Phamacological modulation of epigenetic changes in AML

摘要肿瘤抑制基因的沉默通过异常的表观遗传改变，即启动子超甲基化和染色质组蛋白去乙酰化是恶性转化的组成部分。长期以来，我们一直致力于开发针对这些异常表观遗传变化的治疗策略，以恢复急性髓性白血病（AML）中造血细胞增殖、分化和凋亡的正常模式。在该提案的最后一个资助周期中，我们成功地确定了一种具有生物学和临床活性的无毒低剂量地西他滨（20mg /m2/天x每4周10天），可诱导未经治疗的老年（60岁至60岁）AML的疾病缓解和甲基化ER基因的高再表达水平，这反过来与疾病缓解的实现直接相关。我们还产生了有趣的初步数据，显示地西他滨修饰mirna的表达，mirna是一类新的非编码基因，控制特定信使RNA靶点的翻译，进而控制相应编码蛋白（如癌蛋白）的水平。mirna的异常表达最近与癌症有关。miRNA 29是一种靶向DNA甲基转移酶（DNMT3a和3b）的microRNA，在未治疗的患者中表达水平较低，在地西他滨治疗后表达水平升高。因此，这些数据表明地西他滨也可能通过沉默mirna的重新表达来干扰异常的DNMT活性。鉴于恶性细胞异常基因沉默中DNA甲基化和染色质低乙酰化之间值得注意的相互作用，我们将地西他滨与组蛋白去乙酰化酶（HDAC）抑制剂丙戊酸（VPA）联合使用。不幸的是，VPA的加入是有毒的，在临床反应或基因再表达方面没有提供任何协同作用。最后，我们获得了未报道的证据，证明蛋白酶体抑制剂硼替佐米（Velcade, PS341）通过降低转录因子Sp1的水平和活性干扰DNMT1基因的反激活。这导致DNMT1水平降低，基因组DNA低甲基化和甲基化基因重新表达。值得注意的是，硼替佐米还诱导了miRNA29b的表达，miRNA29b本身下调Sp1，并抑制了AML中过表达的转录因子NF-B的活性，而转录因子NF-B与Sp1共同参与基因转激活。因此，我们假设硼替佐米通过miRNA29诱导和/或NF-B抑制靶向Sp1的多种机制降低了异常的DNMT活性。为了利用这些新发现，我们建议进行：1)地西他滨作为单药治疗未经治疗的老年AML的II期临床试验；2)药代动力学（PK）和药效学（PD）相关研究，以确定在建议剂量和方案下地西他滨的体内机制表观遗传活性；3)硼替佐米和地西他滨联合治疗难治性/复发性AML的一期剂量递增试验；4)通过PK和PD相关研究来确定硼替佐米在体内的表观遗传活性。公共卫生相关性：急性髓性白血病（AML）是一种造血恶性肿瘤，大多数患者目前的化疗治疗结果令人沮丧，特别是年龄超过60岁的患者，因此迫切需要新的治疗方法。AML发展和维持的机制之一是通过基因组DNA序列的甲基化破坏对骨髓和血液正常活性重要的基因的功能。我们在此建议使用化合物（即地西他滨和硼替佐米）进行临床试验，如果以特定剂量和时间表使用，可以降低DNA甲基化水平，恢复基因活性并导致疾病缓解。除了评估对这些化合物的临床反应外，我们还将分析治疗患者的骨髓和血液样本，以了解硼替佐米和/或地西他滨如何减少白血病细胞中的异常甲基化。这些知识将用于设计更有效的AML治疗方法。

DESCRIPTION (provided by applicant): Abstract Silencing of tumor suppressor genes via aberrant epigenetic changes i.e., promoter hypermethylation and chromatin histone deacetylation are integral to malignant transformation. We have long focused on developing therapeutic strategies that target these aberrant epigenetic changes with the scope to restore normal patterns of hematopoietic cell proliferation, differentiation and apoptotsis in acute myeloid leukemia (AML). In the last funding cycle of this proposal, we were successful in identifying a biologically and clinically active, non-toxic low-dose of decitabine (20 mg/m2/day x 10 days every 4 weeks) that induced disease remission in untreated older (>60years) AML and high re-expression levels of the methylated ER gene, which in turn directly correlated with achievement of disease remission. We also produced intriguing preliminary data showing that decitabine modified expression of miRNAs, a novel class of non-coding genes that control translation of specific messenger RNA targets and in turn, levels of the corresponding encoded proteins (e.g., oncoproteins). Aberrant expression of miRNAs has been recently associated with cancer. miRNA 29, a microRNA that targets DNA methyltransferases (DNMT3a and 3 b), was found expressed at low levels in untreated patients and increased following decitabine treatment. Therefore, these data suggested that decitabine might interfere with aberrant DNMT activity also through re-expression of silenced miRNAs. Given the noteworthy interplay between DNA methylation and chromatin hypoacetylation in aberrant gene silencing in malignant cells, we combined decitabine with the histone deacetylase (HDAC) inhibitor valproic acid (VPA). Unfortunately, the addition of VPA was toxic and failed to provide any synergistic effect in terms of clinical response or gene re- expression. Finally, we obtained unreported evidence that the proteasome inhibitor bortezomib (Velcade", PS341) interfered with transactivation of the DNMT1 gene by reducing level and activity of the transcription factor Sp1. This resulted in reduction of DNMT1 levels, genomic DNA hypomethylation and methylated gene re-expression. Notably, bortezomib also induced expression of miRNA29b, which itself downregulated Sp1, and inhibited the activity of the transcription factor NF-B that is overexpressed in AML and cooperates with Sp1 in gene transactivation. Therefore, we hypothesize that bortezomib reduces aberrant DNMT activity by multiple mechanisms that target Sp1 via miRNA29 induction and/or NF-B inhibition. To capitalize on these novel discoveries, we propose here to conduct: 1) a phase II clinical trial of decitabine as a single agent in untreated elderly AML; 2) pharmacokinetic (PK) and pharmacodynamic (PD) correlative studies to define mechanistically, in vivo, the epigenetic activity of decitabine at the proposed dose and schedule; 3) a phase I dose-escalation trial of bortezomib and decitabine in combination in refractory/relapsed AML; 4) PK and PD correlative studies to define mechanistically, in vivo, the epigenetic activity of bortezomib. PUBLIC HEALTH RELEVANCE: Most of patients with acute myeloid leukemia (AML), a type of hematopoietic malignancies, have dismal outcome with current chemotherapy treatment, especially if older than 60 years, and therefore novel therapeutic approaches are highly needed. One of the mechanisms of development and maintenance of AML is represented by disruption of the function of genes important for normal activity of bone marrow and blood through methylation of their genomic DNA sequences. We are proposing here clinical trials with compounds (i.e., decitabine and bortezomib) that if used at specific doses and schedules, can decrease levels of DNA methylation, restore gene activity and result in disease remission. In addition to assessment of the clinical response to these compounds, we will analyze bone marrow and blood samples from treated patients to understand how bortezomib and/or decitabine reduce abnormal methylation in leukemia cells. This knowledge will be used to design more effective treatment for AML .