ATPR triggers acute myeloid leukaemia cells differentiation and cycle arrest via the RARα/LDHB/ERK‐glycolysis signalling axis

Abstract Acute myeloid leukaemia (AML) remains a therapeutic challenge and improvements in chemotherapy are needed. 4‐Amino‐2‐trifluoromethyl‐phenyl retinate (ATPR), a novel all‐trans retinoic acid (ATRA) derivative designed and synthesized by our team, has been proven to show superior anticancer effect compared with ATRA on various cancers. However, its potential effect on AML remains largely unknown. Lactate dehydrogenase B (LDHB) is the key glycolytic enzyme that catalyses the interconversion between pyruvate and lactate. Currently, little is known about the role of LDHB in AML. In this study, we found that ATPR showed antileukaemic effects with RARα dependent in AML cells. LDHB was aberrantly overexpressed in human AML peripheral blood mononuclear cell (PBMC) and AML cell lines. A lentiviral vector expressing LDHB‐targeting shRNA was constructed to generate a stable AML cells with low expression of LDHB. The effect of LDHB knockdown on differentiation and cycle arrest of AML cells was assessed in vitro and vivo, including involvement of Raf/MEK/ERK signalling. Finally, these data suggested that ATPR showed antileukaemic effects by RARα/LDHB/ ERK‐glycolysis signalling axis. Further studies should focus on the underlying leukaemia‐promoting mechanisms and investigate LDHB as a therapeutic target.

However, the molecular mechanism by which ATPR suppresses AML progression remains to be elucidated. 12 While our understanding of cancer metabolism is still developing, altered metabolism is already recognized as a cornerstone mechanism of tumorigenesis. 13 Glucose metabolic reprogramming from oxidative to aerobic glycolysis, refer as the Warburg effect, is a hallmark of cancer. This metabolic reselection contributes to multidrug resistance and is one of the reasons for the increase in cancer-related mortality. 14 Accumulating evidence suggests that glycolysis plays pivotal roles in tumour proliferation, metabolism, migration and invasion. Therefore, inhibition of glycolysis is a promising anti-tumour strategy. Lactate dehydrogenase (LDH) is a key enzyme in glycolysis that catalyses the mutual conversion of lactate and pyruvate, NAD +, and NADH. 15 LDH has two types of subunits: LDHA and LDHB, and the combination of the two subunits yields five kinds of tetramers in different proportions. LDHA is known to be elevated in a variety of tumour cells and plays an important role in tumour development and maintenance. 16 However, compared with LDHA, the potential regulatory roles and molecular mechanisms by which LDHB affects the development and progression of AML remain largely unknown.
Raf/MEK/ERK signal pathway, also known as ERK signalling pathway, is composed mainly of a three-stage enzyme-linked functional unit, namely Raf, MEK and ERK excitation. 17 The duration of ERK phosphorylation and activation is closely related to cell fate.
Generally, continuous and appropriate activation can promote cell proliferation by promoting protein synthesis and improving protein stability. However, over-activation of the ERK pathway can block the process of cell cycle. Recent studies have reported that PD98059 could block the activation of ERK1/2 and reduce the growth and differentiation of AML cell lines induced by dodecyl gallate acid and gifitinib. 18 U0126 significantly blocked the differentiation of human AML cell lines induced by LukS-PV and pulsatilla saponin A via inhibiting the activation of ERK pathway. 19 Abnormal expression of the Raf/MRK/ERK signalling pathway is closely associated with the development and malignant progression of a variety of malignancies and has been identified as a novel target in AML therapy. Therefore, we hypothesize that LDHB is involved in AML progression via regulating cell metabolism pathways and investigate the underlying mechanisms by which ATPR show the antileukaemic effects via the RARα/LDHB/ERK-glycolysis signalling axis. Furthermore, ATPR may have potential as a chemotherapeutic agent, and LDHB may act as a therapeutic target.

| AML patient samples and ethics statement.
Patients with newly diagnosed AML (n = 15) were recruited from the First Affiliated Hospital of Anhui Medical University. Peripheral blood was collected from patients and mononuclear cells were isolated by standard Ficoll-Hypaque density centrifugation. Cells were washed with RPMI 1640 and subjected to various assays.

| Cell culture
The AML cell lines NB4 and HL-60 were purchased from Shanghai Genechem Co., Ltd. (Shanghai, China) and the KG-1, U937 and MOLM-13 cells were donated by the University of Maryland School of Medicine. The cells were maintained in suspension in RPMI-1640 medium (Hyclone) containing 10% FBS (Biological Industries) in a humidified atmosphere of 5% CO 2 at 37°C.

