# Patent

## Title
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.

## Background
RELATED APPLICATIONS
This application claims benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 60/741,015, filed on Nov. 29, 2005 which is incorporated herein by reference in its entirety.


GOVERNMENT SUPPORT
This invention was made in part with government support under Grant Nos. K08CA097185, P20RR016464, and R01HL087948 awarded by the National Institutes of Health (NIH). The government has certain rights in this invention.


BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates generally to factors associated with the Wnt/β-catenin signaling pathway and, more specifically, to interaction between transcription components of the pathway, including the SALL protein family and OCT4, which are involved in the regulation of embryonic and cancer stem cells, including methods for the diagnosis and treatment of proliferative disorders by targeting such interaction.
2. Background Information
ES cells derived from the inner cell mass (ICM) of the blastocyst are able to undergo self-renewing cell division and maintain their pluripotency over an indefinite period of time. ES cells can also differentiate into a variety of different cell types when cultured in vitro. The Wnt/β-catenin signaling pathway has been associated with the self-renewal of normal human stem cells (HSCs) and the granulocyte-macrophage progenitors (GMPs) of chronic myeloid leukemia (CML). Further, the transcriptional factor, OCT4, has been identified as a key regulator for the formation of ICM during preimplantation development. Moreover, OCT4 protein seems to plays a central role in maintaining the pluripotency of embryonic stem (ES) cells by regulating a wide range of genes.
The role of stem cells has been considered in the etiology of cancer. There has been increasing evidence that tumors might contain such cancer stems cells, i.e., rare cells that account for the growth of tumors. These rare cells with indefinite proliferative potential may account for the resistance observed for cancer cells in response to conventional therapeutic modalities. It is known that stem cells can be identified in adult tissues, where such cells arise from a specific tissue; e.g., hematopoietic cells. As the self renewal property of stem cells is tightly controlled in normal organogenesis, the de-regulation of self-renewal might result in carcinogenesis.
Myelodysplastic syndrome (MDS), for example, is a hematological disease marked by the accumulation of genomic abnormalities at the hematopoietic stem cell (HSC) level leading to pancytopenia, multilineage differentiation impairment, and bone marrow apoptosis.
Mortality in this disease results from pancytopenia or transformation to acute myeloid leukemia (AML). AML is a hematological cancer characterized by the accumulation of immature myeloid precursors in the bone marrow and peripheral blood.
From the analysis of genetic translocation in bone marrow samples from AML patients, it is clear that transcription factors critical for hematopoiesis play an important role in leukemogenesis. The pathogenesis of AML is considered to involve multistep genetic alternations. Because only HSCs are considered to have the ability to self-renew, they are the best candidates for the accumulation of multistep, preleukemic genetic changes and transforming them into so-called “leukemia stem cells” (LSCs).
Alternatively, downstream progenitors can acquire self-renewal capacity and give rise to leukemia. LSCs are not targeted specifically under current chemotherapy regimens yet such cells have been found to account for drug resistance and leukemia relapse.
The SALL gene family, SALL1, SALL2, SALL3, and SALL4, were originally cloned on the basis of their DNA sequence homology to Drosophila spalt (sal). In Drosophila, spalt is a homeotic gene essential for development of posterior head and anterior tail segments. It plays an important role in tracheal development, terminal differentiation of photoreceptors, and wing vein placement. In humans, the SALL gene family is associated with normal development, as well as tumorigenesis. SALL proteins belong to a group of C2H2 zinc finger transcription factors characterized by multiple finger domains distributed over the entire protein. During the tracheal development of Drosophila, spalt is an activated downstream target of Wingless, a Wnt ortholog. It has been demonstrated that SALL1 interacts with β-catenin by functioning as a coactivator, suggesting that the interaction between SALL and the Wnt/β-catenin pathway is bidirectional.
SUMMARY OF THE INVENTION
The present invention relates to SALL4, a human homolog to Drosophila spalt, which is a zinc finger transcriptional factor essential for development. SALL4 and its isoforms (SALL4A, SALL4B, and SALL4C) were cloned and sequenced. The present disclosure demonstrates that SALL4 failed to be turned off in human primary AML. Further, the leukemogenic potential of constitutive expression of SALL4 in a murine model is demonstrated. Moreover, SALL4B-transgenic mice which develop myelodysplastic syndrome (MDS)-like signs and symptoms and subsequent transplantable AML are described.
Increased apoptosis associated with dysmyelopoiesis is evident in transgenic mouse marrow and colony-formation (CFU) assays. Both isoforms are able to bind to β-catenin and synergistically enhance the Wnt/β-catenin signaling pathway. This demonstrates that the constitutive expression of SALL4 causes MDS/AML, and that such expression impinges on the Wnt/β-catenin pathway. In a related aspect, the murine model disclosed provides a platform to study human MDS/AML transformation, and the Wnt/β-catenin pathway's role in the pathogenesis of leukemia stem cells.
In one embodiment, an antibody or antibody fragment is disclosed which binds to a polypeptide that includes an amino acid sequence as set forth in SEQ ID NO: 13.
In another embodiment, a method of treating myelodysplastic syndrome (MDS) in a subject is disclosed, including administering a therapeutically effective amount of an antibody which binds to a polypeptide that includes an amino acid sequence as set forth in SEQ ID NO: 13 to the subject.
In another embodiment, a method of treating myelodysplastic syndrome (MDS) in a subject is provided, including administering to the subject a composition of a polynucleotide having a sequence as 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, a complement of SEQ ID NO: 5, and fragments thereof including 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.
In one embodiment, a method of treating myelodysplastic syndrome (MDS) in a subject is disclosed, including administering to the subject a polypeptide composition having a sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 4 and/or SEQ ID NO: 6.
In a related aspect, the MDS is acute myeloid leukemia (AML).
In one embodiment, a method of diagnosing myelodysplastic syndrome (MDS) in a subject is disclosed, including, providing a biological sample from the subject, 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 detecting the hybridization between the probe and the biological sample, where detecting of hybridization correlates with MDS.
In another embodiment, a method of diagnosing a myelodysplastic syndrome (MDS) in a subject is disclosed, including providing a biological sample from the subject, contacting the biological sample with an antibody which binds to a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6, and detecting the binding of the antibody to the sample, where detecting binding correlates with MDS.
In one embodiment, a method for isolating leukemia stem cells is provided, including 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.
In another embodiment, a transgenic animal having a human SALL4 gene is provided, where 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. In a related aspect, the animal constitutively expresses the inserted SALL4 gene.
In one embodiment, a method of preparing a transgenic animal comprising a human SALL4 gene is disclosed, where 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, including 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, generating a transgenic animal from the cells resulting from step the introduction of the construct, breeding the transgenic animal to obtain a transgenic animal homozygous for the human SALL4 gene, and detecting human SALL4 transcripts from tissue from the transgenic animal.
In one embodiment, a method of modulating the cellular expression of a polynucleotide encoding an amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6 is disclosed, including introducing a double stranded RNA (dsRNA) which hybridizes to the polynucleotide, or an antisense RNA which hybridizes to the polynucleotide, or a fragment thereof, into a cell.
In one embodiment, a method of identifying a cell possessing pluripotent potential is disclosed including 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 determining whether a SALL family member protein is expressed in the cell, where 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.
In one aspect, the SALL family member includes SALL1, SALL3, and SALL4. In a related aspect, SALL4 is SALL4A or SALL4B.
In another aspect, 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. In a related aspect, the SALL family member protein sequence includes SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:22, and SEQ ID NO:24. In another related aspect, the nucleic acid is complementary to a sense strand of a nucleic acid sequence including SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5; SEQ ID NO:21, and SEQ ID NO:23.
In one aspect, the cell is an embryonic stem (ES) cell, an embryonic carcinoma (EC) cell, an adult stem cell, or a cancer stem cell. In a related aspect, the tissues is plasma or a biopsy sample from a subject. In a further related aspect, the subject is a human.
In one embodiment, a method of identifying an agent which modulates the effect of a SALL family member protein on OCT4 expression is disclosed including co-transfecting a cell with a vector comprising a promoter-reporter construct, where the construct comprises an operatively linked OCT4 promoter and a nucleic acid encoding gene expression reporter protein, and a vector comprising a nucleic acid encoding a SALL family member protein, contacting the cell with an agent, and determining the activity of the promoter-reporter construct in the presence and absence of the agent, where determining the activity of the promoter-reporter construct correlates with the effect of the agent on SALL family member protein/OCT4 interaction.
In a related aspect, the promoter region comprises nucleic acid sequence as set forth in SEQ ID NO:26 and the expression reporter protein is luciferase.
In another embodiment, a method of diagnosing a neoplastic or proliferative disorder is disclosed including contacting a cell of a subject with an agent that detects the expression of a SALL family member protein and determining whether a SALL family member protein is expressed in the cell, where 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.
In one aspect, the agent is labeled and the determining step includes detection of the agent by exposing the subject to a device which images the location of the agent. In a related aspect, the images are generated by magnetic resonance, X-rays, or radionuclide emission.
In one embodiment, a method of treating a neoplastic or proliferative disorder, where cells of a subject exhibit de-regulation of self-renewal, is disclosed including administering to the subject a pharmaceutical composition containing an agent which inhibits the expression of SALL4.
In another embodiment, a kit for identifying a cell possessing pluripotent potential is disclosed including an agent for detecting one or more SALL family member proteins, reagents and buffers to provide conditions sufficient for agent-cell interaction and labeling of the agent, instructions for labeling the detection reagent and for contacting the agent with the cell, and a container comprising the components.
A method of detecting cells associated with progression of a proliferative disease or neoplastic cell formation is disclosed including contacting the cells with an antibody directed against SALL4, 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 allowing cells and fluids to pass through the cavity, where antibody bound cells in a fluid mixture are detected by optical detectors, and where voltage is applied to the fluid whereby the voltage assorts the bound cells in one or more collectors.
Exemplary methods and compositions according to this invention are described in greater detail below.



BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1(a-c) illustrate properties of the three SALL4 isoforms (SALL4A, SEQ ID NO: 1[GenBank Acc. No.: AY172738]; SALL4B, SEQ ID NO: 3[GenBank Acc. No. AY170621]); and SALL4C, SEQ ID NO: 5 [GenBank Acc. No. AY170622]. Alternative splicing generates two variant forms of SALL4 mRNA. FIG. 1(a) SALL4A and SALL4B vary in protein length and in the presence of different numbers of characteristic sal-like zinc finger domains. SALL4A (encoding 1,067 amino acids) contains eight zinc finger domains, while SALL4B (encoding 623 amino acids) has three zinc finger domains. SALL4C contains 276 amino acids and lacks the region corresponding to amino acids 43 to 820 of the full length SALL4A. Both variants have exons 1, 3, and 4, and SALL4A contains all exons from 1 to 4. However, SALL4B uses an alternative splice acceptor that results in deletion of the large 3′ portion of exon 2. FIG. 1(b) shows the RT-PCR analysis of SALL4 variants in different tissues. Four exons of SALL4 and their potential coding structures are illustrated, with arrows indicating the primers used for PCR amplification of the SALL4 transcripts (A). Tissue-dependent expression of SALL4 transcripts by RT-PCR (B). A 315-bp expected product that was specific for SALL4A with primers A1 (exon 2) and B1 (exon 4) was amplified with cDNAs of various tissues. Primers D1 (exon 4) and C1 (exon 1) were used to amplify the 1,851-bp expected product of SALL4B. Comparable amounts of cDNA were determined by GAPDH. FIG. 1(c) shows SALL4 protein products, SALL4A, and SALL4B identified by a SALL4 peptide antibody. Lysates from Cos-7 cells transiently expressing His-SALL4B (lane 1), His-SALL4A (lane 2), or control vector (lane 8), or lysates from different human tissues were resolved by 10% SDS-PAGE gel, transferred onto a nitrocellulose membrane, and probed with the N-terminal SALL4 peptide antibody.
FIGS. 2(a-b) demonstrate the expression of SALL4 in human primary AML and myeloid leukemia cell lines. FIG. 2(a) demonstrates the failure of SALL4 to turn off in AML. Real-time PCR quantification of SALL4A and SALL4B normalized to GAPDH showed that both SALL4A and SALL4B were expressed in purified CD34+ cells, but SALL4A was rapidly downregulated and SALL4B turned off in normal bone marrow (N=3) and normal peripheral blood (N=3) cells. In contrast, in 15 primary AML samples and three myeloid leukemia cell lines (Kasumi-1, THP-1, and KG.1), the expression of SALL4A or SALL4B, or both, failed to be down-regulated. The results were calibrated against the expression of SALL4A or SALL4B in purified CD34+ cells. FIG. 2(b) shows constitutive expression of SALL4 protein in human AML (FAB M1-M5, N=81) as demonstrated by immunohistochemical staining. No SALL4 expression was detected in normal bone marrow (A), normal spleen (B), or normal thymus (C). All cell nuclei are stained blue. Nuclei of CD34+ HSC/HPCs showed brown staining indicating SALL4 expression (D); acute myeloid leukemia blasts showed similar staining (E, low power [×4]) in microarray leukemia tissue samples. Each circle represents one leukemia sample. (F) High power (×100) view of one leukemia sample shown in part F. The red arrows indicate positive nuclear staining.
FIGS. 3(a-h) show that SALL4B transgenic mice have an MDS-like/AML phenotype. FIG. 3(a) illustrates the generation of SALL4B transgenic mice: CMV/SALL4B transgenic construct and PCR analysis of transgenic line 507. (A) Schematic diagram of transgenic construct. The approximately 1.8-kb cDNA of SALL4B was subcloned into a pCEP4 vector, and the CMV/SALL4 construct was excited by digestion with SalI. (B) Tissue distribution of SALL4B in transgenic mice. The location of primers used for RT-PCR amplification is indicated by arrows in part A. A primer specific for human SALL4B at the C-terminus was used as a 5′ primer, in combination with SV40-noncoding sequence-specific primers for RT-PCR of various tissues. FIG. 3(b) demonstrates the MDS-like changes in SALL4B transgenic mice. Giemsa staining of peripheral blood from normal, age-matched WT litter mates showed normal neutrophils (A), and normal red blood cells and platelets (B, black arrow). In transgenic mice, neutrophils were hypersegmented (E), and pseudo-Pelger-Huet-like atypical white cells were present (F-H), together with increased numbers of immature cells (I-K). Nucleate red cells (L, red arrow), giant platelets (M, red arrow), and polychromasia (N) were also observed in the transgenic mice. A binucleate dysplastic erythrocyte (O, red arrow) and a dysplastic megakaryocyte with a hypolobulated nucleus (P, red arrow) were found in the cytospin from transgenic mouse bone marrow. An erythroid precursor. (C) and a megakaryocyte (D) from WT control animals are shown for comparison. FIG. 3(c) shows that AML is observable in SALL4B transgenic mice. Blasts were present in the peripheral blood (A, ×600), bone marrow biopsy specimen (B, ×100; C, ×400), bone marrow smear (D, ×600), liver (E, ×100), lymph node (F, ×400), and spleen (G and H, gross view; I, ×100; and the inset ×400). FIG. 3(d) shows the flow cytometric analysis of AML in SALL4B transgenic mice. AML cells were positive for CD45, c-kit, Gr-1, and Mac-1; negative for B220, CD3, and Ter119. FIG. 3(e) demonstrates the serial transplantation of SALL4B-Induced AML to NOD/SCID mice. Gross picture (A&B) and histology (B, C, D, E, ×200) on splenomegaly (3e-A black arrow, 3e-C), hepatomegaly (3e-A double black arrows, 3e-D), lymph node enlargement (3e-B black arrow, 3e-E) and pale kidney (3e-B double black arrows, 3e-F) caused by leukemia infiltration in a NOD/SCID mouse 6 weeks after leukemia transplantation. FIG. 3(f) illustrates the comparison between bone marrow of SALL4B transgenic and control mice. SALL4B transgenic mouse bone marrow showed increased cellularity, myeloid population (Gr-1/Mac-1 double positive), immature population (c-kit positive), and apoptosis (Annexin V positive, PI negative), compared with control WT mice. FIG. 3(g) shows that there are an increased number of immature cells and apoptosis in CFUs from SALL4B transgenic mice. On day 7 of culture, a greater number of immature cells (B, C, and D, red arrows) and apoptotic cells (B, C, and D, double red arrows) were observed in transgenic mouse CFUs than in control CFUs (A). Consistent with this morphologic observation, there was increased apoptosis (Annexin V positive, PI negative, E) and more CD34+ immature cells (F). FIG. 3(h) illustrates the comparison between bone marrow CFUs of SALL4B transgenic and control mice. Percentage of different types of colonies found in CFU assays of SALL4B transgenic and control mice (A). CFUs from SALL4B transgenic mice compared with control mice showed a statistically significant increase in CFU-GM (B) (transgenic: 53.6±10.3, N=13 vs. WT: 38.1±3.1, N=8; P=0.002) and decrease in BFU-E (transgenic: 7.8±3.8, N=13 vs. WT: 14.1±2.7, N=8; P=0.001).
FIGS. 4(a-d) demonstrate the interaction between SALL4 and the Wnt/β-catenin signaling pathway. FIG. 4(a) shows that both SALL4A and SALL4B can interact with β-catenin. Nuclear extracts (lysates) prepared from Cos-7 cells were transiently transfected with HA-SALL4A or HA-SALL4B. (A) Anti-HA antibody recognized both SALL4A (165 kDa) and SALL4B (95 kDa). (B) β-Catenin was detected in the lysates. (C) Immunoprecipitation was performed with the use of an HA affinity resin and detected with an anti-α-catenin antibody. β-Catenin was readily detected in both HA-SALL4A and HA-SALL4B pull-downs. FIG. 4(b) shows the activation of the Wnt/β-catenin signaling pathway by both SALL4A and SALL4B. NIH3T3 cells were transfected with 1.0 μg of either SALL4A or SALL4B plasmid and TOPflash reporter plasmid (Upstate USA, Chicago, Ill.). After 24-h stimulation with Wnt1 or the mock, luciferase activity was measured. FIG. 4(c) demonstrates the presence of SALL4 protein expression at the blast phase (N=12) but not the chronic phase (N=11) of CML as demonstrated by immunohistochemical staining. (A) shows a low-power view of tissue arrays of the CML collection at different phases. SALL4 expression was absent in chronic-phase CML (B), with all cells' nuclei remaining blue (B, ×4; D, ×400). SALL4 expression, however, was present in blast-crisis-phase CML as indicated by the brown nuclear staining of SALL4 (C, ×4; E, ×400). In CML accelerated phase (N=6), in which the blast count is increased but still <15%, only immature blasts were observed to stain positively for SALL4 expression (F; red arrows); mature neutrophils did not stain (F, black arrows, ×600). FIG. 4(d) illustrates a working hypothesis. SALL4 is expressed in human stem cells/progenitors but is absent in mature hematopoietic cells during normal hematopoiesis. Constitutive expression of SALL4 isoforms (failure to turn off SALL4) results in blocked differentiation and constitutive renewal with aberrant expansion of the stem cell pool that lead to leukemic transformation (+, presence of SALL4 expression; −, absence of SALL4 expression).
FIG. 5 illustrates dose-dependent effect of SALL4B on the OCT4 promoter. 0.3 μg of OCT4-Luc construct (PMOct4) was cotransfected with 0.1 μg of renilla plasmid and increasing amounts (0-1.0 μg) of SALL4B or pcDNA3 vector control.
FIG. 6 demonstrates the effect of OCT4 on SALL gene family member promoters. Each (0.3 μg) SALL-Luc promoter construct (i.e., pSALL1, pSALL3, and pSALL4) was co-transfected with 0.9 μg of OCT4 or pcDNA3 vector control in HEK-293 cells. After 24 hr post-transfection, luciferase activity was evaluated for each group.
FIG. 7 shows the effect of SALL4 isoforms A and B on SALL4 promoter activity. 0.3 μg of SALL4-Luc was cotransfected with 0.1 μg of either SALL4A or SALL4B expressing plasmid in different cell lines (HEK-293 or COS-7); pcDNA3 vector was used as the control. Luciferase activity was normalized for renilla reporter activity. The values represent the mean±s.e. of three experiments.
FIG. 8 demonstrates the dose dependent effect of SALL4A on SALL4 promoter activity. In HEK-293 cells, 0.3 μg of the SALL4-Luc was co-transfected with 0.1 μg of renilla plasmid and increasing ratios of the SALL4A construct and the control pcDNA3 vector. The Luciferase activity is normalized for the Renilla reporter activity.
FIG. 9 shows the effect of SALL4 on SALL1 and SALL3 promoter activity. Each (0.3 μg) SALL-Luc promoter construct was transiently co-transfected with 0.9 μg of SALL4A plasmid or pcDNA3 vector (control) in HEK-293 cells.
FIG. 10 shows the effect of OCT4 on the SALL4 promoter in the presence of excess SALL4A. 0.25 μg of SALL4-Luc construct (pSALL4) was transiently co-transfected with equal amounts (0.5 μg) of SALL4A and OCT4 plasmid in the HEK-293 cells. pcDNA3 was used as a control.
FIG. 11 shows the effect of OCT4 on other SALL member promoters in the presence of SALL4. HEK-293 cells seeded in a 24 well plate were transiently co-transfected with a different SALL member promoter reporter (pSALL1 or pSALL3) and OCT4 plasmid and/or SALL4A construct. pcDNA3 was used as a control.
FIG. 12 shows the effect of self promoter interaction on promoter activity for other SALL protein family members. HEK-293 cells were seeded on a 24 well plate and transiently transfected or co-transfected with 0.3 μg SALL1-Luc reporter construct with various amounts of SALL1 plasmid (0.45 and 0.9 μg) SIX1, previously found to activate SALL1 promoter, was used as a positive control. Luciferase activity was normalized for renilla reporter activity.



