【作者】 钱正子；

【导师】 郝希山； Michael Wang； Qing Yi； 王华庆；

【作者基本信息】 天津医科大学 ， 肿瘤学， 2009， 博士

【摘要】 套细胞淋巴瘤（MCI）是一种临床治疗具有挑战性的B细胞恶性淋巴瘤，病程进展快，预后极差。MCL大约占所有非霍奇金淋巴瘤（NHI）的2．8-6％，具有独特的形态学、免疫表型和细胞遗传学特征，主要表现为染色体t（11；14）（q13；q32）易位导致cyclin D1过表达及细胞周期紊乱。尽管MCI患者通常对常规一线治疗有效，但最终极易复发和转移，中位生存期仅3-4年。因此，临床急需高效低毒的新靶向药物和免疫治疗来改善MCL患者的生存及预后。来那度胺（LEN）作为一种新型抗肿瘤药物已被FDA批准用于治疗复发或难治性多发性骨髓瘤。虽然LEN的确切作用机制尚不明确，但研究表明LEN可直接靶向肿瘤细胞及其微环境发挥抗肿瘤活性。LEN对多发性骨髓瘤的显著疗效推动了其在NHL中的扩大应用并显示出初步的抗肿瘤活性。因此，本研究探讨了LEN单药或联合治疗对MCL细胞的抗肿瘤作用，并试图阐明其可能的作用机制，为今后临床应用LEN为基础的联合治疗方案提供了有力的临床前数据。第一部分来那度胺与地塞米松体内外抑制套细胞淋巴瘤细胞增殖及诱导肿瘤细胞凋亡的机制研究目的评价LEN与DEX联合治疗MCI是否具有叠加或协同效应，并阐明其诱导肿瘤细胞凋亡的分子机制。方法LEN、DEX或两药联合（LEN／DEX）体外分别作用于MCL细胞系及从MCL患者骨髓或外周血中分离出的新鲜原代肿瘤细胞，通过分析细胞增殖、细胞周期分布和凋亡情况，判断LEN／DEX方案抗MCL细胞的叠加或协同效应。用Western blot方法探讨其诱导MCL细胞凋亡的机制。建立MCL异种移植物动物模型，用以评价LEN／DEX方案的体内抗MCL活性。结果IEN与DEX可有效抑制MCL细胞系MINO和来源于MCL患者的新鲜肿瘤细胞的增殖以及诱导其凋亡。细胞周期分析表明LEN本身对细胞周期分布无作用，但能够增强DEX介导的G₀／G₁细胞周期阻滞作用。LEN／DEX联合主要通过线粒体途径诱导肿瘤细胞凋亡，表现为促凋亡蛋白p-Bcl-2，Bax和Bad表达上调，进而导致caspase-9、caspase-3活化和PARP表达，且研究发现原代肿瘤细胞对药物的敏感性高于MCL细胞系。此外，LEN／DEX联合还可以明显减缓MCL-免疫缺陷（SCID）鼠体内肿瘤细胞的生长并改善荷瘤鼠的生存时间。结论LEN／DEX方案体内外可有效抑制MCL细胞增殖和诱导其凋亡。本研究结果表明LEN是一种很有前景的治疗MCL的靶向药物，并为LEN／DEX方案治疗复发和难治性MCL的临床试验提供了有利的理论依据。第二部分来那度胺增强美罗华介导的套细胞淋巴瘤细胞杀伤的双重作用机制目的抗CD20单克隆抗体美罗华（RTX）主要通过诱导凋亡和抗体依赖性细胞介导的细胞毒作用（ADCC）可有效治疗MCL，LEN作为一种新型免疫调节剂具有致敏肿瘤细胞及增强ADCC的作用。本研究旨在阐明LEN增强RTX杀伤MCL细胞的作用机制。方法分别用LEN、RTX或两药联合（IEN／RTX）作用于三种MCL细胞系SP53、MINO、Grant519和从3例复发或难治性MCL患者骨髓或外周血中分离出的新鲜肿瘤细胞，以及来源于正常人外周血的单个核细胞（PBMCs），测定肿瘤细胞增殖和凋亡的情况以及RTX诱导的ADCC效应，分析LEN／RTX方案的体外抗MCL的细胞毒作用。采用Western blot方法探讨药物诱导MCL细胞凋亡的信号途径，同时阐明LEN增强RTX诱导的ADCC效应的分子机制。建立MCL异种移植物动物模型，评价LEN／RTX方案的体内抗MCL作用。结果LEN和RTX在体外可有效抑制MCL细胞系和新鲜的原代MCL细胞的生长及诱导其凋亡。研究表明，LEN促进RTX诱导的细胞凋亡作用主要通过上调JNK、Bcl-2磷酸化；增加线粒体细胞色素c（cyto c）的释放；增强caspase-3，-8，-9活化和PARP裂解。同时，LEN能激活自然杀伤（NK）细胞，增加CD56^lowCD16⁺NK细胞的CD16表达，含有NK细胞的全PBMCs在LEN（1μM）作用下可使RTX介导的细胞毒作用提高30％，连续每日给予LEN治疗可导致SCID鼠体内的NK细胞数增加10倍。此外，在MCL-SCID鼠动物模型中，LEN与RTX联合可显著降低荷瘤鼠的肿瘤负荷并延长其生存期。结论LEN与RTX联合可显著促进MCL细胞凋亡，增强RTX依赖性NK细胞介导的细胞毒作用，进而发挥协同的体内外抗MCL效应。本研究表明LEN与RTX联合可作为治疗复发或难治性MCL的一个新的可行性临床试验治疗方案。更多还原

