Anti-tumor Effects And Mechanisms Of MELK Inhibitor OTSSP167 In Chronic Lymphocytic Leukemia

Chronic lymphocytic leukemia(CLL)is a proliferative B lymphocytes malignancy with specific immunophenotype mainly in the middle-aged and elderly population,characterized with accumulation of mature-like small B lymphocytes in peripheral blood,bone marrow,lymph nodes and spleen.It is highly heterogeneous in pathogenesis,clinical courses and therapy efficacy.Clinical parameters,such as TP53 alternation,immunoglobulin heavy chain variable region gene(IGHV)mutational status,clinical stage(Rai stage,Binet stage),?2-microglobulin level and age serve as key prognostic predictors in CLL.Among them,patients with TP53 mutations or deletions have inferior prognosis and fail to response effectively to conventional immunochemotherapy regimens,which is a difficult problem in the treatment of relapsed CLL.At present,novel targeted therapeutic drugs,such as Bruton's tyrosine kinase(BTK)inhibitor ibrutinib,BCL2 inhibitor venetoclax,PI3K inhibitor idelalisib have benefited some patients with high-risk CLL,but some CLL patients still fail to achieve clinical remission.Therefore,novel targeted agents and regimens that effectively regulate p53 are needed to break through the therapeutic bottleneck of CLL and improve the prognosis of CLL patients.Maternal embryonic leucine zipper kinase(MELK),also known as MPK38,is a cell cycle-dependent Snfl/AMPK family member of Ser/Thr protein kinases.Highly expressed in proliferating cells such as embryonic stem cells,hematopoietic stem cells,and cancer stem cells,MELK phosphorylates and regulates the activity of target proteins and participates in cell cycle regulation,stem cell differentiation,cell proliferation,apoptosis,invasion and metastasis.Latest studies identified that MELK could affect the cancer stem cells maintenance by regulating p53 signaling pathway as a key driving gene in tumorigenesis.Moreover,MELK is obvious elevated in some solid tumors such as gliomas,prostate,melanoma,and hematological malignancies such as multiple myeloma(MM),and significantly associated with progressed grade,shortened survival,and reduced chemosensitivity,highlighting as an adverse prognosis indicator and a potential target for cancer therapy.In the era of precision medicine,small molecule targeted drugs have achieved outstanding clinical benefits,and their application in cancer treatment is becoming increasingly important.The potent MELK targeted inhibitor,OTSSP167(IC50 = 0.41 nM),is an oral small molecule inhibitor that acts by blocking phosphorylation of MELK substrates PSMA1 and DBNL.OTSSP167 exerts anti-tumor activities by regulating biological functions such as p53 signaling pathway and cell cycle in some malignancies,such as glioma,ovarian cancer and multiple myeloma.So far,phase?/? clinical trials of OTSSP167 have been conduct in breast cancer and acute myeloid leukemia,and have achieved phased results,indicating broad clinical application prospects.However,the role and mechanism of MELK in CLL is still unclear.Furthermore,whether OTSSP167 has therapeutic potential in CLL remain undefined and worthy of further investigation.In this study,we observed the expression status and prognostic value of MELK in CLL,and further investigated the biological roles and mechanisms of MELK and OTSSP167 in CLL,providing theoretical and experimental basis for risk evaluation,prognosis prediction and targeted therapy in CLL patients.This study is expected to reveal the mechanism of MELK regulation in CLL pathogenesis and targeted intervention,promoting the clinical application of OTSSP167 and guiding novel targeted therapy in CLL.Part I.Biological functions and clinical significances of MELK in chronic lymphocytic leukemiaObjectives:This study aims to detect the expression and clinical significance of MELK expression in CLL cell lines and primary CLL cells by associating with clinical pathological parameters and prognosis information of CLL patients.Besides,to analyze the enrichments of MELK expression in biological processes,signaling pathway and gene sets,and to further predict MELK function in CLL gene expression profiles.Moreover,to determine the effects of MELK regulation by CRISPR/Cas9 genomic editing system mediated MELK knockout,shRNA knockdown and overexpression on the biological functions and mechanisms in CLL tumorigenesis.Materials and Methods:1.Clinical specimen collection;2.Isolation of peripheral blood mononuclear cells;3.Magnetic cell isolation of CD19+ B cells;4.Cell culture;5.Real-time quantitative polymerase chain reaction;6.Protein extraction and Western Blotting;7.Open-access gene expression omnibus(GEO)data extraction,bioinformatics analyses;8.Cell transfection by CRISPR/Cas9 gene editing system-mediated MELK knockout lentiviral virus;9.Cell transfection by MELK shRNA knockdown lentivirus;10.Cell transfection by MELK overexpresses adenovirus;11.Cell proliferation assay by Cell Count Kit-8(CCK-8);12.Cell apoptosis detection assay by Annexin V-PE/7AAD staining;13.Cell cycle detection assay by PI/RNase staining;14.Transwell assay of CXCL12-induced cell chemotaxis,flow cytometry cell counting;15.Statistical analyses.Results:1.Real-time quantitative PCR and Western Blotting results showed that the obvious higher expression level of MELK mRNA