Synergy between BCR signaling and stress response

描述（由申请人提供）：弥漫性大B细胞淋巴瘤（DLBCL）是一种分子异质性疾病，尚未完全了解。在标准免疫化疗后，40%的患者死于疾病，这意味着需要确定新的靶点。热休克蛋白90（Heat shock protein 90，Hsp 90）是一种分子伴侣蛋白，可帮助细胞生长、增殖和存活过程中的信号传导介质正确折叠。肿瘤细胞富集了在多分子伴侣复合物（肿瘤富集的Hsp 90，teHsp 90）中发现的Hsp 90的一部分，所述复合物对Hsp 90抑制剂具有高亲和力。当肿瘤细胞暴露于teHsp 90抑制（teHsp 90 i）时，teHsp 90客户蛋白被耗尽。我们已经证明，PUH 71，一种新型的，高度teHsp 90选择性抑制剂，在不同的DLBCL细胞系和异种移植物中具有有效的活性。通过将PUH 71固定在固体载体上，teHsp 90复合物可以被化学沉淀（CP），使我们能够鉴定teHsp 90客户蛋白。使用这种方法，我们验证了BCL中主要癌蛋白BCL 6是teHsp 90底物蛋白的发现。我们还表明，teHsp 90及其客户BCL 6的组合抑制协同杀死DLBCL。为了更好地理解淋巴瘤特异性teHsp 90的作用机制，并确定单一或联合治疗的新靶点，我们在DLBCL细胞系中进行了PUH 71 CP蛋白质组学。B细胞受体（BCR）途径的许多组分被鉴定为teHsp 90的客户。该途径与淋巴瘤发生和DLBCL存活有关。我们假设teHsp 90分子伴侣BCR信号体的形成和活性以及teHsp 90和BCR通路的联合抑制将协同杀死DLBCL。为了检验这一假设，我们将评估PUH 71处理的细胞在mRNA（qPCR，mRNA-seq）、蛋白质（IB）和磷蛋白（FC）水平上的BCR信号体组分和下游信号传导的变化。将使用全内反射荧光显微术（TIRFM）来描绘teHsp 90 i对BCR信号体形成和稳定性的影响。为了评估teHsp 90 i对BCR信号传导的影响，我们将评估钙释放、CARD 11-Bcl 10-MALT 1复合物形成（IP，WB）和转录活性（qPCR）的变化。我们将用单独的BCR通路抑制剂和与teHsp 90 i组合处理DLBCL细胞系，并使用等效线分析评估累加或协同效应。有效的组合治疗将转化为体内异种移植模型和离体患者样品。拟议的实验将表征teHsp 90的新功能，并为临床上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.