Molecular analysis of glutamatergic neurons derived from iPSCs containing PPM1D truncating mutations found in Jansen de Vries Syndrome

Jansen de Vries 综合征中发现的含有 PPM1D 截短突变的 iPSC 衍生的谷氨酸能神经元的分子分析

基本信息

批准号：
10573782
负责人：
HERBERT M LACHMAN
金额：
$ 21万
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：
2023
资助国家：
美国
起止时间：
2023-01-01 至 2024-12-31
项目状态：
已结题

项目摘要

Program Director/Principal Investigator (Lachman, Herbert M) Abstract Mutations in PPM1D (Protein Phosphatase Magnesium-Dependent 1 Delta), which codes for a member of the protein serine-threonine phosphatase family, have recently been found in individuals with Jansen-De Vries Syndrome (JdVS), a condition characterized by intellectual disability (ID), severe anxiety, oppositional behavior, attention problems, high pain threshold, restricted eating, and gastrointestinal problems. So far, several dozen children with JdVS have been reported in the literature. Remarkably, each has a de novo germline nonsense or frameshift mutation in exon 5 or 6 that predicts the formation of a protein truncated at the C-terminal end. The phosphatase domain is spared. A key question is whether the truncating mutations result in gain- or loss- of PPM1D function in neurons and other brain cells. PPM1D is also a tumor suppressor gene that is over-expressed in a variety of cancer subtypes, contributing to malignant transformation through its action as a negative regulator of the p53-mediated DNA repair pathway. In cancers, somatic, truncating mutations in exons 5 and 6 like those found in JdVS, but somatic in origin, result in gain-of-function (GOF) effects because PPM1D degradation is reduced. Yet, loss-of-function (LOF) variants in the catalytic domain have also been found in some cancers, suggesting that the effects of PPM1D on cellular function could be context-dependent. Whether the germline truncating mutations in children with JdVS cause GOF or LOF is a critical question to address because PPM1D inhibitors being developed by cancer researchers could potentially have therapeutic value in JdVS if the former is correct. On the other hand, if they cause LOF, drugs targeting over-phosphorylated downstream targets caused by reduced PPM1D phosphatase activity, would be a more feasible approach. To address these fundamental questions, we have established an induced pluripotent stem cell (iPSC) model for JdVS using patient-specific and CRISPR-engineered lines, each containing exon 5 or exon 6 truncating mutations. Preliminary RNA-seq and proteomics experiments on glutamatergic neurons derived from iPSCs identified a few functional signatures, including deficits in neurite outgrowth, altered chromatin architecture, and calmodulin kinase 2 (CAMK2) phosphorylation that suggest a GOF effect. However, molecular studies need to be repeated and expanded in additional lines to firmly establish these as legitimate phenotypes, and to identify novel makers that can be used to both test the GOF hypothesis and identify downstream targets for therapeutic intervention. This will be accomplished by a multi- OMICs approach on glutamatergic neurons that includes proteomics, phosphoproteomics, RNA-seq, and ATAC-seq. Considering the uniqueness of the PPM1D mutations in the development of JdVS, we also hypothesize that truncated PPM1D proteins have powerful effects on cellular function that could make them resistant to typical PPM1D inhibitors. To test these hypotheses, we will knockout either the mutant allele or wild type PPM1D allele by introducing a null mutation using CRISPR-Cas9 editing, after which, rescue of dysregulated cellular and molecular phenotypes will be assessed. The ability of small molecule PPM1D inhibitors to rescue these phenotypes will also be evaluated. The experiments will show whether JdVS associated PPM1D variants have a GOF effect on glutamatergic neurons, and whether generalized reduction in PPM1D phosphatase activity or targeted inhibition of the truncated protein will be suitable targets for therapeutic intervention.

项目总监/首席研究员（Lachman，Herbert M）抽象的 PPM1D（蛋白磷酸酶镁依赖性 1 Delta）突变，编码最近在 Jansen-De Vries 患者中发现了蛋白质丝氨酸-苏氨酸磷酸酶家族综合症（JdVS），一种以智力障碍（ID）、严重焦虑、对立为特征的疾病行为、注意力问题、高痛阈、饮食限制和胃肠道问题。迄今为止，文献报道了数十名患有 JdVS 的儿童。值得注意的是，每一个都有一个从头外显子 5 或 6 中的种系无义或移码突变，预测在 C-末端。磷酸酶结构域被保留。一个关键问题是截短突变是否会导致神经元和其他脑细胞中 PPM1D 功能的获得或丧失。 PPM1D也是一种抑癌基因它在多种癌症亚型中过度表达，通过其促进恶性转化作为 p53 介导的 DNA 修复途径的负调节因子。在癌症中，体细胞、截短外显子 5 和 6 的突变类似于 JdVS 中发现的突变，但起源于体细胞，导致功能获得性 (GOF) 由于 PPM1D 降解减少而产生的影响。然而，催化域中的功能丧失（LOF）变体在一些癌症中也发现了 PPM1D，这表明 PPM1D 对细胞功能的影响可能是依赖于上下文。 JdVS 儿童的种系截短突变是否会导致 GOF 或 LOF 这是一个需要解决的关键问题，因为癌症研究人员正在开发的 PPM1D 抑制剂可以如果前者正确的话，JdVS 可能具有治疗价值。另一方面，如果它们导致 LOF，药物针对因 PPM1D 磷酸酶活性降低而导致的过度磷酸化的下游靶标，将是更可行的方法。为了解决这些基本问题，我们建立了一个诱导 JdVS 的多能干细胞 (iPSC) 模型使用患者特异性和 CRISPR 工程设计的细胞系，每个含有外显子 5 或外显子 6 截短突变。初步 RNA-seq 和蛋白质组学实验来自 iPSC 的谷氨酸能神经元鉴定出一些功能特征，包括神经突缺陷生长、染色质结构改变和钙调蛋白激酶 2 (CAMK2) 磷酸化表明 GOF效应。然而，分子研究需要在其他线路中重复和扩展，以坚定地将这些建立为合法的表型，并确定可用于测试 GOF 的新型制造商假设并确定治疗干预的下游目标。这将通过多方合作来完成针对谷氨酸能神经元的 OMIC 方法，包括蛋白质组学、磷酸蛋白质组学、RNA-seq 和 ATAC-seq。考虑到 PPM1D 突变在 JdVS 发展中的独特性，我们还假设截短的 PPM1D 蛋白对细胞功能有强大的影响，可能使它们对典型的 PPM1D 抑制剂具有耐药性。为了检验这些假设，我们将敲除突变等位基因或野生等位基因通过使用 CRISPR-Cas9 编辑引入无效突变来键入 PPM1D 等位基因，然后，拯救将评估失调的细胞和分子表型。小分子PPM1D的能力还将评估挽救这些表型的抑制剂。实验将表明 JdVS 是否相关的 PPM1D 变体对谷氨酸能神经元有 GOF 效应，以及是否普遍减少 PPM1D 磷酸酶活性或截短蛋白的靶向抑制将是合适的靶点治疗干预。