Name: a method for screening and validation of resistant mutations against kinase inhibitors
Uploaded: 2014-12-07T15:00:00.000+00:00
Duration: 12 min 40 s
Description: Watch this Scientific Journal Video about A Method for Screening and Validation of Resistant Mutations Against Kinase Inhibitors at JoVE.com

This study was supported by grants to M.A. from NCI (1RO1CA155091), NHLBI (1R21HL114074) and the Leukemia Research Foundation. M.A. is a recipient of V-Scholar award from the V- Foundation. Authors are thankful to Dr. Sara Rohrabaugh for editing.

注意:在这个协议中的所有程序都根据对动物伦理治疗和护理健康准则国家研究所进行的，并且根据批准的IA​​CUC动物使用的协议。 1.细胞的维护<ol><li>培养BaF3细胞在RPMI-1640培养基补充有10％胎牛血清和青霉素/链霉素（100单位/ ml和100μg/ ml）和WEHI细胞的废培养基。生长HEK293T细胞在DMEM补充有10％胎牛血清和青霉素/链霉素（100单位/毫升和100微克/毫升）。保持在含有5％CO 2的潮湿气氛中的细胞在37℃。 </li></ol> 2.质粒构建<ol><li>克隆小鼠JAK2及其致癌亚型JAK2-V617F中的pMSCV-PURO-GW通过LR使用标准重组克隆过程克隆酶。 </li><li>对于荧光素酶表达载体（的pMSCV -吕克-樱桃-GW）用于建造在-VI沃成像，使用引物（LUC-SD / RP TTACAATTTGGACTTTCCG和LUC-SD / FP-CACCATGGAAGACGCCAAAAAC），随后用克隆在载体pENTR-SD-TOPO生成载体pENTR-吕克，其用于通过PCR扩增来自pLVX-TET-ON载体的萤光素酶通过使用LR克隆酶介导的重组克隆转移荧光素酶基因的逆转录病毒载体，的pMSCV樱桃-GW。 </li></ol> 3.准备随机突变库，筛选和鉴定的突变 3.1）随机突变<ol><li>克隆全长的JAK2野生型和V617F突变成的pMSCV-PURO-GW逆转录病毒载体使用市售的重组克隆技术。 </li><li>使用1 JAK2-V617F质粒DNA微克改造，XL-1红，E.大肠杆菌细胞，这是有缺陷的DNA修复机制从而使他们能够在复制期间掺入随机突变。更具体地说，混合50 - 100纳克的DNA（超过100毫微克的DNA降低转化效率）和100微升感受态细胞中预冷的聚丙烯管中，并在冰上孵育30分钟，轻轻摇动，每5分钟。一个良好的库覆盖，使用感受态细胞的四到六管中。 注:超过100毫微克DNA的降低转化效率</li><li>浸入管在水浴中于42℃45秒的热冲击，并在冰上孵育2分钟。接着，加入1 ml的SOC培养基（2.0％胰蛋白胨，0.5％酵母提取物，0.05％的NaCl，10mM的氯化镁，10mM的硫酸镁和0.4％D-葡萄糖）。孵育所述管在37℃下以225-250转摇动90分钟。 </li><li>板细胞上410厘米的LB琼脂平板上含有100微克/毫升氨苄青霉素的每个管中。孵育板16 - 24小时，在37℃下。 </li><li>下面可见菌落，通过刮板用无菌板刮板收集它们。池单元从用市售的质粒提取试剂盒各板及分离质粒DNA。 注:通常菌落较小，并采取18-24小时是可见的，因为这些都是生长缓慢的细菌菌株。在此阶段，评估在库突变通过限制性消化的异质用频繁切割器如Sau3A1或Taq1或测序。 </li></ol>逆转录病毒上清转导和3.2）生产<ol><li>前一天的转染，板4×10 6的HEK 293T细胞在含有10％FCS，青霉素/链霉素和2mM L-谷氨酰胺100厘米菜肴的DMEM。 </li><li>第二天，更换培养基和转染细胞诱变的pMSCV-JAK2-V617F库。对于每个10cm平板，混合10微克的DNA（5微克质粒文库和5微克逆转录病毒包装质粒，pCLEco）与40微升的FuGENE对使用无血清DMEM培养基加入400μl的总体积。孵育DNA和脂质混合20分钟，在室温下进行。 20分钟后，添加明智的DNA-脂质复合物滴在HEK293T细胞的顶部。 </li><li> 6小时后，改转染媒体瓦特第i个新鲜培养基并孵育板48小时，在37℃下进行病毒生产。 48小时后培养，收集通过0.45μm的acrodisc的过滤器过滤的病毒上清。 </li></ol> 3.3）选择抗性克隆的体外 <ol><li>对于耐药JAK2-V617F突变体，转导亿BAF3用100ml具有0.5-1×10 6病毒滴度的病毒上清的细胞。更具体地说，混合10 8个细胞用100ml的病毒上清以使用RPMI培养基300毫升的总体积，含有10％血清和polyberene的24微克/毫升（ployberene的终浓度为8微克/毫升）。发布这些细胞搅拌器在多个6孔板（4-5毫升，每孔）。 </li><li>离心板在1250×g离心90分钟，在25℃。