The Interaction And Mechanism Of The GLI1 And PI3K/AKT Pathways In AML Cells

Background and Objectives: Acute myeloid leukemia(AML)is a hematological malignancy with high incidence,mortality rates and short survival period,which seriously threatened people’s health and life.Over the years,with the rapid development of medical and scientific technology,few breakthroughs have been made in the treatment of acute myeloid leukemia,but the overall situation is still not optimistic.The current treatment strategy for AML mainly includes conventional chemotherapy and hematopoietic stem cell transplantation(HSCT).Conventional chemotherapy has serious side effects,high risks,and it is difficult to fundamentally remove leukemia stem cells,resulting in approximately 50% of acute myeloid leukemia patients ultimately experience relapse/resistance after achieving complete remission.HSCT,as a fast-developing and curable strategy for AML treatment,is attracting broad attention from health care and AML patients.However,it is tough to be widely implemented for some limitations,such as,expensive treatment,insufficient donor sources,transplant intoler ance,and post-transplant rejection.Therefore,exploring the mechanism that mediates AML relapse/resistance,thereby targeting these mechanisms to improve the chemotherapy efficacy and prognosis of leukemia has become a new strategy to treat AML.Our previous studies have confirmed that Hedgehog(Hh)and PI3K/AKT signaling pathways are activated in relapsed/refractory AML patients.Moreover,GLI1,a key molecule of the Hedgehog(Hh)pathway,could reduce AML drug sensitivity through PI3K/AKT pathway.However,the cross-talk between GLI1 and PI3K/AKT pathway remains unclear.Here,GLI1overexpression/ knockdown stable AML cell lines were used to examined the different chemosensitivity of tumor cell caused by GLI1,PI3K/AKT signaling pathway,cyclins and study the specific mechanism of GLI1 regulating PI3K/AKT signaling pathway.Detecting the effect of GLI1 selective inhibitor combined with CDK4/6 selective inhibitor on the cell viability and chemosensitivity of AML cell lines and primary cells,and simultaneously evaluating the toxic side effects of inhibitors on hematopoietic stem cells.In addition,by constructing a mouse tumorigenic model to verify the ability of GLI1 to promote AML cell tumorigenesis in mice and the protein expression of p-AKT,CDK4 and Cyclin D3 in tissues.This proposed study will contribute to our knowledge of the mechanism of AML drug resistance,find potential targeted drugs/small molecule compounds for AML therapy and assess the feasibility and safety of targeting GLI1 and CDK4/6 in the treatment of AML.In the near future,By targeting or combined targeting of GLI1 is expected to significantly improve AML treatment and provide new strategy for AML targeted therapy.Methods:1.To compare the differences in gene expression and pathway enrichment in AML patients,bone marrow samples from clinically relapsed/refractory and remission AML patients were collected for RNA-seq.TCGA database was used to compare the expression of the key molecules of Hedgehog(Hh)and PI3K/AKT pathway in AML patients and normal healthy people,and Kaplan-Meier curves survival curve analysis was performed.2.In order to explored the roles of GLI1 in AML cell lines by establishing the overexpression(GLI1/OE)and knockdown(sh GLI1)stable AML cell lines.Empty vector or vector carrying scramble sequences were used as controls(MOCK,Scramble).Then,to evaluate the cell response to different drugs,cell viability was determined by Cell Counting Kit-8(CCK-8),and the cell cycle status was analyzed using a flow cytometry.3.By analyzing the data from TCGA to quest the correlation of GLI1 expression and cyclin kinase expression in AML patients.In addition,the cyclin kinase expression levels in GLI1/OE and sh GLI1 cells were detected by Western Blot and Q-PCR.4.We verified the mechanism of oncogenic GLI1 in AML cell by the following methods.Firstly,using the TCGA database to explore the correlation between GLI1 and the molecules of PI3K/AKT pathway in AML patients.Secondly,using the PIP3 Mass ELISA Kit to assess the