Enhancing SHP-1 expression with 5-azacytidine may inhibit STAT3 activation and confer sensitivity in lestaurtinib (CEP-701)-resistant FLT3-ITD positive acute myeloid leukemia

Tumor-suppressor genes are inactivated by methylation in several cancers including acute myeloid leukemia (AML). Src homology-2 (SH2)-containing protein-tyrosine phosphatase 1 (SHP-1) is a negative regulator of the JAK/STAT pathway. Transcriptional silencing of SHP-1 plays a critical role in the development and progression of cancers through STAT3 activation. 5-Azacytidine (5-Aza) is a DNA methyltransferase inhibitor that causes DNA demethylation resulting in re-expression of silenced SHP-1. Lestaurtinib (CEP-701) is a multi-targeted tyrosine kinase inhibitor that potently inhibits FLT3 tyrosine kinase and induces hematological remission in AML patients harboring the internal tandem duplication of the FLT3 gene (FLT3-ITD). However, the majority of patients in clinical trials developed resistance to CEP-701. Therefore, the aim of this study, was to assess the effect of re-expression of SHP-1 on sensitivity to CEP-701 in resistant AML cells. Resistant cells harboring the FLT3-ITD were developed by overexposure of MV4-11 to CEP-701, and the effects of 5-Aza treatment were investigated. Apoptosis and cytotoxicity of CEP-701 were determined using Annexin V and MTS assays, respectively. Gene expression was performed by quantitative real-time PCR. STATs activity was examined by western blotting and the methylation profile of SHP-1 was studied using MS-PCR and pyrosequencing analysis. Repeated-measures ANOVA and Kruskal–Wallis tests were used for statistical analysis. The cytotoxic dose of CEP-701 on resistant cells was significantly higher in comparison with parental and MV4-11R-cep + 5-Aza cells (p = 0.004). The resistant cells showed a significant higher viability and lower apoptosis compared with other cells (p < 0.001). Expression of SHP-1 was 7-fold higher in MV4-11R-cep + 5-Aza cells compared to parental and resistant cells (p = 0.011). STAT3 was activated in resistant cells. Methylation of SHP-1 was significantly decreased in MV4-11R-cep + 5-Aza cells (p = 0.002). The restoration of SHP-1 expression induces sensitivity towards CEP-701 and could serve as a target in the treatment of AML. Our findings support the hypothesis that, the tumor-suppressor effect of SHP-1 is lost due to epigenetic silencing and its re-expression might play an important role in re-inducing sensitivity to TKIs. Thus, SHP-1 is a plausible candidate for a role in the development of CEP-701 resistance in FLT3-ITD+ AML patients.


Background
Acute myeloid leukemia (AML) is a hematological malignancy that occurs as a result of genetic aberrations in hematopoietic progenitor cells [1,2]. Epigenetic silencing due to DNA hypermethylation is a frequent mechanism of inactivation of tumor suppressor genes (TSG) in variety of human cancers including AML [3]. 5-Azacytidine (5-Aza) is a chemotherapeutic agent that induces DNA demethylation by inhibition of DNA methyltransferase (DNMT) enzymes [4,5]. The suppression of DNMTs in cancer cell lines induces hypomethylation resulting in decreased viability [6]. CEP-701 is a tyrosine kinase inhibitor (TKI) that potentially inhibits FLT3 tyrosine kinase and induces hematological remission in patients with AML. However, the majority of AML patients have only moderate and transient responses to tyrosine kinase inhibitors (TKIs) [7][8][9].
Transcriptional silencing of SHP-1 due to promoter methylation has been reported in lymphoma and leukemia as well as in many hematopoietic cell lines [12,22,23]. Epigenetic silencing of SHP-1 in myeloproliferative neoplasms and K562 cells results in constitutive activation of JAK/STAT signaling [24]. The restoration of SHP-1 expression by a demethylating agent such as 5-Aza-2-deoxycytidine (5-Aza2dc) resulted in decreased JAK3, p-JAK3, and p-STAT3 but not STAT3 protein [25]. STAT3 and STAT5 are constitutively activated in myeloid tumors [26]. Resistance to imatinib in chronic myeloid leukemia is conferred by the activation of STAT3 signaling, and the sensitivity is restored by STAT3 inactivation [27].
We hypothesized that JAK/STAT negative regulators may lose their tumor suppression function in TKIresistant AML cells due to epigenetic silencing, and the re-expression of these genes could re-induce sensitivity to CEP-701. Therefore, gene expression and methylation profiling of SHP-1 and its downstream targets were studied in FLT3-ITD positive AML cells resistant to CEP-701 before and after treatment with 5-Aza.

