The Study of SALL4 Gene and BMI-1 Gene Expression in Acute Myeloid Leukemia Patients

Abstract

Background

In acute myeloid leukemia (AML), many genes have been studied as prognostic markers. SALL4 is expressed constitutively in human leukemia cell lines and primary AML cells. BMI-1 is expressed highly in purified hematopoietic stem cells (HSCs), and its expression declines with differentiation.

Objective

To study the expression levels of SALL4 and BMI-1 and their clinical significance in patients with AML.

Methods

The study was performed with 60 patients newly diagnosed with AML and 50 control individuals. SALL4 and BMI-1 expression detection were performed using real-time polymerase chain reaction (PCR).

Results

The expression of SALL4 and BMI-1 was significantly higher in cases of AML and showed a strong association with failure to achieve complete remission (CR) or with relapse (P = .02, P = .03, respectively). In multivariate analysis, these genes were the most powerful independent predictors of poor prognosis (P = .01 for SALL4, P = .02 for BMI-1).

Conclusion

SALL4 and BMI-1 are significant prognostic factors in AML and could be strong targets for novel types of therapy.

AML, SALL4, BMI-1, prognosis, real-time PCR, oncogenes

Acute myeloid leukemia (AML) is a heterogeneous disease that remains challenging to treat because of patient factors (age and coexisting diseases) and intrinsic biologic factors.¹ Cytogenetics² and mutational³ data are used to divide patients into subgroups defined according to prognostic factors.^4–7

Mutated genes were classified into 1 of 9 functional categories: transcription factor fusions, the NPM1 gene, tumor suppressor genes, DNA methylation-related genes, signaling genes, chromatin-modifying genes, myeloid transcription-factor genes, cohesin complex genes, and spliceosome complex genes.⁸

AML development is considered to be a multistep process that requires the collaboration of at least 2 classes of mutations to reach full-blown leukemia. Almost a decade ago, Gilliland and Griffin⁹ presented a paradigm model for this process, designated the 2-hit model. It comprises class I mutations that activate signal transduction pathways and confers a proliferation advantage on hematopoietic cells, and class II mutations targeting transcription factors and primarily serving to impair hematopoietic differentiation.^10–11

As an example of class II mutations, BMI-1 and SALL4 are oncogenes that modulate stem-cell pluripotency and play a role in leukemogenesis. The SALL4 zinc-finger transcription factor a member of the SALL gene family, was originally cloned based on sequence homolog to Drosophila spalt(sal). Alternative splicing resulted in 2 variant forms of human SALL4 messenger RNA (mRNA), SALL4A and SALL4B, each having a different tissue distribution.

Recently, SALL4 has been shown to play an important role in maintaining embryonic stem cell (ESC) pluripotency and self-renewal properties. It was demonstrated¹² that SALL4 is expressed constitutively in human leukemia cell lines and primary AML cells. The identification of downstream targets of SALL4 or factors that regulate BMI-1 in leukemogenesis is of significant interest. It was suggested that the SALL4 expression level can potentially be used to guide decision making in the treatment of myelodysplastic syndrome (MDS) and other hematological malignant neoplasms.¹²

The BMI-1 polycomb gene plays an essential role in regulating adult, self-renewing hematopoietic stem cells (HSCs) and leukemia stem cells and is highly expressed in purified HSCs; its expression declines with differentiation. The expression of BMI-1 appears to be important in the accumulation of leukemic cells. We were interested to learn that inhibiting self-renewal in tumor stem cells after deleting BMI-1 can prevent leukemic recurrence. BMI-1 expression is an important marker for predicting the development of MDS and disease progression to AML. Targeting BMI-1 activity might offer more curative success for the hematologic malignant neoplasms associated with its aberrant activity.^13,14 The aims of this study were to investigate the expression levels of SALL4 and BMI-1 genes and their clinical significance in patients with AML.

Materials and Methods

The study was performed with the cases of 60 patients recently newly diagnosed with AML. These patients had presented to the Department of Hematology and the Bone Marrow Transplantation Unit. We also assembled a control group consisting of 50 age- and sex-matched healthy individuals.

