The Role And Mechanism Of IGF2BP2 And YAP1 In The Pathogenesis And Drug Resistance Of Acute Leukemia

PART1The m6A reader IGF2BP2 mediates the development of T-cell acute lymphoblastic leukemia by stabilizing NOTCH1BackgroundT-cell acute lymphoblastic leukemia(T-ALL)is an aggressive haematological neoplasm that frequently occurs in children and adolescents worldwide.T-ALL originates from genomically altered and/or epigenetically changed transformation of immature T cells.Although the current cure rates increased to 80%in children and 60%in adults,patients with primary resistant T-ALL frequently fail to obtain a complete haematological remission or relapse after the initial response.This clinical challenge has led researchers to decipher the molecular mechanism of T-All transformation and progression,and develop alternative drugs to treat this malignancy.Insulin-like growth factor 2 messenger RNA-binding protein 2(IGF2BP2)is a highly conserved single-stranded RNA-binding protein.Initial studies have found that IGF2BP2 could assist mRNA localization and regulate the post-transcriptional translation of various genes including IGF2,participate in the transcription,processing,translation,and stability stages of mRNA,and play an important role in body growth and development,neural differentiation,lipid metabolism and insulin resistance.Recently,several studies have confirmed that IGF2BP2 plays the role of oncogenic genes in a variety of malignant solid tumors including hepatocellular carcinoma,esophageal adenocarcinoma,ovarian cancer,breast cancer and glioblastoma.In addition,IGF2BP2 has been shown to be highly expressed in acute myeloid leukemia(AML)and is involved in the proliferation and chemotherapy resistance of leukemia cells.We also found that the expression of IGF2BP2 in T-ALL was significantly elevated by public database,suggesting that it may be involved in the occurrence and development of T-ALL.However,the role and mechanism of IGF2BP2 in T-ALL remain unclear.Posttranscriptional modifications of mRNAs,including N6-methyladenosine(m6A),which is the most common internal RNA modification,is well known for regulating gene expression by altering mRNA splicing,stability,translocation,and translation.The methyltransferase complex,which includes the methyltransferase-like 3 and 14 proteins(METTL3 and METTL14)and their regulator Wilms tumour 1-associated protein(WTAP),deposits m6A modification,which is then removed by the following "erasers" demethylases:fat mass and obesity-associated protein(FTO)and ketoglutarate-dependent dioxygenase AlkB homologue 5(ALKBH5).IGF2BP2,as an m6A "reader",binds RNA through the KH domains,especially the KH3-4 di-domain,which are critical for the binding of IGF2BP2 to m6A-modified RNAs.whether IGF2BP2 mediated m6A modification could affect the development of T-ALL remains to be further studied.Objectives1.We sought to confirm the important effect of IGF2BP2 in T-ALL through clinical specimen analysis,and clarify that IGF2BP2 directly regulate the expression of NOTCH1 to promote the development of T-ALL.2.We sought to explore the underlying mechanism of IGF2BP2 regulated the NOTCH1 expression through m6A methylation modification,screen out a small molecule inhibitor with specific targeting and inhibit the IGF2BP2 activity,and explore the feasibility of targeting IGF2BP2 for T-ALL treatment in vivo and in vitro.Methods1.Through screening public database data to confirm that IGF2BP2 expression is elevated in T-ALL,and verified the result in clinical patient samples.2.Small interfering RNA(siRNA)or lentivirus were used to down/up regulate the expression of IGF2BP2 in T-ALL cell lines Jurkat and Molt4.CCK8 experiment was applied to detect cell proliferation,cell cycle experiment was applied to detect cell cycle distribution,drug sensitivity experiment and flow cytometry apoptosis experiment were applied to detect the sensitivity of leukemia cells to chemotherapy drugs.3.IGF2BP2 knockdown and wild-type T-ALL mice models were constructed by using IGF2BP2 knockdown cell lines and control cell lines.Periodically observe the mouse models:coat color,body weight,peripheral blood counts,etc.After the onset of the T-ALL mice,smears of bone marrow and peripheral blood,H&E staining of liver,spleen and bone marrow,flow cytometry were accessed to detect the burden of leukemia cells.Additionally,the survival of the two groups of mice was observed.4.The downstream genes and pathways of IGF2BP2 were screened by iRIP-sequence and RNA-sequence.And the direct