Targeting of sall4 for the treatment and diagnosis of proliferative disorders associated with myelodysplastic syndrome (mds)

Abstract

The present invention discloses nucleic acids, proteins, and antibodies for SALL4 (including isoforms SALL4A, SALL4B, and SALL4C), a zinc finger transcriptional factor. Further, methods are disclosed which demonstrate that constitutive expression of SALL4 increases leukemogenic potential in cells of model animal systems. Moreover, constitutive expression of select isoforms (e.g., SALL4B) in transgenic mice demonstrate that these animals develop myelodysplastic syndrome (MDS)-like signs and symptoms, including subsequent acute myeloid leukemia (AML), which is transplantable. The disclosure also provides methods for identifying and purifying embryonic stem cells, adult stem cells, cancer stem cells, including leukemia stem cells, methods for identifying substances which bind to and/or modulate SALL4, methods for diagnosing MDS in a subject, and methods of treating a subject presenting MDS.

1 . An antibody or antibody fragment which binds to a polypeptide consisting essentially of an amino acid sequence as set forth in SEQ ID NO: 13. 
     
     
         2 . A method of treating myelodysplastic syndrome (MDS) in a subject comprising administering a therapeutically effective amount of the antibody of  claim 1  to the subject. 
     
     
         3 . The method of  claim 2 , wherein the MDS is acute myeloid leukemia (AML). 
     
     
         4 . A method of treating myelodysplastic syndrome (MDS) in a subject, comprising administering to the subject a composition comprising a polynucleotide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, a complement of SEQ ID NO: 1, a complement of SEQ ID NO: 3, a complement of SEQ ID NO: 5, and fragments thereof comprising at least 15 consecutive nucleotides of a polynucleotide encoding the amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6. 
     
     
         5 . The method of  claim 4 , wherein the MDS is acute myeloid leukemia (AML). 
     
     
         6 . A method of treating myelodysplastic syndrome (MDS) in a subject, comprising administering to the subject a composition comprising a polypeptide selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO:6. 
     
     
         7 . The method of  claim 6 , wherein the MDS is acute myeloid leukemia (AML). 
     
     
         8 . A method of diagnosing myelodysplastic syndrome (MDS) in a subject comprising: (a) providing a biological sample from the subject; (b) contacting the biological sample with a probe comprising a fragment of at least 15 consecutive nucleotides of a polynucleotide having a sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, a complement of SEQ ID NO: 1, a complement of SEQ ID NO: 3, or a complement of SEQ ID NO: 5 under hybridization conditions; and (c) detecting the hybridization between the probe and the biological sample, wherein detecting of hybridization correlates with MDS. 
     
     
         9 . The method of  claim 8 , wherein the MDS is acute myeloid leukemia (AML). 
     
     
         10 .- 11 . (canceled) 
     
     
         12 . A method for isolating leukemia stem cells comprising: obtaining a sample of cells from a subject; sorting cells that express a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 13 from cells that do not express the amino acid sequence; and selecting, by a myeloid surface marker, leukemia stem cells from the sample of cells that express the polypeptide comprising the amino acid sequence as set forth in SEQ ID NO: 13. 
     
     
         13 . The method of  claim 12 , wherein the step of sorting comprises sorting by fluorescence activated cell sorting (FACS). 
     
     
         14 . The method of  claim 12 , wherein the step of sorting comprises sorting by magnetic bead sorting (MACS). 
     
     
         15 . The method of  claim 12 , wherein the marker is CD34, c-kit, Gr-1, Mac-1, MPO, and/or nonspecific esterase. 
     
     
         16 . The method of  claim 15 , wherein the leukemia stem cells are negative for B-cell, T-cell, megakaryocytic, and erythroid markers. 
     
     
         17 . A transgenic animal comprising a human SALL4 gene, wherein the animal is modified to expresses a sequence of a human SALL4 gene comprising nucleotides encoding an amino acid as set forth in SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6. 
     
     
         18 . The transgenic animal of  claim 17 , wherein the animal constitutively expresses the inserted SALL4 gene. 
     
     
         19 . The transgenic animal of  claim 18 , wherein the SALL4 gene comprises nucleotides encoding an amino acid sequence as set forth in SEQ ID NO: 4. 
     
     
         20 . A method of preparing a transgenic animal comprising a human SALL4 gene, wherein the animal is modified to constitutively express a sequence of a human SALL4 gene comprising nucleotides encoding an amino acid as set forth in SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6, comprising: a) introducing into embryonic cells a nucleic acid molecule a comprising a construct of human SALL4 gene comprising nucleotides encoding an amino acid as set forth in SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6; b) generating a transgenic animal from the cells resulting from step a); c) breeding the transgenic animal to obtain a transgenic animal homozygous for the human SALL4 gene; and d) detecting human SALL4 transcripts from tissue from the transgenic animal. 
     
     
         21 . The method of  claim 20 , wherein the construct of human SALL4 gene comprises nucleotides encoding an amino acid as set forth in SEQ ID NO: 4. 
     
     
         22 . A method of identifying a cell possessing pluripotent potential comprising: a) contacting a cell isolated from an inner cell mass (ICM), a neoplastic tissue, or a tumor with an agent that detects the expression of a SALL family member protein; and b) determining whether a SALL family member protein is expressed in the cell of step (a), wherein determining the expression of the SALL family member protein positively correlates with induction of self-renewal in the cell, whereby such expression is indicative of pluripotency. 
     
     
         23 . The method of  claim 22 , wherein the SALL family member is selected from the group consisting of SALL1, SALL3, and SALL4. 
     
