Preclinical drug development in pancreas cancer

胰腺癌的临床前药物开发

• 批准号: 8349435
• 负责人: Udo Rudloff
• 金额: $ 105.65万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8349435
• 关键词: Aftercare; Animal Model; Apoptosis; Basic Science; Biological Assay; Biological Markers; Breeding; CDKN2A gene; Cancer Biology; Cancer Patient; Cancer cell line; Cell Cycle; Cell Cycle Arrest; Cell Death Induction; Cell Fraction; Cell Line; Cells; Chemicals; Clinical Protocols; Clinical Trials; Common Neoplasm; Complement; Complex; Computing Methodologies; Cytostatics; Data; Dependency; Desmoplastic; Development; Disease; Dose; Drug Sensitization; Environment; Enzymes; Exercise; Exhibits; Extracellular Matrix; Functional Imaging; Future; G0 Phase; G2/M Checkpoint Pathway; Gene Expression; Gene Expression Profiling; Genes; Genetically Engineered Mouse; Genomics; Growth; Homeostasis; Hour; Human; Image; In Vitro; Induction of Apoptosis; Knock-in Mouse; Knockout Mice; Laboratories; Lethal Dose 50; Luciferases; Luminescent Measurements; MAP Kinase Gene; MAP3K8 gene; MEK inhibition; MEKs; Malignant Neoplasms; Malignant neoplasm of pancreas; Measures; Mediating; Metabolism; Mitogen-Activated Protein Kinase Kinases; Modeling; Molecular; Monitor; Mus; Mutation; National Human Genome Research Institute; Normal saline; Oncogenes; Operative Surgical Procedures; Organ; PTEN gene; Pancreatic Ductal Adenocarcinoma; Pathway interactions; Patient Selection; Patients; Phase II Clinical Trials; Phosphotransferases; Pre-Clinical Model; Preclinical Drug Development; Progress Reports; Protein Tyrosine Kinase; RNA Interference; Reactive Oxygen Species; Reporting; Research Priority; Research Project Grants; Resistance; Rest; Role; Route; Screening procedure; Serum; Signal Pathway; Signal Transduction; Small Interfering RNA; Solid; Staining method; Stains; Study models; System; Techniques; Testing; Therapeutic; Transforming Growth Factor beta; Transgenic Mice; Transgenic Organisms; Tumor Suppressor Genes; Tumor Volume; Ultrasonography; United States National Institutes of Health; Validation; annexin A5; base; bench to bedside; biological adaptation to stress; bone morphogenetic protein receptors; cancer cell; cancer therapy; carcinogenesis; cell growth; chemotherapy; discoidin domain receptor 2; discoidin receptor; fight against; gemcitabine; human FRAP1 protein; improved; in vitro activity; in vivo; in vivo Model; inhibitor/antagonist; innovation; mTOR inhibition; mouse model; novel; pancreatic cancer cells; pre-clinical; preclinical evaluation; prevent; prospective; research study; response; small molecule; treatment response; treatment strategy; tumor; tumor growth; tumor progression

