Anti-tumor Effects And Mechanisms Of PLK4 Inhibitor CFI-400945 In Diffuse Large B-cell Lymphoma

Diffuse large B-cell lymphoma(DLBCL)is a class of malignant tumors originating from B lymphocytes with significant heterogeneity in morphology,genetics,immunophenotype and clinical efficacy.While rituximab combined with cyclophosphamide,daunorubicin,vincristine,and prednisone(R-CHOP)significantly improved remission rates and long-term survival in most DLBCL patients,about onethird of the patients still recurred after initial treatment or remission and could not be cured.Therefore,exploring novel R-CHOP-based treatment to improve the therapeutic efficacy and survival outcomes of DLBCL patients has become an attractive topic.Recently,with the improved understanding of biological molecular and cytogenetic characteristics in tumorigenesis,the treatment of DLBCL has entered in the era of molecularly targeted therapies and precision medicine.Novel targeted drugs,including Bruton tyrosine kinase(BTK)inhibitor ibrutinib,immunomodulator lenalidomide,histone deacetylase(HDAC)inhibitor chidamide,B cell lymphoma-2(bcl-2)inhibitor venetoclax,combined with R-CHOP chemotherapy have brought hope to some DLBCL patients.However,there are still some DLBCL patients who failed to benefit from regimen intensification due to age,complications and treatment toxicity.Identification of novel biomarkers and exploration of targeted and combined therapies are urgently needed to provide clinical benefit of DLBCL patients.The cell cycle is an evolutionarily conserved process regulated by the activity of specific proteins and consists of DNA replication and mitosis.Cell-cycle aberrations cause uncontrolled cell division and cell proliferation,leading to tumorigenesis.Therefore,targeting the abnormal cell cycle has been clinically utilized for the treatment of cancer for decades.Microtubule-targeted drugs(such as paclitaxel and vincristine)block tumor cell mitosis by binding and interfering with microtubule dynamics.Although widely effective in various cancers,these drugs do not distinguish between malignant cells and normal cells and are currently limited due to severe side effects and drug resistance.Therefore,exploring cell-cycle regulators that play key roles in the mitosis of tumor cells and developing targeted therapies have become an important topic in the research of cell-cycle targeted therapy.Recent studies have demonstrated that numerous critical cell-cycle regulators were overexpressed in in cancers,including cyclin-dependent kinases,aurora kinases and polo-like kinases(PLKs).These mitotic kinases were identified as strong candidates for therapeutic targets.Polo kinase 4(PLK4)is a member of the PLK family.PLK4 regulates downstream target protein activity mainly through phosphorylation and participates in centriole replication and cell cycle regulation.PLK4 deregulation causes a loss of centrosome numeral integrity,thereby promoting genomic instability and tumorigenesis.Recent studies have emphasized that PLK4 was highly expressed in multiple solid tumors and was significantly associated with tumor staging,metastasis,prognosis and chemosensitivity.PLK4 has multiple roles in tumor pathogenesis:regulating the actin cytoskeleton by interacting with the Arp2/3 complex,which in turn affects tumor cell invasion and metastasis;mediating tumor cell epithelial-mesenchymal transition via the PI3K/Akt signaling pathway;regulating NF-?B transcriptional activity by interacting with IKBKE to mediate the anti-apoptotic effect of tumor cells,etc.Therefore,PLK4 represents a prognostic biomarker and a potential target for tumor therapy.The potent PLK4 targeted inhibitor CFI-400945 is an oral small-molecule inhibitor that selectively inhibits PLK4 in cells,but has no significant inhibitory effect on other PLK family members.CFI-400945 treatment causes aberrant centrosome numbers,resulting in mitotic catastrophe,cell cycle arrest and ultimately cell death.The antitumor