| Quantitative real-time PCR (qRT-PCR)
The total RNA of NB4 cells extracted with TRIzol reagent (Invitrogen Corp., Carlsbad, CA, USA), and the first-strand cDNA was synthesized using a Thermoscript RT-PCR synthesis kit (Fermentas, Canada) according to the manufacturer's instructions. Gene expression was determined using cDNA SYBR-Green real-time PCR Master Mix by quantitative real-time PCR (Takara). The mRNA ratio of the target gene to β-actin was calculated by using the 2 −ΔΔCt formula. The experiments were performed at least three times with three different templates. The primers used were:

| Cell differentiation analysis
Cell maturation was evaluated by cellular morphology and the content of cell surface differentiation-related antigen CD11b and CD14.
Morphology was determined with the Wright-Giemsa staining, and the content of CD11b and CD14 was acquired by CytoFLEX (Becton Dickinson, USA).

| Cell cycle analysis
To analyse the intracellular DNA content, the cells were harvested and washed in cold PBS, and then they were fixed in 75% ethanol/25% PBS at −20°C overnight. A portion of the fixed cell suspension containing 1 × 10 6 cells was washed twice in cold PBS. After then, cells were stained with 500ul of propidium iodide (PI) staining buffer (Beyotime, China), which contains 20μl RNase A, at room temperature for 30min in the dark. The cells were subjected to cell cycle analysis using CytoFLEX (Becton Dickinson, USA), and Modfit software was used to estimate G0/G1/S/G2/M phases of the cell cycle.

| Western blotting analysis
Cultured cells were lysed with RIPA lysis buffer for 30 min for Western blotting (Beyotime, China). Then, it was centrifuged at 12,000 rpm for 40 min, and the supernatants were collected. Protein concentration was measured using a BCA protein assay kit (Boster, China).

| Ki-67 analysis
The AML cells were plated in 6-well plate containing 2 mL RPMI-1640 medium. The plated cells were treated with ATPR (10 -6 M) and

| CFDA-SE cell proliferation assay
Cell proliferation determination was conducted by CFDA-SE probe (Beyotime, China). Briefly, synchronized AML cells were harvested and washed three times with PBS. Afterwards, the cells were stained with CFDA-SE in 6-well plates according to the manufacturer's protocol. Furthermore, the cells were treated with ATPR (1*10 -6 M), and incubated for different times (24 h, 48 h, 72 h). These analyses were performed on the flow cytometer CytoFLEX (Becton Dickinson, USA).

| Tumour xenograft
Six-week-old female NCG mice were obtained from the Nanjing

| Nitroblue tetrazolium (NBT) reduction assay
For the NBT reduction assay, cells were inoculated in a 6-well plate and treated with different time of ATPR (1*10 -6 M). A 10 μl aliquot of NBT solution, composed of 10 mg/ml NBT (Sigma-Aldrich) and 2 µg/ ml PMA(Sigma-Aldrich), was added to each well, and then cells were incubated at 37°C for 30 min, then the proportion of cells containing the precipitated formazan particles in 300 cells was counted by optical microscope.

| Glucose consumption, lactate production and ATP generation assay
The expression of glucose consumption, lactate production and ATP generation in supernatants of AML cells were evaluated using commercial assay kit (Glucose assay kit (F006-1), Lactate colorimetric assay kit II (A019-2), ATP colorimetric assay kit (A095-1), Jiancheng Bioengineering Institute, Nanjing) according to the manufacture's protocol.

| Oxygen consumption rate (OCR) assay
Oxygen consumption rate (OCR) was real time determined using Seahorse Bioscience XF extracellular flux analyzer (Seahorse Bioscience, USA) and experiments were detected according to the manufacturer's protocol.

| Statistical analysis
Experimental data were presented as mean ± SD unless otherwise stated. Statistical significances determined by one-way ANOVA, Student's t test and Bonferroni's test. P < 0.05 was considered to be statistically significant.

| AML cell lines were sensitive to ATPR in a concentration-dependent
To investigate whether AML cells (NB4, HL-60, KG-1, U937 and MOLM-13) were sensitive to ATPR treatment, the CCK8 assay was used. As shown in Figure 1A  Moreover, the NB4 and MOLM-13 were the most sensitive cells to ATPR treatment.