Progress Report 1. Improving anti-MAPK pathway therapy in pancreatic cancer A panel of 70 pancreatic cancer cell lines was profiled for MEK sensitivity using the allosteric small molecule inhibitor AZD6244 which is currently in phase II clinical trials. About 40 percent of profiled pancreatic cancer lines exhibit marked MEK sensitivity according to their half growth inhibitory concentration (GI50) of less than 1 micromolar. Sensitivity was determined by luminescent measurement of intracellular ATP concentration after 72 hours treatment with AZD6244. Induction of apoptosis was measured by determining lethal dose values of AZD6244 using the same assay. Overall, response to MEK inhibition in sensitive cell lines is a cytostatic growth arrest effect rather than mediated by induction cell death as described for MEK therapy in other solid organ cancers. To improve efficacy of MEK treatment the following progress has been made: a. A high-throughput siRNA screen has been carried out in the MEK-resistant cell line YAPC: To identify intracellular signaling pathways and targets which are used or switched on by pancreas cancer cells to escape MAPK pathway blockade and MEK inhibition a synthetic lethality drug sensitization screen in the cell line YAPC inhibited with AZD6244 has been carried out by the RNAi Screening Center, NIH Chemical Genomics Center, NIH Center for Translational Therapeutics, NHGRI/NIH. Targets validated in two independent screens mediating resistance to MEK inhibition which are promising opportunities for future effective combinational targeted therapy strategies are e.g. MAP3K8 (COT kinase of the MAPK pathway), PKMYT1 (regulating kinase of the G2-M cell cycle checkpoint), or GSK3B (regulator of the intracellular Wnt signaling pathway) among others. b. Gene expression arrays from sensitive and resistant cell lines have been carried. Using a variety of computational methods including gene set enrichment analysis (GSE), prioritization of networks of intercorrelated expression, and linkage of dynamic gene expression a gene classifier predicting sensitivity and resistance to MEK inhibition is developed. Validation is carried out on a xenobank of human pancreatic cancer xenotransplants established from patients operated on for pancreatic cancer at the Surgery Branch/NCI. c. Addition of the novel Erk2 inhibitor NCGC00242487-01 to improve anti-MAPK therapy To further improve shutdown of the MAPK pathway the novel Erk2 inhibitor NCGC00242487-01 was tested in a panel of MEK-resistant pancreas cancer lines. Addition of NCGC00242487-01 can induce apoptosis rather than cell cycle arrest in some cell lines implying a superior treatment strategy compared to MEK inhibition in a subset of pancreatic cancers. The cause for this different therapeutically potentially valuable mechanism of action is currently investigated. 2. Targeting the PI3K-Akt pathway in pancreas cancer Nearly all pancreatic cancer lines treated with BEZ235 displayed marked sensitivity to PI3K/mTOR inhibition when judged on their GI50 values in the low nanomolar range. It was therefore decided to use induction of apoptosis after treatment with BEZ235 rather than the absolute GI50 value to determine response to dual PI3K/mTOR inhibition. Using a combination of Annexin V staining and cell cycle analysis in cell lines exposed