离心后，孵育所述板在37℃下24小时。 </li><li>第二天，凝聚细胞一起，并与Ruxolitinib和介质含有软琼脂混合并铺在SIX孔板中。对于每一个药物浓度，混合10毫升转导的BaF3细胞（10-20个细胞）与Ruxolitinib在50ml管适量的。补足体积至用含有20％血清的RPMI 40毫升在6孔板中加入10 mL 1.2％琼脂的细胞，mixcarefully和板。 </li><li>孵育所述板在37℃下两周。挑用0.2毫升吸管尖端和亚培养它们分别在24孔板3-4天的抗性菌落。 </li><li>通过离心收获抗性的BaF3细胞在450×g离心，在室温下5分钟。隔离使用市售的基因组DNA分离试剂盒的基因组DNA。 PCR扩增全长的JAK2基因从100ng的基因组DNA使用引物（mJAK2FP-ATGGCCTGCCTTACAATGACAG和mJAK2RP-GGTTCTGGAAGTCTGCTTGG）和长模板扩大高保真PCR系统。 </li><li>分离的PCR产物通过琼脂糖凝胶电泳。切除的DNA条带的3.4 kb的代表JAK2编码框架和隔离DNA使用市售的凝胶提取试剂盒。序列的cDNA的使用8个内部引物（P1 - P8）全长跨越的编码序列，并分析序列使用商业软件。 </li></ol>耐药突变的4 体外验证许多细胞克隆携带一个​​以上的突变，为了测试在抗性表型的每个突变的贡献，产生所选的变体通过使用的pMSCV-JAK2V-617F质粒为模板位点定向诱变。 <ol><li>执行使用商业突变试剂盒和设计创造的点突变寡核苷酸的pMSCV-JAK2-V617F质粒定点突变。通过测序证实突变克隆的身份。 </li><li>转导的BaF3细胞与逆转录病毒用突变质粒，随后用选择使用嘌呤霉素在1微克/毫升制成。 </li><li>板10 4 BAF3-JAK2-V617F细胞（50微升的RPMI 10％FCS）中加入96孔板的每个孔中。另LY添加50微升含ruxolitinib媒体的横跨板的孔中（最终浓度:0，1，3，5，10，和20微米），并孵育板60小时，在37℃。 </li><li>通过加入10微升的WST-1试剂向每个井与在37℃下进行2-4小时，评估细胞生存力。在A450用酶标仪记录吸光度。完成所有实验一式三份和情节均对吸收INCB018424浓度。执行使用非线性曲线拟合算法的最佳拟合S形曲线。得分导致50％细胞生存力的细胞的IC 50中的药物浓度。 </li><li>接着，板6000000 BaF3细胞表达JAK2变种在6孔板中含有10％血清的增加ruxolitinib的浓度和在37℃下温育4-6小时的RPMI培养基。 </li><li>收集细胞，离心，在450×g离心在4℃下5分钟。在20mM Tris-CL一次用PBS，并裂解洗涤细胞（pH7.5）中，50mM氯化钠，1％NP-40，0.1％SDS，5mM的EDTA，1mM的EGTA，1mM的氟化钠，2mM的钒酸钠，和5％的甘油补充有蛋白酶抑制剂混合物和磷酸酶抑制剂。超声处理的细胞悬浮液1分钟，随后加入5倍的凝胶上样缓冲液（350毫摩尔Tris盐酸[pH为6.8]，500mM的DTT，15％SDS的10mM苯甲脒，5mM的EDTA，5mM的EGTA，5mM的钒酸钠，蛋白酶鸡尾酒，50％甘油，和0.001％溴酚蓝）和变性在70℃下5-10分钟。 </li><li>解决在8％SDS聚丙烯酰胺凝胶中的蛋白质变性条件下。执行与鼠单克隆抗phopsho STAT5免疫印迹。剥离印迹，并再次探测与兔多克隆抗SATA5抗体。使用可视化ECL试剂根据供应商的建议波段。 </li></ol> 5. 在体内验证<ol><li>转导BAF3细胞表达荧光素酶和樱桃（转导自体pMSCV - 吕克 - 雪儿做逆转录病毒RY GW）病毒表达JAK2-V617F和抗药性的变种。选择细胞存活嘌呤选择用于JAK2和其电阻变体的表达。 </li><li>注入经尾静脉注射携带JAK2-V617F及其耐药变异体在200μl的PBS中，以6只Balb / C小鼠2000000活跃生长BAF3吕克/樱桃细胞。 </li><li>后细胞移植的三天，每天两次两周注射小鼠Ruxolitinib（100毫克/千克）。 </li><li>与成像系统执行的荧光素酶为基础的生物发光成像。 。 <ol><li>简言之，通过将300毫克至25ml无菌去离子水，分装在较小体积和存储制备荧光素溶液。 注:储存荧光素在-80℃下并避光保护直到使用。 </li><li>注入250μl到每只小鼠通过IP来实现在每只小鼠125毫克/公斤。麻醉从一起使用，在密封箱吸入异氟烷（1.5％-2.0％），同一组中的小鼠。适用于日兽医药膏Ë眼睛，防止干涩。 注:采取小鼠的睡眠时间（10-15分钟）使荧光素活性峰值。保持时间的组之间是一致的，以避免变形。 </li></ol></li><li>马上转移的小鼠成像室阶段，在成像过程维持麻醉在1.5-2％异氟醚。图像小鼠顺序0.1，0.5，1，5，30，60和300秒的曝光。捕捉图像，并使用图像采集和分析软件定量生物发光强度。继成像返回老鼠回到笼子里。 </li><li>为， 在体内嵌合收获总骨髓细胞。安乐死的小鼠用CO 2进行5分钟，随后用颈椎脱位。收获的骨头和4毫升粉碎冷PBS以收集骨髓。分析荧光显微镜下的百分樱桃阳性细胞和量化使用FACS。收集从每个样品两万事件。 </li></ol>

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No conflicts of interest declared.