PI(3,4,5)P3(PIP3)level in GLI1/OE and GLI1/MOCK cells,and by Western Blot to detect the protein levels of p-pi3 k and p-AKT in the PI3K/AKT in GLI1/OE and sh GLI1 cells.Thirdly,Enzymatic activity analysis were performed using the Dual-Glo Luciferase Assay,and the protein level of GLI1 bound to PIK3R1 was detected by coimmunoprecipitation.Then,After treated with PI3K/AKT inhibitors in OE/MOCK cells,the cell growth rate were determined by CCK8 and cyclin kinase expression were detected by Western Blot.Lastly,Protein-protein interaction networks functional enrichment analysis.5.To evaluate the antitumor effects of GLI1 inhibitors and CDK4/6inhibitors in AML cells,we detected the viability of AML cell lines,AML primary cells,normal peripheral blood lymphocyte lines and normal hematopoietic stem cells,treated with GLI1 inhibitor and/or CDK4/6 inhibitor at different concentrations and times,by CCK-8.6.The cells were further inoculated subcutaneously in nude mice to check the colony formation capability of GLI1 in vivo,and the tumor growth curve,tumor mass,p-AKT,Cyclin D3,CDK4 and Ki67 levels were compared and detected.Results:1.The signature genes of the Hh signaling pathway e.g.,GLI1,PTCH1,SMO,and the PI3K/AKT pathway e.g.,phosphoino sitide-3-kinase,regulatorys-ubunit1(PIK3R1),AKT1,AKT2,were up-regulated in AML-RR patients compared to AML-CR patients.And the expression levels of those genes were higher in AML patients than in healthy people.The Sufu,GLI1,PIK3R1 and AKT3 expression was inversely correlated with overall survival(OS)among AML patients.2.GLI1 overexpression promotes cell proliferation and reduces chemotherapy sensitivity to cytarabine and doxorubicin in THP-1 and U937 cell lines,while knocking down GLI1 has the opposite effect;In addition,GLI1 overexpression significantly increases the percentage of AML cells in S+G2 phase and led to elevated protein levels of cyclins and cyclin-dependent kinases(CDKs)in AML cells.3.The expression level of GLI1 was positively correlated with some cyclin-dependent kinase and negatively correlated with some cyclin-dependent kinase inhibitors in AML patients.The protein expressions levels of GSK3A/B,Cyclin D,CDK4/6 were up-regulated in GLI1 overexpression cell lines and down-regulated in GLI1 knockdown cell lines.4.In AML patients,the expression level of GLI1 was positively correlated with the expression levels of PIK3R1,AKT3,PDK1,and so on.GLI1 overexpression increased the protein expression levels of PIP3,p-pi3 k,and p-AKT,while knocking down GLI1 suppressed the expression of p-pi3 k and p-AKT.GLI1 directly bound to the PIK3R1.Targeting PI3K/AKT reversed the proliferation-promoting effect of GLI1 on AML cell lines.AKT inhibitor offset the ability of GLI1 to up-regulate GSK3A/B and CDK4 protein expression.Furthermore,targeting GSK3A/B influenced the m RNA level of cyclins.5.The CDK4/6 inhibitor,PD 0332991,could abolish the increased proliferation induced by GLI1 overexpression in AML cell lines,and it has no obvious cytotoxicity on normal lymphoid AHH-1 cell lines at a concentration of less than 10 μM.PD 0332991 enhanced the GANT61-mediated cytotoxicity in AML cell lines and AML primary cells but not in normal lymphocyte lines and hematopoietic stem cells.Moreover,GANT61 and PD 0332991 have a synergistic effect on promoting drug sensitivity and reducing cell viability in AML cell lines and AML primary cells.6.Overexpression GLI1 significantly enhanced the capability of tumorigenicity in nude mice.Besides,the protein expression level of Cyclin D3,p-AKT,CDK4,and Ki67 were increased in tumor tissues of nude mice injected with GLI1/OE cells.Conclusions:In AML cells,GLI1 activates the PI3K/AKT signaling pathway by directly acting on PIK3R1,and the hyperactivation of the PI3K/AKT pathway subsequently upregulates GSK3A/B expression and further mediates the expression of cyclin-dependent kinases(CDKs).The upregulated expression of CDKs ultimately promotes cell proliferation,cycle progression and induces drug resistance in AML cells.Combinatory targeting GLI1 and CDK4/6 could safely and effectively suppress the viability of AML cells and enhance their sensitivity to cytarabine.