The diagnosis of AML was based on the recent 2016 World Health Organization (WHO) updated diagnostic criteria based on morphology, immunophenotyping, and genetics. Patients harboring other hematological or nonhematological malignant neoplasms were excluded from our study. Cytogenetics studies and NPM1 and FLT3-ITD mutation analysis by polymerase chain reaction (PCR) were performed in all cases; we excluded from our study all patients with AML who had any chromosomal abnormality by karyotyping or gene mutations, as detected by PCR, to eliminate the effects of these abnormalities on the course of disease, response to treatment, and prognosis; to be able to better assess the effect of SALL-4 and BMI-1 expression on the disease course; and to avoid any interference with the expression levels of our target genes by these cytogenetic abnormalities. The patients were followed up for a period of between 12 and 18 months.

Written informed consent was obtained from all subjects. Also, the ethical committee for Human Research in Alexandria Faculty of Medicine provided their approval.

All patients had a full medical history taken, along with complete clinical examination and laboratory investigations, including complete blood count (CBC) using the 5-part differential ADVIA 2120i automated blood-cell counter (Siemens AG), bone marrow aspiration, and immunophenotyping (performed for patients only) using a FACS caliber flow cytometer (serial number E34297300591) equipped with Cell Quest software (Becton Dickinson and Company). These tests were performed initially for the diagnosis of the patients and on scheduled intervals for follow-up, to evaluate the response of the patients by detection of minimal residual disease by flowcytometric analysis if present, together with peripheral blood, bone marrow, and clinical assessment. SALL4 and BMI-1 detection by real-time polymerase chain reaction (RT-PCR) for patients (performed initially at diagnosis) and controls.

Immunophenotyping of blast cells was performed on bone marrow specimens in dipotassium ethylenediaminetetraacetic acid (K2EDTA) tubes. Briefly, 10 μL of the acute monoclonal panel of antibodies was added to 100 μL of bone marrow blood collected in a K2EDTA tube and incubated for 10 minutes at room temperature, followed by a wash step, in which 2 mL lysing solution was added, mixed, and left for 10 minutes in the dark and then washed twice with phosphate-buffered saline (PBS). After the last wash, an analysis gate was set around the required blast population. The cutoff point of positivity was considered to be when more than 20% of the cells were stained with a particular antibody in excess of the background fluorescence in the negative controls, as set up by our departmental flow-cytometry protocol. On analyzing intracellular antigens, we performed an extra step of fixation and permeabilization.

RNA Extraction and Complementary DNA (cDNA) Synthesis

Purification of total cellular RNA from human whole blood was extracted using QIAamp RNA blood Mini kit (QIAGEN NV). We checked the concentration and purity of RNA using a Nanodrop ND-1000 spectrophotometer (ThermoFisher Scientific Inc.). RT-PCR was performed using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Inc.) according to manufacturer-provided instructions.

Real-Time Quantitative Reverse Transcription–Polymerase Chain Reaction (qRT–PCR)

A relative quantitation of SALL4 and BMI-1 expression was performed using the SensiFast TMSYBR No-ROX Kit RT-PCR assay (Bioline Corporation). In a total volume of 20 µL and 10 µM primer concentration, we used the real-time cycler Rotor-Gene Q (QIAGEN NV) in 40 cycles with initial denaturation of 95°C for 5 seconds and annealed at 60°C for 30 seconds.

The specific primer pairs were as follows: for SALL4: forward primer 5′-TGCAGCAGTTGGTGGAGAAC-3, reverse primer 5′-TCGGTGGCAAATGAGACATTC-3′; for BMI-1, forward primer 5′-TAAGCATTGGGCCATAGT-3′ and reverse primer 5′-ATTCTTTCCGTTGGTTGAA-3′ β2M, 5′-TACACTGAATTCACCCCCAC-3′ (forward) and 5′-CATCCAATCCAAATGCGGCA-3′ (reverse).

The relative quantitation of SALL4 and BMI-1 expression was normalized to the endogenous gene β2 microglobulin using the 2^{(−∆∆CT)} method. The specific amplification of the PCR products was analyzed by melting-curve analysis for greater accuracy.

Statistical Analysis

Statistical analysis was performed using Statistical Package for Social Sciences (SPSS) software, version 20 (IBM Corporation). We recorded arithmatic mean (SD) and number (percentage) values for the data we gathered. For categorized parameters, we used χ ² testing; for comparison of numerical data between 2 groups, we used t-testing. To find the association between 2 variables, Spearman correlation coefficient test was used. The level of significant was a P value of less than .05. We used univariate and then multivariate analysis to detect the powerful predictors of prognosis.

