ATPR Induces Acute Promyelocytic Leukemia NB4 Cells Differentiation And Proliferation Inhibition By Blockade Of SHP2/Rho/ROCK1 Pathway

Acute promyelocytic leukemia（acute promyelocytic leukemia,APL） is a specific biological subtype of acute myeloid leukemia（AML）.Its main feature is the excessive production of immature myeloid cells and the arrest arrest at promyelocytic stage.At present,the differentiation therapy based on all-trans retinoic acid（ATRA）is the first choice for clinical treatment of APL.However,20%patients occur recurrence,drug resistance,and retinoic acid syndrome after treatment.In order to improve the stability and reduce the side effects of ATRA,our group carried out a series of structural modifications and pharmacodynamic screening in vitro,and finally synthesized a new retinoic acid derivatives,4-amino-2-trifluoromethyl phenyl retinoate（ATPR）,which has successfully obtained invention patents of China,the United States and Europe.It has been proved that ATPR has good therapeutic activity against many types of hematological malignant tumors,such as leukemia,lymphoma,myelodysplastic syndrome and so on.According to the study on the tissue distribution of ATPR in rats,it was found that the blood concentration of ATPR in the liver could be maintained at a high level for a long time after intragastric and intravenous injection.A large number of studies have confirmed that ATPR can significantly inhibit the proliferation and promote the differentiation of many kinds of AML cells（NB4,U937,MOLM-13 and THP-1） in vitro,and also show a obvious therapeutic effect in transplanted AML mouse model.However,the molecular mechanism of its role in APL has not been fully revealed.Protein phosphorylation regulated by protein tyrosine phosphatase（PTPs）is a critical post-translational modification,which has an important effect on a variety of physiological and biochemical processes by regulating the activity and function of proteins.More and more studies have shown that PTPs are the key regulatory factors to maintain biological homeostasis,and misregulated PTPs activity has been implicated as causative in the occurrence and development of many tumors.As the first reported protein phosphatase oncogene,the high expression and function-acquired mutation of Src homology region 2-containing protein tyrosine phosphatase-2（SHP2） was found in many kinds of leukemia.And a number of studies have shown that the level of SHP2 is positively correlated with the occurrence of drug resistance in leukemia after treatment,suggesting that it plays a potential role in the occurrence and prognosis of leukemia.In addition,the results of preliminary experiments showed that ATPR could significantly inhibit the expression and phosphorylation of SHP2 in APL cells.Therefore,this study will further clarify the role and potential molecular mechanism of SHP2 in ATPR-induced differentiation and proliferation inhibition of APL cells,thus providing a new theoretical and experimental foundation for preclinical research of ATPR.Objectives:This study aims to explore the role and the potential molecular mechanism of SHP2 in ATPR-induced differentiation and proliferation inhibition of acute promyelocytic leukemia NB4 cells.Methods:1.The effect of ATPR on the expression and activity of SHP2Western blotting was used to detect the difference of SHP2 and p-SHP2 expression in APL patients peripheral blood and APL cell lines compared with normal controls.Western blotting and q RT-PCR were used to detect the expression of SHP2 and p-SHP2in NB4 cells treated with ATPR in different concentrations(10^-5-10^-7M) at different times（0 h,24 h,48 h,72 h）.Then,Western blotting was used to detect the effect of ATPR on the expression of SHP2 and p-SHP2 in non-APL cells（HL-60,THP-1）.2.The influence of sh SHP2 or SHP099 in ATPR induced differentiation and proliferation inhibitionof NB4 cellsFollowing pretreatment with lentivirus containing sh SHP2 or SHP099（the specific inhibitor of SHP2） for 2 h,NB4 cells were then exposed to ATPR(10^-6M) for another72 h.For each treatment group:the expression of cell differentiation related proteins（CD11b,CD14） was detected by western blotting and flow cytometry,and the changes of cell morphology were observed by Wright–Giemsa staining.The expression of cell proliferation related protein（PCNA）and cell cycle related proteins（Cyclin D3,Cyclin A2,p-Rb） was detected by western blotting,the distribution of cell cycle was detected by flow cytometry,and the expression of cell proliferation marker Ki67 was observed by immunofluorescence staining.3.The effect of SHP2 on the Rho/ROCK1 pathwayIn NB4 cells,western blotting was used to detect the expression of Rho and ROCK1 after silencing or inhibiting SHP2 in vitro.By constructing subcutaneous xenograft mouse models,the effect of SHP2 silencing on tumor formation ability of NB4 cells was examined.Then,the expression of Rho and ROCK1 in the tumor tissues was analyzed by western blotting and immunohistochemistry.4.The role of Rho/ROCK1 pathway in ATPR-induced differentiation and proliferation inhibition of NB4 cellsWestern blotting was performed to evaluate the levels of Rho/ROCK1 in NB4 cells incubated with ATPR in different concentrations(10^-5,10^-6,10^-7M) at different times（0 h,24 h,48 h,72 h）.Then,western blotting was used to detect the effect of ATPR on the expression of Rho and ROCK1 in HL-60and THP-1 cells.Following pretreatment with Y-27632（the specific ROCK1 inhibitor）for 2 h,cells were then exposed to ATPR(10^-6M) for another 72 h.For each treatment group:the expression of cell differentiation related proteins（CD11b,CD14）,cell proliferation related protein（PCNA） and cell cycle related proteins（Cyclin D3,Cyclin A2,p-Rb） differentiation related proteins and cell cycle related proteins was detected by western blotting,the changes of surface differentiation antigens and the distribution of cell cycle were detected by flow cytometry,the changes of cell morphology were observed by Wright–Giemsa staining,and the expression of cell proliferation marker Ki67 was observed by immunofluorescence staining.Results:1.In contrast to normal controls,the expression levels of SHP2 and p-SHP2 were substantially raised in APL patients peripheral blood and different cell lines.ATPR inhibited the expression and activity of SHP2 in a concentration and time dependent manner in NB4 cells.ATPR can also significantly inhibit the expression and activity of SHP2 in non-APL cells（HL-60,THP-1）.2.SHP2-silencing or SHP2 inhibitor can promote ATPR-induced NB4 cells differentiation and proliferation inhibition.3.SHP2-silencing or SHP2 inhibitor can prevent Rho/ROCK1 activation,and ATPR can significantly enhance the inhibitory effect of SHP2 on Rho/ROCK1.4.ATPR inhibited the expression of Rho/ROCK1 in NB4,HL-60 and THP-1 cells.Using ROCK1 inhibitor can promoted ATPR-induced cell differentiation and proliferation inhibition of NB4 cells.Conclusion:ATPR significantly lowered expression and activity of SHP2,which then inhibited the Rho/ROCK1 axis,and thereby led to proliferation inhibition and terminal differentiation in acute promyelocytic leukemia NB4 cells.