CN111973590A - 新型hdac抑制剂laq824在治疗弥漫大b细胞淋巴瘤药物中的应用 - Google Patents
新型hdac抑制剂laq824在治疗弥漫大b细胞淋巴瘤药物中的应用 Download PDFInfo
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Abstract
本发明提出一种新型HDAC抑制剂LAQ824在治疗弥漫大B细胞淋巴瘤中的应用。LAQ824在0~0.1μM浓度范围内可有效杀伤DLBCL细胞并表现出药物浓度依赖性，通过抑制DLBCL细胞中Chk2的表达，减弱Chk2对肿瘤细胞DNA损伤的修复，可以增加细胞对药物的敏感性，诱导DLBCL细胞凋亡。
Description
技术领域
本发明涉及分子治疗领域，具体的说是新型HDAC抑制剂LAQ824在治疗弥漫大B细胞淋巴瘤药物中的应用。
背景技术
淋巴瘤是发病率最高的恶性血液疾病，其中弥漫大B 细胞淋巴瘤（diffuse largeB-cell lymphoma, DLBCL）是最常见的非霍奇金淋巴瘤，发病率约占淋巴瘤的30%-50%。DLBCL 是一组具有高度侵袭性及异质性的疾病，在基因改变、临床特征、形态学表现、治疗反应和预后方面均存在差异。根据基因表达谱分析可将DLBCL主要分为两型：生发中心B细胞样淋巴瘤（GCB 型）和活化B细胞样淋巴瘤（ABC 型）；此外还有部分不能确定细胞起源的第三型DLBCL。免疫组织化学则将DLBCL分为GCB及non-GCB（包括ABC 及第三型）两种类型。
R-CHOP（利妥昔单抗+环磷酰胺+多柔比星+长春新碱+泼尼松）方案显著改善了患者的生存，但是仍有约40%的患者耐药和复发，且针对non-GCB 的疗效差于GCB 型。所以不断探索R-CHOP方案联合新型靶向药物的疗效，特别是针对non-GCB或ABC型DLBCL，如联合硼替佐米、来那度胺、BTK 抑制剂伊布替尼、Bcl2抑制venetoclax、PD-1单抗等，尽管对某些亚型可能带来一定的益处，但仍然难以撼动R-CHOP 的地位，R-CHOP 方案仍然是DLBCL的标准一线方案。CAR-T治疗在复发或难治性（relapsed or refractory, R/R）DLBCL患者的治疗中显示出一定的疗效，然而CAR-T治疗后早期进展的患者预后不佳。另外，BTK抑制剂、PI3K抑制剂等靶向药物及与PD-1抑制剂等药物的联合使用也在治疗DLBCL有一定的疗效。尽管目前各种靶向药及免疫治疗等为DLBCL患者提供了更多的选择，但是仍有部分DLBCL患者出现耐药，复发，难治，所以项目组需要探索新的治疗策略和治疗方案来进一步提高DLBCL患者的临床疗效。
发明内容
为了解决上述技术问题，本发明课题组在探索新的治疗策略和治疗方案来进一步提高DLBCL患者临床疗效方面进行了持续的试验和研究。
课题组研究发现，LAQ824可通过诱导抑癌基因 FOXO1 的表达，在体外可有效抑制myc驱动的髓母细胞瘤细胞存活，同时LAQ824还可与PI3K抑制剂协同作用，在体内抑制肿瘤生长；该小分子化合物在急性髓系白血病（acute myeloid leukemia, AML）细胞中可通过上调死亡受体DR5/DR4 mRNA和蛋白的表达引起 flice-inhibitor 蛋白(c-FLIP)的表达降低，同时还可下调Bcl-2、Bcl-x(L)、XIAP 和survivin等凋亡抑制蛋白的表达水平，在诱导急性白血病细胞凋亡方面具有良好的临床应用前景。当细胞内发生DNA损伤反应（DDR）如DNA突变、DNA单/双链断裂、碱基缺失等，细胞周期检测点激酶 Chk2 可直接介导受损DNA的检测和修复，其表达异常会影响 DNA 的损伤修复或复制阻滞。
LAQ824对弥漫大B细胞淋巴瘤是否有效，目前尚未有报导。
为此，课题组对新型HDAC抑制剂LAQ824在弥漫大B细胞淋巴瘤中的作用和机制进行了试验和研究。研究发现，LAQ824在一定浓度范围（0~0.1μM）内可有效杀伤DLBCL细胞并表现出药物浓度依赖性，通过抑制DLBCL细胞中Chk2的表达，减弱Chk2对肿瘤细胞DNA损伤的修复，可以增加细胞对药物的敏感性，诱导DLBCL细胞凋亡。
本发明提出一种新型HDAC抑制剂LAQ824在治疗弥漫大B细胞淋巴瘤中的应用。