伊马替尼的治疗慢性粒细胞白血病的临床成功证明了小分子抑制剂靶向的胭脂激酶不仅潜力，但靶向治疗还发现限制:临床复发和耐药性的突变的靶基因中的出现。耐药突变鉴定有助于更好的临床管理和下一代抑制剂的发展。这个协议描述了一种方法，以确定在目标基因的耐药突变。此方法使用内置在大肠杆菌中随机诱变的质粒文库大肠杆菌 ，表达的突变体蛋白。库，然后在BaF3细胞（由癌基因易被转化）介绍，接着用在化学治疗剂的存在下选择的细胞克隆。从抗性克隆测序靶基因的识别突变抗性。最后，确定了突变位点被重新定向诱变来验证他们耐药性。 使用这种策略，我们定义突变JAK2和BCR / ABL赋予抗Ruxolitinib和伊马替尼，分别的频谱。我们确定了BCR / ABL百余突变。此外，确定患者检测到的所有主要的突变，我们的筛选方法，使我们能够识别内外激酶结构域残新型替代。有趣的是，从我们屏幕上的所有突变已患者样品中确定，但这一过程用了近10年以上15，从而证明了这种屏幕的预期突变，会造成困扰临床耐药性的能力。同时采用随机诱变性筛选提供了相较于其他方法很多优点，也有潜在的隐患要注意以下筛选程序时。对于任何筛选，强烈建议BAF3细胞完全依赖于目标针对其RESI立场是追捧。目标基因上的失败或部分依赖可能不会发展真正的性克隆和最有可能的发展误报。例如，四个不同的研究已经进行抗性筛选针对使用BaF3细胞转导的任一的EPO或MPL受体以促进JAK2-V617F依赖性16-19 JAK2抑制剂。只有八限于激酶域抗性突变从这些画面被确定，虽然众多抗性克隆开发了缺乏的突变，大概是由于误报，这归因于JAK1和JAK3的异二聚细胞因子受体的出现。因此，为了避免JAK2依赖性的损耗，我们进行了以纯的BAF3抗性筛选该显示鲁棒选择针对Ruxolitinib并且所有这些克隆的抗性克隆显示出耐药性突变的存在。根据这些意见，以充分利用的耐药突变的全部范围，我们建议的BaF3细胞s应该是完全依赖于有针对性的激酶，以避免误报的出现，因为一直在对JAK2抑制剂16,17以前进行抗场次一种常态。此外，重要的是避免散装培养条件，使细胞携带不同突变汇集起来，并使其在液体培养基中扩大，因为这可能会导致克隆的几个高度耐药变异体的主导地位。例如，初始选择的诱变BCR-ABL的散装液体培养伊马替尼耐药性时，我们发现只有4人是在第100株，我们测序为代表多次突变形式。因此，用软琼脂筛选允许缓慢生长的克隆用较温和的程度的耐药性被恢复，将不被使用批量培养鉴定的。出于这个原因，建议将前只对14至16小时的选择批量培养生长，随后用无IL3的药物选择在软琼脂。在我们的前PERIENCE，越来越多的文化超越36小时后病毒转导往往是由高度增生的克隆，这对风险失去生长缓慢的克隆（弱变革）为主。同样地，使用的细胞从更早的时间点，如病毒转导后6-8小时是容易选择高度变革或过表达的克隆，从而引起一个偏差和错误陈述抗性克隆的身份和频率。因此，我们建议使用生长14-16小时后病毒转导进行筛选，提供严密的选择单个克隆和克隆阻止通常的优势散装液体培养观察到的细胞。 关键是要确定候选人突变的授予孤立性的能力，因为一些克隆鉴定有多个突变。对于JAK2-V617F的屏幕，我们利用定点mutage重建的大部分来自我们的随机突变库发现的突变NESIS。引入所述分离的突变体成新鲜细胞后，将它们再生长在各种药物的存在下，以确定的IC50为每个突变体。我们还通过监测生物发光，以进一步证实这些单突变引起耐药性在体内的能力测试了在体内抵抗两种不同的抗性变异体。 鉴于在治疗癌症镀锌激酶抑制剂在诊所浪涌抗激酶治疗的成功，这是至关重要的，以确定临床上问题的赋予抗性的突变为改善患者的管理和开发药物，可以针对它们。该筛选策略已被成功地用于鉴定在BCR / ABL，JAK2和FLT3突变20;然而，我们认为，这将是普遍适用于广泛的抗肿瘤剂。

