Preclinical drug development in pancreatic cancer

胰腺癌的临床前药物开发

• 批准号: 8938034
• 负责人: Udo Rudloff
• 金额: $ 115.1万
• 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/8938034
• 关键词: Animal Model; Animals; Apoptosis; Area; B-Lymphocytes; Basic Science; Biological Assay; Biological Markers; CD47 gene; CNKSR1 gene; Cancer Model; Cancer cell line; Cell Cycle Arrest; Cell Cycle Checkpoint; Cell Death; Cell Death Induction; Cell Line; Cells; Clinical Protocols; Clinical Trials; Coculture Techniques; Complex; Cytostatics; DNA Repair; Dactinomycin; Dependence; Development; Disease; Dose; Drug Delivery Systems; Eating; Environment; Evaluation; Exhibits; Future; GTP-Binding Proteins; Gene Expression Profiling; Genes; Genetic; Growth; Head; Heat shock proteins; Human; Immune; In Vitro; Induction of Apoptosis; Inflammation; Institutes; Interleukin-2; Knock-out; Laboratories; Liver; Lung; MAPK Signaling Pathway Pathway; MEK inhibition; MEKs; Malignant Neoplasms; Malignant neoplasm of pancreas; Mediating; Mediation; Metastatic Lesion; Mitochondria; Mitogen-Activated Protein Kinase Kinases; Modeling; Molecular; Molecular Models; Molecular Profiling; Mus; Mutation; NF-kappa B; Neoplasm Metastasis; Nucleotides; Pancreas; Pathway interactions; Peptides; Perfusion; Peritoneum; Pharmaceutical Preparations; Phenotype; Phosphorylation; Phosphotransferases; Population; Pre-Clinical Model; Preclinical Drug Development; Preclinical Drug Evaluation; Proteasome Inhibitor; Protein Isoforms; RNA Interference; Receptor Protein-Tyrosine Kinases; Receptor Signaling; Regimen; Reporter; Resistance; Respiration; Ribosomal Protein S6 Kinase; STK11 gene; Signal Pathway; Signal Transduction; Small Interfering RNA; Solid; Sorting - Cell Movement; Stem cells; System; T-Lymphocyte; Testing; Therapeutic; Toxic effect; Toxicity Tests; Transforming Growth Factor beta; Transgenic Animals; Transgenic Organisms; Up-Regulation; Variant; Xenograft Model; base; bench to bedside; cancer cell; cancer stem cell; cancer therapy; combinatorial; cytotoxic; drug development; fight against; gemcitabine; human FRAP1 protein; human PTPNS1 protein; human TFRC protein; improved; in vitro activity; in vivo; inhibitor/antagonist; innovation; kinase inhibitor; knockout animal; mTOR inhibition; macrophage; molecular modeling; novel; pancreatic cancer cells; pancreatic neoplasm; polypeptide; pre-clinical; preclinical evaluation; prevent; programs; receptor; research study; resistance mechanism; response; scaffold; screening; small molecule; stem; treatment strategy; tumor; tumor growth; tumor progression

Project Summary: 1. Preclinical evaluation of the ITK inhibitor NCGC-00188382 and metarrestin in pancreas cancer: consistent with his anti-cancer stem cell activity, NCGC-00188382 effectively suppresses metastases formation in vivo. It suppresses cancer cell invasion and also shows anti-tumor activity in heterotopic xenograft models. Three of the four top targets of NCGC-00188382 are genes involved in DNA damage repair and cell cycle checkpoint control - TAOK3, CDK7, AURKB - targeting a selective vulnerability of cancer stem cells. Targets show impressive additivity in the induction of cell death explaining the efficacy of the multikinase inhibitor in vitro and in vivo. TAOK3 is a novel target with greater than 3-fold selectivity for the stem cell subpopulation. Derived from a high-throughput combinatorial screen against pancreas cancer stem-like cells treated with NCGC-00188382, and validated in combination experiments with NCGC-00188382 or drugs against targets of the compound, NCGC-00188382 showed dramatic synergies with the alkalyting agent actinomycin D, the heat shock protein inhibitors alvespimycin and AUY-922, and the proteasome inhibitor carfilzomib offering multiple novel new treatment avenues for pancreas cancer to be tested in clinical trials. As part of a trans-institute effort between NCI and NCATS, metarrestin, a novel perinucleolar complex (PNC) inhibitor is developed. Cancer progression is paralleled by an increase of PNC numbers in metastatic lesions, and disruption of the PNC has shown halt of tumor growth. In an orthotopic pancreas cancer model, metarrestin prevented metastases formation in liver, lungs, and peritoneum with no appreciable toxicity. The compound is currently undergoing additional toxicity studies and it scored in the top 10 percent tier of NCI's Experimental Therapeutics (NExT) program for future pre- and clinical development. 