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

理解抗HER2药物耐药机制的主要困难之一是 化疗的同时进行治疗。在本提案中，我们利用了独特的 从一名对抗-HBs首次出现病理完全应答的患者身上获得的样本 HER2抗体曲妥珠单抗随后在远处淋巴结复发超时。深层测序 这些标本的显示，耐药性的获得与外观不谋而合 之前未报道的Akt3基因突变。这一结果完全可以归因于 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 治疗(包括化疗)。 这项研究的目的是调查新发现的Akt3的获得 突变R247C导致HER2阳性乳腺癌患者对抗HER2治疗产生抵抗。 我们计划调查这种突变(或其他假定激活的突变)的流行率 来自对抗HER2治疗无效或变得无效的患者的样本中的Akt基因)。 此外，我们计划研究Akt3R247C的内在激酶活性和后续的获得 抗性表型。最后，我们将评估Akt的催化抑制，单独或在 联合抗HER2治疗能有效地抑制肿瘤细胞的增殖和生长 携带Akt3R247C突变的细胞/肿瘤。 我们已经从HER2收集了20多对活组织检查(治疗前和治疗后) 最初对曲妥珠单抗或拉帕替尼(一种小剂量)有反应的阳性乳腺癌患者 HER2的分子酪氨酸激酶抑制剂)，但最终进展到治疗超时。这个 通过Impact对这些样本进行分析，这是一个允许对约300种癌症进行外显组测序的平台- 相关基因，将揭示这些患者中有多少人获得了Akt基因或 在其他与PI3K/Akt/mTOR通路相关的基因中。 由于Akt 3的R247C突变从未被报道过，我们的目标是研究它在 激活酶，增强对抗HER2药物的抵抗力。将表达AKT 3 R247C HER2阳性细胞对曲妥珠单抗和拉帕替尼敏感。在细胞培养方面，我们计划进行 在2D和3D培养中的长期活性分析，以研究这些稳定克隆的能力 逃避这些制剂的抑制活性。在体内也将采取类似的方法， 同时表达野生型和突变Akt3的BT474细胞(致瘤)将被注射到裸鼠体内 小鼠形成异种移植，将单独或联合使用抗HER2药物治疗。最后， 我们将评估催化Akt抑制对稳定表达Akt3的细胞再敏化的效果 R247C到抗HER2治疗。特别是，我们将监测对肿瘤生长抑制的反应 治疗，并进行药效学研究，以确认抑制的途径。

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.