| ATPR inhibited proliferation of AML cells
To profile the effects of ATPR on the proliferation of AML cells, we chose NB4 and MOLM-13 cells to conduct the following experiment. As shown in Figure 1B, the growth curves of NB4 and MOLM-13 cells treated with ATPR showed a time-and concentration-dependent inhibition. A significantly decreased proliferation was determined by observing the expression of the proliferation antigen, Ki-67 antigen, after ATPR treatment in a time-dependent ( Figure 1C). The CFDA-SE dye showed that the cells treated with ATPR significantly decreased the average number of cells in CFDA-SE profiles for 1 division after 48h and 72h as compared to 0h and 24h ( Figure 1D). Flow cytometry showed that a time-and concentration-dependent accumulation of cells in G0/G1 phase was observed after ATPR treatment. However, the percentage of cells in S phase was reduced ( Figure 1E). As shown in Figure 1F

| ATPR induced the expression of target genes in AML cells containing a functional PML-RARα
To identify whether RARα signalling was functional in AML cells treated with ATPR, we analysed the expression of retinoic acid receptor (RARα, RARβ, RARγ) and PML-RARα by Western blotting in both NB4 and MOLM-13 cells. We found that RARα and RARβ levels were elevated, and PML-RARα levels were reduced ( Figure 2A). This change was maintained until 72 h after treatment. However, unlike RARα, the expression levels of RARγ were little changed by ATPR treatment (P > 0.05). The expression of bona fide CRABP2 targets such as CRABP2 itself, CYP26A1 and RARβ, which promoters contain a RARE element, was detected by qRT-PCR. All targets to be induced in cells treated with ATPR confirming a transcriptional activation ( Figure 2B). Interestingly, RARα activation could also be observed in MOLM-13 cells without PML-RARα ( Figure 2C, D). These findings suggested that ATPR activated RARα signalling and degraded PML-RARα protein.

| ATPR showed antileukaemic effect with RARα dependent
To further define whether RARα mediated the antileukaemic ef-

| The glycolysis situation of AML cells
To profile the glycolysis situation of AML cells, cells were stimulated with the glycolytic inhibitor, 2-DG. CCK-8 showed that 2-DG inhibited the viability of AML cell lines in a concentration-dependent manner (72 h). NB4 was the most sensitive, while KG-1a was less sensitive to 2-DG ( Figure 2H). To investigate the effect of ATPR on the glycolysis rate of AML cells, the levels of glucose consumption, lactate production and ATP generation were tested. Our results showed that ATPR inhibited the levels of glucose consumption, lactate production and ATP generation only in NB4 and MOLM-13 cells ( Figure 2I). qRT-PCR results showed that the expression of LDHB, LDHA, HK2, ENO1 and GAPDH was reduced after ATPR treatment, except GAPDH in both NB4 and MOLM-13 cells ( Figure 2J).
Furthermore, the oxygen consumption rate (OCR) of NB4 and MOLM-13 cells after ATPR treatment was evaluated by Seahorse XF.
The results showed that ATPR treatment significantly decreased the rate of glycolysis and glycolytic capacity of both NB4 and MOLM-13 cells ( Figure 2K). Taken together, these results suggested that ATPR might exert antileukaemic effects by mediating glycolysis.

| Modulation of ATPR-inhibited LDHB expression by RARα-selective agonist
To define the role of LDHB in AML, we analysed the expression of LDHB in normal human PBMC, AML patients PBMC and AML cell lines by Western blotting. We found that the expression of LDHB was increased in AML patients PBMC compared with normal human PBMC ( Figure 3A). Furthermore, the expression of LDHB in NB4 cells was highest among different AML cell lines ( Figure 3B). These indicated that the malignant phenotype of AML may be related to its glycolysis level.
To investigate whether the effect of ATPR on LDHB expression, we first examined the expression of LDHB in response to ATPR. In the present study, proteomics analysis identified 795 differentially expressed proteins after treatment with ATPR compared with control group. A total 6 down-regulated proteins that had changes of < 0.677fold were found. Results of 6 down-regulated proteins are displayed in Table 1. As shown in Figure 3C, D, after ATPR treatment, LDHB levels were decreased in a concentration-and time-dependent manner.
To confirm the direct involvement of the RARα signalling pathway in the modulation of ATPR-inhibited LDHB expression, AML cells were treated with ATPR in the absence or in the presence of the RARαselective antagonist Ro 41-5253. When AML cells were treated with Ro 41-5253 prior to the addition of ATPR, the blockade of RARα signalling partially reduced the response of LDHB to ATPR ( Figure 3E). These results confirmed that the expression of LDHB was regulated through the binding of ATPR to a RARα ligand-binding domain.

