Molecular Biology of Pediatric Tumors

小儿肿瘤的分子生物学

基本信息

批准号：
8350056
负责人：
LEE J. HELMAN
金额：
$ 38.25万
依托单位：
DIVISION OF CLINICAL SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
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/8350056
关键词：
Alveolar Alveolar Rhabdomyosarcoma Apoptotic Area Back Biological Assay Biological Factors Biological Markers Biology CRKL gene Candidate Disease Gene Cell Line Cells Childhood Chimeric Proteins Clinic Clinical Research Collaborations Combined Modality Therapy Critical Pathways Data EWS-FLI1 fusion protein Embryonal Rhabdomyosarcoma Ewings sarcoma Gene Fusion Genes Goals Growth Human Immunohistochemistry In Vitro Laboratories Lead Libraries Luciferases Manuscripts Mass Spectrum Analysis Mining Modeling Molecular Molecular Biology Molecular Target Monitor NR0B1 gene Natural Product Drug Oncogenes Oncology Group Pathway interactions Patient Selection Patients Pediatric Neoplasm Plicamycin Protein Tyrosine Kinase Reaction Reporter Resistance Rhabdomyosarcoma Role Screening procedure Selection Criteria Signal Pathway Signal Transduction Small Interfering RNA Sorting - Cell Movement Specimen Techniques Testing Therapeutic Time Tumor Cell Line Work Xenograft procedure addiction base cell growth follow-up high throughput screening improved in vivo inhibitor/antagonist knock-down mTOR Inhibitor mouse model neoplastic cell novel osteosarcoma preclinical study promoter receptor density response sarcoma small hairpin RNA src-Family Kinases transcription factor tumor tumor growth

项目摘要

Work over the past year has focused on 3 major areas, IGFIR signaling, the identification of oncogene addiction pathways in rhabdomyosarcomas (RMS) using shRNA screening techniques, and screening drug and natural product libraries to identify inhibitors of the EWS-FLI-1 fusion protein in Ewings sarcoma. We have continued to study IGF signaling in pediatric sarcomas now focusing on trying to identify relevant biomarkers that would allow us to enrich patients entered onto clinical studies using IGFIR Abs. We have shown that RMS tumor specimens as well as cell lines have variable IGFIR levels, and that very low levels predict lack or response to IGFIR blockade, as expected. We have shown that compared to immunohistochemistry, the use of single reaction monitoring mass spectrometry is a much more quantitative and accurate assay for determining IGFIR receptor density on tumors and cell lines. We are now working with the Childrens Oncology Group to develop quantitative IGFIR assays that might be used in clinical studies for enrichment strategies in clinical studies of IGFIR blockade. However, we still have not identified markers predictive of response, and work is ongoing in this area. Currently, in collaboration with Dr. Paul Meltzer and SARC, we are evaluating tumor specimens obtained for Ewings sarcoma patients who responded to IGFIR Ab therapy and specimens from those who did not respond. Analysis is almost complete and hopefully will lead to testable hypothesis regarding selection criteria for response to IGFIR Ab treatment. In preclinical studies, in collaboration with Dr. Liang Cao we have found that there may be some RMS tumor cells that use the IGFIR pathway for both proliferation as well as anti-apoptotic signaling, and these tumors are particularly sensitive to IGFIR Ab treatment. Ongoing studies in xenografts are attempting to model optimal timing and combinations of combination therapy of IGFIR Ab and mTOR inhibitors to pick an optimal way to test these combinations in the clinic. We are also attempting to develop mouse models of acquired resistance to IGFIR Ab therapy to better understand what we have observed in our clinical studies. We have continued to mine our data from our high throughput shRNA screening to identify critical pathways for survival of human RMS cell lines. Using an inducible shRNA library containing specific barcodes for clone identification in collaboration with Dr. Lou Staudt, we screened an alveolar and an embryonal RMS cell line to identify specific RNAs that when knocked-down with shRNA would lead to growth arrest. We have identified a number of candidate genes that appear to be critical for survival of these tumor cells. The first gene we have just completed our analysis of confirms that CrkL is required for RMS survival and tumor growth both in vitro and in vivo. We have most recently demonstrated that CrkL signaling in RMS is independent of PI3K-Akt signaling but appears to be via Src family kinase (SFK) signaling, thus identifying a new potential critical signaling pathway for RMS. We have identified YES as the Src family kinase directly involved in CRKL signaling and have shown that targeting SFK with small molecular inhibitors leads to inhibition of RMS cell growth both in vitro and in vivo. We have just submitted a manuscript describing these findings. We are currently working to better understand the mechanism of action of CRKL in maintaining growth of RMS tumor cells. We have also identified Bub1b, a spindle assembly checkpoint gene, as critical for growth of RMS genes. We are currently studying this pathway in RMS to understand its importance in growth of these tumors. We have also used this screen to identify genes whose expression is necessary for survival only in the presence of the Pax-3-Foxo1 gene fusion found in alveolar RMS. We have identified TNK2, a cytoplasmic tyrosine kinase as necessary for survival of alveolar RMS and work is ongoing to study the mechanism of activity. An additional list of 12 other genes has been identified and we are confirming these with follow-up screens. We have developed and utilized a high-throughput screen to evaluate more than 50,000 compounds for inhibition of EWS-FLI1 activity in collaboration with Drs. Woldemichael and McMahon at the Molecular Targets laboratory. We used a cell based luciferase reporter screen utilizing the EWS-FLI1 downstream target NR0B1 promoter and a gene signature secondary screen employing a novel list of more than 10 downstream targets to sort and prioritize the compounds. We identified a lead compound, mithramycin that appears to have specific activity against the EWS-FLI-1 transcription factor, have confirmed its activity in mouse models and hope to test it in the clinic within the next 6 months.

