Preclinical drug development in pancreatic cancer

胰腺癌的临床前药物开发

• 批准号: 8763424
• 负责人: Udo Rudloff
• 金额: $ 108.98万
• 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/8763424
• 关键词: Animal Model; Animals; Apoptosis; Area; Basic Science; Biological Markers; CDKN2A gene; Cancer Patient; Cancer cell line; Cell Cycle Arrest; Cell Death; Cell Death Induction; Cell Line; Cells; Chemicals; Clinical Markers; Clinical Protocols; Clinical Trials; Common Neoplasm; Complex; Cytostatics; Dependence; Development; Dextrans; Disease; Drug Kinetics; Drug Sensitization; Enhancers; Environment; Ephrin B Receptor; Exhibits; Future; Gene Expression; Gene Expression Profiling; Genes; Genetic; Genetically Engineered Mouse; Genomics; Genotype; Goals; Growth; Head; Human; IRAK4 gene; In Situ; In Vitro; Induction of Apoptosis; Interleukin-10; Knock-in Mouse; Knock-out; Knockout Mice; Laboratories; Libraries; Lysine; MAP Kinase Gene; MAP3K8 gene; MAPK Signaling Pathway Pathway; MEK inhibition; MEKs; Malignant Neoplasms; Malignant neoplasm of pancreas; Measures; Mediating; Mitogen-Activated Protein Kinase Kinases; Modeling; Molecular; Mutate; Mutation; National Human Genome Research Institute; Nucleotides; Oncogenes; Operative Surgical Procedures; Organ; Pancreas; Pancreatic Ductal Adenocarcinoma; Pathway interactions; Patients; Perfusion; Phase II Clinical Trials; Phenotype; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Population; Pre-Clinical Model; Preclinical Drug Development; Process; Progress Reports; Protein Isoforms; Protein Kinase; Protocols documentation; RNA Interference; Reporting; Research Priority; Resistance; Ribosomal Protein S6 Kinase; Role; Route; 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; Tumor Volume; United States National Institutes of Health; Validation; Variant; base; bench to bedside; cancer cell; cancer stem cell; cancer therapy; carcinogenesis; cellular targeting; dextran; fight against; gemcitabine; genetic variant; 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; next generation; novel; pancreatic cancer cells; peptide analog; polypeptide; pre-clinical; preclinical evaluation; prospective; research study; response; screening; small molecule; stem; synergism; treatment response; treatment strategy; tumor

Progress Report 1. Improving anti-MAPK pathway therapy in pancreatic cancer A 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 and activity area over the curve. Overall response to MEK inhibition in sensitive cell lines constitutes a cytostatic growth arrest effect rather than induction of cell death as described for MEK therapy in other solid organ cancers. To improve efficacy of future MEK treatment in pancreas cancer the following progress has been made: a. A high-throughput siRNA screen identified genes mediating 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). Gene expression levels of CNKSR1 appear to directly correlate with resistance to MAPK inhibition making CNKSR1 both an attractive target as well as possible predictor of response to MEK inhibition in pancreas cancer. b. Next