Lestaurtinib (CEP-701)
CEP-701 was purchased from LC Laboratories (Woburn, MA, USA) and dissolved in DMSO before use. The stock preparation was 1 mM, which was stored at −20°C according to the manufacturer's protocol.

Development of resistant cells
MV4-11, an AML cell line with FLT3-ITD, was obtained from the Department of Haematology, Universiti Sains Malaysia (USM), having originally been purchased from American Type Culture Collection (ATCC). The cells were cultured with RPMI 1640 (Life Technologies, Grand Island, NY, USA) supplemented with 10 % fetal bovine serum (FBS; Life Technologies, Grand Island, NY, USA) at a density of 5 × 10 4 cells/mL in a humid incubator with 5 % CO 2 at 37°C. A subclone of this line that was resistant to CEP-701, termed MV4-11R-cep, was developed according to the protocol described previously [28]. Briefly, log phase growing MV4-11 cells were cocultured at a starting dose of 20 nM CEP-701 followed by a step-wise increase in concentration of 10-20 nM for 12 months until the cells were able to survive at the IC 50 dose of CEP-701 on parental MV4-11. The resistant cell lines were grown in normal medium without CEP-701 for at least 48 h before starting the experiments.

5-Azacytidine treatment
5-Azacytidine (5-Aza; Sigma-Aldrich Corp., MO, USA) was dissolved in RPMI-1640 and the stocks at 500 μM were prepared for immediate use or stored at −20°C, to be used within 2-3 days. Resistant cells were sub-cultured in working solution (5 μM) and incubated in a humidified incubator with 5 % CO 2 at 37°C for 4-5 days until confluent. MV4-11R-cep + 5-Aza cells were then sub-cultured in normal medium without treatment for at least one passage before re-treatment with CEP-701.

Growth inhibition assay
MV4-11 cells were seeded in 96-well culture plates at a density of 1 × 10 4 viable cells/100 μL/well in triplicates, and were treated with CEP-701. Colorimetric CellTiter 96 AQueous One Solution Cell Proliferation assay (MTS assay; Promega, Madison, WI, USA) was used to determine the cytotoxicity. The IC 50 values were calculated using GraphPad Prism 3.02 (San Diego, California, USA). Each experiment was performed in triplicate.

Apoptosis assay
Annexin V-FITC binding assay (BD Pharmingen, San Diego, California, USA) was used as recommended by the manufacturer and analyzed by flow cytometry (BD FACSCanto™, San Jose, California, USA). Analysis was performed with Diva software (FACS Diva, 6.1.2, San Jose, California, USA). Each experiment was performed in triplicate.

Quantitative reverse transcriptase PCR (RT-qPCR)
High Capacity RNA-to-cDNA kit (Applied Biosystem, Foster City, California, USA) was used to synthesize cDNA according to the manufacturer's protocol. Taq-Man Gene Expression assays (Applied Biosystems) were performed on an Applied Biosystem 7500 Fast Real-Time PCR System according to the manufacturer's protocol. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. ABI 7500 software v2.0.6 (Applied Biosystem) was used to perform relative quantification of 5 target genes, SHP-1, SOCS-1, SOCS-3, STAT5a, and JAK2 using the comparative threshold cycle (Ct) method.

