| ObjectiveAcute myeloid leukemia (AML) refers to a genetically and biologically heterogeneous group of myeloid leukaemias that are characterized by clonal expansion of immature myeloid progenitor stages in blood, bone marrow, or other tissues. Therefore, differentiation therapy is regarded as a effective method for treatment of AML in clinic.All-trans retinoic acid (ATRA) is the biologically active form of vitamin A, which was successfully employed in the treatment of acute promyelocytic leukemia (APL), a distinct subtype of acute myeloid leukemia (AML). However, the current differentiation drugs still have some limitations, such as toxicity and resistance. Furturemore, the use of ATRA as a single agent has not been approved for the clinical management of leukemia other than APLs. It is believed that one possible means to overcome these problems might be the combination of ATRA with other agents.Human epidermal growth factor receptor 2 (HER2) is a member of the ErbB family which plays a fundamental role in regulation of mammalian cell survival, proliferation, adhesion, and differentiation. Several studies have demonstrated that inhibition of HER2 pathway may be a potential therapeutic for leukemia. Mubritinib (TAK165) is a selective inhibitor of HER2, which is under development by Takeda for the treatment of cancer. Studies show that TAK165 exhibits antitumor effect on a serious of human cancer cells including AMLs by inducing apoptosis. Studies on the effects of TAK165 induced differentiation in AML cells were rarely reported. Our study was the first to evaluate TAK165 and ATRA synergy in AML cell differentiation and gain insight into the mechanisms underlying the synergism of TAK165 and ATRA, thus suggesting TAK165and ATRA combination as a promising approach for future differentiation therapy.Methods1) Human acute myeloid leukemia cells HL60 and. NB4 were treated with TAK165 and ATRA. Cell growth curve and surviving curve was performed by trypan blue staining with manual counting each day. Cell cycle distribution was detected by flow cytometry. Cell differentiation-related biochemical function was evaluated by NBT-reducing activity. Cell surface marker CD11b was detected by flow cytometry. Cell morphology was assessed by Wright-Giemsa staining. The transcriptional level of PU.1, C/EBPB and C/EBPE was measured through Real-time PCR. Western-blot analysis for the detection of protein level (c-Myc, p21, p27, C/EBPβ, PU.1).2) Human acute myeloid leukemia cells HL60 and NB4 were used to examine the molecular mechanisms of TAK165 enhanced ATRA induced myeloid differentiation. Cell cycle distribution was detected by flow cytometry. Cell differentiation-related biochemical function was evaluated by NBT-reducing activity. Cell surface marker CD lib was detected by flow cytometry. The transcriptional level of RARB, PXN was measured through Real-time PCR. Cell morphology was assessed by Wright-Giemsa staining. Western-blot analysis for the detection of protein level (HER2, RARa, STAT1, p-STAT1 (S727), MEK, p-MEK, ERK, p-ERK, p38, p-p38, JNK, p-JNK). Herceptin was used to explore the involvement of HER2 in TAK165 and ATRA-induced AML differentiation. Lentivirus-mediated pCCL-RARa transfected into HL60R cells to investigate the role of RARa activation in TAK165 and ATRA combination. Lentivirus-mediated shRNA-STAT1 transfected into NB4 cells to evaluate the role of STAT1 in the combination treatment induced differentiation of AML cells. MAPK inhibitors (PD98059, U0126, SP600125 and SB203580) were used to further explore the involvement of MAPK pathway in activation of RARa/STAT1 axis.Results1) TAK165 inhibited cell proliferation and induced G0/G1 cell cycle arrest in AML cellsTrypan blue exclusion staining showed that TAK165 can significantly inhibit proliferation of HL60 and NB4 cells without any cell death, which was a concentration-dependent and time-dependent process. Cell cycle profile was detected by flow cytometry, an increase in the proportion of cells in G0/G1 phase was observed in HL60 and NB4 cells when treated with TAK165. Furturemore, TAK165 significantly decreased the protein expression of c-myc, p21 and p27.2) TAK165 sensitizes AML cells to ATRA-induced differentiationTrypan blue exclusion staining showed that TAK165 significantly enhanced ATRA-induced growth arrest of HL60 and NB4 cells in a dose-dependent manner.Consistently, TAK165 also strengthened the accumulation