DETAILED DESCRIPTION OF THE INVENTION
Before the present composition, methods, and culturing methodologies are described, it is to be understood that this invention is not limited to particular compositions, methods, and experimental conditions described, as such compositions, methods, and conditions may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only in the appended claims.
As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, references to “a nucleic acid” includes one or more nucleic acids, and/or compositions of the type described herein which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, as it will be understood that modifications and variations are encompassed within the spirit and scope of the instant disclosure. All publications mentioned herein are incorporated herein by reference in their entirety.
Homeobox and homeotic genes play important roles in normal development. Some homeobox genes, such as Hox and Pax, also function as oncogenes or as tumor suppressors in tumorigenesis or leukemogenesis. The important role of SALL4, a homeotic gene and a transcriptional factor, in human development was recognized because heterozygous SALL4 mutations lead to Duane Radial Ray syndrome. In a related aspect, SALL4's oncogenic role in leukemogenesis is described herein.
In one embodiment, the present disclosure identifies two SALL4 isoforms, SALL4A and SALL4B. In a related aspect, the disclosure provides an analysis of SALL4 nucleic acids and proteins as tools for diagnosing and treating patients having proliferation disorders such as hematologic malignancies and other tumors involving constitutive expression of SALL4 nucleic acid and protein. In a related aspect, SALL4 serves as a malignant stem cell marker for diagnosis and treatment of cancers.
For example, during normal hematopoiesis, SALL4 isoforms are expressed in the CD34+ HSC/HPC population and rapidly turned off (SALL4B) or down-regulated (SALL4A) in normal human bone marrow and peripheral blood. In contrast, SALL4 is constitutively expressed in all AML samples (N=81) that were examined, and failed to turn off in human primary AML and myeloid leukemia cell lines. In a related aspect, the leukemogenic potential of constitutive expression of SALL4 in vivo was directly tested via generation of SALL4B transgenic mice. Such transgenic mice exhibit dysregulated hematopoiesis, much like that of human MDS, and exhibited AML that was transplantable. The MDS-like features in these SALL4B transgenic mice do not require cooperating mutations and are observed as early as 2 months of age. The ineffective hematopoiesis observed in these mice is characterized, as it is in human MDS, by hypercellular bone marrow and paradoxical peripheral blood cytopenias (neutropenia and anemia) and dysplasia, which are probably secondary to the increased apoptosis noted in the bone marrow. While not being bound to theory, a reason for the late onset of leukemia development in these transgenic mice may be the accumulation of additional genetic damage during the ≧8 months of replicative stress. Late onset of disease may also be a consequence of SALL4-induced genomic instability.
Further, specific, recurrent chromosomal translocations characterize many leukemias, which can result from a breakdown in the normal process of immunoglobulin or T-cell receptor gene rearrangement, causing inter-chromosomal translocations rather than normal intra-chromosomal rearrangement. The flow of genetic information from genes at chromosomal translocation breakpoints to proteins has several points which therapeutic reagents could intervene. Sequence specific binding elements that exploit zinc-finger binding protein domains can be used to create de novo sequence specific binding elements that could act as gene switches which can target chromosomal fusion junctions to turn off expression of aberrant gene fusion products.
In one embodiment, SALL4 can be used as a component of a fusion protein which targets chromosomal fusion junctions as a gene switch to modulate the expression of gene fusion products. Production of recombinant fusion protein is well known in the art (see, e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).
In one embodiment, SALL4 proteins and/or nucleic acids are detected for diagnosing subpopulations of lymphomas and leukemias or other types of cancers. In another embodiment, the detection of the SALL4 proteins and nucleic acids can be used to identify a subject, including, but not limited to, a human subject, at risk for developing/acquiring a proliferative disease.
In a further embodiment, methods for identifying compounds which alter SALL4 protein and nucleic acid levels are disclosed. In a related aspect, SALL4 can serve as a therapeutic target, where blocking SALL4 function can inhibit tumor development and progression.
In another aspect, investigation of the potential mechanism of SALL4 involvement in leukemogenesis demonstrates that both SALL4A and SALL4B interacted with β-catenin, an essential component of the Wnt signaling pathway involving self-renewal of HSCs. In addition, both are able to activate the Wnt/β-catenin pathway in a reporter gene assay, consistent with SALL family function in Drosophila and humans. Furthermore, similar to the situation with β-catenin, SALL4 expression in CML varied at different phases of the disease: SALL4 expression being absent in the chronic phase, became detectable in the accelerated phase only in immature blasts, and is strongly positive in the blast phase.
On the basis of these studies, a working hypothesis is disclosed (e.g., see FIG. 4d). While not being bound to theory, constitutive expression of SALL4 in AML may enable leukemic blasts to gain stem cell properties, such as self-renewal and/or dedifferentiation, and thus become LSCs. This hypothetical model would parallel what is seen in the case of β-catenin. For example, in normal myelopoiesis, β-catenin is only activated in HSCs bearing a self-renewal property. In the blast phase of CML, β-catenin gains function by becoming activated in the GMPs, resulting in leukemic transformation.
In another aspect, the oncogene SALL4 plays an important role in normal hematopoiesis and leukemogenesis. SALL4B transgenic mice exhibit MDS-like phenotype with subsequently AML transformation that is transplantable. Few animal models are currently available for the study of human MDS. The SALL4B transgenic mice that were generated by the methods described herein provide a suitable animal model for understanding and treating human MDS and its subsequent transformation to AML. The interaction between SALL4 and the Wnt/β-catenin signaling pathway not only provides a plausible mechanism for SALL4 involvement in leukemogenesis but also advances the understanding of the activation of the Wnt/β-catenin signaling pathway in CML blastic transformation.
As disclosed herein, the identification of SALL4 isoforms and their constitutive expression in all human AML were examined. The direct impact of SALL4 expression in AML was tested in vivo. The disclosure demonstrates that constitutive expression of SALL4 in mice is sufficient to induce MDS-like symptoms and transformation to AML that is transplantable. The disclosure also demonstrates that SALL4 is able to bind β-catenin and activate the Wnt/β-catenin signaling pathway. SALL4 and β-catenin share similar expression patterns at different phases of CML.
In one embodiment, an isolated polynucleotide comprising a sequence encoding an amino acid sequence as set forth in SEQ ID NO: 2 (GenBank Acc. No. AAO44950), SEQ ID NO: 4 (GenBank Acc. No. AAO16566), or SEQ ID NO: 6 (GenBank Acc. No. AAO16567) is provided. In a related aspect, such sequences comprise a nucleic acid sequence as set forth in SEQ ID NO: 1 (GenBank Acc. No. AY172738), SEQ ID NO: 3 (GenBank Acc. No. AY170621), SEQ ID NO: 5 (GenBank Acc. No. AY170622), or complements thereof. In another related aspect, a vector comprising such polynucleotides are also disclosed, including, but not limited to, expression vectors which are operably linked to a regulatory sequence which directs the expression of the polynucleotide in a host cell.
In another embodiment, an isolated polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6 is disclosed. In one aspect, a method of treating a myelodysplastic syndrome (MDS) in an individual including administering such a polypeptide is provided. In another aspect, antibodies or binding fragments thereof which bind to such a polypeptide are also disclosed.
Antibodies that are used in the methods disclosed include antibodies that specifically bind polypeptides comprising SALL4, or their isoforms as set forth in SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6. In one aspect, a fragment of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6 is used to generate such antibodies. In a related aspect, such a fragment consists essentially of SEQ ID NO: 13.
In one embodiment, a method of identifying a cell possessing pluripotent potential is disclosed including 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 determining whether a SALL family member protein is expressed in the cell, where 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.
In one aspect, the SALL family member includes SALL1, SALL3, and SALL4. In a related aspect, SALL4 is SALL4A or SALL4B.
In another aspect, 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. In a related aspect, the SALL family member protein sequence includes SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:22, and SEQ ID NO:24. In another related aspect, the nucleic acid is complementary to a sense strand of a nucleic acid sequence including SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5; SEQ ID NO:21, and SEQ ID NO:23.
In one aspect, the cell is an embryonic stem (ES) cell, an embryonic carcinoma (EC) cell, an adult stem cell, or a cancer stem cell. In a related aspect, the tissues is plasma or a biopsy sample from a subject. In a further related aspect, the subject is a human.
As used herein, “pluripotent potential” means the ability of a cell to renew itself by mitosis.
As used herein “positively correlates” means affirmatively associated with the phenomenon observed. For example, induction of SALL4A or SALL4B is associated with increased cell renewal ability.
As used herein, “neoplasm,” including grammatical variations thereof, means new and abnormal growth of tissue, which may be benign or cancerous.
As used herein “consisting essentially of” includes a specific molecular entity (e.g., but not limited to, a specific sequence identifier) and other molecular entities that do not materially affect the properties associated with the specific molecular entity. For example, a fusion protein comprising SEQ ID NO: 13 and an adjuvant, for generating an immunogenic response against SEQ ID NO: 2, SEQ ID NO: 4, and/or SEQ ID NO: 6, would consist essentially of SEQ ID NO: 13.
Antibodies are well-known in the art and discussed, for example, in U.S. Pat. No. 6,391,589. Antibodies of the invention include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab′) fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), and epitope-binding fragments of any of the above. The term “antibody,” as used herein, refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen. The immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or subclass of immunoglobulin molecule.
Antibodies of the invention include antibody fragments that include, but are not limited to, Fab, Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a VL or VH domain. Antigen-binding antibody fragments, including single-chain antibodies, may comprise the variable region(s) alone or in combination with the entirety or a portion of the following: hinge region, CH1, CH2, and CH3 domains. Also included in the invention are antigen-binding fragments also comprising any combination of variable region(s) with a hinge region, CH1, CH2, and CH3 domains. The antibodies of the invention may be from any animal origin including birds and mammals. In one aspect, the antibodies are human, murine (e.g., mouse and rat), donkey, sheep, rabbit, goat, guinea pig, camel, horse, or chicken. Further, such antibodies may be humanized versions of animal antibodies (see, e.g., U.S. Pat. No. 6,949,245). The antibodies of the invention may be monospecific, bispecific, trispecific or of greater multi specificity.
The antibodies of the invention may be generated by any suitable method known in the art. Polyclonal antibodies to an antigen-of-interest can be produced by various procedures well known in the art. For example, a polypeptide of the invention can be administered to various host animals including, but not limited to, rabbits, mice, rats, etc. to induce the production of sera containing polyclonal antibodies specific for the antigen. Various adjuvants may be used to increase the immunological response, depending on the host species, and include but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Such adjuvants are also well known in the art. Further, antibodies and antibody-like binding proteins may be made by phage display (see, e.g., Smith and Petrenko, Chem Rev (1997) 97(2):391-410).
Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example; in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporated by reference in their entireties). The term “monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology. The term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.
In one embodiment, a method for isolating leukemia stem cells using such antibodies is provided, including obtaining a sample of cells from a subject, sorting cells that express 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 amino acid sequence as set forth in SEQ ID NO: 13. In a related aspect, the step of sorting includes sorting by fluorescent activated cell sorting and/or magnetic bead sorting.
In another related aspect, the marker is CD34, c-kit, Gr-1, Mac-1, MPO, and/or nonspecific esterase. In a further related aspect, wherein the leukemia stem cells are negative for B-cell (B220 and CD19), T-cell (CD4, CD8, CD3, and CD5), megakaryocytic (CD41), and erythroid (Ter119) markers.
In one embodiment, a kit for identifying a cell possessing pluripotent potential is disclosed including an agent for detecting one or more SALL family member protein markers, reagents and buffers to provide conditions sufficient for agent-cell interaction and labeling of the agent, instructions for labeling the detection reagent and for contacting the agent with the cell, and a container comprising the components.
One identifies stem cells according to the method of the disclosure by first sorting, from a population of cells, cells that are positive for expression a marker comprising SEQ ID NO: 13 from cells that are not. One then selects from the positive marker cells the stem cell of interest; this is performed by sorting cells by their expression of a known cell marker. Any marker that is known to be associated with the stem cells of interest may be used.
Any population of cells where stem cells are suspected of being found may be sorted according to the methods disclosed. In one aspect, cells are obtained from the bone marrow of a non-fetal animal, including, but not limited to, human cells. Fetal cells may also be used.
Cell sorting may be by any method known in the art to sort cells, including sorting by fluorescent activated cell sorting (FACS) (see, e.g., Baumgarth and Roederer, J Immunol Methods (2000) 243:77-97) and Magnetic bead cell sorting (MACS). The conventional MACS procedure is described by Miltenyi et al., “High Gradient Magnetic Cell Separation with MACS,” Cytometry 11:231-238 (1990). To sort cells by MACS, one labels cells with magnetic beads and passes the cells through a paramagnetic separation column. The separation column is placed in a strong permanent magnet, thereby creating a magnetic field within the column. Cells that are magnetically labeled are trapped in the column; cells that are not pass through. One then elutes the trapped cells from the column. In one embodiment, an antibody directed against SALL4 is used in cell sorting to isolate embryonic stem cells, adult stem cells and/or cancer stem cells. In another embodiment, an antibody directed against SALL4 is used in flow cytometry analysis to detect cells expressing SALL4, where such cells are associated with proliferative disease progression or neoplastic cell formation. In a related aspect, SALL4 is SALL4A or SALL4B.
In one embodiment, a method for detecting the presence or absence of the polynucleotide comprising a nucleic acid sequence encoding SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6 in a biological sample is disclosed including, but not limited to, contacting the biological sample under hybridizing conditions 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, or SEQ ID NO: 5, or a complement of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5, and detecting hybridization between the probe and the sample, where hybridization is indicative of the presence of the polynucleotide.