2. Modulation of the tumor environment to improve efficacy of anticancer therapy: Treatment of the transgenic Kras p16 knockout (KO) pancreas cancer animal model Pdx-Cre; LSLKrasG12D; Ink4a/Arflox/lox with the 10-mer peptide 10N, shown to suppress inflammation in a variety of non-cancer models, suppressed in combination with gemcitabine tumor growth and extended survival of animals. FACS sorting of tumors treated with 10N suggests an immune-mediated mechanism with a steep decrease in tumor-associated macrophages and smaller increase in reactive B cells with no increase of cell death observed in tumors treated with 10N. Improved molecular modeling of 10N implies the 'don't eat me' signaling axis CD47-SIRP(alpha), or CD206, and NF-kappaB signaling as possible targets which are currently evaluated in in vitro co-culture experiments. Treatment of transgenic Kras p16 and Kras TGFbeta receptor II knockout animals with the TGFbeta inhibitor LY2109761 increases perfusion of pancreatic head tumors several-fold in the p16 model compared to control but not in the Kras TGFbeta RII knockout model. Tumors in both animal models are significantly smaller upon treatment with the combination of LY2109761 and gemcitabine suggesting mechanism independent of gemcitabine delivery and anti-cancer cell toxicity. Thus, for gemcitabine to be effective dosing below the currently one used in cytotoxic regimens may suffice, and the combination of anti-TGFbeta signaling and gemcitabine respresents a novel treatment approach. 3. Improving anti-MAPK kinase 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 selumetinib. About 40 percent of profiled pancreatic cancer lines exhibit marked MEK sensitivity according to their half growth inhibitory concentration (GI50) and activity area. 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 organcancers. As means of improving efficacy of future MEK treatment in pancreas cancer the following findings have been made: a. Mutations in the Kras mutation isoform G12R, the G-protein alpha regulator GNAS, or when combined, mutations in tyrosine kinase receptor signaling predict response to MEK inhibition (p0.05). b. The scaffolding kinase CNKSR1 is a major regulator of resistance to MEK inhibition in pancreas cancer. Cell lines resistant to MEK inhibition convert to a sensitive phenotype upon silencing of CNKSR1. Reporter assays imply CNKSR1-mediated NF-kappaB signaling in mediation of resistance, a finding confirmed in xenotransplanted human pancreatic tumors. 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. 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 with maintained p90-RSK phosphorylation. 4. Targeting the PI3K-Akt pathway in pancreas cancer. 20 percent of cell lines treated with clinically achievable concentrations of BEZ235 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 heterotopic xenotransplant models established from sensitive and resistant pancreas cancer cell lines. Results of mutation testing revealed novel single nucleotide variants in intronic regions associated with response to PI3K/mTOR inhibition (p0.05). Upregulation and decoupling of mitochondrial respiration from PI3K-Akt control upon treatment with BEZ235 via activation of AMPK signaling is a common mechanism of resistance in cell lines resistant to PI3K/mTOR inhibiton. Loss of AMPK signaling through RNAi silencing or pharmacological inhibition sensitizes resistant cell lines to BEZ235 treatment. Resistant cell lines show upregulation of the AMPK regulator LKB1/STK11 on expression profiling and increased phospho-AMPK levels compared to sensitive cell lines, a finding also seen in xenotransplanted human pancreas cancers resistant to PI3K/mTOR inhibition. These results suggest AMPK as an attractive co-target in PI3K/mTOR inhibition to increase number of cancers sensitive to this strategy.