for 72 hours to BEZ235, about 20 percent of cell lines showed a greater than 2.5-fold induction of apoptosis which was a match to prior LD50 determination in the lethal dose assay. In vitro activity of dual PI3K/mTOR inhibition was confirmed in vivo using a heterotopic xenotransplant models established from sensitive and resistant cell lines. Tumor volume was markedly decreased in mice growing tumors from sensitive cell lines after treatment for 21 days. Results of gene expression profiling are currently combined with the expression levels of the negative PI3K-Akt pathway regulator PTEN as well as the mutation status of common oncogenes in pancreas cancer to develop a clinically readily applicable biomarker for predicting response to dual PI3K/mTOR inhibition. For prospective validation of such a biomarker a xenobank from human xenotransplants from patients with pancreatic cancer operated on at the Surgery Branch/NCI is currently expanded. 3. Preclinical evaluation of the novel Myrk/Dyrk1B inhibitor NCGC00185981-02 in pancreas cancer Pancreatic cancer lines Panc1 and SU86.86 have been previously shown to be dependent on the function of Myrk/Dyrk1B kinase when resting in the G0 phase. Using a proliferation assay under serum-starved conditions to enhance the fraction of cells in G0, cell growth was significantly reduced in the presence of NCGC00185981-02. Testing in additional cancer biology assays is currently ongoing and will be followed by - testing for synergy when combined with gemcitabine or choroquine to augment ROS stress response and Myrk/Dyrk1B dependency - in vivo models 4. The impact of the tumor environment on the efficacy of anticancer therapy in ductal adenocarcinoma of the pancreas The role of the microenvironment cannot be studied in an in vitro cell system. To evaluate the complex interactions of the various cellular components, the tumor micro-vasculature, and the extracellular matrix of the tumor microenvironment as possible targets for novel treatment strategies in PDAC requires an in vivo model: Transgenic/knockout mice who develop pancreatic cancer are well-established models for studying possible modulators of carcinogenesis. These models contain conditional knock-in mutations of the Kras oncogene which is present in >85% of PDAC in combination with knock-outs of the common tumor suppressor genes CDKN2A and Smad4 which are lost in >50% of PDAC. These genetically engineered mouse models resemble the human genomic landscape of PDAC which is driven by alterations in one of these genes in >95% of cases: Pdx-cre; LSL-KrasG12D; LSL-p53R172H Pdx-cre; LSLKrasG12D; Ink4a/Arflox/lox Pdx-cre; LSL-KrasG12D; Ink4a/Arflox/lox; Smad4lox/lox The efficacy of the following three anti-pancreas cancer treatments/strategies: 1. Control (normal saline) 2. Gemcitabine 3. MEK inhibition (AZD6244) is tested in combination with treatment of the microenviroment using the following compounds / small molecules: 4. TGFRbeta inhibitors LY2109761 and LY2157299 5. BMPR