and protein in primary cells of 55 de novo CLL patients and CLL cell lines(MEC1 and EHEB)compared with B-cells from healthy volunteers(p<0.001).2.MELK high expression was associated with increased white blood cell counts,advanced Binet stage,high-risk Rai stage,elevated LDH level,higher ?2-MG level,unmutated IGHV gene,ZAP-70 positive and chromosome 17p13 deletion in CLL patients.MELK expression was significantly correlated with reduced overall survival of patients(HR=6.189,p<0.001).In addition,the inferior prognostic significance of MELK in CLL patients was validated in Microarray database GSE22762.3.In the public database,the functional enrichment analyses of MELK-related genes indicated that MELK was closely related to CLL cell proliferation,cell cycle regulation and drug sensitivity.KEGG results suggested that MELK were enriched in the regulation of p53 pathway.GSEA analysis implicated that MELK was associated with CLL cell proliferation,apoptosis,G2/M cell cycle regulation and cell migration function.4.MELK knockdown lentivirus(shMELK)and negative control lentivirus were stably transfected MEC1 and EHEB cells.Compared with the control transfected group,CLL cells transfected with shMELK showed obvious decreased proliferation(p<0.01),significantly increased apoptosis(p<0.05),G2/M cell cycle arrest,and attenuated CXCL12-induced migration.Besides,RNA interference(RNAi)of MELK enhanced the sensitivity of CLL cells to fludarabine and ibrutinib.5.Adenovirus mediated MELK vectors(Adv-MELK)were utilized to enforce MELK expression.Compared with the control transfected group,CLL cells transfected with Adv-MELK presented significantly promoted cell viability,elevated CXCL12-induced chemotaxis,and reduced sensitivity of CLL cells to fludarabine and ibrutinib.6.CRISPR/Cas9 genomic-editing system mediated MELK knockout(sgMELK)lentivirus was transfected to MEC1 cell to deplete MELK expression.CLL cells with MELK silence showed significantly reduced proliferation,increased apoptosis and G2/M phase cell arrest.Additionally,Western blotting results showed that p21 protein activation was obviously revealed in MELK knockout cells,suggesting that MELK promoted CLL cell survival by abrogating cell cycle independent of p53.Conclusions:Expression level of MELK is aberrantly increased in CLL cells and predicts inferior prognosis in CLL patients.MELK potentially contributed to CLL tumorigenesis via regulating biological processes such as cell proliferation,apoptosis,cell cycle,and p53 signaling pathway.The above results confirmed that MELK knockout and knockdown reduced CLL cells proliferation,increased apoptosis,G2/M cell cycle arrest,decreased cell chemotaxis,and enhanced sensitivity to fludarabine and ibrutinib.Enforced MELK promoted cell viability,elevated CXCL12-induced chemotaxis,and reduced drug sensitivity.The results provides novel theoretical basis for CLL pathogenesis,precise prognosis assessment and specific targeted therapy.Part ?.Anti-tumor effects and mechanisms of OTSSP167 in chronic lymphocytic leukemiaObjectives:This study aims to examine the effects of MELK inhibitor OTSSP167 in proliferation,apoptosis,cell cycle regulation,chemotaxis and drug sensitivity of chronic lymphocytic leukemia(CLL)cells,and to further investigate the regulatory mechanisms of MELK suppression by OTSSP167 in CLL.Materials and Methods:1.Clinical specimen collection;2.Isolation of peripheral blood mononuclear cells;3.Magnetic cell isolation of CD 19+ B cells;4.Cell culture;5.Protein extraction and Western Blotting;6.Cell immunofluorescence assay;7.Open-access gene expression omnibus(GEO)data extraction,bioinformatics analyses;8.Cell transfection by FoxMl shRNA knockdown lentivirus;9.Cell transfection by FoxMl overexpresses lentivirus;10.Cell proliferation assay by Cell Count Kit-8(CCK-8);11.Cell apoptosis assay by Annexin V-PE/7AAD staining;12.Cell cycle assay by PI/RNase staining;13.Transwell assay of CXCL12-induced cell chemotaxis,flow cytometry cell counting;14.Statistical analyses.Results:1.MELK inhibition by OTSSP167 impeded viability of CLL primary cells,MEC1 and EHEB cells,increased CLL cell apoptosis,induced G2/M cell cycle arrest,reduced cell chemotaxis,and promoted drug sensitivity of CLL cells to fludarabine and ibrutinib.2.Western blotting results revealed that the expression of p-AKT and p-ERK in CLL cells decreased significantly with OTSSP167 incubation,and no significant differences were observed in the total expression of AKT and ERK1/2 expression.3.Western blotting and confocal assay showed that OTSSP167 down-regulated the expression of FoxMl,p-FoxMl and downstream targets cyclinBl and CDK1,and enhanced the expression of p53 and p21.Moreover,FoxMl knockdown and the FoxMl inhibitor Thiostrepton reduced significantly proliferation of MEC1 cells.Notably,the declined viability of MEC1 cells by OTSSP167 treatment were implicated to be rescued in part by FoxMl overexpression.Conclusions:The results demonstrated that OTSSP167 abrogated CLL cell proliferation,induced apoptosis and cell cycle arrest,attenuated cell chemotaxis and promoted CLL sensitivity to fludarabine and ibrutinib.Furthermore,OTSSP167 exerted anti-leukemic efficacy via modulating expression and phosphorylation of FoxM1,and represents a promising strategy to formulating a novel treatment paradigm in CLL.