The possible role of the novel Akt 3 R247C mutation in opposing to anti-HER2 ther

新型 Akt 3 R247C 突变在对抗抗 HER2 治疗中的可能作用

• 批准号: 8747894
• 负责人: Maurizio Scaltriti
• 金额: $ 8.76万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-16 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8747894
• 关键词: Affect; Aftercare; Amino Acids; Antibodies; Appearance; Biological Assay; Biopsy; Cancer Patient; Candidate Disease Gene; Cell Culture Techniques; Cells; Clinical; DNA analysis; Detection; Diagnosis; Disease; Disease Progression; Disease remission; Distant; Drug resistance; ERBB2 gene; Enzymes; Gene Mutation; Gene Proteins; Genes; In complete remission; Laboratories; Lead; Lesion; Lymph; Malignant Neoplasms; Missense Mutation; Monitor; Monoclonal Antibodies; Morphologic artifacts; Mutate; Mutation; Neck; Neoplasm Metastasis; Nodal; Nude Mice; Oncologist; Pathway interactions; Patients; Pharmacodynamics; Phenotype; Phosphotransferases; Positioning Attribute; Pre-Clinical Model; Prevalence; Prevalence Study; Protein Isoforms; Refractory; Relapse; Reporting; Research; Resistance; Role; Sampling; Specimen; Testing; Therapeutic; Tissues; Trastuzumab; Tyrosine Kinase Inhibitor; Up-Regulation; Xenograft procedure; base; chemotherapy; deep sequencing; design; exome sequencing; human FRAP1 protein; insight; lapatinib; lymph nodes; malignant breast neoplasm; neoplastic cell; novel; pressure; public health relevance; research study; resistance mechanism; response; small molecule; tumor; tumor growth; tumorigenic

One of the main difficulties in understanding the mechanisms of resistance to anti-HER2 agents is the concomitant treatment with chemotherapy. In this proposal we took advantage of unique samples obtained from a patient who first achieved a pathological complete response to the anti- HER2 antibody trastuzumab but then relapsed in a distant lymph node overtime. Deep sequencing of these specimens revealed that the acquisition of drug resistance coincided with the appearance of a previously unreported mutation in the Akt 3 gene. This result is imputable entirely to the pharmacological pressure of trastuzumab as this patient did not receive any other concomitant therapies (including chemotherapy). The objective of this study is to investigate whether the acquisition of the newly identified Akt 3 mutation R247C leads to resistance to anti-HER2 therapy in HER2 positive breast cancer. We plan to investigate the prevalence of this mutation (or other putatively activating mutations of the Akt genes) in samples from patients who are or became refractory to anti-HER2 therapy. Moreover, we plan to study the intrinsic kinase activity of Akt 3 R247C and consequent acquisition of the resistance phenotype. Finally, we will assess whether catalytic inhibition of Akt, alone or in combination with anti-HER2 therapy, is efficacious in limiting proliferation and tumor growth in cells/tumors bearing the Akt 3 R247C mutation. We have already collected more than 20 paired biopsies (pre- and post-treatment) from HER2 positive breast cancer patients who initially responded to either trastuzumab or lapatinib (a small molecule tyrosine kinase inhibitor of HER2) but eventually progressed to therapy overtime. The analysis of these specimens by IMPACT, a platform that allows to exome sequence ~300 cancer- related genes, will reveal how many of these patients have acquired a mutation in the Akt genes or in other genes related to the PI3K/Akt/mTOR pathway. Since the R247C mutation of Akt 3 was never reported, we aim to study its potential role in activating the enzyme and confer resistance to anti-HER2 agents. Akt 3 R247C will be expressed in HER2 positive cells sensitive to trastuzumab and lapatinib. In cell culture, we plan to perform long term viability assays, both in 2D and 3D culture, to study the capacity of these stable clones to escape the inhibitory activity of these agents. A similar approach will be taken in vivo, where BT474 cells (tumorigenic) expressing both wild-type and mutated Akt 3 will be injected in nude mice to form xenografts that will be treated with anti-HER2 agents, alone or in combination. Finally, we will assess the efficacy of catalytic Akt inhibition in re-sensitize cells stably expressing Akt 3 R247C to anti-HER2 therapy. In particular, we will monitor tumor growth inhibition in response to the treatments and perform pharmacodynamics studies to confirm inhibition of the pathways.

理解对抗HER2药物抗性机制的主要困难之一是 化学疗法的伴随治疗。在此提案中，我们利用了独特的优势 从患者获得的样本，该患者首先获得了对抗抗的病理完全反应 HER2抗体曲妥珠单抗，但随后在远处的淋巴结中复发。深度测序 在这些标本中，表明耐药性的获取与外观一致 Akt 3基因中先前未报告的突变。这个结果完全可以完全归咎于 曲妥珠单抗的药理压力，因为该患者没有接受任何其他伴随 疗法（包括化学疗法）。 这项研究的目的是研究新确定的AKT 3的获取是否是 突变R247C导致HER2阳性乳腺癌中对抗HER2治疗的抗性。 我们计划研究该突变的流行率（或其他推定的激活突变 来自抗HER2治疗的患者的样品中的Akt基因）。 此外，我们计划研究Akt 3 R247C的固有激酶活性，然后获得的获取 电阻表型。最后，我们将评估单独还是单独的AKT催化抑制 结合抗HER2治疗，在限制增殖和肿瘤生长方面有效 带有AKT 3 R247C突变的细胞/肿瘤。 我们已经从HER2收集了20多个成对的活检（治疗前后） 最初对曲妥珠单抗或拉帕替尼做出反应的乳腺癌阳性患者 HER2的分子酪氨酸激酶抑制剂），但最终会加班。这 通过影响分析这些标本，该平台允许外部序列〜300个癌症 - 相关基因将揭示其中有多少患者在Akt基因或 在与PI3K/AKT/MTOR途径有关的其他基因中。 由于从未报道过Akt 3的R247C突变，因此我们旨在研究其潜在的作用 激活酶并赋予对抗HER2药物的耐药性。 AKT 3 R247C将被表达 在HER2阳性细胞中，对曲妥珠单抗和拉帕替尼敏感。在细胞培养中，我们计划执行 长期的生存力分析，包括2D和3D培养物，以研究这些稳定克隆的能力 避免这些药物的抑制活性。在体内将采取类似的方法 表达野生型和突变Akt 3的BT474细胞（肿瘤）将在裸露中注射 小鼠形成异种移植物，这些异种移植物将单独或组合用抗HER2药物处理。最后， 我们将评估催化AKT抑制在重新敏感细胞中稳定表达Akt 3的功效 R247C至抗HER2治疗。特别是，我们将根据 治疗和进行药效学研究以确认抑制途径。