In another embodiment, a method for detecting a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6 present in a biological sample is disclosed including, but not limited to, providing an antibody that binds to the polypeptide, contacting the biological sample with the antibody, and determining the binding between the antibody to the biological sample, where binding is indicative of the presence of the polypeptide.
In one embodiment, a method of treating myelodysplastic syndrome (MDS) in a subject is described, including administering to the subject a polynucleotide having a nucleic acid sequence as 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, a complement of SEQ ID NO: 5, or 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. In a related aspect, the method includes administering a polynucleotide as set forth in SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5. In one aspect, the MDS is acute myeloid leukemia (AML).
In one embodiment, a method of identifying an agent which modulates the effect of a SALL family member protein on OCT4 expression is disclosed including co-transfecting a cell with 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 a vector comprising a nucleic acid encoding a SALL family member protein, contacting the cell with an agent, and determining the activity of the promoter-reporter construct in the presence and absence of the agent, where determining the activity of the promoter-reporter construct correlates with the effect of the agent on SALL family member protein/OCT4 interaction.
In a related aspect, the promoter region comprises nucleic acid sequence including but not limited to, SEQ ID NO:26, and the expression reporter protein is luciferase.
In another embodiment, a method of treating a neoplastic or proliferative disorder, where cells of a subject exhibit de-regulation of self-renewal, is disclosed including administering to the subject a pharmaceutical composition containing an agent which inhibits the expression of SALL4.
In another embodiment, a method of identifying a substance which binds to a polypeptide including an amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6 is provided, where the method comprises contacting the polypeptide with a candidate substance and detecting the binding of the substance to the polypeptide.
In one embodiment, a method of identifying a substance which modulates the function of a polypeptide including an amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6 is disclosed, where the method includes contacting the polypeptide with a candidate substance and determining the activity of the polypeptide, and where a change in the activity in the presence of the candidate substance is indicative of the substance modulating the function of the polypeptide.
In another embodiment, a method of diagnosing myelodysplastic syndrome (MDS) in a subject is described including, but not limited to, providing a biological sample from the subject, contacting the biological sample with a probe having a fragment of at least 15 consecutive nucleotides of a polynucleotide 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 detecting the hybridization between the probe and the biological sample, where detecting of hybridization correlates with MDS. In one aspect, the MDS is acute myeloid leukemia (AML).
In another embodiment, a method of diagnosing a myelodysplastic syndrome (MDS) in a subject is described, including, but not limited to, providing a biological sample from the subject, contacting the biological sample with an antibody which binds to a polypeptide comprising an amino acid as set forth in SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6, and detecting the binding of the antibody to the sample, where detecting binding correlates with MDS. In one aspect, the MDS is acute myeloid leukemia (AML).
In one embodiment, a method of diagnosing a neoplastic or proliferative disorder is disclosed including contacting a cell of a subject with an agent that detects the expression of a SALL family member protein and determining whether a SALL family member protein is expressed in the cell, where 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.
In one aspect, the agent is labeled and the determining step includes detection of the agent by exposing the subject to a device which images the location of the agent. In a related aspect, the images are generated by magnetic resonance, X-rays, or radionuclide emission.
In one embodiment, a method of modulating the cellular expression of a polynucleotide encoding a zinc finger transcriptional factor which is constitutively expressed in primary acute myeloid leukemia cells, including introducing a double stranded RNA (dsRNA) which hybridizes to the polynucleotide, or an antisense RNA which hybridizes to the polynucleotide, or a fragment thereof, into a cell. In a related aspect, the modulating is down-regulating.
In one embodiment, a transgenic animal is disclosed. In a general aspect, a transgenic animal is produced by the introduction of a foreign gene in a manner that permits the expression of the transgene. Methods for producing transgenic animals are generally described by Wagner and Hoppe (U.S. Pat. No. 4,873,191; which is incorporated herein by reference), Brinster et al. (1985); which is incorporated herein by reference in its entirety) and in “Manipulating the Mouse Embryo; A Laboratory Manual” 2nd edition (eds., Hogan, Beddington, Costantimi and Long, Cold Spring Harbor Laboratory Press (1994); which is incorporated herein by reference in its entirety).
Typically, a gene is transferred by microinjection into a fertilized egg. The microinjected eggs are implanted into a host female, and the progeny are screened for the expression of the transgene. Transgenic animals may be produced from the fertilized eggs from a number of animals including, but not limited to reptiles, amphibians, birds, mammals, and fish.
DNA clones for microinjection can be prepared by any means known in the art. For example, DNA clones for microinjection can be cleaved with enzymes appropriate for removing the bacterial plasmid sequences, and the DNA fragments electrophoresed on 1% agarose gels in TBE buffer, using standard techniques. The DNA bands are visualized by staining with ethidium bromide, and the band containing the expression sequences is excised. The excised band is then placed in dialysis bags containing 0.3 M sodium acetate, pH 7.0. DNA is electroeluted into the dialysis bags, extracted with a 1:1 phenol:chloroform solution and precipitated by two volumes of ethanol. The DNA is redissolved in 1 ml of low salt buffer (0.2 M NaCl, 20 mM Tris, pH 7.4, and 1 mM EDTA) and purified on an Elutip-DTM column. The column is first primed with 3 ml of high salt buffer (1 M NaCl, 20 mM Tris, pH 7.4, and 1 mM EDTA) followed by washing with 5 ml of low salt buffer. The DNA solutions are passed through the column three times to bind DNA to the column matrix. After one wash with 3 ml of low salt buffer, the DNA is eluted with 0.4 ml high salt buffer and precipitated by two volumes of ethanol. DNA concentrations are measured by absorption at 260 nm in a UV spectrophotometer.
The present invention also provides pharmaceutical compositions comprising at least one compound capable of treating a disorder in an amount effective therefor, and a pharmaceutically acceptable vehicle or diluent. The compositions of the present invention may contain other therapeutic agents as described, and may be formulated, for example, by employing conventional solid or liquid vehicles or diluents, as well as pharmaceutical additives of a type appropriate to the mode of desired administration (for example, excipients, binders, preservatives, stabilizers, flavors, etc.) according to techniques such as those well known in the art of pharmaceutical formulation.
Pharmaceutical compositions employed as a component of invention articles of manufacture can be used in the form of a solid, a solution, an emulsion, a dispersion, a micelle, a liposome, and the like, where the resulting composition contains one or more of the compounds described above as an active ingredient, in admixture with an organic or inorganic carrier or excipient suitable for enteral or parenteral applications. Compounds employed for use as a component of invention articles of manufacture may be combined, for example, with the usual non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions, and any other form suitable for use. The carriers which can be used include glucose, lactose, gum acacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn starch, keratin, colloidal silica, potato starch, urea, medium chain length triglycerides, dextrans, and other carriers suitable for use in manufacturing preparations, in solid, semisolid, or liquid form. In addition auxiliary, stabilizing, thickening and coloring agents and perfumes may be used.
Invention pharmaceutical compositions may be administered by any suitable means, for example, orally, such as in the form of tablets, capsules, granules or powders; sublingually; buccally; parenterally, such as by subcutaneous, intravenous, intramuscular, or intracisternal injection or infusion techniques (e.g., as sterile injectable aqueous or non-aqueous solutions or suspensions); nasally such as by inhalation spray; topically, such as in the form of a cream or ointment; or rectally such as in the form of suppositories; in dosage unit formulations containing non-toxic, pharmaceutically acceptable vehicles or diluents. The present compounds may, for example, be administered in a form suitable for immediate release or extended release. Immediate release or extended release may be achieved by the use of suitable pharmaceutical compositions comprising the present compounds, or, particularly in the case of extended release, by the use of devices such as subcutaneous implants or osmotic pumps. The present compounds may also be administered liposomally.
In addition to primates, such as humans, a variety of other mammals can be treated according to the method of the present invention. For instance, mammals including, but not limited to, cows, sheep, goats, horses, dogs, cats, guinea pigs, rats or other bovine, ovine, equine, canine, feline, rodent or murine species can be treated. However, the method can also be practiced in other species, such as avian species (e.g., chickens).
The subjects treated in the above methods, in which cells targeted for modulation is desired, are mammals, including, but not limited to, cows, sheep, goats, horses, dogs, cats, guinea pigs, rats or other bovine, ovine, equine, canine, feline, rodent or murine species, and preferably a human being, male or female.
The term “therapeutically effective amount” means the amount of the subject compound that will elicit the biological or medical response of a tissue, system, animal or human that is being sought by the researcher, veterinarian, medical doctor or other clinician.
The term “composition,” as used herein, is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts. By “pharmaceutically acceptable” it is meant the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
The terms “administration of” and or “administering a” compound should be understood to mean providing a compound of the invention to the individual in need of treatment.
The pharmaceutical compositions for the administration of the compounds of this invention may conveniently be presented in dosage unit form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active ingredient into association with the carrier which constitutes one or more accessory ingredients. In general, the pharmaceutical compositions are prepared by uniformly and intimately bringing the active ingredient into association with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation. In the pharmaceutical composition the active object compound is included in an amount sufficient to produce the desired effect upon the process or condition of diseases.
The pharmaceutical compositions containing the active ingredient may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs.
Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. They may also be coated to form osmotic therapeutic tablets for control release.
Formulations for oral use may also be presented as hard gelatin capsules where the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules where the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin, or olive oil.
Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxy-propylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl, p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.
Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.
Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, may also be present.
Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative and flavoring and coloring agents.
The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleagenous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butane diol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.
The compounds of the present invention may also be administered in the form of suppositories for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials are cocoa butter and polyethylene glycols.
For topical use, creams, ointments, jellies, solutions or suspensions, etc., containing the compounds of the present invention are employed. (For purposes of this application, topical application shall include mouthwashes and gargles).
Nucleic acid according to the present disclosure, encoding a polypeptide or peptide able to interfere with SALL4 may be used in methods of gene therapy, for instance in treatment of individuals with the aim of preventing or curing (wholly or partially) a tumor e.g., in cancer, or other disorder involving loss of proper regulation of the cell-cycle and/or cell growth, or other disorder in which specific cell death is desirable.
Vectors such as viral vectors have been used in the art to introduce nucleic acid into a wide variety of different target cells. Typically the vectors are exposed to the target cells so that transfection can take place in a sufficient proportion of the cells to provide a useful therapeutic or prophylactic effect from the expression of the desired polypeptide. The transfected nucleic acid may be permanently incorporated into the genome of each of the targeted tumour cells, providing long lasting effect, or alternatively the treatment may have to be repeated periodically.
A variety of vectors, both viral vectors and plasmid vectors, are known in the art, see U.S. Pat. No. 5,252,479 and WO 93/07282. In particular, a number of viruses have been used as gene transfer vectors, including papovaviruses, such as SV40, vaccinia virus, herpesviruses, including HSV and EBV, and retroviruses. Many gene therapy protocols in the art have used disabled murine retroviruses.
As an alternative to the use of viral vectors other known methods of introducing nucleic acid into cells includes electroporation, calcium phosphate co-precipitation, mechanical techniques such as microinjection, ballistic methods, transfer mediated by liposomes, and direct DNA uptake and receptor-mediated DNA transfer.
Receptor-mediated gene transfer, in which the nucleic acid is linked to a protein ligand via polylysine, with the ligand being specific for a receptor present on the surface of the target cells, is an example of a technique for specifically targeting nucleic acid to particular cells.
In the treatment of a subject where cells are targeted for modulation, an appropriate dosage level will generally be about 0.01 to 500 mg per kg patient body weight per day which can be administered in single or multiple doses. Preferably, the dosage level will be about 0.1 to about 250 mg/kg per day; more preferably about 0.5 to about 100 mg/kg per day. A suitable dosage level may be about 0.01 to 250 mg/kg per day, about 0.05 to 100 mg/kg per day, or about 0.1 to 50 mg/kg per day. Within this range the dosage may be 0.05 to 0.5, 0.5 to 5 or 5 to 50 mg/kg per day. For oral administration, the compositions are preferably provided in the form of tablets containing 1.0 to 1000 milligrams of the active ingredient, particularly 1.0, 5.0, 10.0, 15.0, 20.0, 25.0, 50.0, 75.0, 100.0, 150.0, 200.0, 250.0, 300.0, 400.0, 500.0, 600.0, 750.0, 800.0, 900.0, and 1000.0 milligrams of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated. The compounds may be administered on a regimen of 1 to 4 times per day, preferably once or twice per day.
It will be understood, however, that the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing therapy.
The following examples are intended to illustrate but not limit the invention.
EXAMPLE 1
Methods
Molecular Cloning.
Plasmid construction and DNA sequencing were performed in accordance with standard procedures. For cloning of SALL4 isoforms, PCR primers were designed, based on the genomic clone RP5-1112F19 (SEQ ID NO: 25) (GenBank accession no. AL034420). SALL4 isoforms were cloned with the use of the Marathon-Ready cDNA library derived from human fetal kidney (BD Biosciences Clontech, Palo Alto, Calif.), according to the supplier's protocol. The amplified PCR products were cloned into a TA Cloning vector (Invitrogen Corp., Carlsbad, Calif.), and the nucleotide sequences were determined by DNA sequencing. The GAL4-SALL4B construct was generated by PCR with the use of a 5′ primer and a 3′ primer with a restriction enzyme site, BamHI, at each end:








