Preclinical drug development in pancreatic cancer

我的实验室旨在通过开发新的治疗方法来解决胰腺癌患者未满足的更有效治疗的医疗需求。美国癌症协会估计，2016年美国有53070例胰腺癌新病例和41780例胰腺癌死亡，并预测到2030年，胰腺癌将在所有癌症相关死亡中排名第二。高达90%的胰腺癌患者在确诊的第一年内死于这种疾病。目前的化学疗法和分子疗法都不能延长患者的生存时间（超过几个月），也没有持续肿瘤消退或治愈的希望。在我的实验室进行的不同药物开发工作的总体研究目标和科学目标是开发胰腺癌的新疗法。这些研究包括从早期结构活性、体外、细胞和体内评估，到IND启用研究和临床开发。为了减少后期的失败，所有药物开发工作都以假设驱动的作用机制研究为指导，包括在原位、患者来源的异种移植或转基因动物模型中的早期疗效、毒性和药代动力学研究。过去一年的科研成果包括：甲氧他雷斯汀是一种新型的小分子抑制剂，对癌细胞的转移表型具有选择性活性。它在胰腺癌转移模型中具有令人印象深刻的活性。我在NCIs NExT项目中共同提交的药物开发项目“Metarrestin，一种治疗转移的新方法”被评为“top tier，在28个申请中排名第2，平均分2.0”。甲脲素项目的近期目标是：1 .完成IND可行性研究，包括GLP对猪（小型猪）的长期毒性。向FDA提交IND申请，并在CCR开始该药物的I期安全性和MTD临床研究。该项目目前已进入临床前后期阶段，预计将于2017年进入临床试验。“IND候选化合物甲他芮素在临床前小鼠胰腺导管腺癌模型中的疗效和生物分布评估”提案已被CCR领导批准并纳入CCR CAPRs组合。在转基因KPC小鼠中进行的大量啮齿动物药理学和PK研究表明，该药物具有良好的血浆组织渗透性：肿瘤AUC比超过1:10，在无毒剂量水平下，肿瘤内药物水平接近100uM。人体配方的研制已经完成。猪（迷你猪）的PK试验研究已经开始，随后将进行长期的GLP毒性研究。根据良好生产规范（GMP）生产的2kg甲氨脲素已开始生产，用于IND支持GLP毒性研究和人体研究。2. “生物仿制药”抗癌肽RP-182的临床前开发我们实验室在小鼠胰腺肿瘤中发现了RP-182的强抗癌活性，并将该肽作为新型有效的癌症治疗方法申请了专利(国际PCT专利申请号：PCT / US15/55305)。我的实验室已经证明，RP-182结合CD206并靶向CD206阳性M2肿瘤相关巨噬细胞，并通过减少和重编程这种通常免疫抑制的免疫细胞群，增加肿瘤内免疫力，使其向抗肿瘤M1表型转变。此外，RP-182下调小鼠胰腺癌癌细胞上PD-1L的表达，添加抗PD-1L免疫检查点抑制剂可延长RP-182治疗动物的生存期。总的来说，结果表明RP-182在胰腺癌中抑制先天免疫抑制信号，作为“生物类似物”（巨噬细胞上CD206甘露糖结合受体识别的生物有机体的甘露糖分子），并诱导这些肿瘤中CD206阳性免疫细胞的死亡和重编程。RP-182的体内测定的PK测定现在可以用来询问RP-182的药理学，这将把靶向先天免疫系统的CD206轴的计划作为抗癌联合免疫治疗方法的一部分，进入临床前的后期开发。3. 干细胞抑制剂-8382的临床前开发在我的实验室开发的这个项目的科学目标是显示这种小分子抑制剂的选择性抗癌干细胞活性，它有效地抑制转移的形成，与吉西他滨化疗相比，在基于细胞和体内的干细胞分析中。一系列球体克隆原性实验、体内肿瘤起始研究和使用流式细胞术测量的干细胞性，包括侧群（SP）分析实验，证实了与吉西他滨相比，该抑制剂具有选择性的抗癌干细胞功能。两种新的靶标化合物——TAOK3和CDK7——也能促进癌症干细胞靶标。该分子协同作用于不同的DNA损伤修复机制，这是癌症干细胞的一个弱点。4. 这个项目的主要目标是继续进行我们发现的KRAS G12R突变异种异种移植细胞系和患者来源的异种移植模型的敏感性增加的临床翻译。无偏基因表达分析及信号转导节点功能丧失；弗雷德里克[Frederick]证实了不同的RAS基因型与不同的基因表达谱和选择信号转导依赖性相关。CCR科学领导委员会和CTEP已经批准了一项针对肿瘤含有G12R KRAS异构体的患者的20例II期试验，该试验验证了KRAS突变异构体是否代表抗mek治疗作为胰腺癌二线治疗新疗法的整体生物标志物，这是我们实验室工作的直接转化。5. 抗pi3k /mTOR分子治疗胰腺癌的研究大量胰腺癌细胞系的体细胞突变状态与对PI3K/mTOR抑制的药物表型抗性（用NVP-BEZ235治疗后未诱导细胞凋亡）的相关性显示，a. ARID1A或其他染色质重塑复合物SNF/SWI基因的变异与b. 11个基因APC、AXIN1、CCND1、CCND3、CTNNB1， CREBBP, EP300， MYC, RAC1, SMAD4， TP53的98个基因参与WNT信号传导。以及对PI3K/mTOR抑制的抗性。上述细胞系和经PI3K/mTOR抑制剂处理的患者源异种移植模型（PDX）的磷酸化蛋白谱显示，与敏感的PDX肿瘤相比，对PI3K/mTOR抑制剂有抗性的肿瘤中AMPK活性增加。由于AMPK是细胞能量稳态的主要调节因子，我们比较了抗性细胞系和敏感细胞系的代谢谱，发现敏感细胞系对营养物质的代谢依赖（包括线粒体基础呼吸和最大呼吸和备用能力的增加）和抗性细胞系糖酵解速率（包括糖酵解能力和糖酵解储备）的显著差异。目前正在进行PI3K/mTOR抑制剂和糖酵解抑制剂在PI3K/mTOR耐药细胞中的联合药物反应评估，以测试糖酵解阻断存在下PI3K/mTOR抑制的致敏性，作为一种新的治疗胰腺癌的方法。

My laboratory aims to address the unmet medical need of more effective treatments for pancreas cancer patients by developing new treatment approaches. The American Cancer Society estimates 53,070 new cases and 41,780 deaths from pancreatic cancer in the United States during 2016 and predicts that pancreatic cancer will rank 2nd of all cancer-related mortalities by the year 2030. Up to 90% of pancreatic cancer patients succumb to the disease within the first year of diagnosis. Neither current chemotherapy nor molecular therapy provides patients with an extension of survival measured by more than a few months, or the hope for sustained tumor regressions or cure. The overall research goals and scientific objectives of the different drug development efforts conducted in my laboratory are the development of novel therapeutics in pancreas cancer. These include studies from early structure-activity, in vitro, cell-based and in vivo evaluations, to IND enabling studies and clinical development. To reduce later failure all