generation genomic sequencing of genes most commonly involved in cancer identified genetic variants associated with response to MAPK pathway inhibition. Interestingly, among several, specific isoforms of the RAS oncogene, which is mutated in more than 85 percent of pancreas cancers, are associated with MEK sensitivity. The mutation isoform G12R is exclusively identified in MEK sensitive cell lines and might represent a readily applicable clinical marker predicting response to MEK inhibition. c. The Erk2 inhibitor VTX-11e (NCGC00242487-01) is superior to MEK inhibition in a subset of pancreatic cancers The 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. Both MEK- and Erk inhibition equally effectively inhibit MAPK pathway signaling as determined by reduction of phospho Elk levels. 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 cancer Nearly 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 were classified as sensitive to PI3K-Akt inhibition. 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 andAKT genes, and others, 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 patients operated on at the Surgery Branch/NCI has been established. 3. Preclinical evaluation of the ITK inhibitor NCGC-00188382 in pancreas cancer In a high-throughput pharmacological screen against pancreas cancer stem-like cells, the 'ITK' inhibitor NCGC-00188382 was found to be the most active compound. Cell-based and in situ kinase screens in the pancreas cancer line Panc1 showed that the compound inhibits a number of kinases (CDK7, IRAK4, CLK1, CLK2, CaMMK2, TAOK3, aurora B, Ephrin receptor B2). These novel targets were validated in secondary screens. siRNA silencing studies probing into the polypharmacological mechanism of action of this novel compound identified 'intrinsic synergism' of some of these targets. The compound was tested in vivo and showed a strong anti-metastatic phenotype. Current studies focus on improving the selectivity and pharmacokinetic profile of this promising new molecule, and to further understand its mechanism of action. 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 as possible targets for novel treatment strategies in pancreas cancer 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 pancreas cancer in combination with knock-outs of the common tumor suppressor genes CDKN2A (p16) and Smad4 which are lost in 50% of cases. These genetically engineered mouse models resemble the human genomic landscape of pancreas cancer 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 several-fold compared to control as measured by increased dextran perfusion and intratumoral gemcitabine. Additionally, the combination of the novel interleukin 10 peptide analogue 10N with gemcitabine led to a dramatic decrease in tumor volume compared to control as well as to a survival advantage in the treated animals. This anti-tumor effect is independent of increased delivery of gemcitabine to the tumor and suggests a novel, yet undioscovered synergistic effect between 10A and gemcitabine. Current studies are aiming to identify