Molecular mechanisms of drug resistance and disease progression in acute myeloid leukemia.

先天性免疫应答的异常激活被假设是导致白血病发生的原因。 骨髓增生异常综合征（MDS）和急性髓性白血病（AML）。MDS患者描述为 这些突变组成性地激活了这些途径。我们的研究表明， 紧急（应激）粒细胞生成（EG）终止也是一种可能的机制。EG是一种 感染性攻击期间快速、间歇性粒细胞（PMN）产生，是先天性 免疫反应我们先前确定了Triad 1，一种E3泛素（Ub）连接酶，是EG-1的必需蛋白。 终止与持续EG反应在白血病发生中的作用相一致，我们发现Triad1功能 作为AML的白血病抑制剂，具有增加的同源域转录因子表达。不良 克隆性髓系恶性肿瘤的预后亚组，包括伴有MLL1/KMT2A重排的MDS/AML， 其特征在于这些蛋白质的一组（例如HoxB3、B4、A9 - 11、Cdx 1和2、Meis 1）的过表达。 移植表达白血病相关M111融合蛋白的骨髓的小鼠在移植后发生AML 几个月的滞后时间;这表明白血病的发生需要积累的突变，除了那些 涉及MLL1/KMT2A-重排。我们发现在白血病发生过程中， 在骨髓中表达MII 1融合蛋白的小鼠。我们还发现，Triad1基因的敲除 或EG发作加速了这些小鼠的AML发展。我们证明HoxA10增强，但HoxA9 抑制，Triad1基因转录。而且，Triad1重新表达挽救了Hoxa10-/-小鼠中的EG终止。 我们进行了筛选以鉴定具有Triad1依赖性Ub的蛋白质。除了炎症介质 和RTKs，我们鉴定了参与整合应激反应的蛋白质（ISR; Gcn1，eIF2B4和eIF4G1）。 ISR通过调节翻译来防止持续炎症过程中的代谢衰竭和细胞死亡 以纠正代谢缺陷并在缺陷得到纠正后增强增殖。gcn1作为主要的 通过激活Gcn2/eIF2B4调节该过程。我们发现在骨髓细胞中敲除Triad1改变了 进行翻译的mRNA谱。然而，在这些细胞中，Triad1和Gcn1的联合敲低 逆转了参与细胞应激反应、细胞DNA应答 损伤、细胞周期进程、翻译、蛋白质代谢和ISR终止与Triad1敲低 一个人我们发现Gcn1基因敲低可以延缓骨髓移植小鼠AML的发展， 一种Mll 1融合癌蛋白，逆转了Triad 1敲低对加速白血病发生的作用。 我们假设Triad1对ISR的抑制促进了紧急粒细胞生成（EG）终止 并抑制Hox表达增加的疾病中的白血病发生。这将通过三个目标来实现。 目的1：明确HoxA10/Triad1抑制ISR在终止紧急粒细胞生成中的作用。 将研究紧急粒细胞生成（EG）的小鼠模型中HoxA10/Triad1介导的Ub在 Gcn1在EG终止、翻译组的调节以及与此过程相关的下游途径中的作用。 目的2：研究Hox/Triad1介导的ISR调控对突变和白血病发生的影响。 使用MLL1/KMT2A重排、预后不良的AML小鼠模型，我们将研究 HoxA10/Triad1介导的Gcn1在白血病发生过程中翻译组和突变积累上的Ub 目的3：确定Hox/Triad 1对人类炎症通路和白血病发生的影响 MDS/AML。将研究来自MDS/AML人类受试者的骨髓和外周血CD34+细胞 Hox/Triad1表达与炎症通路激活和不良结局的相关性。分子 将通过RNA-Seq、全外显子组测序和在鼠异种移植物中研究其机制。 目标是将具有Triad 1/ISR相关事件的人类MDS/AML的分子子集识别为分子 标志物和可能的治疗靶点持续炎症和突变的髓性白血病。

Aberrant activation of the innate immune response is hypothesized to contribute to leukemogenesis in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Patients with MDS were described with mutations that constitutively activated such pathways. Our studies suggested that mutations which impair termination of emergency (stress) granulopoiesis (EG) are also a possible mechanism. EG is the process for rapid, episodic granulocyte (PMN) production during infectious challenge and a key component of the innate immune response. We previously determined that Triad1, an E3 ubiquitin (Ub) ligase, was essential for EG- termination. Consistent with a role for a sustained EG response in leukemogenesis, we found Triad1 functioned as a leukemia suppressor for AML with increased expression of homeodomain transcription factors. An adverse prognosis subset of clonal myeloid malignancies, including MDS/AML with MLL1/KMT2A rearrangements, is characterized by overexpression of a group of these proteins (e.g HoxB3, B4, A9-11, Cdx1 and 2, Meis1). Mice transplanted with bone marrow expressing leukemia-associated Mll1-fusion proteins develop AML after a lag time of months; suggesting leukemogenesis requires accumulation of mutations in addition to those involving MLL1/KMT2A-rearrangement. We found Triad1 expression decreased