SALL4 in Normal and Leukemic Stem Cells
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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. MDS. Myelodysplastic syndromes (MDS) are a group of diseases

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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 diagnosedin 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) a key role in the development of MDS and drives the progression of MDS to AML.

  • 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.


Leukemia stem cells lscs exhibit functions similar to those of normal hematopoietic stem cells hscs l.jpg
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.

HSC=hematopoietc stem cells

CMP=common multipotent progenitor

cells

GMP=granoloctye macrphage progenitor

cells


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SALL4 those of normal hematopoietic stem cells (HSCs)

  • 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 those of normal hematopoietic stem cells (HSCs)

  • We demonstrated that SALL4 failed to be turned off in human primary AML (N=96) 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 β-catenin and synergistically enhanced the Wnt/β-catenin signaling pathway, an important pathway involving self-renewal of HSC.


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Publications and Grant Submission those of normal hematopoietic stem cells (HSCs)

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/β-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, 86:235A.

      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 those of normal hematopoietic stem cells (HSCs) Has Two Isoforms


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Constitutive Expression of those of normal hematopoietic stem cells (HSCs)SALL4 protein in Human Acute Myeloid leukemias


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What is Role of SALL4 in Leukemogenesis? those of normal hematopoietic stem cells (HSCs)

Is SALL4 an oncogene?

Gain-function assay in mice


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MDS-like Features in SALL4B Transgneic Mice those of normal hematopoietic stem cells (HSCs)



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Significance Transgenic Mice

  • 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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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?

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.


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SALL4B expression in human from normal through the preleukemic stages (MDS) and then to acute leukemia using our mouse model?

HSC/HPC

0.18

0.15

0.12

0.09

%GAPDH

0.06

0.03

0

PBSC

CD34+

CD34+CD38-

CD34+CD38+

HSC=hematopoietc stem cells (CD34+ CD38-)

HPC=hematopoietic progenitor cells

(CD34+CD38+)

GAPDH =Glyseraldehyde-3-phosphate

dehydrogenase


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HPC=hematopoietic progenitor cells from normal through the preleukemic stages (MDS) and then to acute leukemia using our mouse model?

CMP=common multipotent progenitor cells

GMP=granoloctye macrphage progenitor cells

MEP=megakaryocyte 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

HSC=hematopoietc stem cells

HPC=hematopoietic progenitor cells

C-kit population=HSC + HPC

HPC=CMP, GMP and MEP


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Comparison of HSCs and HPCs in WT and (MDS) SALL4B transgenic mice

HPC=CMP, GMP and MEP

SALL4B leukemic transplantation

SALL4Bpre-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, N=10; Pre-leukemia SALL4B: 0.38  0.03, N=10, P=0.035; HPC WT 2.8  0.53, N=13; Pre-leukemia SALL4B : 4.65 0.63, N=12, P=0.027; GMP WT 0.23  0.034, N=13, Preleukemia SALL4B: 0.32  0.086, N=12, P=0.0039; CMP WT: 0.2  0.048, N=13, Pre-leukemia SALL4B: 0.28  0.13, N=12, P=0.008; MEP WT 0.485  0.085, Pre-leukemia SALL4B: 0.75  0.28, N=12, P=0.037


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What do we learn from our studies using SALL4 mouse model? (MDS)

  • 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 andthe β‑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 β-catenin signal transduction cascade. Overexpression of β-catenin, a downstream activator of the Wnt-signaling pathway, expands the transplantable GMP pool in leukemia.


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β-catenin signal transduction cascade (

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


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Up-regulation of Bmi-1 and Wnt/β-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 target genes, C-myc and cyclin D1 in SALL4B Transgenic mice associated with disease progression.


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Working hypothesis (SALL4 in leukemic stem cells) target genes, C-myc and cyclin D1 in SALL4B Transgenic mice associated with disease progression.

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 β-catenin

Self-renewal

?Gene amplification

? mutation

SALL4B

( +++ )

GMP


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Future target genes, C-myc and cyclin D1 in SALL4B Transgenic mice associated with disease progression.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 target genes, C-myc and cyclin D1 in SALL4B Transgenic mice associated with disease progression.

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 target genes, C-myc and cyclin D1 in SALL4B Transgenic mice associated with disease progression.

Questions


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