第一部分 TOSO通过活化SYK促进慢性淋巴细胞白血病细胞生存的作用机制研究 第二部分 60岁以下成人Ph/BCR-ABL阳性急性淋巴细胞白血病疗效分析—单中心BDH ALL 2000/02方案临床研究结果

【作者】 章艳茹；

【导师】 邱录贵；

【作者基本信息】 北京协和医学院 ， 内科学（专业学位）， 2014， 博士

【摘要】 背景和目的：慢性淋巴细胞白血病(CLL)是一种老年患者常见的血液肿瘤。本研究组前期研究发现TOSO在我国CLL患者中特异性高表达,是CLL的不良预后标志,但是其生物学功能尚不清楚；国外研究提示在T细胞肿瘤、单核巨噬细胞TOSO可能参与NF-κB、MAPK等信号通路活化,而NF-κB、MAPK信号通路的异常活化在CLL发病中发挥重要作用。基于以上内容,我们旨在探讨TOSO在CLL中的作用机制,为进一步研究CLL的发病机制提供新途径。方法：1)TOSO表达慢病毒感染B淋巴瘤细胞系,应用流式细胞术检测细胞周期和凋亡,观察细胞生长,以明确上调TOSO后对细胞的影响；WB检测上调TOSO后对抗凋亡蛋白BCL-2及NF-κB、MAPK等信号通路影响；2)瞬转TOSO siRNA至CLL患者原代细胞,48小时后检测TOSO及上述信号通路蛋白表达水平和细胞凋亡情况,以明确下调TOSO后对CLL原代细胞的影响；3)使用免疫共沉淀蛋白胶条液相串联质谱(IP-LCMS)鉴定与TOSO相互作用的蛋白,并分析其中影响NF-κB、MAPK等信号通路相关的蛋白；在CLL原代细胞及TOSO过表达B淋巴瘤细胞系,利用IP验证TOSO与IP-HTMS分析蛋白有无相互作用；WB检测TOSO对IP验证蛋白的表达、活化的影响；4)使用IP验证蛋白特异抑制剂处理细胞48小时后WB检测NF-κB、MAPK等信号通路相关的蛋白；5)脂多糖(LPS)处理细胞,WB检测TLR4通路下游NF-κB、MAPK等相关蛋白变化,明确上调TOSO后B细胞对LPS刺激的敏感性变化；6)体内实验：建立淋巴瘤NOD/SCID小鼠皮下移植瘤模型,观察上调TOSO表达对小鼠移植瘤生长的影响；7)应用小鼠移植瘤石蜡切片免疫组化的方法,检测移植瘤相关蛋白表达情况；采用TUNEL法共聚焦下检测移植瘤细胞凋亡情况。结果：1)上调TOSO后B淋巴瘤细胞凋亡显著减少,促进细胞生存,但细胞周期无明显变化；上调TOSO后抗凋亡蛋白BCL-2表达上调；上调TOSO后,可促进IKBct、p65、ERK、p38、AKT等蛋白的磷酸化。2)CLL原代细胞沉默TOSO基因48小时后,细胞凋亡显著增加,BCL-2表达下调；IKBα、p65、ERK、p38、AKT等蛋白的磷酸化水平下调。3)利用IP-HTMS鉴定与TOSO相互作用的蛋白,并筛选其中与NF-κB、MAPK等信号通路相关的蛋白：SYK。在CLL原代细胞及TOSO过表达细胞系利用IP成功验证出TOSO与SYK的结合。予以SYK抑制剂R788(Fostamatinib disodium)处理后会削弱TOSO与SYK的结合。另外发现上调TOSO可促进SYK的磷酸化活化,CLL原代细胞沉默TOSO抑制SYK的磷酸化活化。