5′ primer:









(SEQ ID NO: 7)









5′-TTATCAGGATCCTGGTCGAGGCGCAAGCAGGCGAAACCC-3′;



and


 


3′ primer:








(SEQ ID NO: 8)









5′-CCAGGATCCTTAGCTGACCGCCAATCTTGTTTC-3′.







The GAL4-SALL4B construct was expected to encode 93 amino acids of minimal GAL4 DNA-binding domain and the full length of SALL4B, except for the first amino acid, methionine.
Determination of Alternative Splicing Patterns in Different Tissues.
Reverse transcription (RT)-PCR was used to evaluate mRNA expression patterns of SALL4 in adult tissues. A panel of eight normalized first-strand cDNA preparations, derived from different adult tissues, was purchased from BD Biosciences Clontech. PCR amplification was performed in a 50-μl reaction volume containing 5 μl of cDNA, 10 mM Tris HCl (pH 8.3), 50 mM KCl, 2 mM MgCl2, 9.2 mM dNTPs, and 1.25 U of Taq DNA polymerase (PerkinElmer Life Sciences, Boston, Mass.). After an initial denaturation at 94° C. for 10 min, amplification was performed for 30 cycles under the following conditions: 30-sec denaturation at 94° C., 30-sec annealing at 55° C., and 30-sec extension at 72° C. The last cycle was followed by a final 7-min extension at 72° C.
Amplification of glyceraldehyde phosphate dehydrogenase (GAPDH) mRNA was used to control for template concentration loading. The primer pairs selected specifically for SALL4 isoforms were the following:








1) SALL4A primers (sense primer:



5′-ATTGGCACCGGCAGTTACCACC (SEQ ID NO: 9);


antisense primer: 5′-AGTACTCGTGGGCATATTGTC-3′


(SEQ ID NO: 10))


and


 


2) SALL4B primers (sense primer:


5′-ATGTCGAGGCGCAAGCAGGCGAAAC-3′ (SEQ ID NO: 11);


antisense primer: 5′-TTAGCTGACCGCAATCTTGTTTTCT-3′


(SEQ ID NO: 12)).






