Phamacological modulation of epigenetic changes in AML

描述(申请人提供)：摘要通过异常的表观遗传学改变使肿瘤抑制基因沉默，即启动子超甲基化和染色质组蛋白去乙酰化是恶性转化所必需的。长期以来，我们一直专注于开发针对这些异常的表观遗传学变化的治疗策略，以恢复急性髓系白血病(AML)的造血细胞增殖、分化和凋亡的正常模式。在这项提案的上一个资金周期中，我们成功地确定了一种具有生物学和临床活性、无毒的低剂量地西他滨(20 mg/m2/天×每4周10天)，可诱导未经治疗的老年(&gt；60岁)AML患者的疾病缓解和甲基化ER基因的高再表达，这反过来又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，表明地西他滨改变了miRNAs的表达，miRNAs是一类新型的非编码基因，控制特定信使RNA靶标的翻译，进而控制相应编码蛋白(例如，癌蛋白)的水平。MiRNAs的异常表达最近被认为与癌症有关。MiRNA29是一种针对DNA甲基转移酶(DNMT3a和3b)的微RNA，在未经治疗的患者中低水平表达，经地西他滨治疗后表达增加。因此，这些数据表明，地西他滨可能还通过重新表达沉默的miRNAs来干扰DNMT的异常活动。鉴于DNA甲基化和染色质低乙酰化在恶性肿瘤细胞异常基因沉默中的显著相互作用，我们将地西他滨与组蛋白脱乙酰酶(HDAC)抑制剂丙戊酸(VPA)联合使用。不幸的是，VPA的加入是有毒的，在临床反应或基因重新表达方面没有提供任何协同效应。最后，我们获得了未见报道的证据，即蛋白酶体抑制剂Bortezomib(VELCADE，PS341)通过降低转录因子Sp1的水平和活性来干扰DNMT1基因的反式激活。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，Bortezomib还诱导了miRNA29b的表达，而miRNA29b本身下调了Sp1，并抑制了在AML中过度表达并与Sp1协同进行基因反式激活的转录因子NF-B的活性。因此，我们假设Bortezomib通过多种机制减少异常的DNMT活性，这些机制通过miRNA29诱导和/或抑制NF-B来靶向Sp1。为了充分利用这些新发现，我们建议进行：1)作为单一药物治疗未经治疗的老年AML的地西他滨的II期临床试验；2)药代动力学(PK)和药效学(PD)相关研究，以在体内从机制上确定地西他滨在建议的剂量和时间表下的表观遗传学活性；3)硼替佐米和地西他滨联合治疗难治性/复发性AML的I期剂量递增试验；4)PK和PD相关研究，从体内机械性地确定Bortezomib的表观遗传学活性。公共卫生相关性：急性髓系白血病(AML)是一种血液系统恶性肿瘤，大多数患者在目前的化疗中效果不佳，特别是在60岁以上的患者，因此迫切需要新的治疗方法。AML的发生和维持机制之一是通过其基因组DNA序列的甲基化来破坏对骨髓和血液的正常活动至关重要的基因的功能。我们在这里建议对化合物(即地西他滨和硼替佐米)进行临床试验，如果以特定的剂量和时间表使用，可以降低DNA甲基化水平，恢复基因活性，并导致疾病缓解。除了评估这些化合物的临床反应外，我们还将分析接受治疗的患者的骨髓和血液样本，以了解Bortezomib和/或Decitabine如何减少白血病细胞中的异常甲基化。这些知识将被用于设计更有效的急性髓细胞白血病治疗方法。

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 .