Background
The altered expression of genes, such as
WT1,
SCL, and
Notch1, that play crucial roles in the regulation of hematopoietic progenitor cell proliferation is frequently found in leukemia [
1‐
7]. Increasing data show that the genes involved in hematopoietic stem/progenitor cell (HSPC) proliferation change their expression pattern during leukemogenesis [
8].
SALL4 (sal-like protein 4), a
SALL gene family member that is a newly identified zinc-finger transcription factor, was originally cloned based on its sequence homology to
Drosophila spalt (
sal) [
9‐
12]. Alternative splicing generates two variant forms of human
SALL4 mRNA,
SALL4A and
SALL4B, and each has a different tissue distribution [
9,
13]. Recently,
SALL4 has been shown to play an important role in maintaining ES cell (ESC) pluripotency and self-renewal properties.
SALL4 is involved in the self-renewal of leukemic initiation and HSPC [
14]. Moreover, recent data have shown that
SALL4 plays an essential role in myeloid leukemogenesis.
SALL4 is constitutively expressed in human leukemia cell lines and primary acute myeloid leukemia (AML) cells [
9,
13]. Transgenic mice that ubiquitously overexpress
SALL4B exhibit myelodysplastic syndrome (MDS)-like symptoms and subsequently develop transplantable AML [
9,
13], while
SALL4 knockdown in leukemia cell lines triggers apoptosis [
15].
BMI-1 is a member of the polycomb group of proteins, and it was initially identified in
Drosophila as a repressor of homeotic genes [
9,
16‐
18]. The
BMI-1 gene was initially isolated as an oncogene that cooperates with c-myc in retroviral-induced B and T cell leukemia [
19,
20]. In humans,
BMI-1 is highly expressed in purified HSCs, and its expression declines with differentiation [
9,
21], and it plays an essential role in regulating adult, self-renewing HSPC and leukemia stem cells [
9,
21‐
27]. Knockout of the
BMI-1 gene in mice results in the progressive loss of all hematopoietic lineages [
9,
25].
BMI-1 expression appears to be important for the accumulation of leukemic cells. Interestingly, inhibiting tumor stem cell self renewal after
BMI-1 deletion can prevent leukemic recurrence. Recently,
BMI-1 expression has been used as an important marker for predicting MDS development and the progression to AML [
9,
28].
BMI-1 overexpression was also observed in a significant number of nasopharyngeal carcinoma tumors that correlated with advanced tumor progression, invasive stage and poor prognosis [
19,
29].
BMI-1 was recently demonstrated to be a direct
SALL4 target gene. The induction of
SALL4 expression is associated with increased levels of histone H3–K4 and H3–K79 methylation in the
BMI-1 promoter, indicating a novel connection between
SALL4 and polycomb group proteins in leukemogenesis and a mechanism whereby aberrant
SALL4 expression can directly alter BMI-1 expression [
9].
Moreover,
SALL4 expression was higher in drug resistant primary acute myeloid leukemic patients than those from drug-responsive cases. In addition,
SALL4 expression was enriched in the SP when compared to the non-SP counterpart. Recently, it is reported that
SALL4 could promote the expression of the ABC transporter genes, such as ATP binding cassette transporter A3 (
ABCA3), suggesting that
SALL4 can contribute to the SP phenotype by regulating the expression of
ABCA3 and
ABCG2[
15].
ABCA3 is a member of the ATP-binding cassette (ABC) family of transport proteins and is required for perinatal respiratory adaptation. Mutations in
ABCA3 resulted in fatal neonatal lung disease [
30,
31].
ABCA3 is highly expressed in AML and ALL patient samples and its expression is associated with unfavorable clinical treatment outcome. Furthermore, the expression of
ABCA3 is enriched in leukemic SP cells and has been linked to multidrug resistance by facilitating lysosomal sequestration of drugs in AML primary cells and cell lines [
15,
32‐
35]. RNAi specific for
ABCA3 led to a decrease of
ABCA3 expression in T-ALL cell line such as CCRF-CEM and Jurkat cells. Consequently, a significant sensitization of cells to cytostatic drugs was achieved [
35]. Moreover, both pharmacological blockade and the silencing of
ABCA3 enhanced susceptibility of target B-cell lymphoma cells to anti-CD20 antibody-mediated lysis. Mechanisms of cancer cell resistance to drugs and antibodies are linked in an
ABCA3-dependent pathway of exosome secretion [
36].
Little is known about the expression pattern of the SALL4, ABCA3 and BMI-1 genes in patients with myeloid leukemia and patients that achieved complete remission after chemotherapy. In this study, we determined the expression characteristics of the SALL4, ABCA3 and BMI-1 genes in de novo AML and CML and complete remission samples.
Discussion
BMI-1 and
SALL4 are stem cell genes that modulate stem cell pluripotency and play a role in leukemogenesis. Dysregulated expression of both genes may have a cooperative effect in leukemogenesis [
37]. Patients with RA and RARS who have a higher percentage of BMI-1+ cells showed disease progression to RAEB, suggesting that
BMI-1 is a novel molecular marker that predicts the progression and prognosis of MDS [
28]. In this study, we analyzed
BMI-1 and
SALL4 expression in primary AML and CML at diagnosis and those in complete remission.
