Preclinical drug development in pancreatic cancer

胰腺癌的临床前药物开发

• 批准号: 8553072
• 负责人: Udo Rudloff
• 金额: $ 121.28万
• 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/8553072
• 关键词: Adenocarcinoma; Animal Model; Apoptosis; Basic Science; Biological Assay; Biological Markers; CDKN2A gene; Cancer Patient; Cancer cell line; Cell Cycle; Cell Cycle Arrest; Cell Death; Cell Death Induction; Cell Line; Cells; Chemicals; Clinical Protocols; Clinical Trials; Common Neoplasm; Complex; Cytostatics; Data; Desmoplastic; Development; Dextrans; Disease; Dose; Drug Sensitization; Ductal; Enhancers; Environment; Exercise; Exhibits; Extracellular Matrix; G2/M Arrest; Gene Expression Profiling; Genes; Genetically Engineered Mouse; Genomics; Genotype; Growth; Head; Human; IRAK4 gene; In Vitro; Induction of Apoptosis; Inhibitory Concentration 50; Knock-in Mouse; Knock-out; Knockout Mice; Laboratories; Lethal Dose 50; Libraries; Link; Lysine; MAP Kinase Gene; MAP3K8 gene; MEK inhibition; MEKs; Malignant Neoplasms; Malignant neoplasm of pancreas; Measures; Mediating; Mediation; Mitogen-Activated Protein Kinase Kinases; Modeling; Molecular; Mutation; NF-kappa B; National Human Genome Research Institute; Nucleotides; Oncogenes; Operative Surgical Procedures; Organ; Pancreas; Pathway interactions; Patients; Perfusion; Phase II Clinical Trials; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Population; Pre-Clinical Model; Preclinical Drug Development; Process; Protein Kinase; Proto-Oncogene Proteins c-akt; Protocols documentation; RNA Interference; Reporter Genes; Reporting; Research Priority; Resistance; Ribosomal Protein S6 Kinase; Role; Route; Screening procedure; Signal Pathway; Signal Transduction; Small Interfering RNA; Solid; Specimen; Study models; System; Testing; Therapeutic; Transforming Growth Factor beta; Transgenic Animals; Transgenic Mice; Transgenic Organisms; Translating; Tumor Suppressor Genes; United States National Institutes of Health; Validation; Variant; base; bench to bedside; bone morphogenetic protein receptors; cancer cell; cancer stem cell; cancer therapy; carcinogenesis; cellular targeting; chemotherapeutic agent; dextran; fight against; gemcitabine; high throughput screening; human FRAP1 protein; human KSR protein; human TFRC protein; improved; in vitro activity; in vivo; in vivo Model; inhibitor/antagonist; innovation; mTOR Inhibitor; mTOR inhibition; mouse model; novel; pancreatic cancer cells; pancreatic neoplasm; polypeptide; pre-clinical; preclinical evaluation; prevent; prospective; research study; response; small molecule; treatment response; treatment strategy; tumor

Progress Report1.Improving anti-MAPK pathway therapy in pancreatic cancerA panel of 70 pancreatic cancer cell lines was profiled for sensitivity to MEK inhibition 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. Overall response to MEK inhibition in sensitive cell lines constitutes a cytostatic growth arrest effect rather than mediation of induction of 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 identified genes overcoming resistance to MAPK pathway inhibition in pancreas cancer: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 secondary screens mediating resistance to MEK inhibition are CNKSR1 (connector enhancer of kinase suppressor of Ras 1), WNK2, (WNK lysine deficient protein kinase 2), MAP3K8 (COT kinase of the MAPK pathway), and RPS6KA5 (ribosomal protein S6 kinase, 90kDa, polypeptide 5). b.Multiplexed gene reporter assays identified NF-kB signaling as the main escape pathway switched on upon MEK inhibition. Current experiments aim to link the identified genes mediating MEK resistance in the siRNA screen to NF-kB activation and