4)SYK抑制剂R788B可部分削弱在B淋巴瘤细胞系上调TOSO对SYK、IKBκ、p65、ERK、p38、AKT等蛋白促磷酸化激活作用。5)B淋巴瘤细胞系上调TOS0可增强B细胞对LPS刺激的反应,促进TLR4通路下游NF-κB、MAPK、AKT信号活化。6)体内实验：在小鼠Z138细胞皮下移植瘤模型中,第17天开始Z138TOSO组肿瘤体积明显大于Z138CON组,具有显著性差异。7)Z138TOSO组和Z138CON组小鼠异位移植瘤模型组织切片免疫组化均显示Cyclin D1阳性。小鼠异位移植瘤组织切片TUNEL法检测肿瘤凋亡结果显示,较之对照组,移植瘤凋亡减少。结论：1.在体内、体外实验,上调TOSO均可减少细胞凋亡,促进B细胞淋巴瘤的生长：体外实验显示沉默TOSO可显著促进CLL原代细胞凋亡。2.在慢性淋巴细胞白血病,TOSO可通过与SYK结合,促进SYK磷酸化从而促进下游NF-κB、MAPK、AKT等通路活化,促进细胞生存。3.上调TOSO表达可增强B细胞对LPS刺激的反应。目的评价BDH ALL2000/02方案治疗60岁以下成人Ph染色体/BCR-ABL融合基因阳性(简称Ph+)急性淋巴细胞白血病(ALL)的疗效,并比较分析不同亚组的治疗结果。方法2001年1月至2011年6月就诊的42例成人Ph+ALL(年龄15～60岁)患者纳入本前瞻性、非随机BDH ALL2000/02方案临床研究。患者接受标准VDCP+L+伊马替尼(IM)诱导治疗,缓解后予以改良Hyper-CVAD/MA方案强化巩固,有合适供者的患者在第一次完全缓解(CRl)期行异基因造血干细胞移植(allo-HSCT)。无合适供者的患者序贯强化巩固治疗±M,获得分子学缓解(MCR)者可选择接受自体造血干细胞移植(ASCT), ASCT后接受2年MM±VP+IM维持治疗；其他患者继续完成再诱导、巩固、维持治疗。分析患者的无病生存(DFS),总生存(OS)和复发率(RR)。结果39例(92.9%)患者获得CR,其中1疗程CR率为83.3%。随访至2012年4月30日,28例规范治疗的患者,中位随访26.5(8-75)个月,中位DFS和OS期分别为(22.013.5)个月和(37.0±5.3)个月,累积RR率为(43.7±9.7)%。化疗组(7例)均复发；ASCT组(5例)中3例移植前后未使用IM者在移植后1年内均复发,另2例应用IM者持续MCR已达移植后35和12个月； allo-HSCT组(16例)移植相关死亡率(TRM)为12.5%,5例复发；allo-HSCT、ASCT和化疗组中位OS期分别为未到达、22个月和14个月,预期3年OS率分别为(66.7±12.2)%、(25.0±21.7)%和(16.7±15.2)%(P=0.014)；三组的中位DFS期分别为未达到、24和10个月(P=0.002),预期3年DFS率分别为(56.3±12.4)%、(26.7±±22.6)%和0%(P=0.002)。结论IM联合化疗及Hyper-CVAD/MA强化治疗治疗Ph+ALL可提高缓解率和缓解质量,增加移植的可行性。allo-HSCT仍然是年轻、有合适供者患者的首选治疗选择。对于无合适供者而获得MCR的患者,ASCT联合IM治疗值得进一步探讨。更多还原

