Therapeutic targeting of IRAK4 in MDS

MDS 中 IRAK4 的治疗靶向

• 批准号: 10696208
• 负责人: Daniel Starczynowski
• 金额: $ 51.75万
• 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/10696208
• 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; IRAK4 gene; Immune; Immune signaling; In Vitro; Induced Mutation; Inflammatory; Lead; Leukemic Cell; Leukemic Hematopoietic Stem Cell; Link; Lysine; MAP Kinase Gene; Mediating; Mutation; N-terminal; Nature; Oncogenic; Outcome; Pathway interactions; Patients; Phosphotransferases; Prognosis; Protein Isoforms; Protein-Serine-Threonine Kinases; Publishing; RNA Splicing; Recurrent disease; Regulation; Role; Sampling; Serine; Signal Pathway; Signal Transduction; Spliceosomes; TRAF6 gene; Therapeutic; Therapeutically Targetable; Ubiquitination; Work; Xenograft procedure; biomarker driven; clinical predictors; clinically relevant; efficacy evaluation; efficacy testing; in vivo; inhibitor; kinase inhibitor; knock-down; leukemia; mutant; novel; overexpression; patient derived xenograft model; pharmacologic; 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.

项目总结/摘要 剪接体突变和RNA剪接的改变在骨髓增生异常综合征中很常见 (MDS)然而，由于这些改变引起的特定致癌变化尚未完全确定。 我们证明了丝氨酸/苏氨酸激酶活性亚型IRAK 4的过表达， 在MDS中可见。活性长IRAK 4同种型（IRAK 4-L）由N-末端死亡结构域组成 (DD)和C-末端激酶结构域（KD），其与Myddosome组装 复合物并导致先天免疫信号传导途径的最大激活。包含外显子 4在长同种型中由突变剪接因子（SF）U2 AF 1介导，其导致致癌的 MDS/AML中的信号传导和不良预后（Smith等，Nature Cell Biology，2019）。重要的是， 抑制IRAK 4-L在体外和体内消除白血病生长。这些发现确立了 U2 AF 1突变诱导治疗靶向的“活性”IRAK 4亚型的表达， 并为MDS中慢性先天免疫信号的激活提供遗传联系。我们的初步 在原发性MDS样品中的RNA测序研究揭示（i）剪接因子SF 3B 1突变 也通过外显子6的保留导致活性IRAK 4-L亚型，并且（ii）相当大的比例 的剪接因子（SF）野生型MDS患者也表现出IRAK 4-L的表达。我们假设 致癌IRAK 4-L亚型对SF突变体（即U2 AF 1和SF 3B 1）的功能至关重要 和SF-野生型MDS疾病传播细胞的子集。因此，这项建议将 综合评价SF 3B 1和SF-wild中IRAK 4-L调控的机制基础 通过使用表达SF 3B 1 K700 E突变体的等基因细胞系和通过 改变剪接调节剂PRMT 5的表达。目标2将决定下游监管机构 IRAK 4亚型在MDS中的致癌激活。除检测NF-κB/MAPK外， 信号，我们将进行一个公正的磷酸蛋白质组学筛选，以确定直接底物 IRAK 4-L，并检查其在造血功能障碍中的作用。目标3将决定疗效 通过临床级IRAK 4激酶抑制剂在MDS中抑制IRAK 4-L。体外敏感性和 将在U2 AF 1-和SF 3B 1-突变体和SF野生型MDS细胞中进行分化测定， 进行。将对具有U2 AF 1/SF 3B 1突变体和SF野生型MDS的患者来源的异种移植物进行免疫组化。 用IRAK 4抑制剂处理以检查疾病传播细胞的抑制。我们 还将确定IRAK 4-长亚型的过表达是否是对免疫应答的预测。 正在进行的IRAK 4抑制剂临床试验。综合起来，这些研究将确定 剪接突变诱导的IRAK 4-L亚型在MDS病理生物学中的作用。重要的是，这些研究 导致在MDS中使用临床相关的IRAK 4抑制剂的潜在靶向临床试验。