蛋白激酶是细胞内的信号转导通路的调节似乎每个细胞功能调控的关键酶。激酶介导的信号的适当控制是至关重要的稳态和发展，这主要是依赖于激酶，磷酸酶及其降解适当调控UPS（泛素蛋白酶体系统）。放松管制的激酶是许多癌症的中心舞台，并牵连到主机人类疾病1。人类基因组编码超过500蛋白激酶已被链接，直接或间接地，以400〜人类疾病2。这些观察结果支持这一概念用于激酶的小分子抑制剂3-5治疗靶向。 ABL的激酶抑制剂，如伊马替尼的论证，慢性粒细胞白血病（CML）的治疗提供了概念证明这种方法6,7。这一观察，不仅彻底改变了蚂蚁的i-激酶疗法而且执行的想法，以确定遗传病变的其他肿瘤疾病的治疗为目标，这导致发现的致癌基因突变的从真性红细胞增多症（PV）和患者的骨髓增生性肿瘤（MPN）的JAK2。这一发现产生的靶向JAK2小分子激酶抑制剂治疗MPNS极大的兴趣。现在，JAK2抑制剂几乎一打的临床试验，其中一人已被批准用于骨髓纤维化的治疗。而致癌激酶在癌症的小分子抑制剂特异性靶向携带有为结果如何，还患有显影抵抗的治疗。实际上，到目前为止，患者大多通过获得突变的激酶域的药物靶向8-10治疗激酶抑制剂，如伊马替尼，吉非替尼，埃罗替尼和达沙替尼抗药性的突变。阻力基因突变的结果凸显了限制Ø˚F靶向单一疗法对抗致癌激酶，并且表示在以往更成功的癌症化学疗法的发展下一个挑战。耐药机理和功能的后果应提供选择和药物开发的免费化合物的设计理念。通过在体外筛选鉴定的突变，都表现出与在患者中发现的相关程度高。因此， 在体外筛选赋予识别的电阻图案，有可能导致临床复发耐药在临床或临床前开发协助一个给定的药物靶标对突变。这些突变体形式的识别是不仅在监测患者对药物的反应和复发，还必需为更健壮的下一代抑制剂的设计有帮助的。例如，下一代BCR / ABL抑制剂，尼罗替尼和Ponatinib的发展，分别因为更大MEC成为可能hanistic理解的突变，结构和功能的研究中获得。 此前，我们曾报道我们的屏幕采用随机突变BCR / ABL的揭示突变赋予抗抑制剂如伊马替尼11,12，PD166326 12和AP24163 13的频 ​​谱的结果。结果不但确定授予临床耐药和疾病复发的基因突变，但也提供了耐药性和管理的激酶功能11,14原理的机械理解。在这里，我们提供额外的方法的细节，使用Ruxolitinib和JAK2作为药物靶标对，使这一筛选策略的更广泛的应用。

<table><tbody><tr><td>Cell and Tissue culture&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>BaF3 Cells</td><td>ATCC</td><td></td><td></td></tr><tr><td>HEK293T cells</td><td>ATCC</td><td></td><td></td></tr><tr><td>pMSCV-JAK2-V617F-puro.GW</td><td>A gift from Ross Levine</td><td></td><td></td></tr><tr><td>pMSCV-JAK2-V617F/Y931C.GW</td><td>Made in house</td><td></td><td></td></tr><tr><td>pMSCV-JAK2-V617F/L983F.GW</td><td>Made in house</td><td></td><td></td></tr><tr><td>pMSCV-JAK2-V617F/P58A.GW</td><td>Made in house</td><td></td><td></td></tr><tr><td>pMSCV-V617F-Cherry.GW</td><td>Made in house</td><td></td><td></td></tr><tr><td>pMSCV-JAK2-V617F/Y931C-cherry.GW</td><td>Made in house</td><td></td><td></td></tr><tr><td>pMSCV-JAK2-V617F/L983F-cherry.GW</td><td>Made in house</td><td></td><td></td></tr><tr><td>pMSCV-Luciferase-puro.GW</td><td>Made in house</td><td></td><td></td></tr><tr><td>RPMI</td><td>Cellgro (corning)</td><td>15-040-CV</td><td></td></tr><tr><td>DMEM</td><td>Cellgro (corning)</td><td>15-013-CV</td><td></td></tr><tr><td>Penn/Strep</td><td>Cellgro (corning)</td><td>30-002-CI</td><td></td></tr><tr><td>FBS</td><td>Atlanta biological</td><td>S11150</td><td></td></tr><tr><td>Trypsin EDTA 1X</td><td>Cellgro (corning)</td><td>25-052-CI</td><td></td></tr><tr><td>1x PBS</td><td>Cellgro (corning)</td><td>21-040-CV</td><td></td></tr><tr><td>L-Glutamine</td><td>Cellgro (corning)</td><td>25-005-CL</td><td></td></tr><tr><td>Puromycin</td><td>Gibco (life technologies)</td><td>A11138-03</td><td></td></tr><tr><td>Protamine sulfate</td><td>Sigma</td><td>P3369</td><td>5 mg/ml stock in water</td></tr><tr><td>Trypan Blue solution (0.4%)</td><td>Sigma</td><td>T8154</td><td></td></tr><tr><td>DMSO</td><td>Cellgro (corning)</td><td>25-950-CQC</td><td></td></tr><tr><td>INCB018424 (Ruxolitinib)</td><td>ChemieTeK</td><td>941678-49-5</td><td></td></tr><tr><td>WST-1</td><td>Roche</td><td>11644807001</td><td></td></tr><tr><td>0.45 micron disc filter</td><td>PALL</td><td>2016-10</td><td></td></tr><tr><td>70 micron nylon cell strainer</td><td>Becton Dickinson</td><td>352350</td><td></td></tr><tr><td>Bacterial Culture</td><td></td><td></td><td></td></tr><tr><td>XL-1 red &lt;em&gt;E. coli&lt;/em&gt; cells</td><td>Agilent Tech</td><td>200129</td><td></td></tr><tr><td>SOC</td><td>New England Biolabs</td><td>B90920s</td><td></td></tr><tr><td>Ampicillin</td><td>Sigma</td><td>A0166</td><td>100 mg/ml stock solution&amp;nbsp;</td></tr><tr><td>Bacto agar</td><td>Difco</td><td>214050</td><td></td></tr><tr><td>Terrific broth</td><td>Becton Dickinson</td><td>243820</td><td></td></tr><tr><td>Agarose</td><td>Genemate</td><td>E-3119-500</td><td></td></tr><tr><td>Kits</td><td></td><td></td><td></td></tr><tr><td>Dneasy Blood&amp;amp; tissue kit</td><td>Qiagen</td><td>69506</td><td></td></tr><tr><td>Expand long template PCR system</td><td>Roche</td><td>1168134001</td><td></td></tr><tr><td>Wizard Sv gel and PCR clean up system</td><td>Promega</td><td>A9282</td><td></td></tr><tr><td>Pure Yield plasmid mini prep system</td><td>Promega</td><td>A1222</td><td></td></tr><tr><td>PCR Cloning System with Gateway Technology with pDONR 221 &amp;amp; OmniMAX 2 Competent Cells</td><td>Invitrogen</td><td>12535029</td><td></td></tr><tr><td>Gateway&amp;nbsp; LR Clonase Enzyme mix&amp;nbsp;</td><td>Invitrogen</td><td>11791019</td><td></td></tr><tr><td>Mouse reagents</td><td></td><td></td><td></td></tr><tr><td>Vivo-Glo Luciferin in-vivo Grade</td><td>Promega</td><td>P1043</td><td></td></tr><tr><td>1/2 cc Lo-Dose u-100 insulin syringe 28 G1/2</td><td>Becton Dickinson</td><td>329461</td><td></td></tr><tr><td>Mortor pestle</td><td>Coor tek&amp;nbsp;</td><td>60316 and 60317</td><td></td></tr><tr><td>Isoflorane (Isothesia TM)</td><td>Butler Schien</td><td>29405</td><td></td></tr><tr><td>Instruments</td><td></td><td></td><td></td></tr><tr><td>NAPCO series 8000 WJ CO2 incubator</td><td>Thermo scientific</td><td></td><td></td></tr><tr><td>Swing bucket rotor centrifuge 5810R</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>TC-10 automated cell counter</td><td>Bio-RAD</td><td></td><td>This is not necessary, one can use standard hemocytomemetr for cell counting</td></tr></tbody></table>

