Study Of The Anti-Tumor Effect Of Wee1 Kinase Inhibitor MK-1775 In Acute Lymphoblastic Leukemia

Background:Acute lymphoblastic leukemia (ALL) is a common hematological malignancy that does great harm to the health of human being. Adults with ALL usually have bad outcomes. Combined chemotherapy is usually accompanied by serious side effects such as infection, bleeding events and organs damage. Allogenic hematopoietic stem cell transplant (Allo-HSCT) is a therapeutic regimen of high risk and fragile patients are not applicable to it. In addition,some patients suffer from graft versus host disease (GVHD) or leukemia relapse after Allo-HST that has adverse impacts on survival of patients. So it is of great significance to explore and develop new agents with high efficiency and low toxity.Cell cycle checkpoint plays a crucial role in the regulation of mitosis. It is responsible for repair of damaged DNA and maintaining of gene structure integrity. Weel is a member of serine/threonine kinase superfamily that acts as a checkpoint kinase that regulates the G2/M transformation. Furthermore,it plays a critical role in the synthesis of hisone and repair of DNA damage. Weel is high expressed in many types of tumors. Inhibition of Weel expression showed great anti-tumor activity by inducing cell cycle disturbance,proliferation inhibition and cell apoptosis. It may be a novel target of tumor treatment.PI3K/AKT/mTOR and Notch are two signal pathways that play crucial roles in the pathogenesis of ALL.They are responsible for the proliferation,differentiation and invasion of leukemia cells.The relationship between Weel and PI3K/AKT/mTOR or Notch is not clear currently.Objective:Detect the levels of Weel expression in ALL patients,study the influence of Weel inhibitor MK-1775 on cellular viability,cell cycle and apoptosis and explore its mechanism of anti-ALL.Materials and methods:1.Bone marrow samples of ALL patients and healthy controls were collected. Quantitive RT-PCR was used to detect the Weel mRNA levels in both groups.2.MTT was used to detect the influence of Weel inhibitor MK-1775 and adriamycin on the cellular viability of ALL cells. ALL cells Nalm-6,Jurkat,Sup-T1,Molt-4 and bone marrow stromal cell HS-5 were cultured with different concentration of MK-1775, adriamycin or the combination for 24,48 and 72 hours. MTT was used to detect the cellular viability and IC50 was calculated.3.Cell cycle analysis. Nalm-6 and Jurkat were cultured with MK-1775 of various concentration for 24 hours.Cells were collected and stained with PI then the DNA content was analyzed with flow cytometry and cell cycle was calculated.4.Apoptosis detection. Nalm-6 and Jurkat were cultured with MK-1775, adriamycin or the combination for indicated time.Cells were collected and stained with PI and Annexin V-FITC then the apoptotic cells were analyzed with flow cytometry.5.Western blot. Nalm-6 and Jurkat were cultured with MK-1775, adriamycin or the combination for 24 hours.Total protein was extracted and western blot was used to detect the relative levels of CDK1,p-CDK1,p-H3,PARP1,γ-H2AX,AKT,mTOR,p-mTOR,bcl-2,Notchl and Hesl.Results:1.The expression of Weel in ALL patients and healthy controls.The relative mRNA level of Weel in ALL patients was 0.15±0.08 that was higher than the control group 0.09±0.05(p<0.05).2.The effect of Weel inhibitor MK-1775 on cellular viability.(1)MK-1775 supressed the viability of ALL cells on a time and dose dependent manner. The IC50 of Nalm-6,Molt-4,Jurkat and Sup-T1 was 45.88±3.90 nM,67.60±31.80 nM,137.39±33.99 nM and 347.37±95.47nM repectively with 48 hours of treatment.