effects of CFI-400945 have also been confirmed in vivo.It is well tolerated in a variety of mouse xenograft models.It can be quickly absorbed after oral administration and has a long-lasting effect.Currently,CFI-400945 is undergoing a series of clinical trials.However,the latest clinical findings demonstrated that although CFI-400945 have shown good safety and tolerance in patients with advanced solid tumors,the overall response rate of monotherapy is low.Therefore,there is an urgent need to evaluate disease-specific populations and develop efficient combined treatment options.To date,the functional role of PLK4 in DLBCL remains unclear.Whether CFI400945 treatment has antineoplastic effects in DLBCL remains to be elucidated.In the present study,we examined the expression and clinical significance of PLK4 in DLBCL,and explored the anti-tumor effects and mechanisms of PLK4-targeted small-molecule inhibitor CFI-400945 in DLBCL.We provided theoretical basis and experimental basis for the pathogenesis of DLBCL and novel targeted therapy.Moreover,the synergistic anti-tumor effects and related mechanism of the combined therapy with doxorubicin were further explored.This study provides novel insights in the potential mechanism of PLK4 targeted inhibitors in combination with genotoxic agents and is expected to promote the clinical application of CFI-400945 and CFI-400945-based combined therapy in DLBCL and other tumors.Part ?.Biological functions and clinical significances of PLK4 in diffuse large B-cell lymphomaObjectives:This study aims to detect the expression of PLK4 in diffuse large B cell lymphoma(DLBCL)cell lines and DLBCL tumor tissues,and to further explore the clinical prognostic significance of PLK4 in DLBCL patients by associating with clinical pathological parameters of DLBCL patients.Moreover,the objective is to analyze the biological process,related cell signaling pathway and prognostic significance of PLK4 in DLBCL based on gene expression profiles.Material and methods:1.Clinical specimen collection,2.Isolation and collection of peripheral blood mononuclear cells,3.Cell recovery,cell culture and cell cryopreserving,4.Real-time quantitative polymerase chain reaction(RT-PCR),5.Protein extraction and Western Blot,6.Immunohistochemistry(IHC),7.Bioinformatic analysis of published gene expression datasets,8.Establishment of PLK4 knockdown cell lines by stable cell transfection with PLK4 shRNA knockdown lentivirus,9.Cell proliferation assay by Cell Count Kit-8(CCK-8),10.Statistical analysis.Results:1.Expression of PLK4 was analyzed in 5 DLBCL cell lines(LY1,LY3,LY8,U2932,VAL).Western Blot showed that DLBCL cell lines had higher levels of PLK4 protein expression over normal peripheral blood mononuclear cells from healthy volunteers.Elevated mRNA expression of PLK4 was also observed in DLBCL cell lines by Real-time quantitative PCR.2.Immunohistochemical staining showed that the expression level of PLK4 was significantly upregulated in DLBCL tissues.PLK4 positive rate was 75%(49 of 65)in DLBCL patients,whereas 35%(7 of 20)in reactive hyperplasia.Clinical correlation analysis of PLK4 in DLBCL patients demonstrated that PLK4 high expression was associated with the IPI score in DLBCL patients.The prognostic significance of PLK4 in DLBCL was examined by bioinformatic analysis of public datasets.The results showed that patients with high PLK4 mRNA levels exhibited a shorter overall survival,compared to those with low PLK4 expression.3.Functional enrichment analysis of PLK4 in DLBCL was performed based on public microarray datasets.Results from GO analysis indicated that PLK4 was positively correlated with nucleosome assembly,cell cycle regulation,DNA damage repair and mitotic nuclear division.GSEA analysis implicated that PLK4 was associated with G2/M cell cycle regulation,DNA damage repair and mitotic nuclear division.4.To further confirm the role of PLK4 in chemosensitivity in DLBCL,LY8 cells were stably transfected with PLK4 knockdown