binding of IGF2BP2 and downstream gene were verified by IGF2BP2 RIP-qPCR and Western Blot(WB)experiments.5.The regulatory mechanisms and specific site of IGF2BP2,as an m6A methylation"reader",were determined by the iRIP-sequence and MeRIP-sequence.And the regulatory mechanism and specific site of IGF2BP2 were verified through MeRIP-qPCR,RNA-Pull down,RNA stability and WB experiments.6.The novel specific targeting small molecular drugs was designed based on IGF2BP2 structure.First,the KH3-4 domain of IGF2BP2(the KH3-4 domain to mediate the interaction between IGF2BP2 and RNA)was used for computer virtual screening to find small molecular compounds that may interact with IGF2BP2,and then small molecule inhibitors that can bind and inhibit IGF2BP2 activity were further screened by in vitro experiments.7.The effect of small molecular drugs on cell biological functions were evaluated in IGF2BP2 overexpression or knockdown T-ALL cell lines,and the level of IGF2BP2 mRNA level in the cells was verified by qPCR,and the effects of small molecular drugs in T-ALL mice model was verified.Results1.Through the T-ALL related dataset,IGF2BP2 was significantly overexpression in the T-ALL,and the results of RT-qPCR and WB experiments in clinical patient samples have also confirmed that IGF2BP2 is increasing in T-ALL.2.The IGF2BP2 downregulation of T-ALL cell lines Jurkat and Molt4 could significantly inhibit cell proliferation and slow the DNA replication rate,resulting in cell cycle blocking in stage of G0/G1,and increase the cell apoptosis ratio.Instead,the overexpression of IGF2BP2 in T-ALL cells could promote cell proliferation,increase the proportion of cell apoptosis,enhance the sensitivity of leukemia cells to chemotherapy drugs.3.Compared with wild-type T-ALL mice,IGF2BP2 knockdown T-ALL mice showed less leukemia infiltration in bone marrow,peripheral blood,liver and spleen.The leukemia burden was significantly decreased,and the median survival time of mice was significantly shortened.4.Based on IRIP-sequence and RNA-sequence data analysis,we screened out the downstream gene NOTCH 1.IGF2BP2 RIP-qPCR,and WB experiments confirmed that IGF2BP2 could positively regulate NOTCHI expression.And the recovery experiment also further confirmed above result.5.IGF2BP2 RIP-qPCR,MeRIP-qPCR,and RNA stability experiments showed that IGF2BP2 directly regulates the expression level of NOCTH1 through m6A methylation.iRIP-sequence and MeRIP-sequence assays confirmed that IGF2BP2 positively regulated the expression of NOTCH1 mRNA by the m6A modification sites,which exsited in the CDS region of IGF2BP2 mRNA.RNA-pull down experiment also confirmed this conclusion.6.Twenty-two small molecular drugs,directly combined with IGF2BP2,were selected through virtual screening based on molecular docking in the SPECS database.CCK8 experiments,fluorescence titration experiments,cell apoptosis experiments were performed to confirm that JX5 could inhibit cell proliferation and promote cell apoptosis.7.The drug sensitivity experiments of JX5 was performed in IGF2BP2 overexpression T-ALL cell lines to further explore the direct combination and inhibition effect of JX5 on IGF2BP2.In addition,the leukemia burden of T-ALL mice was significantly decreased by small molecular drugs JX5.Conclusions1.IGF2BP2 is highly expressed in T-ALL.And IGF2BP2 promote cell survival,block the cell cycle in G0/G1 phase,and impair cell chemotherapy sensitivity through regulate NOTCH1 mRNA transcription and activation in T-ALL.In addition,IGF2BP2 knockout weaken the leukemia burden and prolong the survival time of T-ALL mice.2.IGF2BP2 enhances the stability of NOTCH1 mRNA by regulating the m6A methylation modification,thereby promote the expression level of NOTCH1.3.The small molecule drug JX5,which specifically targets IGF2BP2,can inhibit the cell proliferation and promote the cell apoptosis of T-ALL cells.And JX5 can significantly reduce the leukemia burden and effectively prolong the occurrence of T-ALL mice.PART 2Deacetylation of YAP1 promotes the resistance to chemo-and targeted therapy in FLT3-ITD+AML cellsBackgroundAcute myeloid leukemia(AML)represents a group of highly heterogeneous myeloid malignancies characterized by acquired genetic abnormalities in hematopoietic progenitors.Among these recurrent mutations,internal tandem duplications(ITDs)of the FMS-like receptor tyrosine kinase 3(FLT3)are the most frequent,presenting in 25%to 30%AML patients.Patients with FLT3-ITD mutant have a high risk of relapse and low cure rates.Despite several