Results

The patients enrolled in our study were 28 males and 32 females, with a mean (SD) age of 38.17 (17.33) years. In the control group, 21 were male and 29 were female, with a mean (SD) age of 39.12 (13.68) years. All demographic and clinical data are mentioned in the Table 1.

Table 1.

Demographic and Clinical Data of the 2 Studied Groups

Variable	Patients (n = 60)	Control (n = 50)	P Value
Age, mean (SD)	38.17 (17.33)	39.12 (13.68)	.37
Sex (male/female)	28/32	21/29	.13
BMI-1 expression
2^-∆∆CT	59.40 (56.82)	1.34 (1.17)	.001a
SALL4 expression
2^-∆∆CT	34.40 (28.25)	1.20 (0.71)	.001a
AML subtypes
M1	12	…
M2	14	…
M4	5	…
M5	29	…
Clinical findings
No lymphadenopathy or organomegaly	24	…
Only 1 system affected	14	…
2 systems affected	13	…
All	9	…
Outcome
No remission/relapse	35	…
Complete remission	17	…
Death	8	…
CBC (mean [SD])
Hemoglobin (g/dL)	7.97 (2.57)	12.78 (1.35)	.001a
WBCs (× 109/L)	51,222.30 (51538.8)	72,07.00 (2070.44)	.001a
PLTs (× 109/L)	80,266.70 (102233.5)	286,940.00 (110315.2)	.001a
ALT (U/L)	35.25 (23.16)	40.06 (9.96)	.09
AST (U/L)	27.97 (17.60)	24.00 (8.55)	.07
BUN (mg/dL)	19.93 (19.49)	12.36 (3.86)	.048a
Creatinine (mg/dL)	0.94 (0.71)	0.71 (0.21)	.07

AML, acute myeloid leukemia; … , nonapplicable; CBC, complete blood count; WBCs, white blood cells; PLTs, platelets; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen.

^aStatistically significant at P ≤.05.

The expression of SALL4 was found to be significantly higher in our case individuals with AML, compared with the control group (P = .001). Regarding BMI-1, its expression was also significantly increased, compared with the controls (P = .001). When studying the correlation between SALL4 and BMI-1 with all other laboratory and clinical parameters, no significant correlation was found between any of the studied parameters, nor between SALL4 and BMI-1 (P = .13).

We studied the response of the patients to treatment and the relationship of that response with genetic expression. Our results revealed a strong association between SALL4 and BMI-1 and not achieving CR, or relapse (P = .02 and P = .03, respectively). In our patients, the relationship between gene expression and AML subtypes revealed a significant increase in BMI-1 and SALL4 expression in M4 and M5, compared with in M1 and M2 (P = .01 and P = .02, respectively).

We studied the flow-cytometric parameters in our case individuals and tried to find any association with our studied genetic expression; we found no association except for cluster of differentiation (CD)34, in which we found positive CD34 expression on the leukemic blasts in 42 patients, of which 18 tested negative for CD24. A significant increase in SALL4 and BMI-1 expression was found in case individuals that tested CD34 positive, compared with case individuals who tested CD34 negative (P < .001 and P = .001, respectively).

We used age, sex, hemoglobin concentration, percentage of blasts in the peripheral blood and in the bone marrow, white blood cell (WBC) count, CD34 expression on the blast cells, platelet count, BMI-1 expression, and SALL4 expression in our univariate and then our multivariate analysis against the outcome. In univariate analysis, BMI-1 and SALL4 expression and age were strong predictors for unfavorable outcome and worse prognosis. In multivariate analysis, BMI-1 and SALL4 expression were the most powerful independent predictors for unfavorable prognosis (P = .01 and P = .02, respectively). On studying the overall and disease-free survival rates, the disease-free survival rates were significantly lower in patients expressing higher BMI-1 and SALL4 (P = .03 and P = .01, respectively); overall survival was not significantly affected.

Discussion

Our finding that expression of SALL4 and BMI-1 were significantly higher in our case individuals with AML, compared with the control group (P = .001), matches the findings of Tang et al,¹⁵ that the expression of SALL4 mRNA and BMI-1 in AML were significantly higher than their expression in B-cell acute lymphoblastic leukemia (B-ALL) and in the control participants. BMI-1 was positively correlated with SALL4, as proven by Tang et al¹⁵; this finding was further elucidated by Guo et al,¹⁶ who found that BMI-1 expression was positively regulated by Sp1, Twist1, FoxM1, ZEB1, E2F1, SALL4, Myc-N, c-Myc, and HDACs transcription factors, and that SALL4 and Sp1 transcription factors bind to a specific region of the BMI-1 promotor and upregulate its expression in healthy and malignant cells. However, in our study results, no significant positive correlation was found (P = .13).

