SALL4 Has Two Isoforms
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Constitutive Expression of SALL4 protein in Human
Acute Myeloid leukemias
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What is Role of SALL4 in Leukemogenesis?

• Is SALL4 an oncogene?
• Gain-function assay in mice

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MDS-like Features in SALL4B Transgneic Mice
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Acute Leukemia Occurs After Ages 8 Months in
SALL4B Transgenic Mice
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Significance

• We are the first to demonstrate that SALL4 acts
  as an oncogene in MDS and AML transformation.
• We have developed the first murine model with
  SALL4B transgenic mice that resembles human MDS
  and AML and exhibits the transition from normal
  to preleukemia and AML transformation
• To our knowledge, no other transgenic mouse model
  is available to investigate MDS and MDS
  progression to AML. Most existing mouse models
  address myeloproliferative disorders and are
  retroviral vector-based or bone marrow
  transplantation-based.

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Specific Aim I Characterize the MDS/AML
phenotype in SALL4 transgenic mice (Progress).
The study of our model may provide clues how
the disease progresses from MDS to a fatal late
stage, AML.

• What is the order of events that occurs in the
  progression from normal through the preleukemic
  stages (MDS) and then to acute leukemia using our
  mouse model?

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HSChematopoietc stem cells (CD34
CD38-) HPChematopoietic progenitor cells
(CD34CD38) GAPDH Glyseraldehyde-3-phospha
te dehydrogenase
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HPChematopoietic progenitor cells CMPcommon
multipotent progenitor cells GMPgranoloctye
macrphage progenitor cells MEPmegakaryocyte
erythrocyte progenitor cells
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C-kit population (HSC and HPC) is expanded in
preleukemic (MDS) and leukemic stages of SALL4B
Transgenic Mice
Wide Type mice
SALL4BTransgenic mice
Pre-leukemia stage
Leukemia stage
9.4
1.7
26.2
C-kit
HSChematopoietc stem cells HPChematopoietic
progenitor cells
C-kit populationHSC HPC
HPCCMP, GMP and MEP
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Comparison of HSCs and HPCs in WT and SALL4B
transgenic mice
HPCCMP, GMP and MEP
SALL4B leukemic transplantation
SALL4B pre-leukemia
SALL4B leukemia
2.8
4.7
6.4
0.27
7.9
0.38
2.54
0.31
HSC
HSC
HSC
GMP
0.32
GMP
4.12
0.67
GMP
0.23
CMP
0.28
0.24
CMP
CMP
0.2
0.14
0.75
MEP
MEP
0.99
0.99
0.41
MEP
0.23
HSC WT 0.27?0.07, N10 Pre-leukemia SALL4B
0.38 ? 0.03, N10, P0.035 HPC WT 2.8 ? 0.53,
N13 Pre-leukemia SALL4B 4.65 ?0.63, N12,
P0.027 GMP WT 0.23 ? 0.034, N13, Preleukemia
SALL4B 0.32 ? 0.086, N12, P0.0039 CMP WT 0.2
? 0.048, N13, Pre-leukemia SALL4B 0.28 ? 0.13,
N12, P0.008 MEP WT 0.485 ? 0.085, Pre-leukemia
SALL4B 0.75 ? 0.28, N12, P0.037
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What do we learn from our studies using SALL4
mouse model?

• SALL4B transgenic mice have exhibited MDS with
  ineffective hematopoiesis and subsequent AML
  transformation associated with selective
  expansion of granulocyte/macrophage progenitors
  (GMPs)
• SALL4B may confer properties of LSCs to committed
  GMP destined to undergo maturation and or
  apoptotic cell death

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What are biochemical pathways that underlie
expansion of GMP (granoloctye macrphage
progenitor cells) in SALL4B transgenic mice ?
Specific Aim II (Progress)
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Biochemical Pathways in Regulating LSC
Self-Renewal

• Two families of proteins related to
  self-renewal have been the most studied the
  polycomb gene Bmi-1 and the ß-catenin signal
  pathway proteins.
• 1. The polycomb gene Bmi-1 plays an essential
  role in regulating adult self-renewing
  hematopoietic stem cells and LSCs. Inhibiting
  self-renewal in tumor stem cells after deleting
  Bmi-1 could prevent leukemic recurrence. Bmi-1
  expression has been used as an important marker
  for predicting progression of MDS and disease
  progression to AML.
• 2. Another group of genes involved in
  self-renewal are those involved in the ß-catenin
  signal transduction cascade. Overexpression of
  ß-catenin, a downstream activator of the
  Wnt-signaling pathway, expands the transplantable
  GMP pool in leukemia.

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ß-catenin signal transduction cascade

• Both isoforms (SALL4A and SALL4B) were able to
  bind to ß-catenin and synergistically enhanced
  the Wnt/ß-catenin signaling pathway, an important
  pathway involving self-renewal of HSC.

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Up-regulation of Bmi-1 and Wnt/ß-catenin
down-stream target genes, C-myc and cyclin D1 in
SALL4B Transgenic mice associated with disease
progression.
Note 1,2 normal bone marrows 3,4 preleukemic
samples from SALL4B transgenic mice 5, 6
leukemia samples from SALL4B transgenic mice
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Dose-dependent activation of the Bmi-1 promoter
by SALL4B
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Working hypothesis (SALL4 in leukemic stem cells)
Hematopoietic Stem Cells (HSC) Hematopoietic
progenitor cells (HPC) Hematopoietic
cells

erythrocytes
Neutrophils platelets
Normal SALL4B expression Constitutive
expression of SALL4B
( ) ( ) ( --
-- -- )
Normal hematopoiesis
Self-renewal
Leukemia blasts
Self- renewal
( )
X
Differentiation block
( )
HSCs
Up-regulate Bmi-1 Coactivated ß-catenin
Self-renewal
?Gene amplification ? mutation
( )
SALL4B
GMP
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Future Research Directions

• Clinical significance of constitutive
  overexpression of SALL4 in AML.
• Can overexpression of SALL4 confer a poor
  prognosis of AML?
• Can SALL4 transgenic mice used as an animal model
  for MDS for disease progressive to AML?
• Test novel therapeutic agents in animal
  models
• Plan submit the second NIH R01 at the end of
  the next year SALL4 in leukemia stem cells
• PA Stem Cells and Cancer (R21 and
  R01)

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Conclusions
Benefits from NV INBRE
SALL4 in Normal and Leukemic Stem Cells
Mentors
Grantsmanship and Research Support
INBRE Cores
Networking Scientists and Resources to
Strengthen Biomedical Research in Nevada
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The End
Questions
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