a method for screening and validation of resistant mutations against kinase inhibitors

BCR / ABL的发现作为驱动基因在慢性骨髓性白血病（CML）导致了伊马替尼，其中，实际上，证明有效地治疗慢性粒细胞白血病患者靶向激酶在癌症的潜力的发展。这一观察革命性的药物开发目标牵连其他各种恶性肿瘤，如，表皮生长因子受体，B-RAF，KIT和PDGFRs致癌激酶。然而，抗激酶疗法的一个主要的缺点是耐药突变渲染目标的出现，已经减弱或消失亲和性的药物。了解了耐药变异不仅有助于开发下一代抑制剂也采用个性化医疗带动了临床管理使用的机制。我们报道了逆转录病毒载体基于筛选策略来识别抗性赋予在BCR / ABL，突变这在开发下一代BCR / ABL抑制剂帮助的频谱。使用Ruxoli替尼和JAK2作为药物靶标对，在这里我们将介绍在利用表达JAK2激酶的随机突变库鼠标BAF3细胞的体外筛选方法。

基因突变的出现带来了针对性的抗激酶治疗的巨大挑战。突变的研究，除了提供那些有助于选择和下一代药物开发的机械设计和功能的见解，也可以更好地临床管理，可能在未来成为个性化的治疗更有帮助。在这个实验中，我们显示筛选ruxolitinib抗性突变JAK2-V617F激酶（ 图1）。我们构建了通过引入全长小鼠JAK2-V617FcDNA成的pMSCV樱桃-GW的pMSCV-JAK2-V617F-cherry.gateway载体。它建议使用的细菌宿主（ 大肠杆菌 XL-1红色株）在最流行 ​​的选择方法，这是PCR中，由于其与序列偏置，以及较大的基因片段相关联的限制是难以扩增开发随机突变。随机诱变的DNA文库转染到HEK293T细胞进行逆转录病毒的生产。这些突变VIR用途分别用，转导中的选定菌落生长在软琼脂在不存在IL-3与1或5 Ruxolitinib的μM的BaF3细胞。在这些条件下，菌落从细胞携带JAK2V-617F的cDNA表达的激酶的功能和耐变仅出现。后10 - 14天，以及分离各个菌落，其变化的大小，并在液体培养扩展它们。基因组DNA，从这些细胞中分离。前病毒被收回的直接救援或PCR。将回收的原病毒进行测序，以确定突变（ 图1）。序列分析采用DNASTAR包LASERGENE的执行。 因为许多细胞克隆进行多个突变，我们强烈的体 ​​外和体内测定法推荐验证这些突变在隔离双方（ 图2和3）。为了验证变种的活动隔离，通过网站生成变种选择执导的JAK2V-617F序列的突变。这些变体再引入BaF3细胞，并测量在不同剂量Ruxolitinib的细胞增殖的能力来评估IC 50（IC 50值， 图2A）。生化测定法执行用于磷酸酪氨酸或磷酸STAT5通过免疫印迹分析在BaF3细胞的蛋白裂解物即孵育随Ruxolitinib的剂量，以排除任何脱靶介导的抗性（ 图2B）。突变是在较高浓度Ruxolitinib增强参展IC50值和持续磷酸化从而证实是耐药变异。因为许多抗性突变表明可变剂量反应，因此有必要以测试是否这些突变会在体内阻力赋予为好。一般，我们建议以用于体内实验测试仅2 -3不同变体，因为它们是昂贵的并且费力。为了验证体内阻力，BaF3细胞被工程化以表达JAK2-V617F变体和萤光素酶/樱桃，使在体内跟踪。小鼠注射1-2百万樱桃阳性细胞（表达JAK2变体和萤光素酶），接着Ruxolitinib注射两周。两周后，小鼠通过测定樱桃阳性细胞的荧光（ 图3）成像为萤光素酶催化的生物发光（ 图3），并且还对BAF3嵌合于骨髓和外周血。小鼠表达JAK2-V617F是Ruxolitinib敏感，而耐变体显示逐行增量在生物发光以上的治疗时期，从而表明BaF3细胞表达JAK2-V617F变体是抗Ruxolitinib治疗体内 。 <img alt="图1" src="/files/ftp_upload/51984/51984fig1highres.jpg" /> <strong&gt;图1:方案筛选抗性突变对激酶抑制剂Ruxolitinib。 <img alt="图2" src="/files/ftp_upload/51984/51984fig2highres.jpg" /> 图2:A方案显示在体外验证使用的剂量依赖性细胞增殖实验（A）和Western印迹（B） <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51984/51984fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/51984/51984fig3highres.jpg" /> 图3: 在体内验证使用荧光素酶的示意图催化的生物发光测量。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51984/51984fig3large.jpg" target="_blank">请点击此处查看大图</a>版本这个数字。

