Inducing transcriptional reprogramming of leukemic B-cells by facilitating transcription factors binding to nascent decondensed chromatin

项目摘要/摘要 B细胞急性淋巴细胞性白血病(B-ALL)由白血病原始细胞(LBC)组成，在 B细胞发育的不同阶段，因为潜在的转录和表观遗传变化 编程。无法区分赋予了无限自我更新的能力，并增加了 增殖能力。根据全反式维甲酸和砷治疗APL白血病的成功经验， 导致细胞成熟和衰老，基于分化的治疗成为一种流行的治疗方法，但很少 成功的治疗策略。我们认为，这一战略之所以不成功，是因为 关于转录重编程发生的潜在机制的知识。我的新战略 是基于我们小组最近的观察，正常造血祖细胞(HPC)的分化 依赖于它们在诱导时瞬间解聚其复制后染色质的能力 特定细胞因子的谱系。在DNA复制的早期阶段，新生染色质的这种解聚状态 为谱系特异性转录因子(TF)提供了一个机会之窗，以克服 核小体在被抑制的基因上的浓缩结构，并结合和激活它们的靶点。初步 结果表明，B-ALL LBC失去了打开新生染色质的固有能力，从而形成了一种屏障 在他们的转录重新编程中，基于分化的治疗无法克服。在这项建议中， 克服这个浓缩的新生染色质的障碍，并重新编程LBC，我将利用以下内容 接近。首先，我将通过抑制H3K27me3的药物作用，使新生染色质解聚 H3K27me3组蛋白甲基转移酶EZH1/2。第二，我会用小分子激活诱导型 转录因子，然后可以很容易地结合到他们的目标基因，由于解缩结构 新生的染色质。众所周知，癌症会在可诱导的转录因子和受体中积累突变；因此， 将对各种转录因子/受体的小分子诱导剂进行筛选，以确定可能的最佳方案 B-ALL不同亚型的诱导剂。初步结果提供了强有力的迹象表明，由新的小分子诱导 分子配体/诱导剂可能导致B-ALL LBC转录重编程，从而导致几乎 这些细胞的活性完全丧失。这一战略在提高疗效方面也显示出了巨大的前景。 属于并有助于减少类固醇激素等临床标准护理药物的有效剂量。 此外，我的结果表明，B-ALL细胞进入S期的同步性大大增加 我们重新编程策略的效率。总体而言，我们在概念上的进步在于，我们没有试图 诱导LBC向造血细胞分化，而不是通过解聚新生染色质诱导 转录重编程到异常谱系，这可能导致终端成熟，从而 衰老，增殖减少，细胞死亡。

Project Summary/Abstract B cell acute lymphoblastic leukemia (B-ALL) consist of Leukemic Blast Cells (LBCs) that become arrested at distinct stages of B cell development because of underlying alterations in transcriptional and epigenetic programming. The inability to differentiate confers a capacity for unlimited self-renewal and increased proliferative capability. Based on the successes in the treatment of the APL leukemia with ATRA and arsenic, leading to cell maturation and senescence, the differentiation-based therapies became a popular, but rarely successful, therapeutic strategy. We propose that this strategy has been unsuccessful due to a gap in the knowledge of the underlying mechanism through which transcriptional reprogramming occurs. My new strategy is based on our group’s recent observations that differentiation of normal hematopoietic progenitor cells (HPCs) is dependent on their ability to transiently decondense their post-replicative chromatin upon induction with lineage specifying cytokines. At the early stages of DNA replication, this decondensed state of nascent chromatin offers a window of opportunity for lineage-specific transcription factors (TFs) to overcome the barrier of the condensed structure of nucleosomes at repressed genes and to bind and activate their target sites. Preliminary results indicate that B-ALL LBCs have lost the inherent ability to open nascent chromatin, thus creating a barrier in their transcriptional reprogramming that differentiation-based therapies cannot overcome. In this proposal, to overcome this barrier of condensed nascent chromatin and to reprogram LBCs, I will utilize the following approach. First, I will pharmacologically ablate H3K27me3 to decondense nascent chromatin by inhibiting H3K27me3 histone methyltransferases EZH1/2. Second, I will use small molecules to activate inducible transcription factors, which can then readily bind to their target genes due to the decondensed structure of nascent chromatin. Cancers 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 provide strong indications that induction by novel small molecule ligands/inducers may lead to transcriptional reprogramming in B-ALL LBCs, which results in an almost complete loss of viability of these cells. This strategy has also shown significant promise in increasing the efficacy of and in decreasing the effective dosages of clinical standard of care drugs, such as steroid hormones. Furthermore, my results indicate that synchronization of B-ALL cells into the entry of the S-phase greatly increase the efficiency of our reprogramming strategy. Overall, our conceptual advance is in that we are not attempting to induce hematopoietic differentiation of LBCs, but rather through decondensing nascent chromatin induce transcriptional reprogramming to aberrant lineages, which may lead to terminal maturation, and thus to senescence, reduced proliferation, and cell death.