FLT3 inhibitors,such as sorafenib,midostaurin,gilteritinib and quizartinib(AC220)have been approved for targeted therapy in clinical usage,a large number of FLT3-ITD mutant AML patients only achieve a transient clinical response and gradually become resistant to monotherapy or in combination with conventional chemotherapy.It is now widely accepted that the defects in the core machinery of DNA damage pathway contribute to drug resistance.Moreover,we have previously shown the important function of DNA damage repair gene poly(ADP-ribose)polymerase 1(PARP1)in the progression of AML.Thus,exploring novel effective therapeutic agents is urgently needed to prevent FLT3-ITD mutant AML drug resistance and improve prognosis of FLT3-ITD mutant AML patient.Therefore,identifying molecular mechanisms involved in FLT3-ITD mutant AML cell maintenance and drug resistance maybe the key to provide novel treatment strategy.Yes-associated protein 1(YAP1)is one of the key transcriptional effectors in the Hippo pathway,which is a crucial regulator of tissue homeostasis,organ size,and cell proliferation.Once cells proliferate to a high density and Hippo pathway is activated,phosphorylated YAP1 will promote its cytoplasmic retention and subsequent degradation by ubiquitin proteasome pathway.Relatively,unphosphorylated YAP1 entering into the nucleus where YAP1 activates transcription factors,alters the expression of genes involved in cell proliferation and apoptosis YAP1 has been demonstrated to play critical role in the development and progression of cancers.YAP1 have recently been defined to limit cancer transformation in response to DNA damage.It behaves as an oncogene in several solid tumors and hematologic malignances,including chronic myeloid leukemia(CML).Nevertheless,YAP1 was also found to be a tumor suppressor gene.Specifically,restoring the level of YAP 1 significantly inhibits proliferation and increases chemosensitivity of hepatic carcinoma cells.Therefore,the role of YAP 1 is cell context-dependent and its effect on FLT3-ITD mutant AML maintenance needs to be further clarified.Aberrant histone deacetylases(HDACs)expression and the association with poor prognosis are well reported in several types of human solid cancers and specifically in AML.Histone modifications have been central in the understanding of post-translational modifications and their effects on the regulation of gene expression.Therefore,it is not surprising that aberrant activity of HDACs can cause deregulated gene expression and protein function,which may serve as pivotal mechanisms to promote tumorigenesis and drug resistance.In the case of AML,mutations in HDACs genes have not been detected,but interestingly,it has been described that oncogenic fusion proteins can recruit HDACs to specific gene promoters to drive leukemogenesis.For instance,AML1-ETO chimeric fusion protein,recruits HDAC1,HDAC2 and HDAC3,silencing AML1 target genes,and therefore leading to differentiation arrest and transformation.Moreover,HDAC3 enhances the DNA damage repair capability of leukemia cells through activating AKT,and thereby protecting leukemia cells from chemotoxicity,suggesting that HDACs can participate in drug resistance by facilitating DNA damage repair.However,no reports have been published regarding the role of HDACs in the regulation of YAP 1 in leukemia.Our preliminary study demonstrated that HDACs inhibitor chidamide treatment concomitantly facilitated the nuclear localization of YAP1 and enhanced its acetylation level in FLT3-ITD mutant AML cells,which led us to conduct the current study to determine whether and how dysregulation of YAP1 could contribute to chidamide-induced inhibitory effects in human AML.Objectives1.We sought to confirm that YAP1 plays an important role in the pathogenesis and drug resistance of FLT3-ITD mutant AML,and clarify that YAP1 can affect the chemoresistance of FLT3-ITD mutant AML by regulating the expression of DNA damage repair protein PARP1.2.We sought to explore the specific mechanism of HDAC10-mediated deacetylation modification regulating YAP1 expression,and further explore the feasibility of deacetylase inhibitor chidamide targeting YAP1 was used in the treatment of FLT3-ITD mutant AML.Methods1.Differential genes were screened in the public database,and YAP1 expression was verified in FLT3-ITD mutant AML patients.2.The FLT3 inhibitor sorafenib was used to induce the FLT3-ITD mutant AML cell lines MV4-11 and MOLM13 to establish a drug-resistant cell lines,and RT-qPCR and WB was used to