The Role And Mechanisms Of CTP Synthase 2 And M6A Demethylase FTO In Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia(CLL)is the most common hematological tumors of adults in western countries,characterized by mature small B-cells with specific immunophenotype.The biological behavior and clinical outcome are highly heterogeneous in CLL patients.It has been previously reported that the prominent characteristics of CLL disease includes the defect of apoptosis and abnormal activation of oncogenic signaling pathways such as B cell receptor pathway.The small molecular targeted inhibitors have been gradually developed in recent years,including BCL-2 inhibitor(e.g.,venetoclax)and BTK kinase inhibitor(e.g.,ibrutinib).With the development of targeted therapy in CLL,the effective rate of CLL treatment has been further improved.However,there are still refractory/relapsed CLL patients who are unable to achieve long-term survival even after receiving salvage therapy including novel targeted drugs and high-intensity combination chemotherapy.Since the specific pathogenesis of CLL is still not clear,it is important to aim at the key nodes in the pathogenesis of CLL and explore new targets for CLL treatment and prognosis,so as to further improve the risk stratification and treatment evaluation of CLL and guide the targeted individualized therapy of CLL.It has been found that DNA damage caused by replication or stress occurs frequently in tumor cells.However,malignant cells can overcome the unchecked replication stress through activation of the DNA damage and response(DDR)pathway,thus facilitating tumor survival.ATM/TP53 mutations are commonly high-risk mutations in CLL and significantly related to genomic instability,clonal evolution and treatment resistance in CLL cells,severely affecting the prognosis of CLL patients.Patients with ATM/TP53 mutations tend to be refractory/relapsed and are associated with aggressive lymphoma(Richter syndrome)transformation.Recent studies have shown that abnormalities in the DDR pathway can reduce the sensitivity of tumor cells to DNA damage-stimulating factors,thereby inducing treatment resistance in CLL and playing a key role in tumor drug resistance.Thus,inhibition of the DDR pathway is a promising combination treatment strategy.It is not only a hot spot in clinical and basic research of CLL,but also with great scientific significance and potential clinical application prospect to explore novel prognostic targets,drug resistance mechanisms and high-efficiency combination regimens.Part Ⅰ CTPS2 Mediates DNA Damage Response to Promote the Development of Chronic Lymphocytic LeukemiaBackgroundCytidine triphosphate synthase 2(CTPS2)is one of the key rate-limiting steps in the synthesis of CTP,which plays a critical role in energy metabolism and biosynthesis of nucleic acids,phospholipids and membranes in eukaryotic cells.There is growing evidence that CTP synthase is abnormally regulated in a variety of cancers.It has been reported that EBV virus can promote the development of lymphoma by increasing CTPS2 expression to meet the metabolic demand for CTP in infected B cells.However,the specific mechanism and clinical significance of CTPS2 in CLL remain elusive.In this study,we provide a reliable theoretical basis that CTPS2 promotes the pathogenesis and development of CLL.CTPS2 not only enhances the proliferation,impedes apoptosis of CLL,but also interacts with BRCA1,resulting in abnormal activation of DDR signaling in CLL.These results open up new insights into the pathogenesis of CLL and provide potential biomarkers to guide treatment options for CLL.ObjectiveThe aim of this study was to investigate the clinical significance and involved molecular mechanism of CTPS2 in the pathogenesis and development of CLL.Integrated analysis of public database and clinical specimens,we prove the oncogenic effect of CTPS2 in the genesis and development of CLL and clarify that CTPS2 regulates DDR pathway through interaction with BRCA1 to promote CLL.Methods1.Bioinformatic analyses of CTPS2 in CLL patients based on public databases such as GEO database and ICGC database.2.Collection of peripheral blood mononuclear cells(PBMCs)samples and clinical information of untreated CLL patients.3.Isolation of CD 19+cells of PBMCs from healthy donors.4.Total RN A extraction and purification,reverse transcription experiment and real-time polymerase chain reaction(qPCR).5.Cell lines and primary cells culture.6.Protein extraction,Western blotting(WB).7.Lentivirus-mediated knockdown of CTPS2.8.Cell counting kit-8(CCK-8)was used to detect the proliferation of CLL cells.9.Cell cycle detected by PI/RNase.10.Apoptosis detected by Annexin V-PE/7AAD staining.11.RNA sequencing(RNA-seq).12.Comet assay.13.Immunofluorescence staining and confocal microscopy.14.Statistical analysis.Results1.The results of qPCR and WB showed that CTPS2 was up-regulated in 96 CLL patients and CLL cell lines MEC-1 and EHEB compared with normal B cells(p<0.01).Aberrant CTPS2 expression in CLL patients was also confirmed in databases GSE5006,GSE22529,and GSE55288.2.High expression of CTPS2 was significantly associated with elevated serum LDH level(p=0.011),absence of IGHV mutation(p=0.021)and poor FISH prognostic index(11q-and/or 17p-,p=0.038)in CLL patients.In addition,Kaplan-Meier survival analysis showed that CLL patients with high CTPS2 expression had shorter overall survival than patients with low CTPS2 expression(p=0.03).3.Compared with the control group transfected with negative control lentivirus,the proliferation of CLL cells in CTPS2 knock-down group was decreased(p<0.001 at 48h and 72h),G2/M cell cycle arrest(p<0.05)and apoptosis rate was increased(p<0.001).4.Overexpression of CTPS2 and exogenous CTP could partially reverse