Preclinical drug development in pancreas cancer

1. 胰腺癌MAPK和PI3K-Akt双通路抑制及逃逸通路的临床前药物开发-评估MEK和PI3K联合抑制在胰腺癌临床前模型中的疗效-绘制长期MEK和PI3K抑制剂治疗后肿瘤耐药的逃逸途径-通过将潜在基因型与途径激活和对双途径抑制的反应相关联，为该策略开发可靠的预测因子（基因标记）。在胰腺癌（PDAC）中联合使用小分子MEK和PI3K抑制剂有一个非常合理的理由：-由于活化的Kras直接激活了p85 PI3K亚基，大约60%的病例中PDAC中PI3K- akt通路上调。使用MEK和PI3K联合抑制，激活的Kras信号（PDAC中最常见的癌基因Kras）的主要流出和逃逸途径被抑制。在20%的PDAC病例中，PI3K- akt通路也被异常的EGFR信号激活。这些包括原发和转移性肿瘤的细胞系。在大多数情况下，这些细胞系中KRAS、CDKN2A、PTEN、TP53、Smad4和TGFRII突变状态的潜在基因型是已知的，并且可以与在体外细胞系模型中观察到的生长抑制相关。MEK和PI3K抑制的抗增殖和凋亡活性将通过三重（活细胞+死亡细胞+凋亡细胞）试验进行研究，该试验读取细胞死亡-细胞存活-凋亡细胞。下面展示了在MEK抑制敏感和抗性细胞系中使用MEK抑制剂的胰腺癌细胞系的生长抑制曲线的两个例子：ASUC动物实验（SB-211）题为胰腺癌和其他上消化道恶性肿瘤的转化基因型导向药物开发的体内平台，允许从外科分会和合作机构的患者切除的胰腺肿瘤中创建异种移植物。细胞模型的体内生长抑制活性将通过以下方法在异种移植物模型中得到证实：预计上述发现将转化为II期临床试验，胰腺癌患者将根据基因谱或肿瘤预测对MEK抑制的反应和逃逸途径的存在，结合MEK和PI3K进行治疗。肿瘤环境对胰腺导管腺癌抗癌治疗效果的影响目的：-评估PDAC中针对肿瘤微环境的靶向治疗是否能改善药物传递和抗癌治疗的疗效。理由：最近的研究，包括NCI外科分会的报告表明，治疗微环境可以显著提高PDAC中其他抗癌治疗的疗效。抗微环境超音hedgehog基因抑制剂IPI-926可提高吉西他滨在PDAC转基因小鼠模型中的抗肿瘤效果。这些数据提示，致密的促结扎微环境阻碍了胰腺癌常用化疗药物吉西他滨到达实际癌细胞发挥有意义的抗肿瘤作用。本研究的目的是在模拟人类PDAC最常见遗传背景的不同转基因小鼠模型中，评估不同微环境治疗策略对PDAC抗肿瘤治疗效果的影响。方法：微环境的作用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：发展为胰腺癌的转基因/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，在超过85%的PDAC中存在，同时在超过50%的PDAC中缺失常见的肿瘤抑制基因CDKN2A和Smad4。这些基因工程小鼠模型类似于PDAC的人类基因组景观，在95%以上的病例中，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5种抗pdac治疗/策略的疗效：对照组（生理盐水）吉西他滨3。4. MEK抑制MEK - PI3K联合抑制用以下化合物/小分子处理微环境后的周期蛋白依赖性激酶抑制：Sonic hedgehog抑制剂舒尼替尼、莱诺度胺和顺铂。血小板衍生生长因子受体（PDGFR）或血管内皮生长因子受体的抑制剂。肿瘤坏死因子α 11。利妥昔单抗与Toll细胞受体（TLR-9）激动剂研究终点：肿瘤生长和肿瘤生长抑制将通过体内红外成像进行评估。在CCR/NCI的小鼠成像设施（MIF）中，功能成像将辅以正式的CT和MR解剖横断面成像，以评估针对微环境的各种策略对肿瘤发展和进展的影响。对不同组进行分析的最终终点是荷瘤小鼠的总生存率。3. 胰腺癌基因组中功能性非编码区在癌症发生和进展中的作用目的：-评估已知癌症基因的体细胞突变调控单元及其对肿瘤发生和进展的贡献。理论依据：对于胰腺癌中涉及的许多致癌途径，其激活/失调的确切原因尚不清楚。例如，虽然EGFR/HER2信号在超过60%的胰腺癌中被异常激活，但在许多情况下，没有发现激活体细胞突变、基因扩增或其他来自侧接信号的异常输入。这一观察结果提出了一种可能性，即在PDAC中，由于重要癌基因的启动子/功能性非编码区域的改变，必要的生长和增殖途径被失调。方法：该项目涉及在Illumina平台上使用第二代全基因组测序的大规模测序工作。该项目由NCI与完整基因组公司合作进行，将从三个匹配的肿瘤和正常样本开始。筛选最相关的癌症基因（PDAC中的PI3K， EGFR, Kras, Braf）的调控区域（上游-4,000bp的启动子区域），以确定是否存在提示功能改变的体细胞突变/基因组改变（例如，已知转录因子结合位点的改变）。表明功能改变的突变启动子序列（例如，由于转录起始改变而获得功能）将被克隆到报告质粒中，并在体外测试其对细胞生长和增殖的影响。在胰腺中识别调节区域的改变作为癌症促进和驱动事件可以[摘要截断为7800个字符]

1. Preclinical drug development dual MAPK and PI3K-Akt pathway inhibition in pancreatic cancer and identification of escape pathways Aim: - To evaluate the efficacy of combined MEK and PI3K inhibition in a preclinical model of pancreas cancer - To map escape pathways of tumor resistance following prolonged MEK and PI3K inhibitor treatment - To develop a reliable predictor (gene signature) for this strategy by correlating the underlying genotype to pathway activation and response to dual pathway inhibition Rationale: There is a very sound rationale for the combination of small molecule MEK and PI3K inhibitors in pancreatic cancer (PDAC): - The PI3K-Akt pathway is upregulated in PDAC in about 60% of cases due to direct activation of the p85 PI3K subunit by activated Kras. Using a combination of MEK and PI3K inhibition, the major outflow and escape pathway of activated Kras