INNO-406 inhibits the growth of chronic myeloid leukemia and promotes its apoptosis via targeting PTEN

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm. INNO-406 is a novel tyrosine kinase inhibitor (TKI) that possess specific Lyn kinase inhibitory activity with no or limited activity against other sarcoma (Src) family member kinases. The present study aimed to confirm the anti-tumor effect of INNO-406 on CML cells, and elucidate the underlying molecular mechanism. CML cells were treated by INNO-406 at the concentration of 5, 25, 50, 100 μM at the indicated time. Cell proliferation was measured by MTT. Cell apoptosis were detected by Western blot and flow cytometry, respectively. As suggested by the findings, INNO-406 significantly inhibited the proliferation and induced apoptosis of CML cells. In addition, INNO-406 promoted the expression level of PTEN. Rescue experiment revealed that PTEN knockdown reversed the effect of INNO-406 which indicated the correlation between INNO-406 and PTEN. Further study determined that PTEN inhibited the phosphorylation of AKT and 4EBP1 and subsequently altered the expression of apoptotic proteins including bax, cytoplasmic cytochrome c (cyto-c), cleaved caspase3 and bcl-2. In vivo study further confirmed that INNO-406 inhibited the growth of CML cells by targeting PTEN. Based on the above findings, this work extended our understanding of INNO-406 in the therapy of CML and its molecular mechanism.


Introduction
Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by the formation of the fusion gene consist of Abelson murine leukemia (ABL) gene and breakpoint cluster region (BCR) which encodes a constitutively active Bcr-Abl tyrosine kinase [1][2][3]. Tyrosine kinase inhibitors (TKIs) targeting BCR-ABL1 tyrosine kinase have made great development in the treatment of CML over the decades which improved 10 year overall survival from approximately 20% to 80-90% [4,5]. However, about 25% of the children and half of the adults are still insensitive to chemotherapy or will relapse. Moreover, side effects, such as skin toxicity and allergic reaction are still clinical challenges which limited application of TKI [6,7]. Thus, a better understanding of the molecular mechanisms involved in the progression of CML is urgently needed.
Bafetinib (INNO-406) is a novel tyrosine kinase inhibitor designed based on the chemical structure of imatinib for treating Bcr-Abl + leukemia [8,9]. It is a potent dual function inhibitor of both Bcr-Abl kinase and Src kinase Lyn which is developed to overcome imatinib resistance [10,11]. According to the previous study, INNO-406 exerted a higher potency in inhibiting Abl than imatinib; moreover, it inhibited Lyn with higher selectivity than other SFK/Abl inhibitors. INNO-406 may be effective in the CML treatment with possible application to central nervus system leukemia and also be less liable to cause unfavorable side effects than other therapeutic agents that target multiple kinases, such as SFK inhibitors [12,13]. The limitation of chemoresistance on the therapeutic effect of chemotherapy drugs makes the research on enhancing drug sensitivity or inhibiting drug resistance as important as the development of new drugs.
In the present study, we tried to elucidate the effect and the underlying mechanism of Bafetinib on the proliferation and apoptosis of CML cells. We found that Bafetinib is capable of inhibiting proliferation and inducing apoptosis of CML cells. In addition, it notably promotes the expression of PTEN and modulates the downstream AKT/4EPB1 signal pathway.

Cell culture and reagents
The human chronic myeloid cell lines (K562 and KCL22) were purchased from the Chinese Academy of Sciences. The culturing of cells was carried out in Dulbecco's Minimum Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Hyclone, USA). Each cell line was cultured at 37 °C with 5% CO 2 . In addition, the cells were treated with the indicated concentration of INNO-406 (Selleck, USA), whereas the control cells received an equivalent amount of DMSO. The final concentration of DMSO is below 0.1%. The siRNA for knocking down of PTEN was synthesized by Genepharma (Shanghai, China).

MTT assay
The detection of the cell viability was carried out with MTT assay (Sigma-Aldrich, USA). The K562 and KCL22 cells were seeded onto 96-well plates followed by treating with 5, 25, 50 and 100 μM of INNO-406 (Bio-techne Corporation, MN, USA) for 24 h. Thereafter, the addition of 10 μl MTT to the cells was done, followed by incubating at a temperature of 37 °C for 2 h. Optical density was measured with the use of a microplate reader at 490 nm. Moreover, the calculation of the inhibition rates was carried out as well. Each trial was conducted at least 3 times.

Cell apoptosis
After being treated with different concentrations of INNO-406, the collection of both K562 and KCL22 cells was done, which were further washed with PBS 3 times. Concerning the cell apoptosis analysis, cells were stained with annexin V-FITC/propidium iodide following the guidelines of the manufacturer (Sungene, Tianjin, China). Cell apoptosis was detected by a BD FACS Calibur flow cytometry system (Becton Dickinson, NJ, USA). Each trial was conducted at least 3 times.