verify the expression level of YAP 1.3.Lentivirus was used to upregulate the expression of YAP1 in the FLT3-ITD mutant AML cell lines MV4-11 and MOLM13,CCK8 experiment analyzed the cell proliferation,flow cytometry experiment detected cell apoptosis ratio,drug susceptibility experiment tested the sensitivity of cells to chemotherapy drugs.4.Protein mass spectrometry experiment was used to screen out the downstream genes and pathways of YAP 1,and RT-qPCR and WB experiments were used to verify the expression of downstream genes.Recovery experiments were performed to further confirm that YAP1 regulate the cell proliferation depended on the PARP1 expression level by using PARP1 inhibitors and YAP1 siRNA.5.RNA-sequence data analyzed whether HD AC 10 was associated with drug resistance of FLT3-ITD mutant AML cells.And the WB experiments verified the HDAC10 expression.The deacetylase inhibitor chidamide was used to inhibit HD AC 10,and CCK8 experiments were performed to detect cell proliferation and drug-induced cell apoptosis were performed to verify its effect on the cell sensitivity of FLT3 inhibitors.6.Chidamide or small interfering RNA was used to downregulate HD AC 10 expression,and HD AC 10 and YAP1 expression was detected by WB.The membrane protein and nuclear protein of MV4-11 cells were isolated.And the acetylated modification and specific site of nuclear YAP1 were verified by WB and co-immunoprecipitation(CO-IP)experiments.7.The combination of chidamide and FLT3 inhibitors were used to treat primary cells of newly diagnosed and resistant FLT3-ITD mutant AML patients,and to explore the feasibility of chidamide combined with FLT3 inhibitor for the treatment of FLT3-ITD mutant AML.Results1.Through the AML related database,we found that the expression of YAP1 in AML patients was aberrantly inhibited,and YAP1 was significantly positively correlated with the survival of AML patients.2.RT-qPCR and WB experiments verified that the expression of YAP1 in FLT3 inhibitor resistant MV4-11 and MOLM13 cells were lower than control.Clinical patient samples also showed that YAP1 expression was inhibited in resistant FLT3-ITD mutant AML.3.Upregulation YAP1 expression in the FLT3-ITD mutant AML cell lines MV4-11 and MOLM13 significantly inhibited cell proliferation,promoted cell apoptosis,and enhanced the drug sensitivity of cells to FLT3 inhibitors and traditional chemotherapy drugs.4.Protein mass spectrometry experiment screened out the downstream pathway of YAP1,DNA damage repair protein PARP1.In addition,rescue experiments using PARP1 inhibitors and YAP1 small interfering RNA further confirmed that the YAP1 regulated the cell proliferation depended on the PARP1 expression.5.RNA-sequence data screened out HD AC 10,a key gene related to drug resistance of FLT3-ITD mutation AML cell,and WB experiments confirmed that HD AC 10 was poorly expressed in drug-resistant FLT3-ITD mutant AML cells.The deacetylase inhibitor chidamide can negatively regulate the HDAC10/YAP1 axis,thereby enhancing cell susceptibility to FLT3 inhibitors.6.Chidamide or HDAC10 siRNA related experiments confirmed that HD AC 10 could negatively regulate the expression of YAP 1 through deacetylation modification.CO-IP and cell membrane/nuclear protein experiments proved the specific site of nuclear YAP1 acetylation modification.7.The combination regimen of deacetylase inhibitor chidamide and FLT3 inhibitor could significantly promote cell apoptosis ratio of patients with newly diagnosed or resistant FLT3-ITD mutant AML,demonstrated that chidamide combined with FLT3 inhibitor were an optional regimen in the treatment of FLT3-ITD mutant AML.Conclusions1.The expression level of YAP1 in AML patients was significantly lower than that of normal control,the low expression of YAP 1 was positively correlated with the survival and chemoresistance of FLT3-ITD mutant AML.2.YAP1 overexpression could inhibit the cell proliferation,promote cell apoptosis and enhance cell sensitivity to chemotherapy drugs of FLT3-ITD mutant AML by regulating the expression level of DNA damage repair protein PARP1.And HD AC10 could inhibit the transcription and translation of YAP1 by regulating its acetylation modification of H3K27 sites in the YAP1 histone,thereby inhibiting the expression of YAP1.3.The deacetylase inhibitor chidamide enhanced the sensitivity of FLT3-ITD mutant AML cells to FLT3 inhibitors and traditional chemotherapy drugs,and the combination regimen also showed significant efficacy in primary cells of FLT3-ITD mutant AML patients.