PCR products were electrophoretically separated on 1% agarose gel. DNA sequencing was also used to confirm amplification products.
Antibody Generation.
The peptide MSRRKQAKPQHIN (SEQ ID NO: 13) of human SALL4 was chosen for its potential antigenicity (amino acids 1-13) and used to prepare an antipeptide antibody. This region is also identical to that of mouse SALL4 so that the generated antibody could be expected to cross-react with mouse SALL4. SALL4 antipeptide antibody was produced in rabbits in collaboration with Lampire Biological Laboratories Inc. (Pipersville, Pa.).
Gel Electrophoresis and Western Blot Analysis.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out in SDS 10% w/v polyacrylamide slab gels according to Laemmli, and the proteins were then transferred to nitrocellulose membranes. Immunoblotting of rabbit immune serum with the SALL4 antipeptide antibody (1:100) was performed with an electrochemiluminescence detection system as described by the manufacturer (Amersham Biosciences, Piscataway, N.J.).
Leukemia and Normal Tissues.
Leukemia and normal samples, either in paraffin blocks or frozen in dimethylsulfoxide (DMSO), were collected from the files of The University of Texas M.D. Anderson Cancer Center, Houston, Tex., and the Dana-Farber Cancer Institute, Boston, Mass., between 1998 and 2004 under approved Institutional Review Board protocols. The diagnosis of all tumors was based on morphologic and immunophenotypic criteria according to the FAB Classification for Hematopoietic Neoplasms. CD34+ fresh cells were purchased from Cambrex.
Real-Time Quantitative RT-PCR.
TaqMan 5′ nuclease assay was used (Applied Biosystems, Foster City, Calif.) in these studies. Total RNA from purified CD34+ HSCs/HPCs from normal bone marrow and peripheral blood, 15 AML samples, and three leukemia cell lines was isolated with the RNeasy Mini Kit and digested with DNase I (Qiagen). RNA (1 μg) was reverse-transcribed in 20 μL with the use of Superscript II reverse transcriptase and a poly(dT) 12-18 primer (Invitrogen). After the addition of 80 μL of water and mixing, 5-μL aliquots were used for each TaqMan reaction. TaqMan primers and probes were designed with the use of Primer Express software version 1.5 (Applied Biosystems). Real-time PCR for SALL4 and GAPDH was performed with the TaqMan PCR core reagent kit (Applied Biosystems) and an ABI Prism 7700 Sequence Detection System (PE Applied Biosystems). The PCR reaction mixture contained 3.5 mM MgCl2; 0.2 mM each of deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), and deoxyguanosine triphosphate (dGTP); 0.4 mM deoxyuridine triphosphate (dUTP); 0.5 μM forward primer; 0.5 μM reverse primer; 0.1 μM TaqMan probe; 0.25 U uracil DNA glycosylase; and 0.625 U AmpliTaq Gold polymerase in 1× TaqMan PCR buffer. cDNA (5 μL) was added to the PCR mix, and the final volume of the PCR reaction was 25 μL. All samples were run in duplicate. GAPDH was used as an endogenous control. Thermal cycler conditions were 50° C. for 2 min, 95° C. for 10 min, and 45 cycles of 95° C. for 0.30 min and 60° C. for 1 min. Data were analyzed with the use of Sequence Detection System software version 1.6.3 (Applied Biosystems). Results were obtained as threshold cycle (Ct) values. The software determines a threshold line on the basis of the baseline fluorescent signal, and the data point that meets the threshold is given as the Ct value. The Ct value is inversely proportional to the starting number of template copies. All measurements were performed in duplicate. TaqMan sequences include the following:








GAPDH forward primer:



(5′-GAAGGTGAAGGTCGGAGTC-3′ (SEQ ID NO: 14))


and


 


reverse primer:


(5′-GAAGATGGTGATGGGATTTC-3′ (SEQ ID NO: 15)),


 


TaqMan probe:


(5′-CAAGCTTCCCGTTCTCAGCC-3′ (SEQ ID NO: 16)),


and


 


SALL4 forward primer:


(5′-CCTCCTAATGAGAGTATCTGGGTGAT-3′ (SEQ ID NO: 17))


and


 


reverse primer:


(5′-TTAAAACATACAGCGCATGATTGG-3′ (SEQ ID NO: 18)).