pursue in vivo validation of compensatory NF-kB activation using a xenobank of human pancreatic cancer xenotransplants established from patients operated on for pancreatic cancer at the Surgery Branch/NCI.c.The Erk2 inhibitor VTX-11e (NCGC00242487-01) is superior to MEK inhibition in a subset of pancreatic cancerThe Erk small molecule inhibitor VTX-11e (NCGC00242487-01) induces apoptosis rather than cell cycle arrest in a third of pancreatic cancer cell lines implying a superior treatment strategy compared to MEK inhibition. Cell lines undergoing cell death following treatment with VTX-11e show a greater reduction of phosphorylation of the Erk target p90-RSK (ribosomal protein S6 kinase, 90kDa, polypeptide 1) than cells undergoing cell cycle arrest upon VTX-11e treatment. Gene expression profiling identified multiple novel genes involved in embryological pathways upregulated in ?Erk-resistant? cell lines possibly maintaining p90-RSK phosphorylation which mediates survival despite Erk inhibition. 2.Targeting the PI3K-Akt pathway in pancreas cancerNearly all of 70 pancreatic cancer lines treated with the dual PI3K/mTOR inhibitor BEZ235 displayed marked sensitivity when judged on their GI50 values in the low nanomolar range. When tested for induction of cell death upon PI3K/mTOR inhibition, about 20 percent of cell lines showed a greater than 2.5-fold induction of apoptosis and sensitivity to BEZ235 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 pancreas cancer cell lines. Results of mutation testing revealed novel single nucleotide variants in intronic regions of both the PI3K and AKT genes in cell lines sensitive to PI3K/mTOR inhibition and current studies are examining the functional impact on PI3K signaling of these variants. Gene expression profiling of sensitive and resistant lines showed a number of kinases and phosphatases differently expressed between the two groups. For prospective validation of such a possible biomarker a xenobank from human pancreas cancer specimens from patientsoperated on at the Surgery Branch/NCI has been established. 3.Preclinical evaluation of the ITK inhibitor NCGC00188382 in pancreas cancerThe ITK inhibitor NCGC00188382 was shown to have ?pan-activity? against a large panel of pancreas cancer lines with IC50 values in the 20 ? 200 nanomolar range. A cell-based kinase screen in the pancreas cancer line Panc1 showed that the compound inhibited 5 kinases (CDK7, IRAK4, CLK1, CLK2, CaMMK2) greater than 90% and another 12 kinases greater than 80%. Cell cycle analysis showed a unique G2-M arrest and combination siRNA silencing studies are currently under way to elicit the polypharmacological mechanism of action of this compound aiming to identify the main targets of NCGC00188382. 4.The impact of the tumor environment on the efficacy of anticancer therapy in ductal adenocarcinoma of the pancreasThe 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 (p16) 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.Treatment of the transgenic Kras p16 knockout pancreas cancer animal model Pdx-Cre; LSLKrasG12D; Ink4a/Arflox/lox with the TGFRbeta inhibitor LY2109761increases perfusion of pancreatic head tumors 5-fold compared to control as measured by increased dextran perfusion. Currently, LY2109761 is combined with gemcitabine to see if the anti-microenvironment agent LY2109761 can increase delivery of chemotherapeutic agents to pancreatic tumors. These findings are in the process of being extended to other transgenic animal models to probe into genotype-directed anti-microenvironment treatment strategies. It is aimed to translate positive findings of gemcitabine combinations into the currently ongoing RECLAP trial.