进一步的，所述LAQ824为一种新型的组蛋白去乙酰化酶抑制剂HDACi。
进一步的，新型HDAC抑制剂LAQ824在杀伤DLBCL细胞治疗弥漫大B细胞淋巴瘤中的应用。
进一步的，新型HDAC抑制剂LAQ824在抑制DLBCL细胞中Chk2的表达，减弱Chk2对肿瘤细胞DNA损伤的修复，以增加细胞对药物的敏感性，诱导DLBCL细胞凋亡中的应用。
进一步的，LAQ824有效杀伤DLBCL细胞并表现出药物浓度依赖性的浓度范围为0~0.1μM。
有益效果：LAQ824在0~0.1μM浓度范围内可有效杀伤DLBCL细胞并表现出药物浓度依赖性，通过抑制DLBCL细胞中Chk2的表达，减弱Chk2对肿瘤细胞DNA损伤的修复，可以增加细胞对药物的敏感性，诱导DLBCL细胞凋亡。
本发明试验方法如下：
（1）CC8检测
取不同条件下处理后的DLBCL细胞100u加入96孔板，每组3个复孔，在避光的条件下每孔加10ul CCK8，孵育2-4小时，450nm波长处读取吸光度值。
（2）流式
取5-10万重悬的细胞，离心，弃上清，加入 Annexin V-FITC结合液轻轻重悬细胞，加入5μl Annexin V-FITC和10μl碘化丙啶染色液，轻轻混匀，室温(20-25℃)避光孵育10-20分钟，上机进行流式细胞仪检测。
（3）Western Blot
收集药物处理后的细胞，并用RIPA细胞裂解液裂解后，经离心机以12000rmp离心后收集蛋白，之后经BCA法定量蛋白。随后以每组等量的蛋白经电泳分离后，电转移至PVDF膜，以5％脱脂牛奶封闭一小时后，一抗孵育过夜，后二抗室温孵育一小时。通过ECL上机检测蛋白表达量。
（4）蛋白抗体芯片
为了深入研究LAQ824诱导DLBCL细胞凋亡的分子机制，挖掘LAQ824的作用靶点，本研究拟通过H-Wayen公司的信号通路磷酸化广谱筛选抗体芯片（PEX100，包含1318 种高特异抗体），分别检测31条信号通路中432个信号蛋白的679个磷酸化位点，以实现多条信号通路的同步筛选和具体调变位点的清晰定位。通过Western Blot验证，在DLBCL细胞中敲降或过表达候选基因后观察相应细胞表型的变化。
（5）构建DLBCL小鼠模型
本项目中，项目组通过移植DLBCL细胞株到NOD SCID小鼠体内，建立DLBCL小鼠模型。选取6~8周龄雄鼠，通过前肢腋窝皮下接种U2932细胞，1×107 cells/只。将U2932细胞用PBS重悬调整至适宜密度后，与基质胶按照体积比1：1混合后用于接种。待肿瘤肉眼可见后定期测量肿瘤直径计算肿瘤体积，肿瘤大小计算公式：肿瘤体积（mm3）=0.5×（肿瘤长径×肿瘤短径2）。待肿瘤体积长至100~300mm3时将小鼠随机分为3组，每组 6-10只，设置为阴性对照组、LAQ824（75 mg/kg）。LAQ824予尾静脉注射，每5天给一次药，连续治疗4次。于给药后第3天静脉注射18F-FLT,1 h后进行小动物PET全身静态扫描，使用PMOD勾画包括肿瘤在内的感兴趣区域，获得各组织放射性摄取值。每周2次检测肿瘤和体重，每次间隔2~3天，连续监测28天，并在第28天扫描后剥取肿瘤，拍照并称量肿瘤重量，确定受试物抗肿瘤药效。
（6）18F-FLT PET示踪技术进行抗肿瘤疗效评价
18F-FLT PET是一种显示髙分级淋巴瘤的灵敏的显像方法，对于探测淋巴瘤具有较髙的灵敏度，而且还能够对淋巴瘤的增殖情况进行有效的活体检测。18F-FLT作为一种反映细胞增殖的正电子示踪剂，应用于PET显像可以无创、定量地在分子水平观察机体内肿瘤的增殖情况，提髙肿瘤诊断的特异性，同时也提供了一种检测肿瘤治疗反应的方法。本研究中，项目组对构建的DLBCL模型小鼠予不同条件处理，分别于给药前及给药后第3天静脉注射18F-FLT,1h后进行小动物PET全身静态扫描，使用PMOD勾画包括肿瘤在内的感兴趣区域，获得各组织放射性摄取值，对药物的抗肿瘤疗效进行评估。
附图说明
图1为LAQ824杀伤DLBCL细胞的表型及分子机制图；
其中：
图 1 (A)CCK8检测LAQ824对14种DLBCL细胞系增殖能力的影响；
图 1 (B)流式检测LAQ824对DLBCL细胞凋亡的影响；
图 1 (C,D,F) Western Blot检测相关蛋白表达水平；
图 1 (E)蛋白抗体芯片检测LAQ824处理前后DLBCL细胞相关蛋白表达信号；