the inhibition of proliferation and apoptosis of CLL cells and primary cells caused by Gln deficiency.5.Transcriptome sequencing results indicated that CTPS2 was involved in DNA damage repair in CLL cells.Fluorescence colocalization and Co-IP experiments confirmed that CTPS2 interacted with BRCA1 directly,thus participating in the activation of DNA damage repair pathway.Overexpression of BRCA1 rescued the reduction in markers of DNA damage caused by CTPS2 knockdown,including reduced expression of phosphorylated H2AX and reduced comet assay tail moments.ConclusionThe expression of CTPS2 is obviously upregulated in CLL,which is closely related to clinical characteristics,unfavorable treatment effect and poor prognosis of patients.CTPS2 promotes the development of CLL by participating in the biological processes including cell proliferation,apoptosis,and cell cycle.Targeting CTPS2 can inhibit cell proliferation,promote cell cycle arrest and apoptosis by increasing DNA damage and impeding DNA repair in CLL cells.These results suggest that CTPS2 may become a novel molecular target for CLL therapy and a biomarker for disease progression and prognosis.Part Ⅱ FTO Induced m6A Demethylation of CtBP1 in Chronic Lymphocytic LeukemiaBackgroundm6A is the most abundant methylated modification of mRNA and lncRNA in eukaryotes.As one of the most common epigenetic modifications of RNA,m6A methylation is a dynamic and reversible process,which can regulate RNA localization,transportation,splicing,translation and degradation at the post-transcriptional level.Fat mass and obesity associated(FTO)gene,located on chromosome 16(16q12.2),was the first m6A demethylase to be identified.It has been found that FTO-mediated m6A demethylation is not only related to lipid metabolism and neurogenesis,but also closely related to tumor development and drug resistance.However,the role of FTO is not consistent in different tumors.It has been reported that FTO is significantly overexpressed in acute myeloid leukemia,lung cancer,endometrial cancer et al.,while FTO is downregulated in intrahepatic cholangio-carcinomas.However,the role and molecular mechanism of FTO and its targeted inhibitor FB23-2 in chronic lymphocytic leukemia have not been studied.ObjectiveIn this study,we aimed to investigate the role and mechanism of FTO in the development of CLL by combining the results of in vivo and in vitro experiments based on public databases and clinical specimens,elucidate the molecular mechanism of FTO-mediated DDR pathway to regulate the sensitivity to ibrutinib in CLL,and explore the clinical transformation potential of FTO inhibitor FB23-2 in combination with ibrutinib for the treatment of CLL.Methods1.PBMC samples and clinical information of untreated CLL patients collection.2.Isolation and purification of CD 19+cells.3.RNA extraction,reverse transcription and qPCR experiments.4.CLL cell culture.5.Protein extraction and Western blot(WB).6.Cell counting kit-8(CCK-8)was used to detect the proliferation of CLL cells.7.Cell cycle detected by PI/RNase staining.8.Apoptosis detected by Annexin V-PE/7AAD staining assay.9.Lentivirus-mediated knockdown of FTO.10.RNA sequencing(RNA-seq).11.Construction of CLL xenograft mouse model.12.Immunohistochemical staining(IHC)and hematoxylin eosin(HE)staining.13.In vivo imaging system of mouse.14.Methylated RNA immunoprecipitation(MeRIP)sequencing.15.RNA immunoprecipitation(RIP).16.Statistical analysis.Results1.qPCR and WB results showed that FTO expression was significantly upregulated in 95 CLL patients compared with normal B cells(p<0.05),and was significantly associated with shortened overall survival(p=0.03).2.FB23-2,the targeted inhibitor of FTO,could significantly inhibit the proliferation of CLL cell lines and primary cells in a time and dose dependent manner.Treatment with FB23-2 caused the cell cycle arrested in G1/S phase and the increased apoptosis rate.3.Combined analysis of meRIP-seq and RNA-seq screened CtBP1 as a potential FTO downstream target.Subsequent meRIP-qPCR and RIP-qPCR identified that FTO could directly bind to CtBP1 mRNA thus to mediate its m6A modification.4.Knockdown of FTO significantly affected CtBP1 expression.Subsequent RNA stability experiments confirmed that FTO obviously prolonged the half-life and increased the stability of CtBP1 mRNA in CLL cells by mediating the mRNA m6A demethylation modification of CtBP1.5.Inhibition of FTO significantly increased the drug sensitivity of CLL cells to ibrutinib in vitro.The expression levels of DNA damage markers,p-H2AX and p-ATM,were increased in CLL cells treated with FB23-2.And the addition of lentivirusmediated FTO inhibition further increased the DNA damage induced by ibrutinib.6.FB23-2 exhibited potent synergistic effects with ibrutinib in CLL mice.Compared with the ibrutinib single-agent group,the mice in the two-drug combination group showed prolonged survival,reduced tumor load,decreased proportion of CLL cells in bone marrow and spleen,diminished splenomegaly,and decreased levels of Ki67 expression in spleen tissue.ConclusionFTO is highly expressed in CLL,and its expression level is closely related to the clinical characteristics,treatment effect and poor prognosis of patients.FB23-2,a FTO targeted inhibitor,can inhibit the occurrence and development of CLL by inhibiting the proliferation of CLL cells,increasing apoptosis,mediating G1/S phase arrest and other biological processes.FB23-2 shows potent synergistic effect with ibrutinib in vivo and in vitro.FTO inhibition enhanced the drug sensitivity of CLL cells to ibrutinib by inducing DDR signaling.These findings provide preclinical evidence for the use of FB23-2 in CLL and suggest the clinical transformation potential of FB23-2 in combination with ibrutinib in CLL.