Therapeutic targeting of IRAK4 in MDS

MDS 中 IRAK4 的治疗靶向

基本信息

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
项目类别：
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

项目摘要

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.

项目摘要/摘要夹层突变和RNA剪接的改变在骨髓增生综合征中很常见（MDS），但是由于这些改变而引起的特定致癌变化尚未完全识别。我们证明了丝氨酸/苏氨酸激酶Irak4的活性同工型的过表达在MD中看到。活性长的IRAK4同工型（IRAK4-L）由N末端死亡域组成（DD）和C末端激酶结构域（KD），它们与myddosome随机组装复杂并导致先天免疫信号通路的最大激活。包括外显子 4在长的同工型中是由突变剪接因子（SF），U2AF1介导的，这导致致癌 MDS/AML的信号传导和预后不良（Smith等人，自然细胞生物学，2019年）。重要的是， IRAK4-L的抑制可在体外和体内消除白血病的生长。这些发现建立了 U2AF1中的突变会影响热靶向“主动” IRAK4同工型的表达并提供了MDS中慢性先天免疫信号传导激活的遗传联系。我们的初步 RNA测序研究中的主要MDS样品研究表明（i）剪接因子SF3B1突变还通过保留外显子6导致活性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。我们假设致癌性IRAK4-L同工型对于SF突变剂的功能至关重要（即U2AF1和SF3B1）以及SF-WildType MDS疾病传播细胞的子集。因此，该建议将全面评估SF3B1和SF-WILD中IRAK4-L调节的机理基础 MDS型（AIM 1）通过使用表达SF3B1 K700E突变体的同基因细胞系和改变剪接调制器PRMT5的表达。 AIM 2将确定下游调节器 MDS中的致癌IRAK4同工型激活。除了检查NF-κB/MAPK 信号传导，我们将执行公正的磷酸化 - 蛋白质筛选，以识别 irak4-l并检查它们在造血功能障碍中的作用。 AIM 3将确定效率通过临床级IRAK4激酶抑制剂在MDS中的IRAK4-L抑制作用。体外敏感性和 U2AF1和SF3B1突变剂以及SF WildType MDS细胞的分化测定将是实施。患者使用U2AF1/SF3B1突变剂衍生的异种移植物，SF WildType MD将是用IRAK4抑制剂治疗以检查抑制疾病传播细胞。我们还将确定IRAK4长的同工型的过表达是否可以预测 IRAK4抑制剂正在进行的临床试验。综上所述，这些研究将决定剪接突变在MDS病理学中诱导的IRAK4-L同工型。重要的是，这些研究将导致潜在的靶向临床试验具有MDS中IRAK4临床相关抑制剂的潜在临床试验。