Design and Construction of Tissue Arrays.
Tissue arrays that included triplicate tumor cores from leukemia specimens were sectioned (5 μm thick). A manual tissue arrayer (Beecher Instruments, Silver Spring, Md.) was used to construct the tissue arrays.
Immunohistochemistry.
Immunohistochemical staining was performed according to standard techniques. Briefly, formalin-fixed, paraffin-embedded, 4-μm-thick tissue sections were deparafinized and hydrated. Heat-induced epitopes were retrieved with a Tris buffer (pH 9.9; Dako Corp., Carpinteria, Calif.) and a rapid microwave histoprocessor. After incubation at 100° C. for 10 min, slides were washed in running tap water for 5 min and then with phosphate buffered saline (PBS; pH 7.2) for 5 min. Tissue sections were then incubated with anti-SALL4 antibody (1:200) for 5 h in a humidified chamber at room temperature. After three washes with PBS, tissue sections were incubated with antimouse immunoglobulin G and peroxidase for 30 min at room temperature.
After three washes with PBS, tissue sections were incubated with 3,3′-diaminobenzidine/H2O2 (Dako) for color development; hematoxylin was used to counterstain the sections. Neoplastic cells were considered to be positive for SALL4 when they showed definitive nuclear staining.
Generation of Transgenic Mice.
SALL4B cDNA, corresponding to the entire coding region, was subcloned into a pCEP4 vector (IntroGene; now Crucell, Leiden, The Netherlands) to create the CMV/SALL4B construct for the transgenic experiments. Subsequent digestion with SalI, which does not cut within the SALL4B cDNA, released a linear fragment containing only the CMV promoter, the SALL4 cDNA coding region, the SV40 intron, and polyadenylation signal without additional vector sequences.
Transgenic mice were generated via pronuclear injection performed in the transgenic mouse facility at Yale University. Identification of SALL4B founder mice and transmission of the transgene was determined by PCR analyses. The PCR primers used for the genotyping span the junction of the 5′ SALL4B cDNA to the CMV promoter (sense primer: 5′-CAGAGATGC TGAAGAACTCCGCAC-3′ (SEQ ID NO: 19); antisense primer: 5′-AGCAGAGCTCGTTTAGTGAACCG-3′ (SEQ ID NO: 20)).
Hematologic Analysis.
Complete blood cell counts with automated differentials were determined with a Mascot Hemavet cell counter (CDC Technologies, Oxford, Conn.). For progenitor assays, 1.5×104 bone marrow cells were plated in duplicate 1.25-ml methylcellulose cultures supplemented with recombinant mouse interleukin-3 (IL-3) (10 ng/ml), IL-6 (10 ng/ml), stem cell factor (SCF) (50 ng/ml), and erythropoietin (3 U/ml) (M3434, StemCell Technologies, Vancouver, British Columbia, Canada). Colonies were recorded between days 7 and 14 (CFU-G, CFU-GM, CFU-M, CFU-GEMM, and BFU-E). Peripheral blood, bone marrow smears, and cytospin from pooled CFU cells were stained with Wright-Giemsa stain.
Flow Cytometric Analysis.
Cells were stained with directly conjugated antibodies to Gr-1, Mac-1, B220, Ter119, c-kit, CD34, CD45, CD41, CD19, CD5, CD3, CD4, CD8, propidium iodide (PI) or Annexin V (BD Biosciences Pharmingen, San Diego, Calif.). Ten thousand scatter-gated red cells were acquired on a FACScan and analyzed with CellQuest software (BD Biosciences Clontech).
Statistical Analysis.
Student's t-Test was used for all the statistical analysis, assuming normal two-tailed distribution and unequal variance.
Cell Culture.
HEK-293 cells (derived from human embryonic kidney) and cell lines KG.1, Kasumi-1, and THP-1 were purchased from the American Type Culture Collection (Manassas, Va.). Cells were maintained at 37° C. in a humidified environment with 5% carbon dioxide and 10% fetal serum.
Transfection.
Transfection was performed with FuGENE 6 transfection reagent (Roche Applied Science, Indianapolis, Ind.) according to the manufacturer's instructions. Cells were plated in 24-well plates at a density of  ̃1×105 cells/well. Cells were harvested 24 h after transfection. Plasmid DNA for transient transfection was prepared with the Qiagen Plasmid Midi Kit (Valencia, Calif.).
β-Galactosidase and Luciferase Assays.
The cells were extracted with 100 μl of luciferase cell culture lysis reagent (Promega Corp., Madison Wis.) 24 h after transfection. The β-galactosidase assay, performed with 10 μl of cell extract, used the β-Galactosidase Enzyme Assay System (Promega) and the standard assay protocol provided by the manufacturer (except that 1 M Tris base was used as stopping buffer, instead of sodium carbonate). For the luciferase assay (Promega), 5 μl of extract were used in accordance with the manufacturer's instructions. After subtraction of the background, luciferase activity (arbitrary units) was normalized to β-galactosidase activity (arbitrary units) for each sample.
Promoter Reporter Assays
In general, 0.25-0.3 μg of an OCT4-Luc construct (PMOct4) comprising an OCT4 promoter (SEQ ID NO:26) or SALL-Luc construct containing a SALL family protein (i.e., SALL1, SALL3, SALL4A, or SALL4B) promoter (i.e., SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29, respectively, where SALL4A and SALL4B share the same promoter) was cotransfected with between 0.1 μg and 0.12 μg of renilla plasmid and/or various amounts (0-1.0 μg) of plasmid expressing SALL family proteins or OCT4 protein in HEK-293 or COS-7 cells. Typically, pcDNA3 vector was used as the control. Transfected cells were then monitored for luciferase activity 24 hour s post-transfection.
Results
Molecular Cloning of Two Alternatively Splicing Isoforms of Human SALL4.
Two full-length transcripts of SALL4 were isolated by 5′ and 3′ RACE-PCR (rapid amplification of the 5′ and 3′ cDNA ends-polymerase chain reaction) with the use of fetal human kidney Marathon-Ready cDNAs (BD Biosciences Clontech) as templates.
Sequence analysis of the larger cDNA fragment isolated revealed a single, large open reading frame, designated as SALL4A, that started from a strong consensus initiation sequence and was expected to encode 1,053 amino acids. The other splicing variant of SALL4, designated SALL4B, lacked the region corresponding to amino acids 385-820 of the full-length SALL4A (FIG. 1a). The putative protein encoded by SALL4B cDNA was expected to consist of 617 amino acids.
To rule out the possibility that these two apparent splicing variants might result from artifacts, both variant mRNA sequences with corresponding sequences of the human genome were compared. SALL4A contained all exons (1-4) (FIG. 1a), whereas SALL4B lacked the 3′ large portion of exon 2. Both exon-intron splice sites satisfied the G-T-A-G rule. Both splicing variants had the same translational reading frame, but SALL4B mRNA encoded a protein with internal deletions. SALL4A contained eight zinc finger domains, while SALL4B had three zinc finger domains.
Expression Pattern of the SALL4 Isoforms in Human Tissues.
The alternative splicing patterns of SALL4 were delineated by reverse transcription (RT)-PCR in a variety of human tissues. A fragment of the ubiquitous GAPDH gene cDNA was amplified as a control (FIG. 1b). A 315-bp fragment representing the longer splice variant, SALL4A, was amplified in some tissues, achieving various expression levels. The SALL4B variant was present in every tissue at varying levels of expression. Detailed studies on SALL4 expression in hematopoietic tissues are described in the following results.
Generation of SALL4 Antibody and Identification of SALL4 Protein Products.
To identify SALL4 gene products and confirm the presence of SALL4 variants, a polyclonal antibody against a synthetic peptide (amino acids 1-13) of SALL4 was developed. This region was chosen because it is common to both SALL4 variants. The affinity-purified SALL4 peptide antibody recognized specifically two endogenous proteins in a human kidney total lysate. The two proteins were approximately 165 kDa and 95 kDa, which were identical to the molecular weights of overexpressed SALL4A and SALL4B in Cos-7 cells, respectively (FIG. 1c). Western blotting with this antibody confirmed that the SALL4 isoforms had different tissue distributions that were similar to those observed at the mRNA level (FIG. 1b-B).
Failure of SALL4 to Turn Off in Human Primary AML and Myeloid Leukemia Cell Lines.
Because the chromosome region 20q13, where SALL4 is located, is frequently involved in tumors, SALL4 mRNA expression in AML was examined. Expression of SALL4 was quantitatively investigated by real-time RT-PCR in bone marrow cells derived from AML samples (N=15), myeloid leukemia cell lines (N=3) and compared with that of non-neoplastic hematopoietic cells from a purified CD34+ stem/progenitor pool (HSCs/HPCs purchased from Cambrex), normal bone marrow (N=3), and normal peripheral blood (N=3). With the use of isoform-specific primers (see FIG. 2a), either or both SALL4B and/or SALL4A, failed to be turned off (SALL4B) or down-regulated (SALL4A) in all AML samples and myeloid leukemia cell lines. The data were normalized to the endogenous expression of GAPDH and calibrated against the level of SALL4A or SALL4B expression in purified CD34+ cells. In contrast to the total absence of SALL4B in normal bone marrow, its expression in primary AML failed to be turned off in 13 of 15 AML samples and in all three myeloid leukemia cell lines. The median normalized level of SALL4A in primary AML samples was 40-fold higher than that in normal bone marrow. SALL4A expression levels in the myeloid leukemia cell lines KG.1, Kasumi-1, and THP-1 were, respectively, 8-, 25-, and 240-fold higher than those in normal bone marrow. Interestingly, both SALL4A and SALL4B expression levels were increased in 60% of AML samples and in all three cell lines, compared with those in normal bone marrow. In the remaining 40% of AML samples, either SALL4A or SALL4B failed to be down-regulated.
Constitutive Expression of SALL4 Protein in Human Primary AML.
To investigate whether the observed aberrant SALL4 expression was also present at the protein level, 81 AML samples were examined, ranging from AML classes M1 to M5 (FAB classification): M1 (N=20), M2 (N=27), M3 (N=8), M4 (N=16), M5 (N=3), and AML nonspecified (N=7); several samples of normal bone marrow, thymus and spleen, as well as normal CD34+ HSCs/HPCs.
Normal bone marrow, spleen and thymus showed no detectable SALL4 protein expression, and normal CD34+ HSCs/HPCs exhibited positive but weaker SALL4 protein staining; however, much stronger SALL4 expression was detected in the nuclei of leukemic cells (FIG. 2b-F). All 81 AML samples showed aberrant SALL4 expression, with the strongest staining seen in AML-M1 and -M2. These findings were consistent with SALL4 mRNA expression levels demonstrated by real-time RT-PCR (FIG. 2a). The data suggested that SALL4 was present in CD34+ HSCs/HPCs and down-regulated in mature granulocytes and lymphocytes. As a result, the constitutive expression of SALL4 in leukemia may have prevented the leukemic blasts from differentiating and/or gaining properties that were normally seen in HSCs.
Generation of Transgenic Mice Constitutively Expressing Full-Length Human SALL4B.
To directly test whether constitutive expression of SALL4 is sufficient to induce AML, a SALL4 transgenic mouse model was generated. The CMV promoter was fused to cDNA that encoded the 617 amino acids of human SALL4B (FIG. 3a-A), which was chosen because it was expressed in every tissue previously examined (FIG. 1b-B). The CMV promoter was previously used to ectopically express human genes in most murine organs. RT-PCR amplification was performed to examine the overexpression of wildtype (WT), full-length SALL4B in the transgenic mice.
A SALL4B transcript was detected in a variety of tissues from the transgenic mice, including brain, kidney, liver, spleen, peripheral blood, lymph nodes, and bone marrow (FIG. 3a-B). Abnormal gaits and associated hydrocephalus 3 weeks after birth were observed in 20% of the transgenic mice from multiple lines; 60% had polycystic kidneys. These findings suggest that SALL4B plays an important role in neural and renal development.
MDS-Like Symptoms and AML in SALL4B Transgenic Mice.
Monitoring of hematological abnormalities in a cohort of 14 transgenic mice from all six lines revealed that all mice had apparent MDS-like features at ages 6-8 months. Increased number of immature blasts and many atypical and dysplastic white cells, including hypersegmented neutrophils and pseudo-Pelger-Huet-like cells, were seen on peripheral blood smears (FIG. 3b). Nucleate red blood cells and giant platelets were also present, as well as erythroid and megakaryocyte dysplastic features, such as binucleate erythroid precursors and hypolobulated megakaryocytes.
Six (43%) of these 14 mice eventually progressed to acute leukemia (Table 1).







TABLE 1







Summary of MDS-Like/AML in SALL4B Transgenic Mice

















Outcome and Organs


Mouse ID
Sex
Founder
Age
Phenotype
Involved by AML















25
M
507
 8 M
AML
Sacrificed, AML in BM,







PB, Liver, Spleen, LNs


509
F
509
18 M
AML
Sacrificed, AML in BM,







PB, Liver, Spleen, LNs,







Lungs


87
F
504
 8 M
AML
Sacrificed, AML in BM,







PB, Liver, Spleen, LNs


504
M
504
19 M
MDS-like
Sacrificed due to MDS


506
M
506
19 M
MDS-like
Sacrificed due to MDS


507
F
507
24 M
AML
Died, AML in BM, PB,







Liver, Spleen, LNs


510
F
510
24 M
MDS-like
Sacrificed due to MDS


464
M
464
19 M
MDS-like
Died of MDS


23
M
507
22 M
MDS-like
Sacrificed due to MDS


27
M
507
22 M
MDS-like
Alive


86
F
504
18 M
AML
Sacrificed, AML in BM,







PB, Liver, Spleen, LNs


4
M
464
15 M
MDS-like
Alive


3058
F
25
12 M
AML
Died, AML in BM, PB,







Liver, Spleen, LNs


26
M
507
14 M
MDS
Sacrificed due to MDS









Leukemic infiltration of many organs, including lung, kidney, liver, spleen, and lymph nodes, emphasized the aggressiveness of the disease (FIG. 3c). Leukemia blast cells were considered to be myeloid in origin because they were positive for CD34, c-kit, Gr-1, Mac-1, MPO, and nonspecific esterase; they were negative for B-cell (B220 and CD19), T-cell (CD4, CD8, CD3, and CD5), megakaryocytic (CD41), and erythroid (Ter119) markers (FIG. 3d).
SALL4B-Induced AML was Transplantable.
Aggressive fatal AML with onset at approximately 6 weeks developed in immunodeficient NOD/SCID mice after serial transplantation of SALL4B-induced AML cells by subcutaneous injection. The transplanted disease was characterized by dissemination to multiple organs, with marked splenomegaly and hepatomegaly (FIG. 3e).
Ineffective Hematopoiesis and Excessive Apoptosis in SALL4B Transgenic Mice.
Investigation of hematological abnormalities in younger SALL4B transgenic mice (2-6 months old) revealed that their peripheral blood showed minimal myelodysplastic features but statistically significant leukopenia and neutropenia, as well as mild anemia (Table 2).