signaling (KRAS most common oncogene in PDAC) is inhibited - The PI3K-Akt pathway is also activated by abnormal EGFR signaling in 20% of cases in PDAC Methods: The NCI/Surgery Branch has a panel of greater than 50 pancreatic cancer cell lines available. These include cell lines from primary and metastatic tumors. In the majority of cases, the underlying genotype with respect to KRAS, CDKN2A, PTEN, TP53, Smad4, and TGFRII mutation status in these lines is known, and can be correlated with growth inhibition observed in the in vitro cell line model. Anti-proliferation and apoptotic activity of MEK and PI3K inhibition will be studied with a triplex (viable + dead + apoptotic) assay which reads - cell death - cell survival - apoptotic cells Two examples of growth inhibition curves in pancreatic cancer cell lines using a MEK inhibitor in sensitive and resistant cell lines to MEK inhibition are shown: ASUC Animal Protocol (SB-211) entitled In vivo platform for translational genotype-directed drug development in pancreatic cancer and other upper GI malignancies allows the creation of xenografts from resected pancreatic tumors from patients of the Surgery Branch and collaborating institutions. In vivo activity of growth inhibition in the cell model will be confirmed in vivo in a xenograft model by: - Inhibition of tumor growth at primary site (pancreas) - Inhibition of metastasis using consecutive in vivo imaging It is anticipated that the above findings will be translated into a phase II clinical trial where pancreas cancer patients will be treated, based on the genetic profile or their tumors predicting response to MEK inhibition and presence of escape pathways, with a combination of MEK and PI3K. 2. The Impact of the Tumor Environment on the Efficacy of Anticancer Therapy in Ductal Adenocarcinoma of the Pancreas Aim: - To evaluate if targeted therapy against the tumor microenvironment in PDAC improves drug delivery and efficacy of anti-cancer therapy Rationale: Recent studies, including reports from the Surgery Branch/NCI have shown that treating the microenvironment can dramatically increase the efficacy of other anticancer therapy in PDAC. Treatment with the anti-microenvironment sonic hedgehog inhibitor IPI-926 improved antitumor efficacy of gemcitabine in a transgenic mouse model of PDAC. These data suggest that the dense, desmoplastic microenvironement prevents gemcitabine, the commonly used chemotherapy agent against pancreas cancer, to reach the actual cancer cells to exercise a meaningful antitumor effect. The objective of this study is to evaluate the impact of different treatment strategies against the microenvironment on the efficacy of antitumor cancer therapy in PDAC in different transgenic mouse models simulating the most common genetic backgrounds of human PDAC. Methods: The role of the microenvironent 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: The 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 greater than 85% of PDAC in combination with knock-outs of