of AML cells in the G0/G1 phase of the cell cycle induced by ATRA. We then assessed differentiation using standard assays for myeloid differentiation.As assessed by CD11b expression, TAK165 significantly promoted ATRA-induced differentiation in a dose-dependent manner.A significant enhance in NBT-positive NB4 cells was observed after TAK165 treatment when compared with ATRA alone group. Compared with ATRA or TAK165 alone treated cells, when treated with ATRA and TAK165 in combination had more mature morphology, with an increased cytoplasmic to nuclear ratio and obvious nuclear segmentation. Real-time PCR results showed obvious increasing of CEBPE, CEBPB and PU.1 mRNA levels after combination ATRA and TAK165 treatment. Similarly, protein expression analysis also confirmed the strong up-regulation of C/EBPβ and PU.1 after ATRA and TAK165 co-treatment.3)Mechanisms underlying the synergism of TAK165 and ATRAHER2 protein was undetectable in HL60 and NB4 cells, compired with a breast cancer cell line BT474 which HER2 high-expression. Herceptin was further used to evaluate the role of HER2 during ATRA-induced differentiation of AML cells. Herceptin had no effect to enhance ATRA-induced cell cycle arrest, while TAK165 could do so. Meanwhile, Herceptin also had no effect on ATRA and TAK165combination treatment caused cell cycle arrest. In addition, there were no obvious alterations in the myeloid differentiation driven by either ATRA alone or ATRA and TAK165 combination treatment as assessed by CD11b expression. Our data suggested that HER2 pathway was dispensable for myeloid differentiation induced by TAK165 and ATRA combination treatment.The mRNA level of RARa target genes, RARB and PXN were increased in TAK165 and ATRA co-treated group. in sharp contrast to ATRA-responsive cells, HL60R cells which exhibits relative resistance to RA showed no changes in the mRNA expression of RARB and PXN when exposed to ATRA or ATRA plus TAK165. Besides, no visible differentiation as reflected by CD11b expression was observed in HL60R cells treated with ATRA,ATRA plus TAK165 or even high concentration of ATRA (10μM). Then HL60R cells were transduced with lentiviral-vector pCCL or lentiviral-pCCL-RARa. Compared with HL60R-pCCL cells, significant up-regulation of RARa target gene mRNA expression and strong increase in CDllb-positive cells were both detected in HL60R-RARa cells treated with ATRA or ATRA plus TAK165. These findings indicated that the activation of RARa was critical in TAK165 and ATRA induced differentiation of AML cells.Compared with ATRA or TAK165 single treatment groups, significant increased phosphorylation of STAT1 (s727) was observed in TAK165 and ATRA combination treatment group. To further evaluate the role of STAT1, we knocked down STAT1 expression with two shRNAs.. STAT1-knockdown cells showed markedly reduced CD11b expression after ATRA plus TAK165 treatment compared with control cells (80.1%±.21%,42.0% ±6.11%, and 37.6%±1.57% for scramble, shSTAT1#1 and shSTAT1#2). Hence, these results clearly indicated that STAT1 induction contributed to myeloid differentiation upon TAK165 and ATRA treatment.In HL60 and NB4 cells, treatment with TAK165 or ATRA alone led to an increase in phosphorylation of MEK and ERK, and the combination treatment had an even greater effect than monotreatments. In contrast, p-p38 and p-JNK were not further activated in ATRA plus TAK165 cotreated AML cells. Moreover, little change of CDllb expression was detected in all cells treated with JNK inhibitor (sp600125) or p38 inhibitor (SB203580). However, MEK/ERK inhibitors (PD98059 and U0126) significantly inhibited combination treatmentinduced CD11b expression in HL60 and NB4 cells from 82.37%±0.37% to 56.22%±0.68%,36.5%±2.88%, respectly. In addition, the phosphorylation of STAT1 was also strikingly inhibited by PD98059 and U0126 in AML cells, indicating TAK165 and ATRA combination treatment induced activation of STAT1 was dependent on MEK/ERK-cascades activation.ConclusionOur study evaluated the effects of TAK165 to enhance ATRA-induced differentiation ofAML cells. Data presented here demonstrated that TAK165 could combine with ATRA to induce myeloid differentiate ofAML cells via MEK/ERK-dependent activation of RARa/STAT1 axis. We provided the new combination therapy which might help us developing new treatments approaches for human myeloid leukemia. |
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