Western blot analysis
Total proteins were extracted using RIPA-Buffer supplemented with 10 mM PMSF (Beyotime, Shanghai, China). A bicinchoninic acid assay (BCA) was carried out to quantify protein concentrations. The separation of 40 μg proteins was carried out on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by transferring to polyvinylidene fluoride (PVDF) membranes. Thereafter, the membranes were blocked with 5% non-fat milk in Tris-buffered saline and 0.1% Tween 20. Subsequently, the membranes were incubated with primary antibodies at 4 °C overnight. After washing with TBST buffer for 3 times, the membranes were incubated with the secondary antibody for another 2 h at room temperature. An enhanced chemiluminescence (ECL) system kit (Beyotime, Shanghai, China) was employed for the detection of bands. The gray intensity was analyzed by ImageJ software (NIH, Bethesda, MD, USA). Each trial was conducted at least 3 times.

Immunofluorescence
The tumor tissues were fixed in 4% paraformaldehyde for 24 h, followed by dehydrating in a graded alcohol series and embedding in paraffin, followed by cutting into 5 μm sections. The sections were deparaffinized, rehydrated with a graded alcohol series and then incubated in 96 °C with 10 mM sodium citrate buffer for the antigen retrieval. Following the incubation in 5% H 2 O 2 for 2 h, the sections were incubated using primary antibodies overnight at 4 °C. Immunostaining was carried out with the use of streptavidinperoxidase following the manufacturer's instructions (Beyotime, Shanghai, China). Finally, the sections were observed under a fluorescence microscope (Leica, Wetzlar, Germany). injected into the posterior flank region of the nude mice. The long diameter and short diameter were detected every 2 days, together with calculating with the use of the formula, which is as follows: tumor volume = 0.5 × long diameter × short diameter 2 . The mice were injected intraperitoneally with 5 mg/kg INNO-406 (Bio-techne Corporation, MN, USA) every two days since the cell injection. From the first day of cell injection, Adenovirus (Hanbio, Beijing) for silencing the expression of PTEN of 3.5 × 10 7 virus particles was injected by tail vein each time. The administration of adenovirus was once a week and continued for 4 weeks. Finally, the mice were sacrificed followed by excising the tumors. SPSS 17.0 software (SPSS Inc., Chicago, IL, USA) was used to analyze all data for statistical significance. All the data are presented as the means ± SD. One-way ANOVA was used to assess the difference between multiple groups. Differences between two groups were analyzed by the Student's t test. p < 0.05 was considered as statistical significance.

INNO-406 inhibited the proliferation of chronic myeloid leukemia cells
To investigate the effect of INNO-406 on the growth of CML cells including K562, and KCL22, MTT assay was carried out to detect the proliferation of those cells following the treatment of INNO-406 at the dose of 5, 25, 50 and 100 μM for 24 h. As revealed in Fig. 1, INNO-406 of 25, 50 and 100 μM significantly inhibited the proliferation of K562, and KCL22 cells (Fig. 1a). Moreover, we evaluated the growth curve of K562 and KCL22 cells during 72 h under INNO-406 treatment. It was suggested by the findings that INNO-406 notably inhibited the growth of both K562 and KCL22 cells (Fig. 1b, c).

INNO-406 induced apoptosis of CML cells
Apoptosis is well known to be involved in the antitumor effects. We next investigated whether INNO-406 can induce apoptosis of CML cells. Annexin V/PI dual staining and flow cytometry were used to evaluate the apoptosis of CML cells. We found that INNO-406 of 25 and 50 μM induced the apoptosis of CML cells, while 5 μM INNO-406 did not influence the apoptosis of CML cells (Fig. 2a, b). In addition, to confirm our observation, we detected the expression of apoptotic proteins. Accordingly, INNO-406 of 25 and 50 μM promoted the expression of bax, cytoplasmic cyto-c and cleaved caspase3, while inhibited that of bcl-2 (Fig. 2c).