(2) MK-1775 supressed the viability of HS-5 slightly.IC50 of HS-50 was higher than Nalm-6,Molt-4,Jurkat and Sup-Tl(p<0.05).(3) MK-1775 enhanced the anti-ALL activity of adriamycin.With 48 hours of treatment of Nalm-6,IC50 of adriamycin was dropped from 0.058±0.017 mg/ml in the group of single adriamycin treatment to 0.018±0.012mg/ml in the group of adriamycin with additional MK-1775 100nM treatment(p<0.05).Similarly,IC5o of adriamycin was dropped from 0.044±0.017mg/ml to 0.003±0.0005 mg/ml of Jurkat(p<0.05).3.Cell cycle analysis.Nalm-6 and Jurkat were cultured in six-well plates with 0 nM、 100 nM、300nM of MK-1775 for 24 hours and collected for detection of DNA content.Cell cycles were analyzed according to DNA content. Nalm-6 of G1 phase was 39.2±9.7%、34.8±5.0%(p>0.05 compared with control) and 33.3±5.4%(p>0.05 compared with control), S phase was 55.1±7.7%、60.2±9.6%(p>0.05 compared with control) and 61.4±17.2%(p>0.05 compared with control), G2/M phase was 3.3±2.0%、3.9±3.5%(p>0.05 compared with control)and 7.2±9.0%(p>0.05 compared with control) respectively;Jurkat of Gi phase was 42.5±4.1%、43.3±7.5%(p>0.05 compared with control)and 15.6±3.5%(p<0.05 compared with control), S phase was 42.6±6.1% 和 43.3±7.5%(p>0.05 compared with control).62.6±9.6%(p<0.05 compared with control), G2/M phase was 12.6±2.7%、14.0±3.4%(p>0.05 compared with control) 和 27.0±4.9%(p<0.05 compared with control)4. Apoptosis detection.(1)MK-1775 induces cells apoptosis on a dose dependent mode.Cultured with OnM, 100nM and 300nM of MK-1775 for 48 hours,the apoptosis proportion of Nalm-6 was 3.46±2.25%、64.82±18.44%(p<0.05 compared with control) and 88.83±7.65%(p<0.05 compared with control) respectively, and the apoptosis proportion of Jurkat was 2.12±0.47%、41.26±12.30%(p<0.05 compared with control) and 70.08±8.52%(p<0.05 compared with control) respectively.(2) After treatment of 24 hours,the apoptotic rate of Nalm-6 in the control group, 0.05mg/ml ADM group、100nM MK-1775 group and 0.05mg/ml ADM combined with 100nM MK-1775 group was 2.30±1.13%、12.8±5.07%、17.15±7.89% and 49.42±6.82% respectively; Apoptotic rate of Jurkat was 3.01±1.31%、18.50±7.67%、 23.25±10.87、67.16±7.67 respectively. MK-1775 combined with adriamycin induced higher apoptotic rates than each single agent and the control (p<0.05)5.Western blot analysis. After treatment of 24 hours cells of the control group, 0.05mg/ml ADM group、100nM MK-1775 group and 0.05mg/ml ADM combined with 100nM MK-1775 group were collected and total protein was extracted. Western blot analysis reavealed that MK-1775 treatment suppressed the phosphorylation of CDK1,promoted PARP1 cleavage increased the expression of p-H3 and γ-H2AX,decreased the expression of Notch1 and Hesl.AKT and mTOR levels were not altered.Conclusions:1. Weel was high expressed in ALL patients that indicates Weel might play a critical role in the pathogenesis of ALL.2. Weel inhibitor MK-1775 supresses the cellular viability of ALL cells and induces apoptosis on a time and dose-dependent mode.ALL with mutated p53 or the wild type are both sensitive to MK-1775 treatment.MK-1775 enhances the anti-ALL effect of adriamycin.3. MK-1775 has a slight influence on the viability of bone marrow stromal cells.4. MK-1775 shows anti-ALL effect by inducing mitotic catastrophe due to driving cells into mitosis prematurely.It induces apoptosis by supressing DNA repair and increasing DNA lesion.5. The anti-ALL effect of MK-1775 is accompanied by downregulation of Notch1 and Hes1 but no alteration of AKT-mTOR pathway.6. The combination of Weel inhibitor MK-1775 and cytotoxic drugs may be a promising anti-ALL regimine with high effenciency and low side effects.