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A Method for Screening and Validation of Resistant Mutations Against Kinase Inhibitors

针对激酶抑制剂治疗遗传性的出现带来了有效的癌症治疗显著的挑战。对新开发的药物鉴定和耐药突变特性有助于更好的临床管理和下一代药物设计。在这里，我们描述了体外筛选及耐药突变的验证协议。

对于耐药性突变对激酶抑制剂的筛选和验证方法

The discovery of BCR/ABL as a driver oncogene in chronic myeloid leukemia (CML) resulted in the development of Imatinib, which, in fact, demonstrated the ...

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Biology

14.8K 次观看.  Cincinnati Children's Hospital Medical Center. 针对激酶抑制剂治疗遗传性的出现带来了有效的癌症治疗显著的挑战。对新开发的药物鉴定和耐药突变特性有助于更好的临床管理和下一代药物设计。在这里，我们描述了体外筛选及耐药突变的验证协议。 

视频: A Method for Screening and Validation of Resistant Mutations Against Kinase Inhibitors

Establishing Dual Resistance to EGFR-TKI and MET-TKI in Lung Adenocarcinoma Cells In Vitro with a 2-step Dose-escalation Procedure

Drug resistance is a major challenge in cancer therapy. The generation of resistant sublines in vitro is necessary for discovering novel mechanisms to overcome this challenge. Here, a 2-step dose-escalation method for establishing dual-resistance to an epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI), gefitinib, and a MET-TKI, PHA665752, is described. This method is based on simple stepwise dose-escalation of inhibitors for inducing acquired resistance in cell lines. The alternate method for generating resistant sublines involves exposing the cells to high concentrations of the inhibitor in one step. The stepwise dose-escalation method has a higher possibility of successfully inducing acquired resistance than this method. Activating EGFR mutations are biomarkers of a response to treatment with EGFR-TKI, which is an applied first-line treatment for non-small cell lung cancers (NSCLC) that harbor these mutations. However, despite reports of effective responses, the use of EGFR-TKI is limited because tumors inevitably acquire resistance. The major mechanisms behind EGFR-TKI resistance include a secondary mutation at the gatekeeper site, T790M in exon 20 of EGFR, and a bypass signal of MET. Thus, a potential solution for this issue would be a combination of EGFR-TKI and MET-TKI. This combined treatment has been shown to be effective in an in vitro study model. Acquired gefitinib-resistance was established through MET-amplification by stepwise dose-escalation of gefitinib for 12 months, and a cell line named PC-9MET1000 was generated in a previous study. To further investigate the mechanisms of acquired MET-TKI and EGFR-TKI resistance, a MET-TKI, PHA665752, was administered to these cells with stepwise dose-escalation in the presence of gefitinib for 12 months. This protocol has also been successfully applied for a number of combination therapies to establish acquired resistance to other inhibitor molecules.

Establishing Dual Resistance to EGFR-TKI and MET-TKI in Lung Adenocarcinoma Cells In Vitro with a 2-step Dose-escalation Procedure

An in vitro method for establishing dual resistance to an EGFR-TKI and a MET-TKI in cancer cells is described. This method is useful for developing treatments for patients with EGFR-mutations, who exhibit disease progression despite EGFR-TKI treatment with MET-amplification. It can also be modified for inhibitors targeting other molecules.

建立双抗 EGFR-使用 tki 药物治疗和满足使用 tki 药物治疗在肺腺癌细胞体外与 2 步剂量升级程序

Drug resistance is a major challenge in cancer therapy. The generation of resistant sublines in vitro is necessary for discovering novel mechanisms to ...

科研

癌症研究

Assessment of Resistance to Tyrosine Kinase Inhibitors by an Interrogation of Signal Transduction Pathways by Antibody Arrays

Cancer patients with an aberrant regulation of the protein phosphorylation networks are often treated with the tyrosine kinase inhibitors. Response rates approaching 85% are common. Unfortunately, patients often become refractory to the treatment by altering their signal transduction pathways. An implementation of the expression profiling with microarrays can identify the overall mRNA-level changes, and proteomics can identify the overall changes in protein levels or can identify the proteins involved, but the activity of the signal transduction pathways can only be established by interrogating post-translational modifications of the proteins. As a result, the ability to identify whether a drug treatment is successful or whether resistance arose, or the ability to characterize any alterations in the signaling pathways, is an important clinical challenge. Here, we provide a detailed explanation of antibody arrays as a tool which can identify system-wide alterations in various post-translational modifications (e.g., phosphorylation). One of the advantages of using antibody arrays includes their accessibility (an array does not require either an expert in proteomics or costly equipment) and speed. The availability of arrays targeting a combination of post-translational modifications is the primary limitation. In addition, unbiased approaches (phosphoproteomics) may be more suitable for the novel discovery, whereas antibody arrays are ideal for the most widely characterized targets.

Here, we present a protocol for antibody arrays to identify alterations in signaling pathways in various cellular models. These changes, caused by drugs/hypoxia/ultra-violet light/radiation, or by overexpression/downregulation/knockouts, are important for various disease models and can indicate whether a therapy will be effective or can identify mechanisms of drugs resistance.