drug development efforts are guided by hypothesis-driven mechanism of action studies including early efficacy, toxicity, and pharmacokinetic studies in orthotopic, patient-derived xenotransplantation, or transgenic animal models. Scientific achievements in the last year include: 1. Preclinical and clinical development of metarrestin Metarrestin is a novel small molecule inhibitor with selective activity against the metastatic phenotype of cancer cells. It has impressive activity in pancreatic cancer metastasis models. The drug development project 'Metarrestin, a new approach towards metastasis' co-presented by me to NCIs NExT program was evaluated as 'top tier, ranked 2nd out of 28 applications, average score 2.0'. The near term goals of the metarrestin program are i. completion of IND enabling studies, including GLP long term toxicity in swine (miniature pigs) ii. compile and file IND application with FDA and commence phase I safety and MTD clinical studies with this agent at CCR The program has now moved into the late preclinical stage, and expected for 2017, to advance into clinical testing. The proposal 'Assessment of Efficacy and Biodistribution of IND Candidate Compound Metarrestin in Preclinical Murine Models for Pancreatic Ductal Adenocarcinoma' has been approved by CCR Leadership and included into CCR CAPRs portfolio. Extensive rodent pharmacology and PK studies in transgenic KPC mice have shown excellent tissue penetration of the drug with plasma:tumor AUC ratios exceeding 1:10 and intratumoral drug levels close to 100uM at non-toxic dose levels. Development of a human formulation has been completed. A pilot of PK studies in swine (mini pigs) has been started, to be followed by long term GLP toxicity studies. Production of 2kg of metarrestin produced under Good Manufacturing Practices (GMP) to be used for IND enabling GLP toxicity studies and human studies has been started. 2. Preclinical development of the 'biosimilar' anti-cancer peptide RP-182 The discovery of the strong anti-cancer activity of RP-182 in murine pancreatic tumors in our laboratory has led to a patent filing of such peptides as novel effective treatment of cancers (Int'l PCT Patent Application No. PCT/US15/55305). My laboratory has shown that RP-182 binds CD206 and targets CD206 positive M2 tumor-associated macrophages, and increases intratumoral immunity through reduction and reprogramming of this generally immune suppressive immune cell population towards an anti-tumor M1 phenotype. Additionally, RP-182 downregulates in murine pancreatic cancers PD-1L expression on cancer cells, and the addition of an anti-PD-1L immune check point inhibitor extends survival of RP-182-treated animals. Overall, results show RP-182 suppresses innate immune suppressive cues in pancreatic cancers acting as a 'biosimilar' (to mannose moeities of bioorganisms recognized by CD206 mannose-binding receptors on macrophages) and inducing death and reprogramming of CD206 positive immune cells in these tumors. A PK assay for in vivo measurements of RP-182 is now available to interrogate the pharmacology of RP-182 which will move the program of targeting the CD206 axis of the innate immune system, as part of anti-cancer combination immunotherapy approaches, into late preclinical development. 