     
         24 . The method of  claim 23 , wherein the SALL family member is SALL4. 
     
     
         25 . The method of  claim 24 , wherein SALL4 is SALL4A or SALL4B. 
     
     
         26 . The method of  claim 22 , wherein the agent is an antibody directed against the SALL family member protein or a nucleic acid which is complementary to a mRNA encoding the SALL family member protein. 
     
     
         27 . The method of  claim 26 , wherein the nucleic acid is complementary to a nucleic acid encoding SALL family member protein sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:22, and SEQ ID NO:24. 
     
     
         28 . The method of  claim 26 , wherein the nucleic acid is complementary to a sense strand of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5; SEQ ID NO:21, and SEQ ID NO:23. 
     
     
         29 . The method of  claim 22 , wherein the cell is an embryonic stem (ES) cell, an embryonic carcinoma (EC) cell, an adult stem cell, or a cancer stem cell. 
     
     
         30 . The method of  claim 22 , wherein the tissues is plasma or a biopsy sample from a subject. 
     
     
         31 . The method of  claim 22 , wherein the subject is a mammal. 
     
     
         32 . The method of  claim 31 , wherein the subject is a human. 
     
     
         33 . A method of identifying an agent which modulates the effect of a SALL family member protein on OCT4 expression comprising: a) co-transfecting a cell with: i) a vector comprising a promoter-reporter construct, wherein the construct comprises an operatively linked OCT4 promoter and a nucleic acid encoding gene expression reporter protein, and ii) a vector comprising a nucleic acid encoding a SALL family member protein; b) contacting the cell of step (a) with an agent; and c) determining the activity of the promoter-reporter construct in the presence and absence of the agent in step (b), wherein determining the activity of the promoter-reporter construct correlates with the effect of the agent on SALL family member protein/OCT4 interaction. 
     
     
         34 . The method of  claim 33 , wherein the promoter region comprises nucleic acid sequence as set forth in SEQ ID NO:26. 
     
     
         35 . The method of  claim 33 , wherein the nucleic acid of step (a)(ii) encodes a protein sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:22, and SEQ ID NO:24. 
     
     
         36 . The method of  claim 33 , wherein the expression reporter protein is luciferase. 
     
     
         37 . A method of diagnosing a neoplastic or proliferative disorder comprising: a) contacting a cell of a subject with an agent that detects the expression of a SALL family member protein; and b) determining whether a SALL family member protein is expressed in the cell of step (a), wherein determining the expression of the SALL family member protein positively correlates with induction of self-renewal in the cell, whereby such expression is indicative of neoplasia or proliferation. 
     
     
         38 . The method of  claim 37 , wherein the agent is labeled. 
     
     
         39 . The method of  claim 38 , wherein determining step comprises detection of the agent is accomplished by exposing the subject to a device which images the location of the agent 
     
     
         40 . The method of  claim 39 , wherein the images are generated by magnetic resonance, X-rays, or radionuclide emission. 
     
     
         41 . A method of treating a neoplastic or proliferative disorder, wherein cells of a subject exhibit de-regulation of self-renewal, comprising administering to the subject a pharmaceutical composition comprising an agent which inhibits the expression of SALL4. 
     
     
         42 . The method of  claim 41 , wherein the agent is a nucleic acid. 
     
     
         43 . The method of  claim 41 , wherein the agent is an antibody directed against SALL4. 
     
     
         44 . A kit for identifying a cell possessing pluripotent potential comprising: a) an agent for detecting one or more SALL family member proteins; b) reagents and buffers to provide conditions sufficient for agent-cell interaction and labeling of the agent; c) instructions for labeling the detection reagent and for contacting the agent with the cell; and d) a container comprising the components (a), (b), and (c). 
     
     
         45 . The kit of  claim 44 , wherein the SALL family member protein is selected from the group consisting of SALL1, SALL3, and SALL4. 
     
     
         46 . The kit of  claim 44 , wherein the agent is an antibody directed against the SALL family member protein or a nucleic acid which is complementary to a mRNA encoding the SALL family member protein. 
     
     
         47 . A method of isolating cells comprising: a) contacting the cells with an antibody directed against SALL4; b) applying cells bound to the antibody to a surface delimited cavity comprising at least two apertures for ingress and egress of fluids and cells; and c) allowing cells and fluids to pass through the cavity, wherein antibody bound cells in a fluid mixture are detected by optical or magnetic detectors, and wherein voltage or magnetic flux is applied to the fluid whereby the voltage or flux assorts the bound cells in one or more collectors or within the cavity. 
     
     
         48 . The method of  claim 47 , wherein the method is fluorescence activated cell sorting (FACS). 
     
     
         49 . The method of  claim 47 , wherein the method is magnetic bead cell sorting (MACS). 
     
     
         50 . The method of  claim 47 , wherein the cell is an embryonic stem cell, an adult stem cell, or a cancer stem cell. 
     
     
         51 . The method of  claim 47 , wherein SALL4 is SALL4A or SALL4B. 
     
     
         52 . A method of detecting cells associated with progression of a proliferative disease or neoplastic cell formation comprising: a) contacting the cells with an antibody directed against SALL4; b) applying cells bound to the antibody to a surface delimited cavity comprising at least two apertures for ingress and egress of fluids and cells; and c) allowing cells and fluids to pass through the cavity, wherein antibody bound cells in a fluid mixture are detected by optical detectors and wherein voltage is applied to the fluid whereby the voltage assorts the bound cells in one or more collectors.