用抗体阵列对信号转导通路的审问对酪氨酸激酶抑制剂的抗性评价

Cancer patients with an aberrant regulation of the protein phosphorylation networks are often treated with the tyrosine kinase inhibitors. Response rates ...

Methods for Evaluating the Role of c-Fos and Dusp1 in Oncogene Dependence

The demonstration of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) has heralded a new era in cancer therapeutics. However, a small population of cells does not respond to TKI treatment, resulting in minimal residual disease (MRD); even the most potent TKIs fail to eradicate these cells. These MRD cells serve as a reservoir to develop resistance to therapy. Why TKI treatment is ineffective against MRD cells is not known. Growth factor signaling is implicated in supporting the survival of MRD cells during TKI treatment, but a mechanistic understanding is lacking. Recent studies demonstrated that an elevated c-Fos and Dusp1 expression as a result of convergent oncogenic and growth factor signaling in MRD cells mediate TKI resistance. The genetic and chemical inhibition of c-Fos and Dusp1 renders CML exquisitely sensitive to TKIs and cures CML in both genetic and humanized mouse models. We identified these target genes using multiple microarrays from TKI-sensitive and -resistant cells. Here, we provide methods for target validation using in vitro and in vivo mouse models. These methods can easily be applied to any target for genetic validation and therapeutic development.

Here, we describe protocols for the genetic and chemical validation of c-Fos and Dusp1 as a drug target in leukemia using in vitro and in vivo genetic and humanized mouse models. This method can be applied to any target for genetic validation and therapeutic development.

c-fos 和 dusp1 在癌基因依赖性中的作用评价方法

The demonstration of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) has heralded a new era in cancer therapeutics. However, ...

Characterize Disease-related Mutants of RAF Family Kinases by Using a Set of Practical and Feasible Methods

The rapidly accelerated fibrosarcoma (RAF) family kinases play a central role in cell biology and their dysfunction leads to cancers and developmental disorders. A characterization of disease-related RAF mutants will help us select appropriate therapeutic strategies for treating these diseases. Recent studies have shown that RAF family kinases have both catalytic and allosteric activities, which are tightly regulated by dimerization. Here, we constructed a set of practical and feasible methods to determine the catalytic and allosteric activities and the relative dimer affinity/stability of RAF family kinases and their mutants. Firstly, we amended the classical in vitro kinase assay by reducing the detergent concentration in buffers, utilizing a gentle quick wash procedure, and employing a glutathione S-transferase (GST) fusion to prevent RAF dimers from dissociating during purification. This enables us to measure the catalytic activity of constitutively active RAF mutants appropriately. Secondly, we developed a novel RAF co-activation assay to evaluate the allosteric activity of kinase-dead RAF mutants by using N-terminal truncated RAF proteins, eliminating the requirement of active Ras in current protocols and thereby achieving a higher sensitivity. Lastly, we generated a unique complementary split luciferase assay to quantitatively measure the relative dimer affinity/stability of various RAF mutants, which is more reliable and sensitive compared to the traditional co-immunoprecipitation assay. In summary, these methods have the following advantages: (1) user-friendly; (2) able to carry out effectively without advanced equipment; (3) cost-effective; (4) highly sensitive and reproducible.

In this article, we presented a set of practical and feasible methods for characterizing disease-related mutants of RAF family kinases, which include in vitro kinase assay, RAF co-activation assay, and complementary split luciferase assay.

使用一套实用可行的方法,描述英国皇家空军家族激酶与疾病相关的突变体

The rapidly accelerated fibrosarcoma (RAF) family kinases play a central role in cell biology and their dysfunction leads to cancers and developmental ...

Kinase Inhibitor Screening In Self-assembled Human Protein Microarrays

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with clinical implications. Several methodologies have been reported to accomplish such screening. However, each has its own limitations (e.g., the screening of only ATP analogues, restriction to using purified kinase domains, significant costs associated with testing more than a few kinases at a time, and lack of flexibility in screening protein kinases with novel mutations). Here, a new protocol that overcomes some of these limitations and can be used for the unbiased screening of kinase inhibitors is presented. A strength of this method is its ability to compare the activity of kinase inhibitors across multiple proteins, either between different kinases or different variants of the same kinase. Self-assembled protein microarrays generated through the expression of protein kinases by a human-based in vitro transcription and translation system (IVTT) are employed. The proteins displayed on the microarray are active, allowing for measurement of the effects of kinase inhibitors. The following procedure describes the protocol steps in detail, from the microarray generation and screening to the data analysis.

A detailed protocol for the generation of self-assembled human protein microarrays for the screening of kinase inhibitors is presented.

自组装的人类蛋白质微阵列中的激酶抑制剂筛选

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with ...

生物化学

Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for Treatment of Acute Leukemia

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are attractive druggable therapeutic targets. Here we describe an efficient four-step strategy for pre-clinical evaluation of tyrosine kinase inhibitors (TKIs) in the treatment of acute leukemia. Initially, western blot analysis is used to confirm target inhibition in cultured leukemia cells. Functional activity is then evaluated using clonogenic assays in methylcellulose or soft agar cultures. Experimental compounds that demonstrate activity in cell culture assays are evaluated in vivo using NOD-SCID-gamma (NSG) mice transplanted orthotopically with human leukemia cell lines. Initial in vivo pharmacodynamic studies evaluate target inhibition in leukemic blasts isolated from the bone marrow. This approach is used to determine the dose and schedule of administration required for effective target inhibition. Subsequent studies evaluate the efficacy of the TKIs in vivo using luciferase expressing leukemia cells, thereby allowing for non-invasive bioluminescent monitoring of leukemia burden and assessment of therapeutic response using an in vivo bioluminescence imaging system. This strategy has been effective for evaluation of TKIs in vitro and in vivo and can be applied for identification of molecularly-targeted agents with therapeutic potential or for direct comparison and prioritization of multiple compounds.

