Phosphodiesterase 4B as a Therapeutic Target in Lymphoma

描述（由申请方提供）：本研究的主要目的是确定PDE 4 B作为弥漫性大B细胞淋巴瘤（DLBCL）治疗探索的新靶点的作用，DLBCL是成人中最常见的B淋巴恶性肿瘤。DLBCL是一种侵袭性和异质性疾病，其中只有大约40%的患者可以通过标准治疗治愈。更积极和/或剂量强化的化疗方案不提供主要的生存优势，可能具有更高的毒性。因此，为了提高DLBCL的治愈率，我们需要更好地了解其分子基础，并将这些发现转化为针对这些肿瘤特定遗传病变的治疗方法。为了实现这一目标，我们在微阵列上使用了表达谱，并发现了与疾病结果相关的单个基因和途径。在我们的初步研究中，致死性/难治性DLBCL中最显著的过表达基因之一是磷酸二酯酶4 B（PDE 4 B）。这种酶使cAMP失活，cAMP是诱导B淋巴细胞周期停滞和凋亡的第二信使。由于PDE 4 B终止cAMP活性，因此消除了这些负面影响。我们的初步研究证实，在DLBCL中，PDE 4 B通过与PI 3 K/AKT存活途径的独特串扰阻断cAMP介导的细胞凋亡。在这项初步研究中，我们还在体外证实了PDE 4抑制剂治疗这些恶性肿瘤的潜力。在本提案中，我们将在这些初步数据的基础上实现两个具体目标：1。定义cAMP-PDE 4 B调节DLBCL中PI 3 K/阿基活性的机制。2.开发小鼠DLBCL模型，以在体内证实PDE 4抑制剂治疗这种疾病的有效性。 我们的研究计划利用了我们最近产生的一系列工具，包括用逆转录病毒载体重建的具有低内源性PDE 4 B活性的DLBCL细胞系，以表达野生型PDE 4 B或磷酸二酯酶失活突变体。在具体目标一中，我们将利用这些细胞来确定细胞内cAMP/PDE 4 B如何调节PI 3 K活性;我们将专门研究cAMP对调节PI 3 K的蛋白酪氨酸激酶（PTK）和小GT3 Ras的影响。为了精确评估PTKs在这一过程中的作用，我们还将使用缺乏相关上游激酶的DT-40敲除淋巴瘤细胞。为了解决Ras的作用，我们将逆转录病毒产生表达该蛋白质的组成型活性（V12）或显性阴性（N17）形式的DLBCL细胞。在目标二中，我们将在NOD/SCID小鼠中产生人DLBCL异种移植物，以确定PDE 4 B抑制剂在治疗这些肿瘤中的有效性。由于临床级PDE 4抑制剂是可用的，目前在临床试验中的各种条件，从这个建议中得出的结果可以实际导致新的治疗策略DLBCL。

DESCRIPTION (provided by applicant): The major objective of this research is to firmly establish the role of PDE4B as a novel target for therapeutic exploration in diffuse large B-cell lymphoma (DLBCL), the most common B-lymphoid malignancy in adults. DLBCL is an aggressive and heterogeneous disorder in which only approximately 40% of the patients can be cured with standard therapy. More aggressive and/or dose-intensified chemotherapy regimens do not provide major survival advantages may have higher toxicity. Therefore, to improve the cure rate in DLBCL, we need to better understand its molecular basis and translate these findings into therapies that target the specific genetic lesions of these tumors. Towards this goal, we used expression profiling on microarrays and found single genes and pathways that were associated with the disease outcome. In our pilot study, one of the most prominently over expressed genes in fatal/refractory DLBCL was the phosphodiesterase 4B (PDE4B). This enzyme inactivates cAMP, a second messenger which induces cell cycle arrest and apoptosis in B-lymphocytes. Because PDE4B terminates cAMP activity, it abrogates these negative effects. Our preliminary studies confirmed that in DLBCLs PDE4B blocks cAMP-mediated apoptosis, via a unique cross-talk with the PI3K/AKT survival pathway. In this initial study, we also confirmed, in vitro, the potential of PDE4 inhibitors in the treatment of these malignancies. In the present proposal, we will build on these preliminary data to address two specific aims: 1. Define the mechanisms by which cAMP-PDE4B modulate PI3K/AKI activity in DLBCL. 2. Develop murine DLBCL models to confirm, in vivo, the effectiveness of PDE4 inhibitors in the treatment of this disease. Our study plan exploits a series of tools that we have recently generated, including a DLBCL cell line with low endogenous PDE4B activity reconstituted with a retrovirus vector to express wild-type PDE4B or a phosphodiesterase inactive mutant. In specific aim one, we will utilize these cells to define how intracellular cAMP/PDE4B modulates PI3K activity; we will specifically study cAMP effects on protein tyrosine kinases (PTK) that regulate PI3K and on the small GTPase Ras. To precisely evaluate the role of the PTKs in this process, we will also use DT-40 knockout lymphoma cells lacking relevant upstream kinases. To resolve the role of Ras, we will retrovirally generate DLBCL cells expressing constitutive active (V12) or dominant negative (N17) forms of this protein. In aim two we will generate human DLBCL xenografts in NOD/SCID mice to define the effectiveness of PDE4B inhibitors in the treatment of these tumors. Since clinical grade PDE4 inhibitors are available and currently in clinical trials for a variety of conditions, the findings derived from this proposal can realistically lead to novel therapeutic strategies in DLBCL.