during leukemogenesis in a mice with expression of an Mll1-fusion protein in the bone marrow. We also found that either Triad1 knockdown or EG episodes accelerated AML development in such mice. We demonstrated HoxA10 enhanced, but HoxA9 repressed, Triad1 gene transcription. And, Triad1 re-expression rescued EG termination in Hoxa10-/- mice. We performed a screen to identify proteins with Triad1-dependent Ub. In addition to inflammatory mediators and RTKs, we identified proteins involved in the integrated stress response (ISR; Gcn1, eIF2B4 and eIF4G1). The ISR prevents metabolic exhaustion and cell death during sustained inflammation by modulating translation to correct metabolic defects and enhance proliferation once defects are corrected. Gcn1 functions as a primary regulator of this process by activating Gcn2/eIF2B4. We found Triad1-knockdown in myeloid cells altered the profile of mRNAs undergoing translation. However, combined knockdown of Triad1 and Gcn1 in these cells reversed abnormalities in translation of mRNAs involved in cellular response to stress, cellular response to DNA damage, cell cycle progression, translation, protein metabolism and ISR termination with Triad1 knockdown alone. We found Gcn1 knockdown delayed AML development in mice transplanted with bone marrow expressing an Mll1-fusion oncoprotein, and reversed the effect of Triad1-knockdown on accelerating leukemogenesis. We hypothesize that inhibition of the ISR by Triad1 facilitates emergency granulopoiesis (EG)-termination and suppresses leukemogenesis in disorders with increased Hox expression. This will be pursued by 3 Aims. Aim 1: Define the role of ISR inhibition by HoxA10/Triad1 in terminating emergency granulopoiesis. Murine models of emergency granulopoiesis (EG) will be studied for the role of HoxA10/Triad1-mediated Ub of Gcn1 in EG termination, the modulation of the translatome, and downstream pathways relevant to this process. Aim 2: Identify the influence of ISR regulation by Hox/Triad1 on mutagenesis and leukemogenesis. Using the murine models of MLL1/KMT2A rearranged, adverse prognosis AML, we will study the impact of HoxA10/Triad1-mediated Ub of Gcn1 on the translatome and accumulation of mutations during leukemogenesis. Aim 3: Determine the impact of Hox/Triad1 on inflammatory pathways and leukemogenesis in human MDS/AML. Bone marrow and peripheral blood CD34+ cells from human subjects with MDS/AML will be studied for association of Hox/Triad1 expression with inflammatory pathway activation and adverse outcomes. Molecular mechanisms will be investigated by RNA-Seq, whole exome sequencing and in murine xenografts. The goal is to identify a molecular subset of human MDS/AML with Triad1/ISR related events as molecular markers and possible therapeutic targets for sustained inflammation and mutagenesis in myeloid leukemia.