Synergy between BCR signaling and stress response

描述(申请人提供)：弥漫性大B细胞淋巴瘤(DLBCL)是一种尚未完全了解的分子异质性疾病。在标准免疫化疗之后，40%的患者死于他们的疾病，这意味着需要确定新的靶点。热休克蛋白90(Hsp90)是一种伴侣蛋白，可帮助参与细胞生长、增殖和存活的信号介质正确折叠，是一种新兴的癌症治疗靶点。肿瘤细胞对Hsp90抑制剂具有高亲和力的多伴侣复合体(肿瘤富集型Hsp90，teHsp90)中发现的Hsp90的一部分进行了浓缩。当肿瘤细胞暴露于teHsp90抑制(TeHsp90i)时，teHsp90客户蛋白被耗尽。我们已经证明，PUH71是一种新型的、高度teHsp90选择性的抑制剂，在不同的DLBCL细胞株和异种移植瘤中具有很强的活性。通过将PUH71固定在固体载体上，teHsp90复合体可以化学沉淀(CP)，使我们能够识别teHsp90客户蛋白。利用这种方法，我们验证了BCLS中的主要癌蛋白BCL6是一种teHsp90底物蛋白的发现。我们还表明，联合抑制teHsp90及其客户端BCL6，可以协同杀死DLBCL。为了更好地了解淋巴瘤特异性teHsp90的作用机制，并为单一或联合治疗确定新的靶点，我们在DLBCL细胞系中进行了PUH71 CP蛋白质组学研究。B细胞受体(BCR)途径的许多成分被确定为teHsp90的客户。这一途径与淋巴瘤的发生和DLBCL的生存有关。我们假设teHsp90伴随着BCR信号体的形成和活性，并且teHsp90和BCR通路的联合抑制将协同作用杀死DLBCL。为了验证这一假设，我们将评估PUH71处理的细胞BCR信号体成分和下游信号在mRNA(qPCR，mRNA-seq)、蛋白(IB)和磷酸蛋白(FC)水平的变化。全内反射荧光显微镜(TIRFM)将被用来描述teHsp90i对BCR信号体形成和稳定性的影响。为了评估teHsp90i对BCR信号的影响，我们将评估钙释放、CARD11-Bcl10-MALT1复合体形成(IP，WB)和转录活性(QPCR)的变化。我们将用bcr途径抑制剂单独和与teHsp90i联合处理DLBCL细胞，并用等谱图分析评估相加或协同作用。有效的联合治疗将转化为体内异种移植模型和体外患者样本。拟议的实验将表征teHsp90的一种新功能，并为临床将有效的联合治疗转化为患者提供理论基础。

DESCRIPTION (provided by applicant): Diffuse large B cell lymphoma (DLBCL) is molecularly heterogeneous disease that is not yet completely understood. Following standard immunochemotherapy, 40% of patients succumb to their disease, signifying the need to identify new targets. Heat shock protein 90 (Hsp90), an emerging therapeutic target for cancer, is a chaperone protein that assists the correct folding of signaling mediators involved in cell growth, proliferation and survival. Tumor cells are enriched for a fraction of Hsp90 found in multi-chaperone complexes (tumor-enriched Hsp90, teHsp90) that have high affinity for Hsp90 inhibitors. teHsp90 client proteins are depleted when tumor cells are exposed to teHsp90 inhibition (teHsp90i). We have shown that PUH71, a novel, highly teHsp90 selective inhibitor, has potent activity in different DLBCL cell lines and xenografts. By immobilizing PUH71 on a solid support, teHsp90 complexes can be chemically precipitated (CP), allowing us to identify teHsp90 client proteins. Using this method, we validated the discovery that BCL6, the main oncoprotein in BCLs, is a teHsp90 substrate protein. We have also shown that combined inhibition of teHsp90 and its client, BCL6, synergizes to kill DLBCLs. To better understand lymphoma-specific teHsp90 mechanisms of action and to identify new targets for mono- or combination therapy, we performed PUH71 CP proteomics in DLBCL cell lines. Many components of the B cell receptor (BCR) pathway were identified as clients of teHsp90. This pathway has been implicated in lymphomagenesis and DLBCL survival. We hypothesize that teHsp90 chaperones BCR signalosome formation and activity and that combined inhibition of teHsp90 and the BCR pathway will synergize to kill DLBCLs. To test this hypothesis we will assess PUH71 treated cells for changes in BCR signalosome components and downstream signaling at the mRNA (qPCR, mRNA-seq), protein (IB), and phospho-protein (FC) levels. Total internal reflection fluorescence microscopy (TIRFM) will be used to depict the effect of teHsp90i on BCR signalosome formation and stability. To assess effects of teHsp90i on BCR signaling, we will evaluate changes in calcium release, CARD11-Bcl10-MALT1 complex formation (IP, WB), and transcriptional activity (qPCR). We will treat DLBCL cell lines with BCR pathway inhibitors alone and in combination with teHsp90i and evaluate for additive or synergistic effect using isobologram analysis. Effective combination treatments will be translated to in vivo xenograft models and ex vivo patient samples. The proposed experiments will characterize a new function for teHsp90 and provide the rationale for translating effective combination treatments to patients in the clinic.