Receptor tyrosine kinases are ectopically expressed in many cancers and have been identified as therapeutic targets in acute leukemia. This manuscript describes an efficient strategy for pre-clinical evaluation of tyrosine kinase inhibitors for the treatment of acute leukemia.

酪氨酸激酶抑制剂的临床前评价急性白血病的治疗

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are ...

医学

Pooled shRNA Library Screening to Identify Factors that Modulate a Drug Resistance Phenotype

Understanding clinically relevant driver mechanisms of acquired chemo-resistance is crucial for elucidating ways to circumvent resistance and improve survival in patients with acute myeloid leukemia (AML). A small fraction of leukemic cells that survive chemotherapy have a poised epigenetic state to tolerate chemotherapeutic insult. Further exposure to chemotherapy allows these drug persister cells to attain a fixed epigenetic state, which leads to altered gene expression, resulting in the proliferation of these drug-resistant populations and eventually relapse or refractory disease. Therefore, identifying epigenetic modulations that necessitate the survival of drug-resistant leukemic cells is critical. We detail a protocol to identify epigenetic modulators that mediate resistance to the nucleoside analog cytarabine (AraC) using pooled shRNA library screening in an acquired cytarabine-resistant AML cell line. The library consists of 5,485 shRNA constructs targeting 407 human epigenetic factors, which allows high-throughput epigenetic factor screening.

High-throughput RNA interference (RNAi) screening using a pool of lentiviral shRNAs can be a tool to detect therapeutically relevant synthetic lethal targets in malignancies. We provide a pooled shRNA screening approach to investigate the epigenetic effectors in acute myeloid leukemia (AML).

合并的shRNA文库筛选，以确定调节耐药性表型的因素

Understanding clinically relevant driver mechanisms of acquired chemo-resistance is crucial for elucidating ways to circumvent resistance and improve ...

基因组规模筛选，用于识别和比较肿瘤中的治疗靶点

Genetic Profiling and Genome-Scale Dropout Screening to Identify Therapeutic Targets in Mouse Models of Malignant Peripheral Nerve Sheath Tumor

Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are derived from Schwann cells or their precursors. In patients with the tumor susceptibility syndrome neurofibromatosis type 1 (NF1), MPNSTs are the most common malignancy and the leading cause of death. These rare and aggressive soft-tissue sarcomas offer a stark future, with 5-year disease-free survival rates of 34-60%. Treatment options for individuals with MPNSTs are disappointingly limited, with disfiguring surgery being the foremost treatment option. Many once-promising therapies such as tipifarnib, an inhibitor of Ras signaling, have failed clinically. Likewise, phase II clinical trials with erlotinib, which targets the epidermal growth factor (EFGR), and sorafenib, which targets the vascular endothelial growth factor receptor (VEGF), platelet-derived growth factor receptor (PDGF), and Raf, in combination with standard chemotherapy, have also failed to produce a response in patients. 
In recent years, functional genomic screening methods combined with genetic profiling of cancer cell lines have proven useful for identifying essential cytoplasmic signaling pathways and the development of target-specific therapies. In the case of rare tumor types, a variation of this approach known as cross-species comparative oncogenomics is increasingly being used to identify novel therapeutic targets. In cross-species comparative oncogenomics, genetic profiling and functional genomics are performed in genetically engineered mouse (GEM) models and the results are then validated in the rare human specimens and cell lines that are available. 
This paper describes how to identify candidate driver gene mutations in human and mouse MPNST cells using whole exome sequencing (WES). We then describe how to perform genome-scale shRNA screens to identify and compare critical signaling pathways in mouse and human MPNST cells and identify druggable targets in these pathways. These methodologies provide an effective approach to identifying new therapeutic targets in a variety of human cancer types.

作者聚焦:通过基因组规模 shRNA 筛选寻找恶性周围神经鞘瘤的新治疗靶点 

We have developed a cross-species comparative oncogenomics approach utilizing genomic analyses and functional genomic screens to identify and compare therapeutic targets in tumors arising in genetically engineered mouse models and the corresponding human tumor type.

遗传分析和基因组规模的辍学筛选以确定恶性周围神经鞘瘤小鼠模型中的治疗靶点

Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are derived from Schwann cells or their precursors. In patients with the tumor susceptibility syndrome ...

对于耐药性突变对激酶抑制剂的筛选和验证方法

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建立双抗 EGFR-使用 tki 药物治疗和满足使用 tki 药物治疗在肺腺癌细胞体外与 2 步剂量升级程序

用抗体阵列对信号转导通路的审问对酪氨酸激酶抑制剂的抗性评价

c-fos 和 dusp1 在癌基因依赖性中的作用评价方法

使用一套实用可行的方法,描述英国皇家空军家族激酶与疾病相关的突变体

自组装的人类蛋白质微阵列中的激酶抑制剂筛选

酪氨酸激酶抑制剂的临床前评价急性白血病的治疗

合并的shRNA文库筛选，以确定调节耐药性表型的因素

遗传分析和基因组规模的辍学筛选以确定恶性周围神经鞘瘤小鼠模型中的治疗靶点

遗传分析和基因组规模的辍学筛选以确定恶性周围神经鞘瘤小鼠模型中的治疗靶点

遗传分析和基因组规模的辍学筛选以确定恶性周围神经鞘瘤小鼠模型中的治疗靶点