The leukemogenesis induced by SALL4 was demonstrated by Wang et al,¹⁷ who pointed out that SALL4 can negatively affect the DNA damage-repair process. Also, SALL4 can promote cell survival by activating the antiapoptosis gene Bcl2. Regarding the leukemogenic effect of BMI-1, it was found that the expression of BMI-1 is required for maintenance and self-renewal of healthy and leukemic stem cells and progenitor cells, and protects cells against oxidative stress. Also, BMI-1 has an essential role in maintaining the self-renewing capacity of leukemic stem cells.

In our study findings, SALL4 and BMI-1 were significantly associated with failure to achieve CR, or relapse (P = .02 and P = .03, respectively). Jeong et al¹⁸ found that patients with AML who responded to treatment had decreasing SALL4 expression throughout the treatment course, and patients with AML who experienced disease relapse or drug resistance had increasing SALL4 expression that was correlated to the disease progression. Saudy et al¹⁹ concluded that 2-year overall and disease-free survival rates were significantly lower in patients who expressed BMI-1 more strongly. Nishida et al²⁰ hypothesized that a therapeutic strategy targeting BMI-1 could eradicate chemotherapy-resistant AML stem cells. BMI-1 and p53 function in the BMI-1–ARF–MDM2–p53 signaling pathway, and reduced BMI-1 expression could potentially trigger p53-mediated apoptosis. Based on the leukemogenic role of SALL4 and BMI-1 and their impact on prognosis and response to chemotherapy, they are considered to be ideal targets of novel therapy. Nishida et al²⁰ used them as targets in this way, demonstrating that the novel BMI-1 inhibitor PTC596 downregulates MCL-1 and induces mitochondrial apoptosis in a p53-independent manner. PTC596 effectively killed CD34+CD38− AML stem/progenitor cells while sparing normal hematopoietic stem/progenitor cells.

Another key finding was the significant increase of SALL4 and BMI-1 expression in case individuals who tested CD34 positive, compared with those who tested CD34 negative (P <.001 and P = .001, respectively). This finding coincides with the fact that SALL4 and BMI-1 are stem-cell markers, responsible for self-renewal and maintenance of stem cells.However, this phenomenon is extended to leukemic stem cells, preventing apoptosis and providing a chemotherapy-resistance advantage to those cells.²¹

Conclusion

SALL4 and BMI-1 are significant prognostic factors in AML; however, more long-term studies are needed to support this conclusion. Those genes are favorable targets for novel therapy, especially in relapsed/refractory AML. We recommend that more studies be performed on the topic of SALL4 and BMI-1 expression in AML, with larger numbers of all subtypes with normal and abnormal cytogenetic expression, to better stratify the role of those genes in AML, together with the measurement of the protein levels of these genes, to confirm the increase in their expression.

Abbreviations

Abbreviations
 
• AML

  acute myeloid leukemia

 
• mRNA

  messenger RNA

 
• ESC

  embryonic stem cell

 
• MDS

  myelodysplastic syndrome

 
• HSCs

  hematopoietic stem cells

 
• WHO

  World Health Organization

 
• PCR

  polymerase chain reaction

 
• CBC

  complete blood count

 
• RT-PCR

  real-time polymerase chain reaction

 
• K2EDTA

  dipotassium ethylenediaminetetraacetic acid

 
• PBS

  phosphate-buffered saline

 
• cDNA

  complementary DNA

 
• qRT-PCR

  quantitative reverse transcription–polymerase chain reaction

 
• SPSS

  Statistical Package for Social Sciences

 
• CD

  cluster of differentiation

 
• WBC

  white blood cell

 
• B-ALL

  B-cell acute lymphoblastic leukemia

 
• PLT

  platelet

 
• ALT

  alanine aminotransferase

 
• AST

  aspartate aminotransferase

 
• BUN

  blood urea nitrogen

Acknowledgments

We thank all participating patients, nurses, and technicians for their dedicated cooperation, work, and support. Without them, we would never have been able to accomplish this work.