Elucidating Mechanisms of Therapy-Resistance to Interferon-alfa in Myeloproliferative Neoplasm Stem Cells

阐明骨髓增殖性肿瘤干细胞对干扰素-α的治疗耐药机制

• 批准号: 10736872
• 负责人: Ann Mullally
• 金额: $ 71.84万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-15 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10736872
• 关键词: Binding; Biological Assay; Blood; Bone Marrow Aspiration; CD34 gene; CEBPA gene; Cell Nucleus; Cells; Chemicals; Chromatin; Clinical; Clonal Hematopoietic Stem Cell; Coupled; DNA Sequence Alteration; DNMT3a; Data; Development; Disease; Disease remission; Equilibrium; Exhibits; Genes; Genotype; Hematological Disease; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; Human; Immunophenotyping; Individual; Induced Mutation; Interferon alpha; JAK2 gene; Link; Lymphoid; Measures; Mediating; Megakaryocytes; Methods; Modeling; Molecular; Mutate; Mutation; Myelogenous; Myeloproliferative disease; Outcome; Pathology; Pathway interactions; Patients; Production; Proteins; Recombinant Interferon Alfa; Resistance; Role; Sampling; Shapes; Tumor Burden; Up-Regulation; cell type; cohort; effective therapy; fitness; hematopoietic differentiation; hematopoietic stem cell expansion; in vivo; innovation; insight; monocyte; morphogens; mouse model; multiple omics; mutant; mutational status; neoplastic; novel; novel therapeutic intervention; patient subsets; patient variability; progenitor; programs; response; single cell sequencing; single nucleus RNA-sequencing; stem; stem cells; therapy resistant; tool; transcription factor; transcriptome; transcriptome sequencing

PROJECT SUMMARY Recombinant interferon-alpha (IFN) remains a highly effective therapy for patients with myeloproliferative neoplasms (MPN). We recently identified that patients with CALR-mutated MPN frequently exhibit normalization of blood counts (i.e. clinical response), but often do not exhibit a decrease in tumor burden (i.e. molecular response), providing an informative model to decipher the mechanisms of therapy-resistance to IFN. Interrogating the molecular impact of IFN on human MPN stem cells may reveal critical insights into mechanisms of therapy-resistance. Thus, we applied our innovative Genotyping of Transcriptomes (GoT) platform – that captures the mutation status and single-cell whole transcriptomes (scRNA-seq) within the same cells – CD34+ cells from serial bone marrow (BM) aspirates from patients with CALR-mutated MPN treated with IFN. Strikingly, we observed that IFN caused major shifts in the differentiation landscapes, distinctly in the mutated and wildtype progenitors: IFN exposure on wildtype cells resulted in a large expansion of lymphoid progenitors, while the mutated cells, in contrast, displayed an expansion of the granulo-monocytic (GM) progenitors (with a less striking expansion of the lymphoid compartment). Our preliminary data indicate that (1) the GM differentiation bias of CALR-mutated stem cells may underlie therapy-resistance, and that (2) the CALR-mutation induced UPR may prime the mutated stem cells toward the GM lineage and play a role in therapy-resistance. To interrogate these hypotheses, we will determine the transcription factor (TF) networks that govern the IFN-induced differentiation shifts by applying a novel single-cell multi-omics platform that captures RNA-seq, chromatin accessibility and somatic genotyping within the same thousands of single cells (GoT-ATAC) to the same IFN-treated cohort (Aim 1a), and by targeting these TF networks in mouse models (Aim 1b). We will define the role of UPR in therapy- resistance in treated CALR-mutated cells through GoT-ATAC and chromatin binding assays (Aim 2a) and by assessing perturbations to the UPR pathways in mouse models (Aim 2b). Finally, we will determine the impact of co-mutations in DNMT3A or ASXL1 in therapy-resistance to IFN in CALR-mutated MPN via application of single-cell multi-omics platforms to clinical samples (Aim 3a) and interrogation of IFN effects on novel mouse models with double mutations (Aim 3b). The project is centered on a conceptually innovative framework in which we superimpose neoplastic and normal hematopoietic development within the same individuals to define how therapy reshapes differentiation topographies, as a function of mutation status and cell identity. This conceptual innovation is enabled by technical innovations in single-cell multi-omics platforms applied to compelling clinical cohorts, coupled with functional assessments in novel mouse models. These studies have the potential to uncover new insights into the mechanisms of molecular resistance to IFN in MPN, resulting in novel therapeutic approaches.

项目摘要 重组干扰素-Alpha（IFN）仍然是骨髓增生患者的高效治疗 肿瘤（MPN）。我们最近确定患有CALR突变MPN的患者经常暴露于归一化 血液计数（即临床反应），但通常不会表现出肿瘤负担的减少（即分子 响应），提供一个信息的模型，使IFN的治疗耐药机制破译。 询问IFN对人MPN干细胞的分子影响可能会揭示对机制的关键见解 耐治疗。那就是我们应用了转录组（GOT）平台的创新基因分型 - 捕获相同细胞中的突变状态和单细胞全转录组（SCRNA-SEQ） - CD34+ 来自连续骨髓（BM）的细胞从接受IFN治疗的CALR突变MPN患者的患者抽吸。令人惊讶的是， 我们观察到IFN在突变和野外型中显然引起了分化景观的重大变化 祖细胞：IFN暴露于野生型细胞上导致淋巴祖细胞的大量膨胀 相比之下，突变的细胞显示出颗粒单形（GM）祖细胞的膨胀 淋巴区室的膨胀）。我们的初步数据表明（1）GM分化偏差 CALR突变的干细胞可能是抗化治疗的基础，并且（2）CALR诱导的UPR可能 将突变的干细胞朝向GM谱系，并在耐治疗中发挥作用。询问这些 假设，我们将确定控制IFN诱导分化的转录因子（TF）网络 通过应用新颖的单细胞多摩s平台来捕获RNA-seq，染色质可及性和 在相同的IFN处理队列中，相同数千个单元格（got-atac）内的体细胞分型（AIM） 1A），并通过针对鼠标模型中的这些TF网络（AIM 1B）。我们将定义UPR在治疗中的作用 - 通过GOT-ATAC和染色质结合测定法（AIM 2A）和通过 评估小鼠模型中UPR途径的扰动（AIM 2B）。最后，我们将确定影响 通过施用CALR突变MPN的IFN的DNMT3A或ASXL1中的共突变。 单细胞多摩尼克平台到临床样本（AIM 3A）和对新小鼠的影响的审问 具有双重突变的模型（AIM 3B）。该项目以概念上创新的框架为中心 我们将肿瘤和正常的造血发育叠加在同一个人中，以定义如何 治疗重塑分化地形，这是突变状态和细胞身份的函数。这个概念 通过用于引人注目的临床的单电池多摩斯平台的技术创新来实现创新 队列，再加上新型小鼠模型中的功能评估。这些研究有可能 发现对MPN中IFN的抗性机制的新见解，导致新的治疗 方法。