Methylation-specific polymerase chain reactions (MS-PCR)
One microgram of DNA was treated with bisulfite using the EZ DNA Methylation-Gold™ Kit (Zymo Research, Irvine, NY, USA) according to the manufacturer's instructions. MS-PCR was performed as described previously [29] and modified DNA was subjected to two separate PCRs. MS-PCR primers were designed to amplify the methylated (M) or unmethylated (U) alleles. SHP-1 (GeneBank: NM_002831) was amplified using previous designed primers [22]. Universal methylated DNA (Zymo Research, Irvine, NY, USA) was used as a positive control. The 50-μL PCR reaction contained 200 ng of bisulfite-treated DNA, ReddyMix PCR master mix (Bioline Ltd., London, UK) and 0.2 μM of each primer. PCRs were performed in a thermal cycler (PTC-200, Alameda, California, USA). The amplified PCR products were denatured for 2 min at 95°C followed by 40 cycles: 95°C for 25 s, 59°C for 35 s, 52°C and 72°C for 65 s, and extension at 72°C for 5 min. PCR products were electrophoresed on 2 % agarose gels, and visualized by ethidium bromide staining under ultraviolet transillumination. Results from triplicate experiments were used to determine methylation status.

Pyrosequencing analysis
Twenty microliters (1 μg) of purified DNA from each sample were sent to EpigenDx (Hopkinton, MA, USA) for pyrosequencing analysis. The assay was designed to target six CpG islands in the promoter regions of the SHP-1 gene.

Statistical and bioinformatics analysis
Repeated-measures ANOVA and Kruskal-Wallis tests were employed for statistical analyses. All statistical analyses were performed using the SPSS software package (Version 20, SPSS, Armonk, NY, USA) and a p value <0.05 was considered as significant.

Long-term co-culture of MV4-11 cells with low doses of CEP-701 resulted in resistant cells
To verify the resistance of MV4-11R-cep cells to CEP-701, we determined the cytotoxicity and apoptosis of CEP-701 on MV4-11R-cep and parental MV4-11 cells. MV4-11 cells were inhibited by 290 nM CEP-701 whereas the resistant MV4-11R-cep cells were only inhibited by a higher dose (3340 nM). The IC 50 of CEP-701 on MV4-11R-cep was more than 10-fold higher than that on MV4-11 (p = 0.004) ( Fig. 1-a). There was a significant decrease in the percentage of apoptotic cells in MV4-11R-cep compared with parental MV4-11 based on incubation of parental and resistant cells in serial concentrations of CEP-701 (p < 0.001) ( Fig. 1-b).

Activated STAT3 in resistant acute myeloid leukemia cells
The activation status of STAT1, STAT3, and STAT5 proteins were studied using western blot. Although these proteins were expressed in all cell lines, STAT3 was only activated in the resistant cells and not the parental and MV4-11R-cep + 5-Aza cells. In contrast, there was no differences in the activity of STAT1 and STAT5 between resistant and parental or MV4-11R-cep + 5-Aza cells (Fig. 1-c).

