Project 3: Targeting PRMT5 in Mantle Cell Lymphoma

项目3摘要 套细胞淋巴瘤(MCL)是一种以异常遗传和表观遗传为特征的不可治愈的B细胞淋巴瘤 事件。已经使用了各种治疗策略来改善MCL患者的预后，包括 免疫化疗和干细胞移植。靶向B细胞受体(BCR)信号转导途径 伊布鲁替尼的治疗方法显示出了希望，然而，结果数据还不成熟，新出现的耐药性是 很明显。因此，对于治疗MCL的新的治疗方法仍然存在明显的、未得到满足的需求。我们 已经证明PRMT5在MCL中选择性地过度表达，并驱动全球抑制性表观遗传标记 在染色质上沉默调节和肿瘤抑制基因。用shRNA启动子敲除PRMT5 MCL的选择性毒性使我们假设PRMT5是治疗这种疾病的一个有吸引力的靶点。我们有 开发了一种“一流”药物，选择性地抑制PRMT5的活性，IC50在低NM范围内。我们的领先优势 药物对淋巴瘤是有毒的，而不是正常的B细胞，在体内耐受性良好，具有令人印象深刻的抗肿瘤活性 MCL临床前动物模型的建立。PRMT5抑制剂已经被用作化学探针来剖析关键的 MCL肿瘤细胞利用的途径。用PRMT5特异性抗体绘制全基因组图的初步研究 表观遗传标记和整个转录组测序(RNA-Seq，与WCM合作)存在和 PRMT5抑制剂的缺失揭示了广泛的信号、生存和生长途径 由这种酶控制的。我们的初步工作发现，PRMT5直接针对诸如 如PTPROt、TRIB3和PIK3IP1，负向调节BCR和PI3K/AKT通路。停用 结构性AKT活性导致FOXO1去磷酸化，促进其表观遗传调节 癌症基因的表达。我们发现，PRMT5抑制导致多个微抑制基因的直接下调 可能耗尽细胞周期蛋白D1导致Rb激活和Rb/E2F调节恢复的RNAs 网络。因此，PRMT5抑制导致RB/E2F介导的EZH2、SUZ12和EED的沉默， 表观遗传抑制物PRC2复合体的组成部分，是赖氨酸甲基转移酶家族，是 在MCL中频繁突变和激活。因此，通过抑制PRMT5，我们可以直接抑制 抑制性精氨酸表观遗传标记，同时间接耗尽赖氨酸抑制标记，如H3K27(ME3)和 可能是FOXO1的目标。我们抑制像PRMT5这样的核心参与者的战略导致了一种新的方法 在多个水平上恢复对MCL细胞的调控，包括表观基因组、BCR、PI3K/AKT和周期蛋白 D/Rb-E2F通路。我们提出了整合全基因组的方法和策略，利用 研究PRMT5抑制如何规避耐药性的创新临床前MCL模型 在临床试验和建议的临床前工作中遇到新的分子靶向药物 项目1和2。我们提出了一种创新的、直接的和强大的方法来发现基因 在MCL中改变的计划，并将利用这些信息来开发治疗这种不治之症的新策略。

Project 3 Summary Mantle cell lymphoma (MCL) is an incurable B-cell lymphoma characterized by aberrant genetic and epigenetic events. A variety of therapeutic strategies have been used to improve the outcome of MCL patients including immune-chemotherapy and stem cell transplantation. Approaches targeting the B cell receptor (BCR) signaling pathway with ibrutinib have shown promise, however, outcome data is immature and emerging resistance is clearly evident. Thus there remains a clear, unmet need for novel therapeutic approaches for treating MCL. We have shown that PRMT5 is selectively over expressed in MCL and drives global repressive epigenetic marks on chromatin to silence regulatory and tumor suppressor genes. PRMT5 knock down with shRNA promotes selective MCL toxicity which led us to hypothesize that PRMT5 is an attractive target for this disease. We have developed a “first-in-class” drug that selectively inhibits PRMT5 activity with IC50 in low nM range. Our lead drug is toxic to lymphoma, not normal B cells, is well tolerated in vivo with impressive anti-tumor activity in preclinical animal models of MCL. PRMT5 inhibitors have been used as chemical probes to dissect out critical pathways utilized by MCL tumor cells. Preliminary whole genome mapping with antibodies specific for PRMT5 epigenetic marks and whole transcriptome sequencing (RNA-Seq, in collaboration with WCM) in presence and absence of PRMT5 inhibitors has revealed a broad range of signaling, survival and growth pathways to be governed by this enzyme. Our preliminary work has found that PRMT5 directly targets regulatory genes such as PTPROt, TRIB3, and PIK3IP1 which negatively regulate BCR and PI3K/AKT pathways. Inactivation of constitutive AKT activity leads to dephosphorylation of FOXO1, promoting its epigenetic regulation of anti- cancer gene expression. We discovered that PRMT5 inhibition leads to direct de-repression of multiple micro- RNAs that may deplete CYCLIN D1 leading to activation of RB and restoration of the RB/E2F regulatory network. Consequently, PRMT5 inhibition leads to RB/E2F-mediated silencing of EZH2, SUZ12 and EED, components of the epigenetic repressor PRC2 complex, a family of lysine methyltransferase enzymes that are frequently mutated and activated in MCL. Thus, by inhibiting PRMT5, we can achieve direct inhibition of repressive arginine epigenetic marks while indirectly depleting lysine repressive marks like H3K27(Me3) and possibly, targets of FOXO1. Our strategy to inhibit a central player like PRMT5 has led to a novel approach to restore regulation to the MCL cell at multiple levels including the epigenome, BCR, PI3K/AKT, and CYCLIN D/RB-E2F pathways. We have proposed integrative whole genome approaches and strategies utilizing innovative preclinical MCL models to examine how PRMT5 inhibition can circumvent drug resistance encountered with novel molecularly targeted agents examined in clinical trials and preclinical work proposed in Projects 1 and 2. We present an innovative, straight-forward and robust approach to discover the genetic programs altered in MCL and will use this information to develop new strategies to treat this incurable disease.