PARP Inhibitor Targeted Therapy for Breast Cancer

PARP抑制剂靶向治疗乳腺癌

• 批准号: 7481522
• 负责人: Jeffrey T Holt
• 金额: $ 14.9万
• 依托单位: TISSUE GENETICS, INC.
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-03-06 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7481522
• 关键词: Address; Affect; Animal Model; Animals; Antibodies; Apoptosis; BRCA1 Protein; BRCA1 gene; BRCA2 Mutation; BRCA2 Protein; BRCA2 gene; Base Sequence; Biochemical; Biological Markers; Bortezomib; Cancer Family; Cancer Patient; Cancer cell line; Cell Cycle; Cell Line; Cell physiology; Cells; Charge; Clinical Trials; DNA Repair; Data; Defect; Development; Diagnostic; Diagnostic tests; Disease; Engineering; Epithelial ovarian cancer; Estrogens; Family; Family member; Formalin; Funding; Gene Mutation; Gene Transfer; Genes; Goals; Hereditary Breast Carcinoma; Hereditary Malignant Neoplasm; Hormones; Hospitals; Human; Immune response; Immunohistochemistry; In Vitro; Inherited; Inhibitory Concentration 50; Laboratories; Lead; MG132; Malignant - descriptor; Malignant Neoplasms; Malignant neoplasm of ovary; Marketing; Mediating; Medical; Medicine; Messenger RNA; Methylation; Modeling; Molecular; Monoclonal Antibodies; Mus; Mutation; Numbers; Oral Contraceptives; Paraffin; Pathology; Patients; Pharmacologic Substance; Phase; Phase I Clinical Trials; Phase I/II Trial; Phase II Clinical Trials; Phospho-Specific Antibodies; Phosphorylation; Phosphorylation Site; Population; Preventive; Progesterone; Protein Dephosphorylation; Proteins; Public Health; Reagent; Regulation; Research; Research Personnel; Sampling; Serine; Signal Transduction; Societies; Specificity; Speed; Testing; Therapeutic; Threonine; Time; Tissue Sample; Tissues; Toxic effect; Treatment Cost; United States Food and Drug Administration; Western Blotting; Work; Xenograft procedure; base; cancer cell; cancer diagnosis; cellular engineering; chemotherapeutic agent; concept; drug development; drug testing; expression vector; gene function; gene therapy; in vivo; inhibitor/antagonist; interest; malignant breast neoplasm; mouse model; multicatalytic endopeptidase complex; mutant; mutation carrier; protein degradation; research and development; research study; response; tissue fixing; tumor; tumorigenesis

DESCRIPTION (provided by applicant): Mutations in BRCA1 lead to hereditary ovarian cancer in about 5% of ovarian cancer patients. However 60- 90% of epithelial ovarian cancers have markedly decreased levels of BRCA1 protein resulting from various perturbations in BRCA1 gene function spanning acquired mutation, gene methylation, and protein instability. Gene transfer of BRCA1 into ovarian cancer cells inhibits tumorigenesis in cell lines and animal models and lead to an FDA sponsor-investigator IND for our Phase I and Phase II trials for BRCA1 retroviral gene therapy. Problems with immune response and concerns about potential malignant transformation ultimately terminated these trials, but generally supported the concept that elevating BRCA1 expression inhibits ovarian cancer. We have continued to study BRCA1 regulation and have recently discovered that 1) estrogen and progesterone lead to an akt-mediated phosphorylation of BRCA1 presumably at Thr509 which markedly, post-translationally, stabilizes BRCA1 protein, and 2) that BRCA1 is rapidly degraded by the proteasome in the absence of these hormones. Based on these data we hypothesize that phosphorylation of BRCA1 at Thr509 inhibits proteasomal degradation and propose that this explains why some ovarian cancers respond to Bortezomib therapy. Since decreased BRCA1 expression is a clear and important mechanism for ovarian cancer and BRCA1 protein is rapidly degraded by the proteasome, we propose that BRCA1 protein degradation may represent a biomarker for Bortezomib response in ovarian cancer. We hypothesize that ovarian cancers with moderate to high levels of BRCA1 mRNA but low levels of BRCA1 protein will respond to Bortezomib therapy. Phase I Milestones: 1) Treat SKOV3 and ES-2 ovarian cancer cell lines and xenografts (low level BRCA1 protein but moderate mRNA levels) with the proteasomal inhibitors Bortezomib and MG132 and quantitate BRCA1 mRNA and protein levels and extent of tumor inhibition. 2) Construct BRCA1 mutant expression vectors expressing distinct phosphorylation mutants at threonine 509: specifically T509A (non-phosphorylatable); T509E (constitutively charged, presumed active), disease-associated patient mutation R504H (non-phosphorylatable), and wildtype T509T. 3) Transfect SKOV3 cells with Aim 2 expression vectors; analyze phosphorylation and protein degradation in vitro and in vivo and analyze tumor inhibition. These cell lines will permit Phase II Proof- Of-Concept studies to test if decreased BRCA1 protein stability predicts response to proteasomal agents. PUBLIC HEALTH RELEVANCE: This Phase I study will develop the reagents and cell lines for a definitive Proof-of-Principle Phase II Study testing the hypothesis that S3291 BRCA2 phosphorylation predicts responders to PARP inhibitors and that an IHC test using this antibody can predict which patients should receive this therapy. This proposal will create engineered cancer cell lines with either absent or increased S3291 BRCA2 phosphorylation to allow direct testing of PARP inhibitor effects in vitro and in animal tumors. If the hypothesis is correct then an IHC test will be developed and marketed to predict responders to this targeted PARP therapy.

