A functional genomics process for systematic dissection and mutation-specific target discovery in breast cancer PIK3CA hotspot mutations [RNA-seq]

Despite the general understanding that different mutations impart different phenotypic changes within a cell, the majority of targeted therapies in cancer treatment are used to treat all mutations in the same gene regardless of location or phenotypic effects. There is a significant unmet need to distinguish distinct changes in cellular signaling which may provide opportunities for mutation-specific treatment with reduced toxicity to patients. Herein, we describe a series of functional genomic analysis with a unique isogenic cell line panel to accurately identify targetable differences between mutations within the same gene. Using an isogenic cell line model bearing two distinct hotspot PIK3CA mutations found in breast cancer, we were able to identify many gene expression and chromatin accessibility differences. These findings allowed us to identify mutation specific molecular targets, specifically AREG as well as a proximal gene regulatory region, that may provide clinically relevant targets. When disrupted, these targets induce a mutation-specific decrease in proliferation and survival in vivo. These findings suggest new mutation-specific modes of treatment for PIK3CA mutant breast cancer and provide a means with which to find mutation-specific targets for the treatment of other oncogenic mutations. The first stage of our discovery platform is the evaluation of the transcriptional environment of isogenic cells bearing either the PIK3CA E545K or H1047R mutation using RNA-seq. The development of this isogenic model was described in Gustin et al 2009 PNAS.

尽管学界普遍认为不同突变可在细胞内引发各异的表型改变，但当前癌症治疗中的绝大多数靶向疗法，均针对同一基因的所有突变，而不考虑其所在位置或所产生的表型效应。目前仍存在显著的未被满足的临床需求：亟需区分细胞信号通路中的各类差异变化，这可为开发突变特异性治疗方案提供契机，同时降低对患者的毒副作用。本文中，我们将介绍一套基于独特同基因细胞系组合的功能基因组分析流程，以精准鉴定同一基因内不同突变之间的可靶向差异。我们采用携带有乳腺癌中两种不同热点PIK3CA突变的同基因细胞系模型，成功鉴定出大量基因表达与染色质可及性的差异。基于上述发现，我们确定了突变特异性的分子靶点，尤其是AREG及一处近端基因调控区域，这些靶点具备潜在临床转化价值。当这些靶点被干扰后，可在体内诱导出突变特异性的增殖与存活能力下降。本研究结果为PIK3CA突变型乳腺癌提供了全新的突变特异性治疗策略，同时也为其他致癌突变的靶向治疗靶点筛选提供了通用方法。我们的发现平台的第一阶段，是通过RNA-seq（RNA测序）对携带PIK3CA E545K或H1047R突变的同基因细胞的转录组环境进行评估。该同基因细胞系模型的构建方法已在Gustin等人2009年发表于《美国国家科学院院刊》（PNAS）的研究中得到阐述。