inhibitor LDN-193189 6. discoidin domain receptor tyrosine kinase 2 (DDR2) inhibitor 409100 Study endpoint: A novel ultrasound technique has been developed to accurately assess tumor size in mice. 3D-ultrasound is the main cross-sectional technique to sequentially monitor tumor growth and tumor growth inhibition. Ultrasound will be complemented with CT and functional imaging after mice have been bred into ROSA 26 PdxCre mice which allows following and quantification of tumor progression via luciferase activity by Xenogen imaging. Ultimate endpoint for analysis of the impact of the different anti-microenvironment treatments on efficacy of anti-cancer therapy is/are: - increase in intratumoral gemcitabine concentration - tumor size - overall survival of the tumor-bearing mice

进度报告使用变构小分子抑制剂AZD6244对70个胰腺癌细胞系的MEK敏感性进行了分析，该抑制剂目前处于II期临床试验中。约40%的胰腺癌谱系表现出显著的MEK敏感性，根据其一半生长抑制浓度（GI50）小于1微摩尔。用AZD6244处理72小时后，通过发光测量细胞内ATP浓度来确定灵敏度。用同样的方法测定AZD6244的致死剂量值来测定细胞凋亡的诱导作用。总的来说，在敏感细胞系中，对MEK抑制的反应是一种细胞抑制生长效应，而不是像其他实体器官癌症中MEK治疗那样通过诱导细胞死亡介导。为了提高MEK治疗的疗效，目前取得了以下进展：a.在MEK耐药细胞系YAPC中进行了高通量siRNA筛选；为了确定胰腺癌细胞利用或开启的细胞内信号通路和靶点，以逃避MAPK通路阻断和MEK抑制，由RNAi筛选中心、NIH化学基因组学中心、NIH转化治疗中心、NHGRI/NIH对AZD6244抑制的YAPC细胞系进行了合成致死性药物致敏筛选。在两个独立的筛选中验证的靶点介导MEK抑制的抗性，这是未来有效的联合靶向治疗策略的有希望的机会，例如MAP3K8 （MAPK途径的COT激酶），PKMYT1（调节G2-M细胞周期检查点的激酶）或GSK3B（细胞内Wnt信号通路的调节剂）等。b.携带了来自敏感和抗性细胞系的基因表达阵列。利用多种计算方法，包括基因集富集分析（GSE）、相互关联表达网络的优先级排序和动态基因表达的连锁，开发了一个预测MEK抑制敏感性和抗性的基因分类器。在NCI外科分部的胰腺癌手术患者中建立的人类胰腺癌异种移植异种样本库进行了验证。c.添加新型Erk2抑制剂NCGC00242487-01改善抗MAPK治疗，为了进一步改善MAPK通路的关闭，我们在一组mek耐药的胰腺癌细胞系中测试了新型Erk2抑制剂NCGC00242487-01。添加NCGC00242487-01可以在某些细胞系中诱导细胞凋亡而不是细胞周期阻滞，这表明与MEK抑制相比，在胰腺癌亚群中具有更好的治疗策略。目前正在研究这种不同的治疗有价值的作用机制的原因。2. 在低纳摩尔范围内，几乎所有用BEZ235处理过的胰腺癌细胞系都表现出对PI3K/mTOR抑制的显著敏感性。因此，我们决定使用BEZ235治疗后诱导细胞凋亡，而不是绝对GI50值来确定对PI3K/mTOR双重抑制的反应。在暴露于BEZ235 72小时的细胞系中，结合Annexin V染色和细胞周期分析，约20%的细胞系显示出超过2.5倍的凋亡诱导，这与先前致死剂量测定中的LD50相匹配。利用敏感和耐药细胞系建立的异位异种移植模型，在体内证实了PI3K/mTOR双抑制的体外活性。治疗21天后，敏感细胞系生长的小鼠肿瘤体积明显减小。基因表达谱分析的结果目前与PI3K- akt通路负调控因子PTEN的表达水平以及胰腺癌中常见癌基因的突变状态相结合，以开发一种临床易于应用的生物标志物，用于预测PI3K/mTOR双抑制的反应。为了对这种生物标志物进行前瞻性验证，目前正在扩大来自NCI外科分部手术的胰腺癌患者的人类异种移植的异种样本库。3. 新型Myrk/Dyrk1B抑制剂NCGC00185981-02在胰腺癌中的临床前评估胰腺癌系Panc1和SU86.86在G0期休息时依赖于Myrk/Dyrk1B激酶的功能。在血清饥饿条件下进行增殖试验，以提高G0中细胞的比例，NCGC00185981-02的存在显著降低了细胞的生长。目前正在进行其他癌症生物学测试，随后将在体内模型中测试与吉西他滨或氯喹联合使用以增强ROS应激反应和Myrk/Dyrk1B依赖性时的协同作用4。肿瘤环境对胰腺导管腺癌抗癌治疗效果的影响微环境的作用尚不能在体外细胞系统中进行研究。为了评估各种细胞成分、肿瘤微血管和肿瘤微环境的细胞外基质之间复杂的相互作用，作为PDAC新治疗策略的可能靶点，需要一个体内模型：胰腺癌的转基因/基因敲除小鼠是研究可能的致癌调节剂的成熟模型。这些模型包含Kras癌基因的条件敲入突变（存在于85%的PDAC中）以及常见肿瘤抑制基因CDKN2A和Smad4的敲除（在50%的PDAC中丢失）。这些基因工程小鼠模型类似于PDAC的人类基因组景观，在95%的病例中，PDAC是由其中一个基因的改变驱动的：Pdx-cre；LSL-KrasG12D;LSL-p53R172H Pdx-cre;LSLKrasG12D;Ink4a / Arflox / lox Pdx-cre;LSL-KrasG12D;Ink4a / Arflox /液态氧;Smad4lox/lox以下三种抗胰腺癌治疗/策略的疗效：对照组（生理盐水）吉西他滨3。MEK抑制（AZD6244）与微环境处理结合使用以下化合物/小分子进行测试：TGFRbeta抑制剂LY2109761和LY2157299BMPR抑制剂LDN-193189盘状蛋白结构域受体酪氨酸激酶2 （DDR2）抑制剂409100研究终点：一种新的超声技术已经被开发出来，可以准确评估小鼠肿瘤的大小。3d -超声是连续监测肿瘤生长和抑制肿瘤生长的主要横断面技术。在将小鼠培育成ROSA 26 pdxre小鼠后，超声将与CT和功能成像相补充，这使得Xenogen成像可以通过荧光素酶活性跟踪和量化肿瘤进展。分析不同抗微环境治疗对抗癌疗效影响的最终终点是：-肿瘤内吉西他滨浓度增加-肿瘤大小-荷瘤小鼠总生存期