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.

理解抗HER 2药物耐药机制的主要困难之一是 化疗的伴随治疗。在这份提案中，我们利用了独特的 从首先获得对抗-HCV的病理完全应答的患者获得的样品， HER 2抗体曲妥珠单抗，但随后随时间推移在远处淋巴结复发。深度测序 结果表明，耐药性的获得与这些标本的外观一致， Akt 3基因的一个先前未报道的突变。这一结果完全归因于 曲妥珠单抗的药理学压力，因为该患者未接受任何其他伴随治疗 治疗（包括化疗）。 本研究的目的是调查新发现的Akt 3的获得是否 突变R247 C导致HER 2阳性乳腺癌对抗HER 2治疗的抗性。 我们计划调查这种突变的流行率（或其他puerectin激活突变）。 Akt基因）。 此外，我们计划研究Akt 3 R247 C的内在激酶活性和随后的获得， 抗性表型。最后，我们将评估Akt的催化抑制，单独或联合 与抗HER 2疗法组合，在限制肿瘤细胞增殖和肿瘤生长方面是有效的。 携带Akt 3 R247 C突变的细胞/肿瘤。 我们已经收集了20多对来自HER 2的活检（治疗前和治疗后）。 最初对曲妥珠单抗或拉帕替尼有反应的阳性乳腺癌患者（小剂量） HER 2分子酪氨酸激酶抑制剂），但最终随时间推移进行治疗。的 通过IMPACT分析这些标本，IMPACT是一个平台，可以对大约300种癌症进行外显子组测序， 相关基因，将揭示这些患者中有多少人获得了Akt基因突变， 在与PI 3 K/Akt/mTOR通路相关的其他基因中。 由于Akt 3的R247 C突变从未报道过，我们的目的是研究其在以下方面的潜在作用： 激活酶并赋予对抗HER 2剂的抗性。Akt 3 R247 C将在 在对曲妥珠单抗和拉帕替尼敏感的HER 2阳性细胞中。在细胞培养中，我们计划 在2D和3D培养物中进行长期存活力测定，以研究这些稳定克隆的能力， 逃避这些药剂的抑制活性。将在体内采取类似的方法，其中 将表达野生型和突变型Akt 3的BT474细胞（致瘤性）以裸细胞注射到小鼠皮下。 小鼠以形成将用抗HER 2剂单独或组合治疗的异种移植物。最后， 我们将评估催化性Akt抑制在稳定表达Akt 3的再致敏细胞中的功效， R247 C与抗HER 2治疗的关系。特别是，我们将监测肿瘤生长抑制反应， 治疗和进行药效学研究，以确认抑制的途径。