Molecular Biology of Pediatric Tumors

小儿肿瘤的分子生物学

• 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
• 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

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.

过去一年中的工作集中在3个主要领域，IGFIR信号，使用SHRNA筛选技术鉴定横纹肌肉瘤（RMS）的癌基因成瘾途径，以及筛选药物和天然产物库，以鉴定EWINGS SARMAMA中EWS-FLI-1融合蛋白的抑制剂。 我们一直在研究小儿肉瘤中的IGF信号传导，现在专注于尝试鉴定相关的生物标志物，这使我们能够富含使用IGFIR ABS进入临床研究的患者。我们已经表明，RMS肿瘤标本和细胞系具有可变的IGFIR水平，并且非常低的水平预测，这是预期的，预期的是对IGFIR阻断的缺乏或反应。我们已经表明，与免疫组织化学相比，单个反应监测质谱法的使用是确定肿瘤和细胞系上IGFIR受体密度的更具定量和准确的测定。现在，我们正在与儿童肿瘤学组合作，开发定量的IGFIR分析，这些测定可能用于临床研究中，以用于IGFIR封锁临床研究中的富集策略。 但是，我们仍然没有确定预测响应的标记，并且在这一领域正在进行工作。目前，与Paul Meltzer博士和SARC合作，我们正在评估针对对Igfir AB疗法反应的EWINGS肉瘤患者获得的肿瘤标本，并从没有反应的人那里获得了标本。分析几乎是完整的，希望将导致有关响应IGFIR AB治疗的选择标准的可检验假设。在临床前研究中，与Liang Cao博士合作，我们发现可能有一些RMS肿瘤细胞使用IGFIR途径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建模IGFIR AB和MTOR抑制剂组合疗法的最佳时机和组合，以选择一种最佳方法来测试诊所中的这些组合。我们还试图开发出对IGFIR AB疗法的耐药性的小鼠模型，以更好地了解我们在临床研究中观察到的内容。 我们继续从高吞吐量shRNA筛选中挖掘数据，以识别人类RMS细胞系存活的关键途径。使用诱导的shRNA文库，该库中包含特定条形码用于克隆识别的特定条形码，并与Lou Staudt博士合作，筛选了肺泡和胚胎RMS细胞系，以识别特定的RNA，而当用shrna撞倒时，这些RNA会导致生长停滞。我们已经确定了许多似乎对于这些肿瘤细胞存活至关重要的候选基因。我们刚刚完成的第一个基因对RMS存活和体内肿瘤生长都需要CRKL的分析确认。我们最近证明，RMS中的CRKL信号与PI3K-AKT信号无关，但似乎是通过SRC家族激酶（SFK）信号传导，从而识别了RMS的新潜在临界信号传导途径。我们已经确定是的是直接参与CRKL信号传导的SRC家族激酶，并表明用小分子抑制剂靶向SFK会导致体外和体内抑制RMS细胞生长。我们刚刚提交了一个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肿瘤细胞生长中的作用机理。 我们还鉴定出纺锤体组装检查点基因Bub1b对于RMS基因的生长至关重要。我们目前正在研究RMS中的这一途径，以了解其在这些肿瘤生长中的重要性。我们还使用此屏幕来识别仅在肺泡RMS中发现的PAX-3-福克斯基基因融合存在的情况下，其表达对于生存是必要的基因。我们已经确定了TNK2，一种细胞质酪氨酸激酶是肺泡RMS存活的必要条件，并且正在进行研究活性机理。已经确定了其他12个基因的其他列表，我们正在用后续屏幕确认这些基因。 我们已经开发并利用了一个高通量屏幕来评估50,000多种化合物，以抑制与DRS合作的EWS-FLI1活性。 Woldemichael和McMahon在分子目标实验室。我们使用了基于细胞的荧光素酶报告基屏幕，该屏幕利用EWS-FLI1下游目标NR0B1启动子和一个基因签名二级屏幕，该筛选使用了10多个下游目标的新颖列表来对化合物进行排序和优先级。我们确定了一种铅化合物，毛霉素似乎对EWS-FLI-1转录因子具有特定活性，已证实其在小鼠模型中的活性，并希望在未来6个月内在诊所中对其进行测试。