Low methylation of SHP-1 gene in MV4-11R-cep + 5-Aza cells
To relate the expression of SHP-1 with methylation status, MS-PCR and pyrosequencing analysis were performed on DNA from the three cell lines as well as positive and negative controls. MS-PCR revealed methylated SHP-1 in parental MV4-11 and resistant MV4-11Rcep cells but not MV4-11R-cep + 5-Aza cells (Fig. 4-b). The methylation status was confirmed by pyrosequencing analysis, which revealed a significantly lower level of methylation in CpG islands in the promoter region of the SHP-1 gene when treated with 5-Aza compared with untreated MV4-11 and MV4-11R-cep cells (p = 0.023). However, there was no significant difference in the methylation of CpG islands in the promoter region of SHP-1 in MV4-11 cells compared with MV4-11R-cep cells (p = 0.200; Table 1 and Fig. 4-c).
In the present study, FLT3-ITD+ AML cells resistant to CEP-701 were developed by overexposure of parental cells to the drug. Acquired resistance was confirmed by cytotoxicity and apoptosis assays that showed significant differences in parental compared with resistant cells. The IC 50 of CEP-701 on resistant MV-11R-cep was more than 10-fold higher than that of parental MV4-11 cells (p = 0.004) and acquired resistance was associated with low apoptosis (p < 0.001). This is in agreement with previously reported studies on the development of resistant cell lines to ABT-869 and PKC-412 [7,31].
Gene expression analysis revealed low expression of SHP-1 and PRG2 in MV4-11 and MV4-11R-cep cells (data not shown for PRG2 gene). However, after treatment of MV4-11R-cep cells with 5-Aza, we observed reexpression of SHP-1 and PRG2 that was associated with inhibition of STAT3 activity. Moreover, the transcriptional silencing of SHP-1 and PRG2 genes was due to hyermethylation of CpG islands in the promoter regions of both genes in MV4-11 and MV4-11R-cep cells. Transcriptional silencing of TSGs is mediated by DNMTs in tumor cells [43][44][45][46]. The gene expression analysis also showed significant up-regulation of DNMT1, DNMT3a, and DNMT3b (data not shown), which have been reported to regulate the expression of TSGs through methylation of CpG islands in the promoter regions [47,48]. Methylation profiling revealed hypermethylation of CpG islands in the promoter regions of SHP-1 and PRG2 in MV4-11 and MV4-11R-cep cells (data not shown for PRG2 gene). These findings suggest that, the up-regulated DNMTs in the parental and resistant cells methylate the CpG islands of SHP-1 and PRG2 genes, resulting in their transcriptional silencing. Our findings are consistent with previous reports that revealed hypermethylation of SHP-1 and PRG2 in leukemic cell lines [22,38,42,49].
In hematological malignancies and leukemic cell lines, the tumor-suppressing function of SHP-1 is lost because of promoter methylation, resulting in constitutive activation of JAK/STAT signaling [22,24,39,50,51]. Epigenetic silencing of one of the JAK/STAT negative regulators is sufficient for activation of STAT signaling [40]. Methylation of SHP1 is involved in the constitutive activation of STAT3 [50], and a low level of SHP-1 is not sufficient to inhibit activated STAT3 [25]. Transcriptional silencing of SHP-1 also plays a role in the development of resistance to imatinib in BCR-ABL1-positive CML cells [51]. Similarly, constitutive activation of STAT3 and STAT5 are common events in myeloid leukemia and have previously been implicated in resistance to TKIs [7,26,52]. Bewry, et al. [27] suggested that, the activation of STAT3 is an important mechanism of imatinib resistance. Likewise, overexpression of PRG2 in myeloid cells blocked G-CSF-dependent proliferation and increased apoptosis [53]. However, epigenetic silencing of PRG2 is associated with higher proliferation and lowered apoptosis in pancreatic cancer cells [54] and leukemic cells [42]. In the present study, we observed activation of STAT3 in MV4-11R-cep cells but not in MV4-11 cells. The findings suggest that, activated STAT3 could be involved in the acquisition of resistance to CEP-701 in MV4-11Rcep cells, which is consistent with previous reports in which, the activation of STAT3 was associated with acquired resistance to ABT-869 in AML [7], and to imatinib in CML [27,51]. After treatment of MV4-11R-cep with 5-Aza, STAT3 was inactivated and cells showed higher sensitivity to CEP-701. This finding is in accordance with other reports [7,25,51]. Our data suggest a crucial role for STAT3 in the development of resistance to TKIs and inhibition of STAT3 phosphorylation provides an effective means of re-inducing sensitivity.
We also found that, increasing CEP-701 concentration caused a significant increase of apoptosis in 5-Aza-treated resistant cells compared with the untreated resistant cells (p < 0.001) In addition, we observed no significant difference when CEP-701 concentration was increased by 100 nM in untreated resistant cells whereas the 5-Aza-treated resistant cells showed significant differences at each incremental increase in TKI concentration. Taken together, our results indicate that, re-expression of SHP-1 and suppression of STAT3 are associated with induction of apoptosis in TKI-resistant FLT3-ITD+ AML cells. These findings are similar to those previously reported [7,[60][61][62].
Enhanced re-expression of SHP-1 could therefore play role in the management of CEP-701-resistant patients. Further studies are needed to clarify the correlation between the re-expression of the SHP-1 gene with STAT3 inhibition and to confirm the clinical utility of this approach.

Conclusion
The epigenetic silencing of SHP-1 results in loss of its tumor suppressor function and re-expression of SHP-1 by 5-Aza may enhance sensitivity to CEP-701 through inactivation of STAT3.