It has been shown that
BMI-1 overexpression occurs in a variety of cancers including several types of leukemias and lymphomas [
38]. In this study,
BMI-1 was found to be overexpressed in AML and chronic phase CML patient groups; and its expression level was lower in patients who achieved complete remission. Similar results were reported by Sawa, M et al. who found that moderate to high
BMI-1 expression was detected in AML patients, and the AML-M0 subtype showed higher relative expression of the
BMI-1 transcript [
39]. In addition, Merkerova, M et al. demonstrated that
BMI-1 and its significantly higher
BMI-1 transcript level in CML cells seem to play a secondary role in CML transformation [
40]. Our results also indicate that a decreased
BMI-1 expression level is associated with complete disease remission. Interestingly, the
BMI-1 expression level in the CML-BC group appeared to be low in comparison with the de novo CML group, although the difference was not significant. Further investigation is needed using a larger patient cohort to extend our findings. Preliminary results indicate that
BMI-1 may have potential as a therapeutic target for myeloid leukemia. It has been reported that
BMI-1 depletion by RNA interference leads to reduced U937 cell growth and proliferation and increased apoptosis [
41], and an antisense
BMI-1 gene can inhibit the growth of K562 cells and upregulate p16 expression in K562 cells [
42].
Using immunohistochemistry and real-time PCR,
SALL4 was demonstrated to be constitutively expressed in human primary acute myeloid leukemia [
13]. In this study, we found that
SALL4 was overexpressed in different primary AML subtypes, and its expression was lower in the AML-CR patient group. These results are similar to the findings of Jeong, HW et al. who showed that AML patients who responded to treatment had decreasing
SALL4 expression throughout the course of treatment, while AML patients with disease relapse or drug resistance had increasing
SALL4 expression, which was correlated with disease progression [
15]. Interestingly, unlike the
SALL4 expression characteristics in AML, the
SALL4 expression level in the CML-CP and CML-CR groups was lower. Moreover, the
SALL4 expression level in patients with chronic phase CML was significantly lower than that in the CML-CR group. There is no direct evidence demonstrating the
SALL4 expression level in CML-CP and comparing the expression feature to healthy individuals; however, Lu and colleagues have found that the SALL4 protein was overexpressed in CML samples in blast crisis but not those in chronic phase by FACS [
37]. Our results also demonstrated that
SALL4 expression was higher in the CML-BC group in comparison with the CML-CP and CML-CR groups; however, there was no significant difference in comparison with the HI group. Is it possible that
SALL4 is preferentially expressed in leukemic blasts? These results are similar to a report by Cui W et al. who demonstrated that only precursor B-cell lymphoblastic leukemias/lymphomas and AML had detectable
SALL4 in neoplastic tissues [
43]. The different
SALL4 expression patterns in AML and CML suggest that these two disease entities may have different biological characteristics and/or mechanisms of leukemogenesis, at least for the association between
SALL4 and pathogenesis. However, there are not reports comparing the data of SALL4 expression level in CML-CP to healthy individuals, it is difficult to evaluate the significance of this finding. Recently, research from Zhu et al. showed that hematopoietic transcription factor PU.1 expression was significantly lower in newly diagnosed APL patient samples as compared to normal hematopoietic cells, which may relate to the expression level of PML-RARα, and they found that suppression of PU.1 expression occurred concurrently with PML-RARα expression, the authors suggested that low PU.1 expression in APL patients is required for disease initiation and progression [
44]. This finding might provide a direction in farther analysis the correlation of
SALL4 with BCR-ABL in the pathogenesis of CML and to address this question.
In principle, the
BMI-1 and
SALL4 gene expression level should be positively correlated in stem cells [
9]. Little is known about the expression pattern and differences in the
SALL4 and
BMI-1 genes in patients with AML and CML. In this study, we analyzed the correlation between the relative expression levels of
BMI-1 and
SALL4. A positive expression level correlation was found for both genes in HI, AML, chronic phase CML, CML-BC and CML-CR patient groups; however, there was no significant correlation between these genes in patients with AML-CR, leaving their role in this group an open question. These results indicate that a positively correlated expression pattern is a common feature in patients with myeloid leukemia and healthy individuals, and both genes may cooperate during cell proliferation and differentiation.
Based on the different expression features of
SALL4 in AML and CML, we further analyzed its regulating gene
ABCA3, which is a member of the ATP-binding cassette (ABC) family of transport proteins [
30,
31]. Unlike the description by Wult, Norwood and Steinbach groups, who showed that
ABCA3 is highly expressed in acute meyloid leukemia samples and is associated with unfavorable clinical treatment outcome [
24,
33,
45], in the present study, lower expression level of
ABCA3 was found not only in AML but also in CML groups, especially in CML-CP and CR groups. Moreover, the expression level of
ABCA3 lost the correlation with SALL4 expression in leukemia patients. To determine whether these results relate to favorable clinical outcome, further investigation is needed. Additionally, detection of ABCA2, ABCB2 and ABCC10, which were found overexpressed in childhood AML, may be worthy to build the gene regulation network in proliferation of myeloid leukemia cells.
In conclusion, we determined the expression characteristics of the SALL4, ABCA3 and BMI-1 genes in different phases of AML and CML. Further studies will be needed to determine whether BMI-1 and SALL4 are novel therapeutic targets for leukemic stem/initiation cells in primary myeloid leukemia.
Competing interests
The authors declare that they have no potential conflicts of interest.
Authors’ contributions
YQL and YPM contributed to concept development and study design. QS, SCL, SHC and YM performed the real-time PCR. JYH, LJY, BL, XLW, JCY were responsible for collection of clinical data. YQL and QS coordinated the study and helped drafting the manuscript. All authors read and approved the final manuscript.