Project 4

项目4/摘要 NUP 98融合癌基因（例如NUP 98-HOXA 9、-KDM 5A和-NSD 1）与几种儿科疾病相关。 以不良预后为特征的血液学恶性肿瘤（例如，AML; 6-10%;和AEL; 20%）。所有这些 癌基因将核孔蛋白NUP 98的N-末端FG-重复结构域融合到染色质结合结构域 来自多种转录和表观遗传调节因子（例如，KDM 5A的组蛋白结合PHD 3结构域）。 内源性NUP 98主要定位于核孔复合物，具有其内在无序的FG重复序列 结构域（~500个残基）填充中心孔。相反，NUP 98融合癌蛋白（FO）进入细胞内。 细胞核，结合染色质，并导致许多亚微米大小的核斑点的形成。 重要的是，这些斑点将表观遗传和转录调节因子募集到发育调节基因中， 特别是HOX家族，激活异常基因表达，并转化造血细胞。白血病 携带NUP 98 FO的患者用标准的基于AML的缓解诱导疗法治疗， 强化/移植用于次优反应疾病，但预后通常较差。因此，我们认为， 非常需要治疗NUP 98 FO驱动的白血病的新治疗策略。当N- NUP 98 FO的末端结构域被认为是不可药用的，因为它本质上是无序的，它们的折叠C- 末端结构域为治疗开发提供了机会。例如，PHD 3结构域的结合 在NUP 98-KDM 5A FO中，组蛋白3的二甲基化和三甲基化赖氨酸4（H3 K4-Me 2/3）是 造血细胞的表观遗传状态的重新布线及其转化。此外，H 0XA 9的缺失 来自NUP 98-HOXA 9 FO的DNA结合结构域改变核斑点形成并阻止细胞凋亡 转型因此，靶向这些折叠结构域与染色质相互作用的治疗策略 具有抑制NUP 98 FO引起的细胞转化的潜力。在这里，我们建议开发小分子， 结合KDM 5A PHD 3结构域并抑制其与H3 K4-Me 2/3的结合（目的1和2）。我们进一步建议， 使用PROTAC（PROteolysis-Targeting Chimaera）策略调整PHD 3结构域结合剂和抑制剂， 诱导细胞中NUP 98-KDM 5A降解（目的2）。我们开发的抑制剂和PROTAC将被测试 针对鼠和人细胞模型，以及NUP 98-KDM 5A驱动的AML的体内鼠模型。

Project 4/Summary NUP98-fusion oncogenes (eg, NUP98-HOXA9, -KDM5A & -NSD1) are associated with several pediatric hematological malignancies (eg, AML; 6-10%; and AEL; 20%) characterized by poor prognosis. All of these oncogenes fuse the N-terminal FG-repeat domain of the nucleoporin NUP98 to chromatin binding domains from a variety of transcriptional and epigenetic regulators (e.g., the histone binding PHD3 domain of KDM5A). Endogenous NUP98 primarily localizes to the nuclear pore complex, with its intrinsically disordered FG-repeat domain (~500 residues) filling the central pore. In contrast, NUP98 fusion oncoproteins (FOs) enter the nucleus, bind to chromatin and cause the formation of numerous, sub-micron sized nuclear puncta. Importantly, these puncta recruit epigenetic and transcriptional regulators to developmental regulatory genes, notably the HOX family, activate aberrant gene expression, and transform hematopoietic cells. Leukemias harboring NUP98 FOs are treated with standard AML-based remission induction therapy, with intensification/transplantation for suboptimally responsive disease, but prognosis is generally poor. Therefore, there is great need for new therapeutic strategies for treatment of NUP98 FO-driven leukemias. While the N- terminal domain of NUP98 FOs is considered undruggable because it is intrinsically disordered, their folded C- terminal domains offer opportunities for therapeutic development. For example, binding of the PHD3 domain within the NUP98-KDM5A FO to di-and tri-methylated lysine 4 of Histone 3 (H3K4-Me2/3) is required for rewiring of the epigenetic state of hematopoietic cells and their transformation. Further, deletion of the HOXA9 DNA binding domain from the NUP98-HOXA9 FO alters nuclear puncta formation and prevents cell transformation. Thus, therapeutic strategies targeting the interactions of these folded domains with chromatin have potential to inhibit cell transformation by NUP98 FOs. Here, we propose to develop small molecules that bind the KDM5A PHD3 domain and inhibit its binding to H3K4-Me2/3 (Aims 1 & 2). We further propose to adapt PHD3 domain binders and inhibitors using the PROTAC (PROteolysis-TArgeting Chimaera) strategy to induce degradation of NUP98-KDM5A in cells (Aim 2). The inhibitors and PROTACs we develop will be tested against murine and human cellular models, and in vivo murine models of NUP98-KDM5A-driven AML.