过去一年的工作主要集中在 3 个主要领域：IGFIR 信号传导、使用 shRNA 筛选技术鉴定横纹肌肉瘤 (RMS) 中的致癌基因成瘾途径，以及筛选药物和天然产物库以鉴定 EWS-FLI-1 融合蛋白的抑制剂在尤文氏肉瘤中。我们继续研究儿科肉瘤中的 IGF 信号传导，现在专注于尝试识别相关生物标志物，这将使我们能够丰富使用 IGFIR Abs 进入临床研究的患者。我们已经证明，RMS 肿瘤标本以及细胞系具有可变的 IGFIR 水平，并且非常低的水平预示着 IGFIR 阻断的缺乏或反应，正如预期的那样。我们已经证明，与免疫组织化学相比，使用单反应监测质谱法是一种更加定量和准确的测定肿瘤和细胞系上 IGFIR 受体密度的方法。我们现在正在与儿童肿瘤学小组合作开发定量 IGFIR 检测方法，这些检测方法可用于临床研究，以实现 IGFIR 阻断临床研究的富集策略。然而，我们仍然没有确定预测反应的标记物，并且该领域的工作正在进行中。目前，我们与 Paul Meltzer 博士和 SARC 合作，正在评估从对 IGFIR Ab 治疗有反应的尤文氏肉瘤患者获得的肿瘤样本以及从没有反应的患者身上获取的样本。分析已接近完成，希望能够得出有关 IGFIR Ab 治疗反应选择标准的可检验假设。在临床前研究中，我们与梁曹博士合作发现，可能有一些 RMS 肿瘤细胞利用 IGFIR 通路进行增殖和抗凋亡信号传导，并且这些肿瘤对 IGFIR Ab 治疗特别敏感。正在进行的异种移植研究正在尝试模拟 IGFIR Ab 和 mTOR 抑制剂联合治疗的最佳时机和组合，以选择在临床中测试这些组合的最佳方法。我们还尝试开发对 IGFIR Ab 疗法获得性耐药的小鼠模型，以更好地了解我们在临床研究中观察到的情况。我们继续挖掘高通量 shRNA 筛选的数据，以确定人类 RMS 细胞系生存的关键途径。我们与 Lou Staudt 博士合作，使用包含用于克隆识别的特定条形码的诱导型 shRNA 文库，筛选了肺泡和胚胎 RMS 细胞系，以识别特定的 RNA，当用 shRNA 击倒时会导致生长停滞。我们已经确定了许多候选基因，它们似乎对这些肿瘤细胞的生存至关重要。我们刚刚完成的第一个基因分析证实，CrkL 是 RMS 存活和肿瘤体外和体内生长所必需的。我们最近证明 RMS 中的 CrkL 信号传导独立于 PI3K-Akt 信号传导，但似乎是通过 Src 家族激酶 (SFK) 信号传导，从而确定了 RMS 的新的潜在关键信号传导途径。我们已经确定 YES 是直接参与 CRKL 信号传导的 Src 家族激酶，并表明用小分子抑制剂靶向 SFK 可在体外和体内抑制 RMS 细胞生长。我们刚刚提交了一份描述这些发现的手稿。我们目前正在努力更好地了解 CRKL 在维持 RMS 肿瘤细胞生长方面的作用机制。我们还发现 Bub1b（一种纺锤体组装检查点基因）对于 RMS 基因的生长至关重要。我们目前正在研究 RMS 中的这条通路，以了解它在这些肿瘤生长中的重要性。我们还使用此筛选来识别仅在肺泡 RMS 中发现的 Pax-3-Foxo1 基因融合体存在时才需要表达的基因。我们已经确定 TNK2（一种细胞质酪氨酸激酶）对于肺泡 RMS 的生存是必需的，并且正在进行研究其活性机制的工作。已确定了 12 个其他基因的附加列表，我们正在通过后续筛选来确认这些基因。我们与 Drs. 合作开发并利用高通量筛选来评估 50,000 多种化合物对 EWS-FLI1 活性的抑制作用。沃尔德米切尔和麦克马洪在分子靶标实验室。我们使用基于细胞的荧光素酶报告基因筛选，利用 EWS-FLI1 下游靶标 NR0B1 启动子，并使用基因特征二次筛选，利用超过 10 个下游靶标的新列表来对化合物进行排序和优先级排序。我们鉴定出一种先导化合物光神霉素，它似乎对 EWS-FLI-1 转录因子具有特异性活性，并已在小鼠模型中证实了其活性，并希望在未来 6 个月内在临床中对其进行测试。