Targeting GRB7 Signaling in Solid Tumors

描述（由申请人提供）： GRB 7在乳腺癌中扩增并过表达，染色体17 q11 -12扩增。GRB 7可以与多种信号分子相互作用，并与乳腺癌生物学有关。GRB 7可以促进HER-2介导的信号传导和肿瘤形成。包括我们的研究在内的研究表明，GRB 7蛋白的过表达，而不是HER-2的过表达，是乳腺癌的不良预后因素。此外，GRB 7蛋白是三阴性乳腺癌中唯一的不良预后标志物，来自374例研究。 我们假设GRB 7定向信号在染色体17 q11 -12扩增的乳腺癌中具有重要的功能。因此，靶向GRB 7信号传导代表了HER-2阳性和三阴性乳腺癌的重要和新的治疗策略，否则这些乳腺癌在临床上是侵袭性的。 目标是：（1）通过确定GRB 7信号传导功能的抑制是否导致乳腺癌细胞生长的抑制来研究乳腺癌细胞对GRB 7信号传导的依赖性;（2）开发乳腺癌细胞系的同基因对， （3）用覆盖整个“可操作的”激酶组的siRNA文库，用细胞斑点微阵列和新的反向转染技术进行筛选，以鉴定和验证其敲低显示对增殖、凋亡和/或迁移/侵袭的GRB 7依赖性作用的靶标;以及（4）研究候选细胞周期激酶在GRB 7信号传导中的功能参与及其作为治疗靶点的作用。 我们的方法如下：GRB 7敲低已实现了在多个乳腺癌细胞系与染色体17 q11 -12扩增的瞬时siRNA转染。GRB 7蛋白缺失降低细胞系中的细胞增殖，而不管它们对目前可用的HER-2靶向疗法的敏感性如何。通过乳腺癌细胞系中的shRNA表达实现了稳定的GRB 7敲低，并将评估其对培养物和原位小鼠异种移植模型中细胞生长的影响。稳定的GRB 7敲低产生了一个“致敏”的细胞环境，以筛选其信号传导功能依赖于GRB 7表达的治疗靶点和分子。我们将筛选信号分子，其抑制被GRB 7过表达敏化-即诱导的必要性。此外，信号分子的抑制显示不同的敏感性，根据其GRB 7状态将解开乳腺癌中GRB 7信号的功能复杂性。这种新的细胞点芯片结合siRNA反向转染将用于筛选整个人激酶组的可作用靶点。我们能够扩展这种基于细胞的功能性遗传筛选，以包括更多的siRNA构建体，允许组合siRNA分析，并同时获得多参数表型读数和多种细胞类型的比较分析。我们还将对小分子抑制剂文库进行筛选，这些文库包含已充分表征并正在进行早期临床开发的化合物。通过筛选小分子抑制剂和由“可操作的”靶标组成的siRNA文库，我们正在寻找其抑制产生取决于GRB 7表达状态的不同表型的候选物。筛选阳性将通过独立的功能试验以及在其他细胞系和动物模型中进行验证。由于筛选阳性是具有可操作特征的分子或已经进行早期临床开发的化合物，因此它们可以很容易地快速推进药物开发。对这些GRB 7依赖性信号分子和治疗靶点的彻底研究将产生新的治疗方法，并揭示为什么GRB 7表达预测不良预后。可操作的siRNA和小分子抑制剂文库的高通量筛选代表了阐明衔接子分子的功能的新范例，更重要的是，有效地产生特异性针对致癌信号传导分子的临床有用的无毒治疗剂，否则该致癌信号传导分子“难以靶向”。

DESCRIPTION (provided by applicant): GRB7 is amplified and over-expressed in breast cancer with chromosome 17q11-12 amplification. GRB7 can interact with multiple signaling molecules and is implicated in breast cancer biology. GRB7 can facilitate HER-2 mediated signaling and tumor formation. Studies, including that of ours, have shown GRB7 protein overexpression, rather than that of HER-2, is an adverse prognostic factor in breast cancer. Moreover, GRB7 protein is the only adverse prognostic marker, from among 374 studied, in triple negative breast cancer. We hypothesize that GRB7 directed signaling is functionally important in breast cancer with chromosome 17q11-12 amplification. Targeting GRB7 signaling therefore represents an important and novel therapeutic strategy for HER-2 positive and triple negative breast cancers that are otherwise clinically aggressive. The objectives are: (1) to investigate the dependency of breast cancer cells on GRB7 signaling by determining if inhibition of GRB7 signaling functions leads to suppression of breast cancer cell growth; (2) to develop isogenic pairs of breast cancer cell lines with shRNA mediated GRB7 knockdown and perform ""Induced Essentiality"" screening with small molecule inhibitor libraries; (3) to perform screening with a siRNA library that covers the whole "actionable" kinome with cell spot microarrays and a novel reverse transfection technology to identify and validate targets whose knock down displays GRB7-dependent effects on proliferation, apoptosis, and/or migration/invasion; and (4) to investigate the functional involvement of candidate cell cycle kinase(s) in GRB7 signaling and their roles as therapeutic targets. Our approaches are the following: GRB7 knock down has been achieved by transient siRNA transfection in multiple breast cancer cell lines with chromosome 17q11-12 amplification. GRB7 protein depletion decreases cell proliferation in cell lines irrespective of their sensitivityto currently available HER-2 targeted therapies. Stable GRB7 knock down has been achieved by shRNA expression in breast cancer cell lines and its effect on cell growth in culture and in orthotopic mouse xenograft models will be assessed. Stable GRB7 knock down creates a 'sensitized' cellular context to screen for therapeutic targets and molecules whose signaling functions depend on GRB7 expression. We will screen for signaling molecules whose inhibition is sensitized by GRB7 over-expression- i.e. induced essentiality. In addition, signaling molecules whose inhibition displays differential sensitivity according to their GRB7 status will unravel the functional complexities of GRB7 signaling in breast cancer. The novel cell spot microarray coupled with siRNA reverse transfection will be used to screen actionable targets of the whole human kinome. We are able to expand this cell based functional genetic screen to include more siRNA constructs, allow combinatorial siRNA analyses, and obtain multi-parametric phenotypic readouts and comparative analysis of multiple cell types simultaneously. We will also perform screening of small molecule inhibitor libraries that comprise of compounds that are well characterized and undergoing early clinical development. With screening of small molecule inhibitor and siRNA libraries made up by "actionable" targets, we are looking for candidates whose inhibition produces distinct phenotypes that depend on the GRB7 expression status. Screen positives will be validated with independent functional assays and in additional cell lines and animal models. Since screen positives are molecules with actionable features or compounds already undergoing early phase clinical development, they can be readily moved forwards in drug development expeditiously. Thorough investigation of these GRB7 dependent signaling molecules and therapeutic targets will produce new therapies and reveal why GRB7 expression predicts adverse prognosis. High throughout screening of actionable siRNA and small molecule inhibitor libraries represents a novel paradigm to elucidate the function of an adaptor molecule and, more importantly, to effectively produce clinically useful, non-toxic therapeutics specifically directed against an oncogenic signaling molecule that is otherwise "difficult to target".