Phosphodiesterase 4B as a Therapeutic Target in Lymphoma

描述(申请人提供)：这项研究的主要目标是确定PDE4B作为治疗弥漫性大B细胞淋巴瘤(DLBCL)的新靶点的作用。弥漫性大B细胞淋巴瘤是成人最常见的B淋巴样恶性肿瘤。DLBCL是一种侵袭性和异质性疾病，只有大约40%的患者可以用标准治疗治愈。更具侵袭性和/或剂量强化的化疗方案不能提供主要的生存优势，可能会有更高的毒性。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的特定遗传损害的治疗。为了实现这一目标，我们使用了微阵列的表达谱，并找到了与疾病结果相关的单基因和单途径。在我们的初步研究中，在致死性/难治性DLBCL中最显著的过度表达基因之一是磷酸二酯酶4B(PDE4B)。这种酶使cAMP失活，cAMP是诱导B淋巴细胞细胞周期停滞和凋亡的第二信使。由于PDE4B终止cAMP活性，它消除了这些负面影响。我们的初步研究证实，在DLBCL中，PDE4B通过与PI3K/AKT生存通路的独特串扰，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PDE4抑制剂在治疗这些恶性肿瘤方面的潜力。在目前的提案中，我们将基于这些初步数据来解决两个具体目标：1.确定cAMP-PDE4B调节DLBCL中PI3K/AKI活性的机制。2.建立小鼠DLBCL模型，在体内证实PDE4抑制剂治疗DLBCL的有效性。 我们的研究计划利用了我们最近开发的一系列工具，包括用逆转录病毒载体重组的内源性低PDE4B活性的DLBCL细胞系，以表达野生型PDE4B或磷酸二酯酶失活突变体。在具体目标一，我们将利用这些细胞来确定细胞内cAMP/PDE4B如何调节PI3K活性；我们将专门研究cAMP对调节PI3K的蛋白酪氨酸激酶(PTK)和小GTP酶RAS的影响。为了准确评估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基因敲除淋巴瘤细胞。为了解决RAS的作用，我们将逆转录病毒产生DLBCL细胞，表达该蛋白的构成活性(V12)或显性负性(N17)形式。在第二个目标中，我们将在NOD/SCID小鼠体内建立人DLBCL异种移植瘤，以确定PDE4B抑制剂在治疗这些肿瘤中的有效性。由于临床级别的PDE4抑制剂已经上市，目前正在针对各种情况进行临床试验，从这项提议中得出的发现可以实际地引导DLBCL的新治疗策略。

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.