the common tumor suppressor genes CDKN2A and Smad4 which are lost in greater than 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 greater than 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 five anti-PDAC treatments/strategies: 1. Control (normal saline) 2. Gemcitabine 3. MEK inhibition 4. Combined MEK PI3K inhibition 5. Cycline-dependent kinase inhibition after treatment of the microenviroment with the following compounds / small molecule: 7. Sonic hedgehog inhibitor 8. Sunitinib and lenolidomide and metronomic cisplatin 9. Inhibitors of the platelet-derived growth factor receptor (PDGFR) or the vascular endothelial growth factor receptor 10. Tumor necrosis factor alpha 11. Rituximab and Toll cell receptor (TLR-9) agonist 12. Integrin inhibitor Study endpoint: Tumor growth and tumor growth inhibition will be evaluated by in vivo infrared imaging. Functional imaging will be complemented by formal anatomical cross-sectional imaging with CT and MR in the Mouse Imaging Facility (MIF) of the CCR/NCI to assess impact of the various strategies targeting the microenvironment on tumor development and progression. Ultimate endpoint for analysis of the different groups is overall survival of the tumor-bearing mice. 3. Role of functional, non-coding regions in the pancreas cancer genome in cancer development and progression Aim: - To evaluate regulatory units of known cancer genes for somatic mutations and their contribution to tumor initiation and progression Rationale: For many pathways involved in carcinogenesis in pancreas cancer the exact cause of its activation/dysregulation is not known. For example, while EGFR/HER2 signaling is abnormally activated in greater than 60 % of pancreatic cancers, in many cases no activating somatic mutations, gene amplifications, or other abnormal inputs from collateral signaling has beenidentified. This observation raises the possibility that in PDAC essential growth and proliferation pathways are dysregulated because of alterations in the promoter / functional non-coding regions of important oncogenes. Methods: The project involves a large-scale sequencing effort using second generation whole genome sequencing on the Illumina platform. The project is performed within an NCI collaboration with Complete Genomics, Inc, and will start with three samples of matched tumor and normal. The regulatory regions (promoter regions to -4,000bp upstream) of the most pertinent cancer genes (PI3K, EGFR, Kras, Braf in PDAC) are screened for the presence of somatic mutations / genomic alterations suggesting altered function (e.g. altered known binding sites of transcription factors). Mutated promoter sequence(s) suggesting altered function (e.g. gain of function because of altered transcription initiation) will be cloned into a reporter plasmid, and tested in vitro with respect to their impact on cell growth and proliferation. Identifying alterations in regulatory regions as cancer-promoting and driving events in pancreas can [summary truncated at 7800 characters]