the cellular compartment targeted by anti-interleukin 10 and gemcitabine as well as gene differently expressed upon treatment with this combination. These positive findings are in the process of being extended to other transgenic animal models to probe into genotype-directed anti-microenvironment treatment strategies of combining anti-stroma with anti-cancer treatments in pancreas cancer. It is aimed to translate positive findings of gemcitabine combinations into the currently ongoing RECLAP trial.

进展报告 1. 改善胰腺癌的抗 MAPK 通路治疗 使用目前处于 II 期临床试验的变构小分子抑制剂 AZD6244 对一组 70 种胰腺癌细胞系对 MEK 抑制的敏感性进行了分析。根据小于 1 微摩尔的半生长抑制浓度 (GI50) 和曲线上的活性面积，大约 40% 的胰腺癌细胞系表现出显着的 MEK 敏感性。敏感细胞系中对 MEK 抑制的总体反应构成细胞生长抑制效应，而不是如其他实体器官癌中 MEK 治疗所述的诱导细胞死亡。为了提高未来 MEK 治疗胰腺癌的疗效，已取得以下进展：高通量 siRNA 筛选鉴定了介导胰腺癌中 MAPK 通路抑制抗性的基因：鉴定胰腺癌细胞使用或打开的细胞内信号通路和靶标，以逃避 MAPK 通路阻断和 MEK 抑制。RNAi 筛选已在 AZD6244 抑制的细胞系 YAPC 中进行了合成致死药物敏化筛选 中心、NIH 化学基因组学中心、NIH 转化治疗中心、NHGRI/NIH。在两次独立的二次筛选中验证的介导 MEK 抑制抗性的靶点是 CNKSR1（Ras 1 激酶抑制子的连接器增强子）、WNK2（WNK 赖氨酸缺陷蛋白激酶 2）、MAP3K8（MAPK 途径的 COT 激酶）和 RPS6KA5（核糖体蛋白 S6 激酶，90kDa，多肽） 5）。 CNKSR1 的基因表达水平似乎与 MAPK 抑制的抗性直接相关，这使得 CNKSR1 既是一个有吸引力的靶标，又是胰腺癌中 MEK 抑制反应的可能预测因子。 b.对最常参与癌症的基因进行下一代基因组测序，确定了与 MAPK 通路抑制反应相关的遗传变异。有趣的是，在几种中，RAS 癌基因的特定亚型（在超过 85% 的胰腺癌中发生突变）与 MEK 敏感性相关。突变同种型 G12R 专门在 MEK 敏感细胞系中发现，可能代表一种易于应用的临床标记，可预测对 MEK 抑制的反应。 c. Erk2 抑制剂 VTX-11e (NCGC00242487-01) 在部分胰腺癌中优于 MEK 抑制 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停滞，这意味着与 MEK 相比，这是一种更优越的治疗策略 抑制。 MEK- 和 Erk 抑制同样有效地抑制 MAPK 通路信号传导，这通过磷酸 Elk 水平的降低来确定。与 VTX-11e 处理后经历细胞周期停滞的细胞相比，VTX-11e 处理后经历细胞死亡的细胞系显示 Erk 靶点 p90-RSK（核糖体蛋白 S6 激酶，90kDa，多肽 1）磷酸化的减少更大。基因表达谱鉴定出多个参与 Erk 抗性细胞系中胚胎学途径上调的新基因，这些基因可能维持 p90-RSK 磷酸化，尽管 Erk 受到抑制，但 p90-RSK 磷酸化仍能介导存活。 2. 靶向胰腺癌中的 PI3K-Akt 通路 当根据低纳摩尔范围内的 GI50 值进行判断时，几乎所有用 PI3K/mTOR 抑制剂 BEZ235 治疗的 70 个胰腺癌系均显示出显着的敏感性。当测试 PI3K/mTOR 抑制诱导细胞死亡时，约 20% 的细胞系显示出大于 2.5 倍的细胞凋亡诱导，并被归类为对 PI3K-Akt 抑制敏感。使用从敏感和耐药胰腺癌细胞系建立的异位异种移植模型在体内证实了双重 PI3K/mTOR 抑制的体外活性。突变测试结果显示，在对 PI3K/mTOR 抑制敏感的细胞系中，PI3K 和 AKT 基因及其他基因的内含子区域中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。敏感株系和抗性株系的​​基因表达谱显示，许多激酶和磷酸酶在两组之间表达不同。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部/NCI 接受手术的患者的人类胰腺癌样本的异种库。 3. ITK抑制剂NCGC-00188382在胰腺癌中的临床前评估在针对胰腺癌干细胞样细胞的高通量药理学筛选中，发现“ITK”抑制剂NCGC-00188382是最活跃的化合物。对胰腺癌细胞系 Panc1 进行的细胞原位激酶筛选表明，该化合物可抑制多种激酶（CDK7、IRAK4、CLK1、CLK2、CaMMK2、TAOK3、aurora B、Ephrin 受体 B2）。这些新靶标在二次筛选中得到了验证。 siRNA 沉默研究探讨了这种新型化合物的多药理学作用机制，确定了其中一些靶标的“内在协同作用”。该化合物经过体内测试，显示出很强的抗转移表型。目前的研究重点是提高这种有前途的新分子的选择性和药代动力学特征，并进一步了解其作用机制。 4.肿瘤环境对胰腺导管腺癌抗癌治疗疗效的影响微环境的作用无法在体外细胞系统中研究。为了评估作为胰腺癌新治疗策略的可能靶点的各种细胞成分的复杂相互作用，需要一个体内模型：产生胰腺癌的转基因/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% 的胰腺癌中），以及常见肿瘤抑制基因 CDKN2A (p16) 和 Smad4 的敲除（在 50% 的病例中缺失）。这些基因工程小鼠模型类似于人类胰腺癌的基因组图谱，在 95% 的病例中，胰腺癌是由这些基因之一的改变引起的。转基因Kras p16敲除胰腺癌动物模型Pdx-Cre的治疗； LSLKrasG12D；通过增加葡聚糖灌注和肿瘤内吉西他滨测量，与对照相比，Ink4a/Arflox/lox 与 TGFRbeta 抑制剂 LY2109761 使胰头肿瘤的灌注增加数倍。此外，新型白细胞介素 10 肽类似物 10N 与吉西他滨的组合与对照相比，导致肿瘤体积显着减小，并在治疗动物中获得生存优势。这种抗肿瘤作用与吉西他滨向肿瘤的递送增加无关，表明 10A 和吉西他滨之间存在新颖但尚未发现的协同作用。目前的研究旨在确定抗白细胞介素 10 和吉西他滨靶向的细胞区室以及该组合治疗后不同表达的基因。这些积极的发现正在扩展到其他转基因动物模型，以探讨胰腺癌中抗基质与抗癌治疗相结合的基因型导向的抗微环境治疗策略。其目的是将吉西他滨组合的积极发现转化为目前正在进行的 RECLAP 试验。