A novel strategy for transcriptional reprogramming of lymphoid leukemia cells

摘要 类白血病B-ALL和TCLL白血病由在早期阶段停滞的白血病母细胞（LBC）组成。 分化，其表现出高增殖潜力和自我更新能力。诱导的想法 白血病和其他癌细胞的重编程，导致细胞成熟和衰老， 但其临床应用很少成功，主要局限于APL的治疗 用全反式维甲酸和三氧化二砷治疗白血病这一战略可能是不成功的，由于差距， 了解转录重编程发生的机制。我们公布的数据 表明正常造血祖细胞（HPC）经历染色质瞬时去凝聚， 允许谱系特异性转录因子（TF）结合到它们的基因靶点并激活新的转录因子， 程序，导致细胞分化。这种短暂的去凝作用是通过极低的积累发生的 H3 K27 me 3在DNA复制后的DNA上。H3 K27 me 3是最密集的 在基因组中的核小体，并发现在所有抑制基因的调控区。我们的结果表明 测试培养的和初级淋巴B-ALL和TCLL细胞已经失去了这种固有的能力， 染色质，从而为它们的转录重编程创造障碍。在本提案中，我们将测试一种新的 重编程策略，它克服了这些障碍的重编程为基础的治疗，并可能导致 清除白血病细胞。这一新战略的关键特征是第一步，其中包括 药理学抑制H3 K27 me 3组蛋白甲基转移酶（HMT）EZH 1/EZH 2，从而产生去甲基化的H3 K27 me 3组蛋白甲基转移酶（HMT）EZH 1/EZH 2， 在所有基因的调节区域的新生染色质的浓缩结构。第二步，我们将使用 小分子激活内源性诱导型TF，然后由于内源性诱导型TF可以很容易地结合到它们的靶基因， 新生染色质的非浓缩结构。肿瘤细胞，包括白血病细胞， 在可诱导的TF和受体中积累突变;因此， 将进行TF/受体的筛选，以确定不同B-ALL亚型的最佳可能诱导剂。 初步结果表明，小分子诱导剂的诱导导致转录重编程。 细胞系和原代B-ALL和TCLL细胞，其免疫表型和凋亡的变化。而且这 策略强烈抑制小鼠中的淋巴样白血病负荷。该项目的目标是开发一个广泛的 转录重编程和许多类型的细胞活力丧失的适用治疗策略 淋巴白血病细胞为此，我们建议：1.淋巴白血病细胞的扩展和泛化 重编程方法; 2.研究淋巴细胞重编程的机制和生物学结果， 白血病细胞; 3.检查我们的治疗策略在体内的效果。

Abstract Lymphoid B-ALL and TCLL leukemia consists of leukemic blast cells (LBCs) arrested at early stages of differentiation which exhibit high proliferative potential and capability for self-renewal. The idea to induce reprogramming of leukemic and other cancer cells, leading to cell maturation and senescence, gained high popularity, but its clinical applications are rarely successful and are mainly limited to the therapy of the APL leukemia with ATRA and arsenic trioxide. This strategy may have been unsuccessful due to a gap in the knowledge of the mechanisms through which transcriptional reprogramming occurs. Our published data suggest that normal hematopoietic progenitor cells (HPCs) undergo transient de-condensation of chromatin to allow lineage-specific transcription factors (TFs) to bind to their gene targets and to activate new transcriptional programs, leading to cell differentiation. This transient de-condensation occurs through very low accumulation of H3K27me3, on DNA just after DNA replication. H3K27me3 is a mark of the most condensed arrays of nucleosomes in the genome and is found at regulatory regions of all repressed genes. Our results suggest that tested cultured and primary lymphoid B-ALL and TCLL cells have lost this inherent ability to ‘open’ nascent chromatin, thus creating a barrier for their transcriptional reprogramming. In this proposal, we will test a new reprogramming strategy, which overcomes these barriers of reprogramming-based therapies and may lead to elimination of leukemic cells. The key feature of this new strategy is the first step, which includes pharmacological inhibition of the H3K27me3 histone methyltransferases (HMTs) EZH1/EZH2, thus creating de- condensed structure of nascent chromatin at regulatory regions of all genes. At the second step, we will use small molecules to activate endogenous inducible TFs, which can then readily bind to their target genes due to the de-condensed structure of nascent chromatin. Tumor cells, including leukemic cells, are commonly known to accumulate mutations in inducible TFs and receptors; thus, screens of small molecule inducers for a variety of TFs/receptors will be performed to determine the best possible inducer for distinct subtypes of B-ALL. Preliminary results suggest that induction by small molecule inducers leads to transcriptional reprogramming of cell lines and primary B-ALL and TCLL cells, changes in their immunophenotype and apoptosis. Moreover, this strategy strongly suppresses lymphoid leukemia burden in mice. The goal of this project is to develop a widely applicable treatment strategy for transcriptional reprogramming and loss of cell viability for many types of lymphoid leukemic cells. To this end, we propose to: 1. Extend and generalize the lymphoid leukemic cells reprogramming approach; 2. Examine the mechanisms and biological outcomes of reprogramming of lymphoid leukemic cells; 3. Examine the effects of our treatment strategy in vivo.