【Abstract】 Background and objective:Chronic lymphocytic leukemia (CLL) is a common middle-aged and old adult leukemia. Our previous works showed that TOSO was specifically overexpressed in patients with CLL and was an independent indicator for shorter survival.In addition, recent studies have demonstrated that TOSO may involve in the activation of NF-κB, MAPK pathway in other cells, which has been identified as the important abnormal pathways in CLL. But the detailed function of TOSO in CLL has not been well defined. Therefore, we will investigate the function of TOSO in the pathogenesis of CLL.Methods:1) After lentiviral transduction to B cell lymphoma’s cells(Granta-519and Z138), we investigate the effect of enhanced expression of TOSO on the proliferation,the cell cycle and apoptosis by flow cytometry. Western blotting (WB) was performed to detect the status of BCL-2, NF-κB and MAPK pathways.2) TOSO mRNA, Protein status and apoptosis were determined at48h after TOSO siRNA transfected into the primary cells of CLL.3) By immunoprecipitation and liquid chromatographycoupled with tandem mass spectrometry (IP/LCMS), we identified that TOSO might have direct interaction with some protein, which is the crucial activator of NF-κB, MAPK path way.This complex formation will be validated by coimmunoprecipitation (IP) experiments. And we will investigate the effect of TOSO on the expression and activation of this protein.4) After treatment with the IP validated protien inhabitor for48h in control (CON) and TOSO overexpression (TOSO) cells in culture, we detect the key proteins in the activation of NF-κB and MAPK pathways. We want to confirm whether TOSO active NF-κB and MAPK signaling pathway through the IP validated protein.5) Western blot analysis revealed the expression of Toll-like receptor-4(TLR4)-dependent signaling pathway proteins in CON and TOSO cells in culture with the addition of Lipopolysaccharide (LPS)(200ng/mL) for different times.6) CON group and TOSO group of B cell lymphoma’s cell line xenograft tumor model was established in NOD/SCID mouse and tumor growth was monitored.7) The expression of cyclinD1proteins in xenograft tumor tissues were detected by immunohistochemistry. The apoptosis of xenograft tumor tissues was detected by TUNEL analysis.Results:1) Enhanced expression of TOSO in B lymphoma’s cells promotes growth and survival, induces apoptosis decrease, but no effect in cell cycle. Cells overexpressing TOSO stimulate more BCL-2, p-p65,p-IKBa, p-ERK, p-p38, and p-Akt activation than CON cells.2) Silencing of TOSO in CLL primary cells is associated with apoptosis increase and decrease survival, inhibiting the expression of BCL-2, p-p65,p-IKBα, p-ERK, p-p38, and p-Akt.3) By mass spectrometry, we identified that TOSO might have direct interaction with SYK, which is the crucial activator of NF-κB, MAPK pathway. IP studies identity SYK(p-SYK) as a binging parter of TOSO in enhanced TOSO expression cells of B lymphoma and the primary cells of CLL,and abrogation of TOSO/SYK binding following treatment with SYK inhabitor R788in TOSO-expressing cells.In addition, TOSO is a regulator of SYK phosphorylation and activity in over expression TOSO B lymphoma’s cells and the primary cells of CLL4) SYK inhabitor R788can partly impair enhanced phosphorylation of p65, IKBa, ERK, p38, and Akt in overexpressing TOSO cells.5) Overexpression of TOSO enhance the LPS response (TLR4downstream signaling) in enhanced TOSO expression cells of B lymphoma.6) In mouse xenograft model of B lymphoma’s cells, TOSO group promotes tumor growth significantly from17days (P<0.05).7) Immunohistochemical study showed a positive expression of CyclinDl in TOSO and CON group, while TOSO group shown an decrease positive rate for TUNEL staining. Conclusion:1. TOSO decreases apoptosis and promotes the growth and survival of B lymphoma’s cells in vitro and vivo. Silencing of TOSO is associated with apoptosis increase in CLL primary cells.2. By mass spectrometry approach, we have identified SYK as a candidate TOSO-interacting protein. The TOSO-SYK complex formation is validated by coimmunoprecipitation experiments. Importantly, we show that the association of TOSO with SYK leads to enhanced SYK phosphorylation and subsequent activation of the downstream NF-κB, MAPK and AKT pathway in CLL.3. Overexpression of TOSO enhance the LPS response in B lymphoma cells. Objective:To explore the treatment options for younger than60years old adults with Ph/BCR-ABL positive acute lymphoblastic leukemia by comparing efficacy of different therapy.Methods:From January2001to June2012,42adult patients were enrolled in the study. All patients received combined with standard VDCP±L±imatinb(IM) as induction therapy and then received intensive consolidation with modified Hyper-CVAD/MA±IM. After achieved complete remission1(CRi),some patients who had appropriate donor received allogeneic hematopoietic stem cell transplantation (allo-HSCT),the other got sequential intensive consolidation±IM and followed by autologous HSCT(ASCT) in molecular CR(MCR) and received MM±VP±IM as maintenance therapy after ASCT. Overall survival (OS), disease free survival (DFS), relapse rate (RR) were analyzed. With the follow up to April30,2012, the clinical parameters.Results:The CR rate afterl cycle of induction chemotherapy was83.3%.39(92.9%) patients achieved CR. With a median follow-up of26.5(8-75) months, the median DFS and OS were (22±3.5) and (37±5.3) months, and cumulative RI were (43.7±9.7)%. All patients of CT Group relapsed. Two patients with IM therapy pre-and post ASCT maintain MCR for35and12months after ASCT. But the other three ASCT patients without IM died for relapse in one year. The transplant related mortality rate of allo-HSCT group was12.5%. The estimated OS at3year of allo-HSCT (n=16), ASCT (n=5) and CT (n=7) group were (66.7±12.2)%,(25±21.7)%and (16.7±15.2)%respectively (P=0.014). And the estimated DFS at3year of these groups were (56.3±12.4)%,(26.7±22.6)%and0%(P=0.002).Conclusion:IM combined with intensivechemotherapy significantly increased the CR rate and improved the quality of CR, which highly enhanced the feasibility of SCT. But it was poor for easy to relapse. Allo-SCT in CR1can decrease relapse and facilitates favorable OS and DFS of young adults with Ph+ALL. For those who without donor but achieved MCR, ASCT combined IM maybe a treatment option.更多还原