3. Preclinical development of the stem cell inhibitor -8382 The scientific goal of this program developed in my laboratory is to show selective anti-cancer stem cell activity of this small molecule inhibitor, which effectively suppresses metastasis formation, in comparison to gemcitabine chemotherapy in cell-based and in vivo assays of stemness. A series of spheroid clonogenicity assays, in vivo tumor initiation studies, and measures of stemness using flow cytometry including side population (SP) profiling experiments confirm a selective anti-cancer stem cell function of the inhibitor compared to gemcitabine. Two of the compounds new targets - TAOK3 and CDK7 - also promote cancer stemness targets. The molecule targets synergistically different mechanisms of DNA damage repair, a vulnerability of cancer stem cells. 4. Clinical development of RAS mutational isoform-directed anti-MAPK pathway therapy The major objective of this project is to proceed with clinical translation of our discovery of increased sensitivity of KRAS G12R mutational isoform-harboring cell lines and patient-derived xenotransplantation models. Unbiased gene expression analysis as well as loss of function of signaling nodes siREN screening [knockdown of signal transduction nodes; in collaboration with NCIs RAS Initiation of NCI Frederick] confirmed that different RAS genotypes are associated with different gene expression profiles and select signal transduction dependencies. A 20-patient phase II pilot treating patients whose tumor harbor G12R KRAS isoforms has been approved by the CCR Scientific Leadership Committee and CTEP which tests the hypothesis whether KRAS mutational isoforms represent an integral biomarker for response to anti-MEK therapy as novel therapy in 2nd line treatment of pancreas cancer and is a direct translation of our laboratory efforts. 5. Anti-PI3K/mTOR molecular therapy in pancreas cancer: genetic variants and activation of LKB1-AMPK signaling predict response to NVP-BEZ235 Correlation between somatic mutation status of a large panel of pancreatic cancer cell lines and drug phenotype resistance to PI3K/mTOR inhibition (no induction of apoptosis upon treatment with NVP-BEZ235) shows statistically significant associations between a. variants in the ARID1A or other genes of the chromatin remodeling complex SNF/SWI, and b. variants in any of the 11 genes APC, AXIN1, CCND1, CCND3, CTNNB1, CREBBP, EP300, MYC, RAC1, SMAD4, TP53 of the 98 genes involved in WNT signaling. and resistance to PI3K/mTOR inhibition. Phosphoprotein profiling of above cell lines and of patient-derived xenotrans-plantation models (PDX) treated with the PI3K/mTOR inhibitor showed increased AMPK activity in tumors resistant to PI3K/mTOR inhibition compared to sensitive PDX tumors. As AMPK is a major regulator of cell energy homeostasis, we compared metabolic profiles of resistant versus sensitive cell lines and found sharp differences between metabolic reliance on nutrients including increase in mitochondrial basal and maximum respiration and spare capacity in sensitive cell lines, and increased rate of glycolysis including glycolytic capacity and glycolytic reserve in resistant cell lines. The combination drug response evaluations of PI3K/mTOR inhibitors and inhibitors of glycolysis in PI3K/mTOR resistant cells to test for sensitization to PI3K/mTOR inhibition in the presence of glycolysis blockade as a novel therapeutic approach to pancreas cancer are currently ongoing.