lentivirus(shPLK4)and negative control lentivirus(shCON).DLBCL cells transfected with shPLK4 showed enhanced sensitivity to doxorubicin,accompanied by increased y-H2AX expression.Conclusions:The present study identified the aberrantly expression levels of PLK4 in DLBCL cell lines and tumor tissues,which were associated with inferior prognosis.PLK4 may influence tumor progression by participating in biological processes including cell cycle regulation,cell division,and DNA damage.PLK4 knockdown increased the chemosensitivity of DLBCL cells to doxorubicin and increased the DNA damage.Therefore,the results of this study will provide theoretical basis for the pathogenesis of DLBCL,targeted therapy and potential combined treatment strategy.Part ?.Anti-tumor effects and mechanisms of PLK4 inhibitor CFI-400945 in diffuse large B-cell lymphomaObjectives:The study aims to investigate the antineoplastic effects of PLK4 inhibitor CFI-400945 in diffuse large B-cell lymphoma(DLBCL)cells through cell proliferation,cell apoptosis,cell cycle regulation and chemosensitivity,and to further explore the molecular mechanisms of chemical inhibition of PLK4 kinase by CFI-400945 and the combined therapeutic strategy based on mitosis-targeted drugs in DLBCL.Materials and methods:1.Cell recovery,cell culture and cell cryopreserving,2.Protein extraction and Western Blot,3.Cell immunofluorescence staining,4.Immunohistochemistry(IHC),5.Cell proliferation assay by Cell Count Kit-8(CCK-8),6.Detection of cell apoptosis by Annexin V-PE/7AAD double staining,7.Cell cycle analysis by PI/RNase staining,8.Evaluating in vivo anti-tumor effects of PLK4 inhibitor CFI-400945 by DLBCL bioluminescent xenograft model,9.Statistical analysis.Results:1.PLK4 inhibition by CFI-400945 induced growth inhibition,apoptotic deaths,G2/M cell cycle arrest and polyploidy in DLBCL cell lines.2.Confocal assay demonstrated that CFI-400945 treatment induced cytokinesis failure and gave rise to binucleated cells.Western Blot showed the enhanced expression of p-p53,p53 and p21.Moreover,in LY8 cells,CFI-400945 induced an increase of LATS1 phosphorylation and subsequent YAP phosphorylation.Consistently,CFI-400945 down-regulated the expression of nuclear YAP and upregulated the expression of cytosolic YAP.3.Western Blot and confocal assay demonstrated that CFI-400945 resulted in an increase of ?-H2AX expression,with activation of ATM/ATR-Chkl/Chk2 signaling pathway.4.The antiproliferative effects of a combined treatment of CFI-400945 and doxorubicin exceeded that of single agent.CFI-400945 synergized with doxorubicin only in LY8 cells,which harbored TP53 mutation.Compared to single drug,CFI-400945 plus doxorubicin treatment also induced significantly more apoptosis.5.In DLBCL xenograft models,mice treated with CFI-400945 exhibited significant delay in tumor progression compared to those treated with vehicle.Mice treated with combination of CFI-400945 and doxorubicin had significantly lower DLBCL tumor burden after 21 days compared with those treated with vehicle or CFI-400945 or doxorubicin alone.IHC staining exhibited a significant decrease in Ki-67 positive cells and an increase in y-H2AX positive cells after CFI-400945 plus doxorubicin treatment,compared to CFI-40045 or doxorubicin alone.Notably,tumor cells were generally larger,heterogeneous in size and frequently binucleated.Conclusions:In the current study,we demonstrated that the selective PLK4 inhibitor,CFI-400945,suppressed cell proliferation,induced cell apoptosis and cell cycle arrest in DLBCL cell lines.CFI-400945 sensitized DLBCL cells to doxorubicin chemotherapy in vitro and in vivo.Mechanistically,CFI-400945 induced mitotic defects,which was accompanied by increased activation of p53 and Hippo/YAP signaling,followed by DNA damage response.These findings indicate that PLK4 is a promising target for therapeutics in DLBCL,and mitotic defects induced by CFI-400945 may enhance the sensitivity of cancer cells to DNA damaging agents.