描述（申请人提供）：BRCA 1突变导致约5%的卵巢癌患者发生遗传性卵巢癌。然而，60- 90%的上皮性卵巢癌由于BRCA 1基因功能的各种扰动（包括获得性突变、基因甲基化和蛋白质不稳定性）而导致BRCA 1蛋白水平显著降低。将BRCA 1基因转移到卵巢癌细胞中可抑制细胞系和动物模型中的肿瘤发生，并为我们的BRCA 1逆转录病毒基因治疗的I期和II期试验提供了FDA的研究者IND。免疫反应的问题和对潜在恶性转化的担忧最终终止了这些试验，但总体上支持BRCA 1表达升高抑制卵巢癌的概念。我们继续研究BRCA 1的调节，最近发现：1）雌激素和孕激素导致akt介导的BRCA 1磷酸化，可能是在Thr 509，这在产后显著稳定BRCA 1蛋白，2）在没有这些激素的情况下，BRCA 1被蛋白酶体迅速降解。基于这些数据，我们假设BRCA 1在Thr 509的磷酸化抑制蛋白酶体降解，并提出这解释了为什么一些卵巢癌对硼替佐米治疗有反应。由于BRCA 1表达降低是卵巢癌的一个明确而重要的机制，并且BRCA 1蛋白被蛋白酶体迅速降解，我们认为BRCA 1蛋白降解可能代表卵巢癌中硼替佐米反应的生物标志物。我们假设BRCA 1 mRNA水平中到高水平但BRCA 1蛋白水平低的卵巢癌对硼替佐米治疗有反应。第一阶段里程碑：1）用蛋白酶体抑制剂硼替佐米和MG 132处理SKOV 3和ES-2卵巢癌细胞系和异种移植物（低水平BRCA 1蛋白但中等mRNA水平），并定量BRCA 1 mRNA和蛋白水平以及肿瘤抑制程度。2)构建表达苏氨酸509处不同磷酸化突变体的BRCA 1突变体表达载体：特别是T509 A（不可磷酸化）; T509 E（组成型带电，假定有活性），疾病相关患者突变R504 H（不可磷酸化）和野生型T509 T。3)用Aim 2表达载体转染SKOV 3细胞;分析体外和体内磷酸化和蛋白降解，并分析肿瘤抑制。这些细胞系将允许II期概念验证研究测试BRCA 1蛋白稳定性降低是否预示对蛋白酶体试剂的应答。公共卫生关系：这项I期研究将开发用于确定性原理验证II期研究的试剂和细胞系，该研究将测试S3291 BRCA 2磷酸化可预测PARP抑制剂应答者以及使用该抗体的IHC检测可预测哪些患者应接受该治疗的假设。该提案将创建缺乏或增加S3291 BRCA 2磷酸化的工程化癌细胞系，以允许在体外和动物肿瘤中直接测试PARP抑制剂的作用。如果假设是正确的，那么将开发并销售IHC检测，以预测这种靶向PARP治疗的应答者。