Report1进展。使用变构小分子抑制剂AZD6244对70个胰腺癌细胞系的MEK抑制敏感性进行了分析，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。在敏感细胞系中，对MEK抑制的总体反应构成了细胞抑制生长阻滞效应，而不是像其他实体器官癌症中MEK治疗那样介导诱导细胞死亡。为提高MEK治疗的疗效，取得了以下进展：高通量siRNA筛选鉴定了胰腺癌中克服MAPK通路抑制抗性的基因：为了鉴定胰腺癌细胞使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 /国家卫生研究院。在两个独立的二级筛选中证实了介导MEK抑制的靶标是CNKSR1 （Ras 1激酶抑制因子的连接增强子）、WNK2 （WNK赖氨酸缺乏蛋白激酶2）、MAP3K8 （MAPK途径的COT激酶）和RPS6KA5（核糖体蛋白S6激酶，90kDa，多肽5）。b.多重基因报告基因分析发现，NF-kB信号通路是MEK抑制时开启的主要逃避途径。目前的实验旨在将siRNA筛选中鉴定的介导MEK耐药的基因与NF-kB激活联系起来，并利用nci外科分会（Surgery Branch/NCI.c）建立的人类胰腺癌异种移植物异种库，在体内验证代偿性NF-kB激活。Erk小分子抑制剂VTX-11e （NCGC00242487-01）在三分之一的胰腺癌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，其治疗策略更优越。与VTX-11e治疗后细胞周期阻滞的细胞相比，VTX-11e治疗后细胞死亡的细胞系显示Erk靶蛋白p90-RSK（核糖体蛋白S6激酶，90kDa，多肽1）磷酸化的减少幅度更大。基因表达谱鉴定了多个涉及胚胎学途径的新基因，这些基因在erk抗性？细胞系可能维持p90-RSK磷酸化，介导Erk抑制的存活。2.用PI3K/mTOR双抑制剂BEZ235治疗的70种胰腺癌细胞系在低纳摩尔范围内的GI50值判断时，几乎所有的胰腺癌细胞系都显示出明显的敏感性。当对PI3K/mTOR抑制诱导细胞死亡进行测试时，大约20%的细胞系显示出超过2.5倍的细胞凋亡诱导和对BEZ235的敏感性，这与先前致死剂量试验中LD50的测定相匹配。通过建立敏感和耐药胰腺癌细胞系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。突变检测结果显示，在对PI3K/mTOR抑制敏感的细胞系中，PI3K和AKT基因的内含子区域存在新的单核苷酸变异，目前的研究正在研究这些变异对PI3K信号传导的功能影响。敏感系和抗性系的基因表达谱显示，两组之间的激酶和磷酸酶表达存在差异。为了对这种可能的生物标志物进行前瞻性验证，已经建立了来自外科分会/NCI手术患者的人类胰腺癌标本的异种样本库。3.胰腺癌ITK抑制剂NCGC00188382的临床前评价研究显示，ITK抑制剂NCGC00188382具有泛活性。与IC50值在20 ？200纳摩尔范围。胰腺癌细胞系Panc1的细胞激酶筛选显示，该化合物对CDK7、IRAK4、CLK1、CLK2、CaMMK2等5种激酶的抑制作用大于90%，对12种激酶的抑制作用大于80%。细胞周期分析显示出独特的G2-M阻滞和联合siRNA沉默研究目前正在进行中，以引出该化合物的多药理作用机制，旨在确定NCGC00188382的主要靶点。4.肿瘤环境对胰腺导管腺癌抗癌疗效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 （p16）和Smad4的敲除（在50%的PDAC中缺失）。这些基因工程小鼠模型类似于PDAC的人类基因组景观，在95%的情况下，PDAC是由其中一个基因的改变驱动的。转基因Kras p16敲除胰腺癌动物模型Pdx-Cre的治疗LSLKrasG12D;Ink4a/Arflox/lox与TGFRbeta抑制剂ly2109761相比，通过增加葡聚糖灌注来测量胰腺头部肿瘤的灌注增加5倍。目前，LY2109761正与吉西他滨联合研究抗微环境药物LY2109761是否能增加化疗药物对胰腺肿瘤的递送。这些发现正在扩展到其他转基因动物模型，以探索基因型导向的抗微环境治疗策略。其目的是将吉西他滨联合治疗的阳性结果转化为目前正在进行的RECLAP试验。