| Knockdown LDHB expression inhibited proliferation and glycolysis while promoting differentiation of AML cells
To confirm the above data derived from AML patients PBMC and to corroborate the function of LDHB in AML cells, we knocked down LDHB expression using shLDHB in NB4 and MOLM-13 cells.
Treatment with shLDHB significantly decreased the expression of LDHB compared with that in the control group ( Figure 3F, G). The CCK-8 assay results showed that the LDHB knockdown group inhibited cell proliferation more than the NC group ( Figure 3H). Flow cytometry showed that the cell cycle was blocked at G0/G1 phase after shLDHB treatment ( Figure 3I). KI-67 dye showed that ATPR significantly decreased cell proliferation ( Figure 3J). Wright-Giemsa staining, NBT assay and flow cytometry analysis indicated that the level of cell differentiation was increased after shLDHB treatment ( Figure 3K-M). Furthermore, this conclusion was verified using Western blotting ( Figure 3N). Collectively, these data revealed that LDHB potentially contributed to cell proliferation inhibition and differentiation induction in vitro.

| ATPR regulated the Raf/MEK/ERK signalling pathway through LDHB
To test which signal pathway mediated the AML cells proliferation and differentiation in response to ATPR, the expression levels of  Figure 4C). Taken together, these data indicated that ATPR regulated the Raf/MEK/ ERK signalling pathway through LDHB.

| Effects of LDHB on tumour growth in vivo
Our previous study had successfully constructed subcutaneous tumour in NCG mice. Compared with vehicle group, ATPR group inhibited the expression of LDHB in vivo ( Figure 4D). In order to confirm the effect of LDHB on tumour formation in vivo, we first established a stable NB4 and MOLM-13 cell lines using shLDHB.
NCG mice received subcutaneous injections of shLDHB-treated or control cells to establish the tumour model. After 2 weeks, tumours were completely stripped. Photographs and measured volumes of the tumours indicated that LDHB-depleted cells grew much more slowly than control cells ( Figure 4E, F). Moreover, the weights of the tumours from these mice were lower than those from control mice ( Figure 4G). Immunohistochemical staining results indicated that CD11b was elevated and LDHB and KI67 were reduced ( Figure 11H) . Together, these data demonstrated that LDHB knockdown inhibited tumour growth and induced differentiation in vivo by blocking the Raf/MEK/ERK signalling pathway in AML.  The typical MAPK signalling starts from the activation of receptor tyrosine kinase on the cell membrane and spreads through Raf/MEK/ ERK in various biological processes, such as cell proliferation, cycle regulation, differentiation, survival and apoptosis. 26,27 It is well documented that multiple components of this signalling pathway provide attractive therapeutic targets in promoting disease progression and metastasis. 28 However, the lack of success with MAPK components highlights the need for new treatment strategies. We and others have shown that ATPR could also crosstalk with this MAPK signalling to rapidly stimulate or suppress ERK phosphorylation in various cellular contexts. 29,30 We have also shown that ATPR could induce apoptosis via RARβ/RXRβ heterodimers and activation of ER stress involving the MAPK pathway in the breast cancer MDA-MB-231 cells. 31 Furthermore, ATPR could induce cell differentiation in K562 cells, and its mechanism might be related to its ability in regulating the activation of ERK1/2 signalling pathway. 30,32 The present study also provided evidence for ATPR-induced differentiation and proliferation inhibition in AML through regulating RARα/LDHB/ERK-glycolysis signalling axis.

| D ISCUSS I ON
We just investigated the effects of LDHB on differentiation and proliferation, and more studies are needed to research the potential molecular mechanism between LDHB and Raf/MEK/ERK pathway.

| CON CLUS IONS
In summary, the present study suggests that inducing differentiation and inhibiting proliferation of ATPR is, at least partially, mediated F I G U R E 5 ATPR triggers AML cells differentiation and cycle arrest via the RARα/LDHB/ERK-glycolysis signalling axis. ATPR triggers AML cells differentiation and cycle arrest via the RARα/LDHB/ERK-glycolysis signalling axis by RARα/LDHB/ERK-glycolysis signalling axis ( Figure 5). Our finding highlights a novel mechanism underlying the effect of ATPR and discloses potential future therapeutic strategies for AML treatment.

ACK N OWLED G M ENTS
We thank the First Affiliated Hospital of Anhui Medical University (Hefei, China) for providing the human PBMCs.

CO N FLI C T O F I NTE R E S T
The authors declared no potential conflicts of interest concerning the research, authorship and publication of this article.

E TH I C S A PPROVA L A N D CO N S E NT TO PA RTI CI PATE
The study was approved by written informed consent which was ob-

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support the findings of this study are included within the article.