Therapeutic targeting of IRAK4 in MDS

项目摘要/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)，但这些变化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IRAK4的过度表达， 在MDS中可见。激活的长IRAK4亚型(IRAK4-L)由N端死亡结构域组成 (DD)和C-末端激酶结构域(KD)，它与Myddosome随机组装 复杂，并导致最大限度地激活天然免疫信号通路。包含外显子 4是由突变型剪接因子(SF)U2AF1介导的，导致致癌 MDS/AML中的信号和不良预后(Smith等人，自然细胞生物学，2019年)。重要的是 体外和体内抑制IRAK4-L抑制白血病生长。这些发现证实了 U2AF1基因突变诱导具有治疗靶向性的活性IRAK4亚型的表达 并为MDS中慢性先天免疫信号的激活提供了遗传联系。我们的预赛 原发MDS样本中的RNA测序研究表明：(I)剪接因子SF3B1突变 也通过保留外显子6而导致活性IRAK4-L亚型，并且(Ii)相当大的比例 剪接因子(SF)野生型MDS患者也表达IRAK4-L。我们假设 致癌的IRAK4-L亚型对SF突变体(即U2AF1和SF3B1)的功能至关重要 和SF-野生型MDS疾病传播细胞的子集。因此，这项提议将 综合评价IRAK4-L在SF3B1和SF-WARD中调控的机制基础 通过使用表达SF3B1 K700E突变体的等基因细胞系和通过 改变剪接调节子PRMT5的表达。AIM 2将确定下游监管机构 MDS中致癌的IRAK4亚型激活。除检测核因子-κB/丝裂原活化蛋白激酶外 信号，我们将进行无偏见的磷酸蛋白质组筛选，以确定直接底物 IRAK4-L，并研究它们在造血功能障碍中的作用。目标3将决定疗效 临床级别的IRAK4激酶抑制剂对MDS中IRAK4-L的抑制作用。体外敏感度和 对U2AF1和SF3B1突变的MDS细胞以及SF野生型MDS细胞的分化分析将是 指挥。患者来源的具有U2AF1/SF3B1突变和SF野生型MDS的异种移植将是 用IRAK4抑制剂治疗，以检查对疾病传播细胞的抑制。我们 也将确定IRAK4-Long异构体的过度表达是否预示着 正在进行的IRAK4抑制剂的临床试验。综上所述，这些研究将确定 MDS病理生物学中IRAK4-L亚型剪接突变的研究重要的是，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。

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.