TABLE 2







CBC from SALL4B Transgenic Mice and Wild Type Control
















WBC
Neutrophil
Lymphocyte
RBC
Hb
HCT
MCV
PLT



(×103/μL)
(×103/μL)
(×103/μL)
(×106/μL)
(g/dL)
(%)
(fL)
(×103/μL)



















Transgenic
 8.38 ± 3.52
0.93 ± 1.06
6.34 ± 4.62
 8.85 ± 2.08
14.26 ± 3.04
50.52 ± 11.82
57.15 ± 6.42
1616 ± 662


(n = 20)


Control
11.59 ± 5.14
1.51 ± 0.86
9.04 ± 4.06
10.02 ± 1.84
15.66 ± 2.44
55.75 ± 9.62 
55.78 ± 7.54
1384 ± 806


(n = 18)


P value
0.27
0.048
0.029
0.015
0.030
0.038
0.398
0.196









To determine whether the cause of cytopenia in these transgenic mice was related to production problems, their bone marrow was studied. Bone marrow samples showed increased cellularity and an increased myeloid population (FIG. 3f), compared with those of WT controls (Gr-1/Mac-1 double-positive population in SALL4B transgenic mice: 67±16%, N=10 vs. WT: 55.3±4%, N=11; P=0.048).
As excessive apoptosis plays a central role in ineffective hematopoiesis in human MDS, apoptosis in SALL4 transgenic mice in vivo and in vitro was examined next. Increased apoptosis was observed in SALL4B transgenic mice on both primary bone marrow (Annexin V-positive, PI-negative population in transgenic mice: 4.4±2.4%, N=10 vs. WT: 1.86±1.55%, N=7; P=0.03) and day-7 CFUs (Annexin V-positive, PI-negative population in transgenic mice: 20.1±6%, N=10 vs. WT: 10.9±4%, N=7; P=0.002) (FIGS. 3f and g). These findings may account for the fact that despite an increased myeloid population in bone marrow, these transgenic mice had statistically significant low neutrophil counts in the peripheral blood, secondary to an ongoing ineffective myelopoiesis in their bone marrow. An increased population of immature cells was also noted in SALL4B transgenic mice on both primary bone marrow (c-kit-positive population in SALL4B transgenic mice: 10.2±1.3%, N=14 vs. WT: 6.5±2.5%, N=10; P=0.008) (FIG. 3f) and day-7 CFUs (CD34-positive population in SALL4B transgenic mice: 11±2.2%, N=8 vs. WT: 6.3±2.4%, N=7; P=0.002) (FIG. 3g). Similar numbers of total colonies were observed in SALL4B transgenic mice (mean=51, N=10) and WT controls (mean=40, N=6). Increased myeloid and decreased erythroid colony populations (FIG. 3h), however, were found in SALL4B transgenic mouse CFUs compared with those of WT controls, as has been reported in human MDS patients and other MDS mouse models. These observations suggest that the defect in SALL4B transgenic mice lies at the stem cell/progenitor level affecting hematopoietic differentiation.
Binding of SALL4A and SALL4B to β-Catenin in Vitro.
The potential signaling pathway that SALL4 may affect in leukemogenesis was explored next. In Drosophila, spalt (sal) is a downstream target of Wnt signaling. ALL1, another member of the SALL gene family, can interact with β-catenin. The high affinity site for this interaction is located at the C-terminal double zinc finger domain. This region of SALL1 was found to be almost exactly identical to that of SALL4. This finding prompted the investigation of whether SALL4 was also able to bind β-12 catenin. Expression constructs of SALL4A and SALL4B tagged with hemagglutinin (HA) were generated. As shown in FIG. 4a, endogenous β-catenin was pulled down by HA-SALL4A and HA-SALL4B, but not by HA alone.
Activation of the Wnt/β-Catenin Signaling Pathway by Both SALL4A and SALL4B.
To investigate the functional effect of the interaction of the SALL4 isoforms with β-catenin, a luciferase reporter (TOPflash; Upstate USA) containing multiple copies of Wnt-responsive elements to determine the potential of SALL4A and SALL4B to activate the canonical Wnt signaling pathway was used. This reporter construct has been shown to be efficiently stimulated by Wnt1 in a variety of cell lines. TOPflash reporter plasmid was transiently transfected in the HEK-293 cell line, in which both Wnt and its Wnt/β-catenin signal pathways were present. TOPflash reporter plasmid was also cotransfected with SALL4A or SALL4B. Significant activation of the Wnt/β-catenin signaling pathway by both SALL4A and SALL4B was indicated by increased luciferase activity (FIG. 4b).
Similar Expression Patterns of β-Catenin and SALL4 at Different Phases of CML.
Dysregulated Wnt/β-catenin signaling is known to be involved in the development of LSCs. The best evidence for β-catenin's involvement in LSC self-renewal comes from the study of CML blast transformation. It has been demonstrated that Wnt signaling was activated in the blast phase of CML but not the chronic phase, where it was concluded that dysregulated Wnt signaling, such as activation of β-catenin, could confer the property of self-renewal on the GMPs of CML and lead to their blastic transformation.
Given the potential interaction between SALL4 and β-catenin and spalt's position as a downstream target of Wnt signaling in Drosophila, SALL4 protein expression in CMLs in different phases was examined. SALL4 expression was present in blast-phase CML (N=12, 75%) but not the chronic phase (N=11, 100%) (FIG. 4c). In the accelerated phase (N=6, 10%), in which blast counts are increased, immature blasts expressing SALL4 were observed upon a background of nonstaining mature myeloid cells, such as neutrophils.
Effect of SALL4 on OCT4 Promoter.
To identify the effect of SALL4 on OCT 4, cells, OCT4-Luc constructs were co-transfected with renilla plasmids and increasing concentrations of SALL4B (FIG. 5). As the figure shows increasing SALL4B increased OCT4 promoter activity by more than 8 fold.
To determine if OCT4 stimulates the activity of SALL gene member promoters, promoter constructs (pSALL1, pSALL3, and pSALL4) were co-transfected with OCT4 in HEK-293 cells. As can be seen from the data (FIG. 6), after 24 hr post-transfection, the overexpression of OCT4 strikingly stimulated the promoter activities of SALL gene members SALL1, SALL3, and SALL4 when compared with that of the pcDNA3 vector control. Also, this activation was totally blocked by the presence of a small amount of excess SALL4 (FIG. 10).
To determine whether there was any self regulation of SALL promoters by SALL family member proteins, SALL4-Luc was co-transfected with renilla reporter and either SALL4A or SALL4B expression plasmids is HEK-293 and COS-7 cells (FIG. 7). As shown in the figure, SALL4 (both A and β isoforms) suppresses its own promoter activity in different cell lines. Further, this self-suppression is dose dependent (see, FIG. 8). When the ratio of SALL4A with SALL4 promoter reached 6:1, the promoter activity dropped approximately 3.5 fold compared with the basal level. This data indicates that SALL4 bears a self-suppression function. This is not true for all SALL members, for example, SALL1 fails to demonstrate self-suppression of its promoter (FIG. 12).
Data also indicates that SALL1 and SALL3 promoters were strikingly activated by exogenously added SALL4 (See, FIG. 9), indicating that SALL4 is able to regulate other members of the SALL gene family involving embryonic stem cell function.
Since the stimulation of OCT4 on SALL4 promoter can be totally blocked by SALL4 (FIG. 10), SALL4 was examined to determine if it represses the activation of OCT4 on other SALL member promoters. As can be seen in FIG. 11, SALL4 also blocked OCT4 activation of other SALL member promoters.
SALL4 in Adult Stem Cells and Embryonic Carcinoma.
The characterization of tissue stem cell populations remains difficult because of the lack of markers that can distinguish between stem cells and their differentiating progeny. For many tissues, panels of molecular markers have been developed to define the stem cell compartment.
The present data shows that SALL4 is a key regulator of embryonic stem cells in pluripotency and self-renewal. For example, embryonic carcinomas display the phenotype of early embryonic stem cells and possess pluripotent potential. Therefore, the expression of SALL4 protein in this type of tumors by immunohistochemistry was examined. Immunohistochemical data conclusively indicated that all tumor cells of embryonic carcinomas showed a nuclear staining, whereas all non-tumor cells were negative. These observations suggest that SALL4 can be used as a specific marker for normal and malignant embryonic germ cells and embryonic stem cells.
Given that SALL4 was expressed in very early embryonic stem cells, and embryonic carcinoma is reported to arise from transformation of these cells, immunohistochemistry also shows that a) SALL4 positive cells in normal breast lobules, accounted for less than 2% of the epithelium and b) in breast carcinoma samples, SALL4 protein expression in clusters of cells or scattered cells was observed. Further, SALL4 protein was expressed in the nucleus of normal breast epithelial cells and breast carcinoma cells. Moreover, this pluripotent gene expression was observed in other normal adult tissues such as prostate and lung, and carcinoma arising from these tissues with SALL4 antibody. The presence of a small number of SALL4-expressing cells in the broncho-epithelium and prostatic acini, and their stromal cells was observed, as well as the finding that SALL4 was expressed at a similar frequency in normal prostate and lung to that in lobular epithelial cells of breast. In addition, scattered tumor cells in the prostate carcinoma expressed SALL4 protein by immunohistochemistry studies with a SALL4 antibody.
In conclusion, the present examples reveals that (1) immunostaining with anti-SALL4 antibodies are useful diagnostic tools in the identification of embryonic carcinomas, (2) expression of SALL4 is found in several human stem cells and cancer cells; (3) identification of SALL4-expressing cells in human tissues can be used to identify the stem cells, their pre-malignant clones, and malignant cells, and (4) SALL4 represents an ideal marker for embryonic stem cells, adult stem cells and cancer stem cells.
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Although the invention has been described with reference to the above examples, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims.

## Claims

1. A method of diagnosing a myelodysplastic syndrome (MDS) in a subject comprising:
(a) providing a biological sample from the subject;
(b) contacting the biological sample with an antibody which binds to a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6; and
(c) detecting the binding of the antibody to the sample,
wherein detecting binding correlates with MDS.

2. A method of diagnosing acute myeloid leukemia (AML) in a subject comprising:
(a) providing a biological sample from the subject;
(b) contacting the biological sample with an antibody which binds to a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6; and
(c) detecting the binding of the antibody to the sample,
wherein detecting binding correlates with AML.

3. The method of claim 2, wherein the polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 2.

4. The method of claim 2, wherein the polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 4.

5. The method of claim 2, wherein the polypeptide comprising an amino acid sequence as set forth in SEQ ID NO: 6.