Overexpression of SOX4 correlates with poor prognosis of acute myeloid leukemia and is leukemogenic in zebrafish

The SOX4 transcription factor is a key regulator of embryonic development, cell-fate decision, cellular differentiation and oncogenesis. Abnormal expression of SOX4 is related to malignant tumor transformation and cancer metastasis. However, no reports are available regarding the clinical significance of SOX4 in acute myeloid leukemia (AML) and the role of SOX4 in leukemogenesis. In the current study, we found that AML patients with low bone marrow (BM) SOX4 expression had higher remission rates and longer overall survival than those with high SOX4 expression, regardless of age, white blood cell count at diagnosis, karyotype profile and NPM1/FLT3-ITD status. To elucidate the role of SOX4 in leukemogenesis, we generated a transgenic zebrafish model that overexpressed human SOX4 in the myeloid lineage Tg(spi1-SOX4-EGFP). These transgenic zebrafish showed, at 5 months of age, increased myelopoiesis with dedifferentiation in kidney marrow. At 9 months of age, their kidney structure was significantly effaced and distorted by increased infiltration of myeloid progenitor cells. These results suggest that SOX4 is not only an independent prognostic factor of AML, but also an important molecular factor in leukemogenesis.

30 min; DAKO, Carpinteria, CA) was used as the secondary antibody, and proteins were detected using the streptavidin-peroxidase complex (DAKO). Finally, the specimens were counter-stained with hematoxylin. An overall score of 0 to 4 was calculated for each specimen. This overall score was the sum of the staining intensity score (0: none, 1: weak, 2: strong) and a score that described the percentage of positively stained myeloid cells (0: ≤25%, 1: 26-50%, 2: >50%)

Generation and husbandry of transgenic zebrafish
Zebrafish (Danio rerio) embryos, larvae, and adult fish were maintained at 28°C under continuous flow and a 14-hour light/ 10-hour dark cycle 2 . All experiments involving zebrafish were approved by the Institutional Animal Care and Use Committee (IACUC) of the National Taiwan University (NTU). The transgenic founders were created using the Tol2 transposon system as described previously 3 and reared to sexual maturity. The Tol2 construct containing spi-1-controlled SOX4 was generated as described previously 4 . The human SOX4 gene was amplified by PCR (using cDNA from MV4-11 as a template) with the attB1-SOX4-F and attB2-SOX4-R primers but no stop codon. The primer sequences are listed in supplemental Table 1.

Isolation of RNA as well as reverse-transcription-PCR
Total RNA from various tissues or a total number of 30 embryos was isolated using NucleoSpin and TRIzol (Invitrogen, Carlsbad, CA). One microgram RNA was then reverse-transcribed into cDNA with the High Capacity RNA-to-cDNA Kit (Applied Biosystems, Carlsbad, CA). Following the reverse transcription reaction, the cDNA template was amplified by polymerase chain reaction (PCR) with KOD-FX Taq polymerase (TOYOBO, Osaka, JAPAN), in accordance with the manufacturer's instructions. PCR products were then subjected to 2.0% agarose gel electrophoresis, and actin was used as an internal control for the cDNA assay.

Quantitative reverse-transcription polymerase chain reaction (qRT-PCR)
Quantitative reverse-transcription PCR (Q-RT-PCR) was carried out in an ABI 7500 Fast Real-Time PCR system (Applied Biosystems, Foster City, CA) using SYBR green as the detection dye (Power SYBR  Green PCR Master Mix, Applied Biosystems).
PCR conditions consisted of 1 cycle at 50°C for 2 min and 95°C for 10 min, followed by up to 40 cycles at 95°C for 15 s (denaturation) and 60°C for 1 min (annealing/extension). Primer specificity was subsequently confirmed by dissociation curves. The primer sequences of various target genes are listed in Supplemental Table 1. The cycle thresholds (Ct) were determined, and their transcript levels were normalized according to β-actin. Specifically, normalization was calculated using the ΔC T method, as follows: relative expression = 2 -ΔC T , where ΔC T =C T(target) -C T(actin) .

Whole mount in situ hybridization (WISH) and immunohistochemical staining-whole mount (IHC-wm)
For WISH analysis, the partial cDNA (901 bp) sequence of mpo was amplified by PCR with T3-mpo-F and T7-mpo-R primers (supplemental Table 1). The antisense digoxigenin (DIG)-labeled mpo RNA probe used for WISH was generated by an in vitro

Cytological analysis of kidney marrow and peripheral blood
Control wildtype and transgenic fish were euthanized at 5, 9, 12 and 15 months of age. Peripheral blood (PB) was obtained by puncturing the tail using micropipette

Flow cytometric analysis
Collection of PB and KM were performed as previously described 4  Gated populations were as follows: immature and mature erythrocytes, lymphocytes, myelomonocytes, and precursor cells.

Sudan Black staining
Sudan black staining was performed as previously described 6