INNO-406 promoted the expression of PTEN
To elucidate the molecular mechanism underlying the effect of INNO-406 on CML cells, we evaluated the expression of PTEN which is a superstar in anti-cancer research. We detected the level of PTEN under INNO-406 treatment. Interestingly, qPCR (Fig. 3a), western blot (Fig. 3b) and immunofluorescence assay (Fig. 3c)

INNO-406 modulated PTEN expression and the phosphorylation of AKT/4EBP1
As we know, PTEN was involved in the regulation of PI3K/ AKT pathway. We speculated that INNO-406 might regulate the progression of apoptosis through modulating PTEN mediated PI3K/AKT signaling pathway. Western blot demonstrated that INNO-406 inhibited the phosphorylation of AKT as well as 4EBP1 in CML cells (Fig. 5a). PTEN knockdown reversed the effect of INNO-406 significantly (Fig. 5a). Then, apoptotic protein expression was evaluated, we found that PTEN knockdown reversed the effect of INNO-406 on promoting the expression of bax, cytoplasmic cyto-c, cleaved caspase3 and inhibiting the expression of bcl-2 (Fig. 5b).

INNO-406 inhibited the CML cell growth in vivo
In vivo study was carried out to further investigate the impact of INNO-406 on CML. INNO-406 significantly suppressed the tumor growth and reduced the weight of tumors in comparison with control group. Interestingly, this effect could be reversed by knockdown of PTEN by tail vein injection of sh-PTEN adenovirus (Fig. 6a-c).  Being different from the second-generation TKIs, INNO-406 demonstrates specific Lyn kinase activity with no or limited activity against other Src-family member kinases [13][14][15]. However, the potential chemoresistance of INNO-406 will be the key factor limiting its application. Elucidating the molecular mechanism underlying its biological effects will help to alleviate the influence brought by chemoresistance. Previous studies have been carried out to explore the precise mechanism of INNO-406. It was demonstrated that INNO-406 induces apoptosis of Bcr-Abl + leukemic cells by modulating the expression of Bim and, other BH3-only proteins, such as Bad, Bmf and Bik 17510658. In addition, Y Kamitsuji reported that INNO-406 induces programmed cell death (PCD) in CML cell lines through both caspase-mediated and caspase-independent pathways. INNO-406 can also induce autophagy of CML cells, when autophagy was inhibited, INNO-406-induced cell death was enhanced, which indicates that the autophagic response of the tumor cells is protective18617896. Imortantly, it was found that even in the presence of the pan-caspase inhibitor zVAD-fmk, NS-187 still induces apoptosis in some cells, indicating the additional involvement of NS-187 in a caspase-independent apoptotic pathway 19662183.

Discussion
In order to find out the mechanism underlying the effect of INNO-406 on proliferation and apoptosis, we focused on PTEN. PTEN/ PI3K/ Akt is a well known critical signaling axis to regulate the signal transduction of many biological processes including apoptosis, metabolism, and proliferation [16][17][18]. PTEN is a protein phosphatase that can dephosphorize serine and threonine residues [19,20]. It has been previously confirmed that PTEN is capable of inhibiting phosphorylation of AKT [21,22]. In our study, we confirmed that up-regulated PTEN expression by INNO-406 treatment inhibited the phosphorylation of AKT. 4EBP1 is a downstream protein of AKT/mTOR pathway which is crucial for protein synthesis due to its effect of binding to eIF4E and stopping the formation of the cap-dependent translation initiation complex [23,24]. 4EBP1 has been indicated to participate in the regulation of apoptosis by targeting various genes. For instance, 4EBP1 modulate Bcl-2 expression to change apoptosis of the penumbral cortex in cerebral infarction injury [25].    [26].
In the present study, we determined that PTEN inhibited the phosphorylation of AKT and 4EPB1 and influenced the expression of apoptotic proteins such as bax, cytoplasmic cyto-c, cleaved caspase3 and bcl-2. However, the evaluation of p-AKT and p-4EPB1 is not enough to prove the role of AKT/4EPB1 signalling underlying the effect of INNO-406, at least the evaluation of mTOR and s6k should be carried out.
Taken together, we indicated that INNO-406 was capable of inhibiting the proliferation of CML cells through inducing apoptosis. In spite of the effect on inhibition of Bcr/Abl fusion protein tyrosine kinase, these findings may extend out understanding of INNO-406 in the chemotherapy of CML and its molecular mechanism. The underlying mechanism was involved in the regulation of PTEN/AKT/4EPB1 signal pathway. Further understanding of action of INNO-406 in the drug resistance will help for avoiding or solving the problem of drug resistance.
Author contributions SUN J acquired the data, WANG Y helped analysis data, SUN L supervised the project.
Data accessibility All data is available at the request of corresponding author. Fig. 6 INNO-406 inhibited the chronic myeloid leukemia growth in vivo. a The subcutaneous xenograft tumors were generated from CML cells in nude mice (n = 6). b Tumor growth in nude mice was indicated by the curves represented the trend of the tumor size increase. c The weight of the tumors was shown (n = 6). *p < 0.05 vs control, #p < 0.05 vs INNO-406 group