【Abstract】 Mantle cell lymphoma （MCL） is a challenging B-cell lymphoma to treat,with anaggressive clinical course and extremely poor prognosis.It accounts forapproximately 2.8-6% of all non-Hodgkin’s lymphomas （NHLs） and has distinctmorphologic,immunophenotypic,and cytogenetic features,characterized by thet（11;14） （q13;q32） translocation resulting in cyclin D1 overexpression and cell-cycledysregulation.Although MCL patients achieve a high rate of complete response fromfrontline therapies,but relapse almost always occurs with a median overall survival of3-4 years with less than 15% of patients alive at 5 years.Therefore,MCL remainsincurable and novel targeted agents and immunotherapies with improved efficacy andsafety are urgently required to improve the survival of patients with MCL.LEN,a novel anticancer drug,has emerged as a potent treatment with improvedsafety profile for multiple myeloma （MM） and received FDA approval for use incombination with DEX in relapsed or refractory MM patients.Although the exactmechanism of its action remains complicated and unclear,LEN has been shown invarious systems to have striking antitumor activities directly targeting tumor cells andtheir microenvironment.The success of LEN in the treatment of MM has led to itsexpanded use in treating other B-cell malignancies,with impressive results in someNHLs.These encouraging findings prompted us to investigate the therapeuticeffeicacy of LEN alone or in combination on MCL cells and attempt to elucidate itsantitumor mechanism.Our study could provide favorable preclinical data for thefuture use of LEN-containing regimens in clinical trials of MCL.PartⅠLenalidomide plus dexamethasone induces the growth arrestand apoptosis of mantle cell lymphoma cells in vitro and in vivoObjective Our study was undertaken to evaluate the effect of LEN combined withDEX on MCL cells and elucidate the mechanism by which it induces tumor cellapoptosis.Methods Established MCL cells and freshly isolated MCL cells from three relapsedpatients were treated with LEN,DEX individually or in combination of two drugs （LEN/DEX）.Cell proliferation,cell cycle distribution and apoptosis were evaluatedto determine if there was additive or synergistic effect of LEN/DEX regimen.Westernblot analysis was used to elucidate the molecular mechanism by which different druginduced apoptosis in MCL cells.In addition,a MCL-bearing xenograft model insevere combined immune deficiency （SCID） mice was established to examine the invivo efficacy of the regimen to eradicate MCL cells.Results The results showed that LEN and DEX induced growth inhibition andapoptosis of MCL cell lines and freshly isolated patient MCL cells in adose-dependent manner.Cell cycle analysis showed that LEN was able to enhanceDEX-induced G₀/G₁ arrest despite no effect using LEN alone.Apoptosis induced byLEN/DEX was revealed mainly through mitochondrial pathways,as demonstrated byup-regulation of proapoptotic proteins phosphor-Bcl-2,Bax,and Bad,and thesubsequent cleavage of caspase-9,caspase-3,and PARP;This effect was moresignificant in primary MCL cells than established MCL cells.Importantly,LEN/DEXwas also effective to delay tumor growth and improve survival of MCL-bearing SCIDmice inoculated subcutaneously with 1×10⁷ MCL cells.Conclusions LEN/DEX inhibits growth and induces apoptosis of MCL cells invitro and in vivo.Cell apoptosis was induced mainly through mitochondrial pathway.Our results provide favorable preclinical data for the future use of LEN/DEX regimenin clinical trials of MCL.PartⅡDual mechanisms for lenalidomide-enhanced rituximab-mediated killing of mantle cell lymphomaObjective Rituximab （RTX）,a chimeric anti-CD20 antibody,is associated withdirect induction of apoptosis and antibody-dependent cell-mediated cytotoxicity（ADCC） with clinical efficacy in mantle cell lymphoma （MCL）.Lenalidomide （LEN）,a novel immunomodulatory agent,sensitizes tumor cells and enhances ADCC.Ourstudy attempted to elucidate the mechanism of LEN-enhanced RTX-mediated killingof MCL cells.Methods Three human MCL cell lines:SP53,MINO,Grant519;freshly isolated primary MCL cells from three relaped or refractory MCL patients;and normalperipheral blood mononuclear cells （PBMCs） from healthy donors were treated withLEN and RTX,either alone or in combination.Cell proliferation,apoptosis andRTX-induced ADCC were examined to analyse the in vitro cytotoxicity of LEN plusRTX against MCL.Western blot analysis was used to investigate the signal pathwayby which different drug induced apoptosis in MCL cells.Meanwhile,we attempted toelucidate the molecular mechanism by which LEN enhanced RTX-dependent NKcell-mediated cytotoxicity.In addition,MCL-bearing xenograft models using withSCID mice were established to evaluate the in vivo anti-MCL capacity of the LENand RTX combination.Results The results showed that LEN and RTX induced growth inhibition andapoptosis of both cultured and fresh primary MCL cells.LEN enhanced RTX-inducedapoptosis via upregulating phosphorylation of c-Jun N-terminal protein kinases （JNK）,Bcl-2;increasing release of cytochrome-c;enhancing activation of caspase-3,-8,-9and cleavage of PARP.Meanwhile,LEN activated NK cells and increased CD16expression on CD56^lowCD16⁺ NK cells.Whole PBMCs but not NK cell-depletedPBMCs treated with 1 1μM of LEN augmented 30% of RTX-dependent cytotoxicity.Daily treatment with LEN increased NK cells by 10 folds in SCID mice.Moreover,the combination of LEN and RTX decreased tumor burden and prolonged survival ofMCL-bearing SCID mice.Conclusions LEN plus RTX provides a synergistically therapeutic effect on MCLcells by enhancement of apoptosis and RTX-dependent NK cell-mediated cytotoxicityand may be an optimal combination in clinical trial of relapsed or refractory MCL.更多还原