项目概述：1；ITK抑制剂NCGC-00188382与甲骨restin在胰腺癌中的临床前评价：与其抗癌干细胞活性一致，NCGC-00188382在体内有效抑制转移形成。它可以抑制癌细胞的侵袭，并在异位异种移植模型中显示出抗肿瘤活性。NCGC-00188382的四个主要靶点中有三个是参与DNA损伤修复和细胞周期检查点控制的基因——TAOK3、CDK7、AURKB——靶向癌症干细胞的选择性易感性。靶标在诱导细胞死亡方面表现出令人印象深刻的可加性，解释了多激酶抑制剂在体外和体内的功效。TAOK3是一种新的靶标，对干细胞亚群具有超过3倍的选择性。通过对NCGC-00188382处理的胰腺癌干细胞进行高通量组合筛选，并与NCGC-00188382或针对该化合物靶点的药物进行联合实验验证，NCGC-00188382与碱化剂放线菌素D、热休克蛋白抑制剂alvespimycin和AUY-922显示出显著的协同作用。蛋白酶体抑制剂卡非佐米为胰腺癌提供了多种新的治疗途径，即将在临床试验中进行测试。作为NCI和NCATS跨研究所合作的一部分，metarrestin，一种新型核周复合物（PNC）抑制剂被开发出来。癌症的进展与转移性病变中PNC数量的增加是平行的，PNC的破坏表明肿瘤生长停止。在原位胰腺癌模型中，甲氨脲素可阻止肝、肺和腹膜转移形成，且无明显毒性。该化合物目前正在进行额外的毒性研究，并在NCI的实验治疗（NExT）项目中排名前10%，用于未来的前期和临床开发。2. 调节肿瘤环境提高抗癌疗效：转基因Kras p16基因敲除（KO）胰腺癌动物模型Pdx-Cre的治疗LSLKrasG12D;Ink4a/Arflox/lox与10-mer肽10N，在多种非癌症模型中显示抑制炎症，与吉西他滨联合抑制肿瘤生长并延长动物生存期。用10N处理的肿瘤的FACS分选提示免疫介导的机制，肿瘤相关巨噬细胞急剧减少，反应性B细胞增加较小，但未观察到10N处理的肿瘤细胞死亡增加。改进的10N分子模型表明，“不要吃我”信号轴CD47-SIRP（α）或CD206和NF-kappaB信号是目前体外共培养实验中评估的可能靶点。用TGFbeta抑制剂LY2109761处理转基因Kras p16和Kras TGFbeta受体II敲除动物，与对照组相比，p16模型中胰腺头部肿瘤的灌注增加了数倍，但在Kras TGFbeta RII敲除模型中没有。LY2109761联合吉西他滨治疗后，两种动物模型的肿瘤均明显缩小，提示其作用机制不依赖于吉西他滨给药和抗癌细胞毒性。因此，要使吉西他滨有效，剂量低于目前用于细胞毒性方案的剂量可能就足够了，抗tgf - β信号和吉西他滨的联合治疗代表了一种新的治疗方法。3. 改善胰腺癌抗mapk激酶通路治疗。用变构小分子抑制剂selumetinib对70个胰腺癌细胞系的MEK抑制敏感性进行了分析。根据一半生长抑制浓度（GI50）和活性区域，大约40%的胰腺癌细胞系表现出显著的MEK敏感性。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效应，而不是像其他实体器官中MEK治疗那样诱导细胞死亡。作为提高未来MEK治疗胰腺癌疗效的手段，已经取得了以下发现：a. Kras突变异构体G12R、g蛋白α调节因子GNAS的突变，或酪氨酸激酶受体信号的突变联合预测对MEK抑制的反应（p0.05）。b.支架激酶CNKSR1是胰腺癌对MEK抑制的抗性的主要调节因子。耐MEK抑制的细胞系在CNKSR1沉默后转化为敏感表型。报告者试验提示cnksr1介导的NF-kappaB信号通路介导耐药，这一发现在异种移植的人胰腺肿瘤中得到证实。c. Erk2抑制剂VTX-11e （NCGC00242487-01）在胰腺癌亚群中优于MEK抑制剂。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，这意味着与MEK抑制剂相比，这是一种更好的治疗策略。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。基因表达谱分析发现，在维持p90-RSK磷酸化的erk抗性细胞系中，多个涉及胚胎学途径的新基因上调。4. 靶向PI3K-Akt通路在胰腺癌中的作用20%用临床可达到浓度的BEZ235处理的细胞系显示出超过2.5倍的细胞凋亡诱导，并被归类为对PI3K-Akt抑制敏感。在敏感和耐药胰腺癌细胞系建立的体内异位异种移植模型中，证实了PI3K/mTOR双抑制的体外活性。突变检测结果显示，与PI3K/mTOR抑制反应相关的内含子区域出现了新的单核苷酸变异（p0.05）。在对PI3K/mTOR抑制产生抗性的细胞系中，BEZ235通过激活AMPK信号，使线粒体呼吸从PI3K- akt控制中上调和解耦是一种常见的抗性机制。通过RNAi沉默或药物抑制失去AMPK信号使耐药细胞系对BEZ235治疗增敏。与敏感细胞系相比，耐药细胞系显示AMPK调节因子LKB1/STK11在表达谱上上调，磷酸化AMPK水平增加，这一发现也出现在对PI3K/mTOR抑制有抗性的异种移植人类胰腺癌中。这些结果表明AMPK是PI3K/mTOR抑制的一个有吸引力的共同靶点，可以增加对该策略敏感的癌症数量。