Therapeutic targeting of IRAK4 in MDS

MDS 中 IRAK4 的治疗靶向

• 批准号: 10537837
• 负责人: Daniel Starczynowski
• 金额: $ 54.5万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-02 至 2027-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10537837
• 关键词: AML/MDS; Acute Myelocytic Leukemia; Alternative Splicing; Biological Assay; C-terminal; CDK2 gene; Cell Line; Cell physiology; Cells; Cellular biology; Chronic; Clinical; Clinical Trials; Complex; Correlative Study; Data; Death Domain; Disease; Dysmyelopoietic Syndromes; Exhibits; Exons; Functional disorder; Gene Expression; Genes; Genetic; Growth; Hematopoietic; Hematopoietic Neoplasms; Hematopoietic stem cells; IRAK4 gene; Immune; Immune signaling; In Vitro; Induced Mutation; Inflammatory; Lead; Leukemic Cell; Link; Lysine; MAP Kinase Gene; Mediating; Mutation; N-terminal; Nature; Oncogenic; Outcome; Pathway interactions; Patients; Pharmacology; Phosphotransferases; Prognosis; Protein Isoforms; Protein-Serine-Threonine Kinases; Publishing; RNA Splicing; Recurrent disease; Regulation; Role; Sampling; Signal Pathway; Signal Transduction; Spliceosomes; TRAF6 gene; Therapeutic; Ubiquitination; Work; Xenograft procedure; base; biomarker-driven; clinical predictors; clinically relevant; efficacy testing; in vivo; inhibitor; kinase inhibitor; knock-down; leukemia; mutant; novel; overexpression; patient derived xenograft model; phosphoproteomics; potential biomarker; predicting response; response; small hairpin RNA; therapeutic target; transcriptome sequencing

PROJECT SUMMARY/ABSTRACT Spliceosome mutations and alterations in RNA splicing are common in myelodysplastic syndrome (MDS), yet the specific oncogenic changes due to these alterations have not been fully identified. We demonstrated that overexpression of an active isoform of the serine/threonine kinase, IRAK4, is seen in MDS. The active long IRAK4 isoform (IRAK4-L) consists of an N-terminal death domain (DD) and C-terminal kinase domain (KD), which stochastically assembles with the Myddosome complex and results in maximal activation of innate immune signaling pathways. Inclusion of exon 4 in the long isoform is mediated by mutant splicing factor (SF), U2AF1, which results in oncogenic signaling and poor prognosis in MDS/AML (Smith et al., Nature Cell Biology, 2019). Importantly, inhibition of IRAK4-L abrogates leukemic growth in vitro and in vivo. These findings established that mutations in U2AF1 induce expression of therapeutically targetable "active" IRAK4 isoforms and provide a genetic link to activation of chronic innate immune signaling in MDS. Our preliminary RNA-sequencing studies in primary MDS samples reveal that (i) splicing factor SF3B1 mutations also lead to the active IRAK4-L isoforms via retention of exon 6, and that (ii) a sizable proportion of splicing factor (SF) wild-type MDS patients also exhibit expression of IRAK4-L. We hypothesize that oncogenic IRAK4-L isoforms are critical for the function of SF-mutant (i.e. U2AF1 and SF3B1) and a subset of SF-wildtype MDS disease propagating cells. As such, this proposal will comprehensively evaluate the mechanistic basis of IRAK4-L regulation in SF3B1 and SF-wild type MDS (Aim 1) by using isogenic cell lines expressing the SF3B1 K700E mutants and by altering the expression of splicing modulator PRMT5. Aim 2 will determine downstream regulators of oncogenic IRAK4 isoform activation in MDS. In addition to examination of NF-κB/MAPK signaling, we will perform an unbiased phospho-proteomic screen to identify direct substrates of IRAK4-L and examine their roles in hematopoietic dysfunction. Aim 3 will determine the efficacy of IRAK4-L inhibition in MDS by a clinical-grade IRAK4 kinase inhibitor. In vitro sensitivity and differentiation assays in U2AF1- and SF3B1-mutant, and SF wildtype MDS cells will be conducted. Patient derived xenografts with U2AF1/SF3B1-mutant, and SF wildtype MDS will be treated with the IRAK4 inhibitors to examine for suppression of the disease-propagating cells. We will also determine whether overexpression of IRAK4-long isoforms is predictive of response in an ongoing clinical trial of IRAK4 inhibitor. Taken together, these studies will determine the role of splicing mutation induced IRAK4-L isoform in MDS pathobiology. Importantly, these studies will lead to potential targeted clinical trials with clinically relevant inhibitors of IRAK4 in MDS.

项目概要/摘要 剪接体突变和 RNA 剪接改变在骨髓增生异常综合征中很常见 （MDS），但由于这些改变引起的具体致癌变化尚未完全确定。 我们证明丝氨酸/苏氨酸激酶的活性亚型 IRAK4 的过度表达， 见于MDS。活性长 IRAK4 亚型 (IRAK4-L) 由 N 端死亡结构域组成 (DD) 和 C 端激酶结构域 (KD)，与 Myddosome 随机组装 复合物并导致先天免疫信号通路的最大激活。包含外显子 长亚型中的 4 由突变剪接因子 (SF)、U2AF1 介导，从而导致致癌 MDS/AML 中的信号传导和不良预后（Smith 等人，Nature Cell Biology，2019）。重要的是， 抑制 IRAK4-L 可消除体外和体内白血病的生长。这些发现成立 U2AF1 的突变诱导治疗上可靶向的“活性”IRAK4 亚型的表达 并提供与 MDS 慢性先天免疫信号激活的遗传联系。我们的初步 原发性 MDS 样本中的 RNA 测序研究表明 (i) 剪接因子 SF3B1 突变 还通过保留外显子 6 产生活性 IRAK4-L 同工型，并且 (ii) 相当大的比例 剪接因子 (SF) 野生型 MDS 患者也表现出 IRAK4-L 的表达。我们假设 致癌 IRAK4-L 亚型对于 SF 突变体（即 U2AF1 和 SF3B1）的功能至关重要 以及 SF 野生型 MDS 疾病传播细胞的子集。因此，该提案将 全面评估SF3B1和SF-wild中IRAK4-L调控的机制基础 通过使用表达 SF3B1 K700E 突变体的同基因细胞系和通过 改变剪接调节剂 PRMT5 的表达。目标 2 将确定下游监管机构 MDS 中致癌 IAK4 同工型激活的研究。除了检查NF-κB/MAPK 信号传导，我们将进行无偏见的磷酸化蛋白质组筛选，以识别 IRAK4-L 并检查它们在造血功能障碍中的作用。目标3将决定功效 临床级 IRAK4 激酶抑制剂对 MDS 中 IRAK4-L 的抑制作用。体外敏感性和 U2AF1 和 SF3B1 突变体以及 SF 野生型 MDS 细胞的分化测定将 实施。具有 U2AF1/SF3B1 突变体和 SF 野生型 MDS 的患者来源的异种移植物将被 用IRAK4抑制剂处理以检查对疾病传播细胞的抑制。我们 还将确定 IRAK4 长亚型的过度表达是否可以预测 IRAK4 抑制剂正在进行的临床试验。总而言之，这些研究将确定其作用 MDS 病理学中剪接突变诱导的 IRAK4-L 同工型的研究。重要的是，这些研究将 导致在 MDS 中使用临床相关的 IRAK4 抑制剂进行潜在的靶向临床试验。