图2为LAQ824在体内抑制DLBCL肿瘤生长试验结构图；
其中：
图2 (A) LAQ824治疗组及对照组静脉注射18F-FLT后进行PET全身静态扫描；
图2 (B)第28天剥取的LAQ824治疗组及对照组的DLBCL肿瘤。
具体实施方式
试验例1：肿瘤细胞增殖抑制实验
项目组检测了不同处理条件下14 种 DLBCL 细胞系（包括 GCB 和 non-GCB 型）的增殖能力，取对数生长期的DLBCL细胞与LAQ824（0uM，0.01uM，0.1uM）共同孵育24h，48h后，通过CCK8的方法检测肿瘤细胞增值情况，结果显示，LAQ824 处理后，绝大多数DLBCL细胞系的存活率在一定范围内发生显著下调，药物浓度超过0.1 μM后，抑制细胞增殖程度未有显著改变，在24 h 和48 h组中趋势一致（图 1A）。
试验例2：LAQ824诱导DLBCL细胞发生凋亡
取对数生长期的DLBCL细胞与LAQ824（0uM，0.01uM，0.1uM）共同孵育24h，48h后经流式细胞仪检测细胞凋亡情况。流式分析发现，以细胞系U2932、HBL-1为例，LAQ824可诱导DLBCL细胞凋亡，凋亡水平呈浓度/时间依赖性（图 1B）。
试验例3：蛋白抗体芯片（PEX100）挖掘 LAQ824 作用靶点
项目组利用蛋白抗体芯片（PEX100）挖掘LAQ824作用靶点，芯片结果发现LAQ824处理后细胞周期检测点激酶Chk2的表达被抑制（图 1E）。Chk2 直接介导受损 DNA 的修复，抑制Chk2的表达会削弱肿瘤细胞的DNA损伤修复或复制阻滞，提高肿瘤细胞对放、化疗药物的敏感性。
试验例4：Western Blot检测DLBCL中凋亡、乙酰化及DNA损伤相关蛋白的变化
收集药物LAQ824（0uM，0.01uM，0.1uM）处理后的细胞，经Western Blot检测药物处理前后重要凋亡蛋白的表达情况，结果显示活化的凋亡蛋Cleaved-caspase-3/9 以及Cleaved-PARP表达均升高，同时抗凋亡蛋白Bcl-2表达降低，与流式检测结果一致（图 1C）。LAQ824是非常有效的组蛋白去乙酰基酶抑制剂，项目组通过Western Blot检测了组蛋白H3的表达及其乙酰化水平，结果表明，药物处理后组蛋白H3的表达上调，LAQ824促进了DLBCL细胞的组蛋白乙酰化（图 1D）。同时Western Blot检测发现，随着LAQ824浓度升高，Chk2的表达被抑制，同时γH2AX水平上调（图 1F），提示同时通过抑制Chk2表达削弱了肿瘤细胞的DNA损伤修复功能，组蛋白H2AX的Ser-139残基的磷酸化（γH2AX）标志着DNA双链断裂（DSB），被认为是最致命的 DNA 损伤形式之一，严重损害了基因组稳定性，从而显著诱导细胞凋亡，对DLBCL细胞起到有效杀伤作用。
试验例5：LAQ824抑制小鼠体内DLBCL细胞生长
项目组通过移植DLBCL细胞株到NOD SCID小鼠体内，建立DLBCL小鼠模型。将小鼠随机分为2组，为阴性对照组、LAQ824（75 mg/kg）。于给药后第3天静脉注射18F-FLT,1 h后进行小动物PET全身静态扫描，使用PMOD勾画包括肿瘤在内的感兴趣区域，获得各组织放射性摄取值。于每周2次检测肿瘤和体重，连续监测28天，并在第28天扫描后剥取肿瘤，拍照并称量肿瘤重量，确定受试物抗肿瘤药效。18F-FLT PET能够对淋巴瘤的增殖情况进行有效的活体检测。通过18F-FLT PET和肿瘤体积的检测发现LAQ824能够在体内抑制DLBCL肿瘤细胞增殖及生长。
Claims (5)
1.新型HDAC抑制剂LAQ824在治疗弥漫大B细胞淋巴瘤药物中的应用。
2.根据权利要求1所述的应用，其特征在于：LAQ824为一种新型的组蛋白去乙酰化酶抑制剂HDACi。
3.新型HDAC抑制剂LAQ824在杀伤DLBCL细胞治疗弥漫大B细胞淋巴瘤中的应用。
4.新型HDAC抑制剂LAQ824在抑制DLBCL细胞中Chk2的表达，减弱Chk2对肿瘤细胞DNA损伤的修复，以增加细胞对药物的敏感性，诱导DLBCL细胞凋亡中的应用。
5.根据权利要求3所述的应用，其特征在于：LAQ824有效杀伤DLBCL细胞并表现出药物浓度依赖性的浓度范围为0~0.1μM。
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