SALL4 has 2 isoforms. Alternative splicing generates 2 variant forms of SALL4 mRNA. (A) SALL4A and SALL4B vary in protein length and in the presence of different numbers of characteristic sal-like zinc finger domains. SALL4A (encoding 1067 amino acids) contains 8 zinc finger domains, while SALL4B (encoding 623 amino acids) has 3 zinc finger domains. Both variants have exons 1, 3, and 4, and SALL4A contains all exons from 1 to 4. However, SALL4B uses an alternative splice donor that results in deletion of the large 3′ portion of exon 2. (B) RT-PCR analysis of SALL4 variants in different human 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 (i). Tissue-dependent expression of SALL4 transcripts by RT-PCR (ii). 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 1851-bp expected product of SALL4B. Comparable amounts of cDNA were determined by GAPDH. (C) 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.

SALL4 expression in human primary AML and myeloid leukemia cell lines. (A) SALL4 mRNA expression 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 down-regulated 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 3 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. Y-axis: Log scale on the relative quantification. (B) Constitutive expression of SALL4 protein in human AML (FAB M1-M5, n = 81) is demonstrated by immunohistochemical staining. No SALL4 expression was detected in normal bone marrow (i), normal thymus (ii), or normal spleen (iii). All cell nuclei remained blue. Nuclei of CD34⁺ HSCs/HPCs showed brown staining indicating SALL4 expression (iv); acute myeloid leukemia blasts showed similar staining (v, low power [× 4]) in microarray leukemia tissue samples. Each circle represents one leukemia sample. (vi) High-power (× 400) view of one leukemia sample shown in panel vii. The red arrows indicate positive nuclear staining.

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 panel 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.

SALL4B transgenic mice have an MDS-like/AML phenotype. (A) MDS-like changes in SALL4B transgenic mice. Giemsa staining of peripheral blood from normal, age-matched WT littermates showed normal neutrophils (i), and normal red blood cells and platelets (ii, black arrow). In transgenic mice, neutrophils were hypersegmented (v), and pseudo-Pelger-Huet-like atypical white cells were present (vi-viii), together with increased numbers of immature cells (ix-xi). Nucleate red cells (xii, red arrow), giant platelets (xiii, red arrow), and polychromasia (xiv) were also observed in the transgenic mice. A binucleate dysplastic erythrocyte (xv, red arrow) and a dysplastic megakaryocyte with a hypolobulated nucleus (xvi, red arrow) were found in the cytospin from transgenic mouse bone marrow. An erythroid precursor (iii) and a megakaryocyte (iv) from WT control animals are shown for comparison. (B) AML (AML is defined as blast count more than 20% in peripheral blood and/or bone marrow with multiple organ involvements) observed in SALL4B transgenic mice (mouse 25). Blasts were present in the peripheral blood (i, × 600), bone marrow biopsy specimen (ii, × 100; iii, × 400), bone marrow smear (iv, × 600), liver (v, × 100), lymph node (vi, × 400), and spleen (vii-viii, gross view; ix, × 100; and the inset, × 400). (C) Flow cytometric analysis of leukemia in SALL4B transgenic mice. Leukemia cells from bone marrow, spleen, and lymph nodes were positive for CD45 and myeloid markers such as c-kit, Gr-1, and Mac-1, and negative for B cells (B220), T cells (CD3), and erythrocytes (Ter119). Numbers in quadrants indicate the percentage of total cells. (D) Serial transplantation of SALL4B-induced AML to NOD/SCID mice. Gross picture (i-ii) and histology (iii-vi, × 200) on splenomegaly (i [black arrow], iii), hepatomegaly (i [double black arrows], iv), lymph node enlargement (ii [black arrow], v), and pale kidney (ii [double black arrows], vi) caused by leukemia infiltration in a NOD/SCID mouse 6 weeks after leukemia transplantation.

Ineffective hematopoiesis in SALL4B transgenic mice. (A) Comparison of bone marrow of SALL4B transgenic (i) and control mice (ii). 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. (B) Increased number of immature cells and apoptosis in CFUs from SALL4B transgenic mice. On day 7 of culture, a greater number of immature cells (ii-iv, red arrows) and apoptotic cells (ii-iv, double red arrows) were observed in transgenic mouse CFUs than in control CFUs (i). Consistent with this morphologic observation, there was increased apoptosis (annexin V positive, PI negative; v) and more CD34⁺ immature cells (vi). (C) Comparison of 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 (i). CFUs from SALL4B transgenic mice compared with control mice showed a statistically significant increase in CFU-GM (ii) (transgenic: 53.6 ± 10.3 [n = 13] vs WT: 38.1 ± 3.1 [n = 8]; P = .002) and decrease in BFU-E (transgenic: 7.8 ± 3.8 [n = 13] vs WT: 14.1 ± 2.7 [n = 8]; P = .001).

SALL4 and the Wnt/β-catenin signaling pathway. (A) 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. (i) Anti-HA antibody recognized both SALL4A (165 kDa) and SALL4B (95 kDa). (ii) β-Catenin was detected in the lysates. (iii) Immunoprecipitation was performed with the use of an HA affinity resin and detected with an anti-β-catenin antibody. β-Catenin was readily detected in both HA-SALL4A and HA-SALL4B pull-downs. Untransfected cells subjected to the same immunoprecipitation condition as the transfected cells were used as a control. (B) Activation of the Wnt/β-catenin signaling pathway by both SALL4A and SALL4B. HEK-293 cells were transfected with 1.0 μg of either mock alone, or SALL4A or SALL4B plasmid, with or without Wnt1 (including Wnt1, and its coactivators: LRP6, MESD, and F₂5), and TOPflash (TOP) or FOPflash (FOP) reporter plasmid (Upstate USA, Chicago, IL). After 24 hours, luciferase activity was measured. SALL4A or SALL4B alone showed more potent activation of Wnt signaling pathway when compared with the positive control Wnt1. In addition, both SALL4 isoforms demonstrated a significantly synergistic activation of the Wnt signaling pathway with Wnt1. Data represent mean ± SD of 3 independent experiments. (C) Up-regulation of c-Myc and Cyclin D1 expression in SALL4B transgenic mice. RT-PCR analysis was performed on total bone marrow cells from 2 wild-type control mice (lanes 1-2), 2 preleukemic transgenic mice (lanes 3-4), and leukemic bone marrow cells from 2 leukemic transgenic SALL4B mice (lanes 5-6). Both c-Myc and Cyclin D1 expression were significantly up-regulated in SALL4B transgenic mice at both preleukemia MDS and leukemic stages. Beta actin was used as an internal standard.