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-α（IFN）仍然是骨髓增生性骨髓瘤患者的一种高效治疗方法。 肿瘤（MPN）。我们最近发现，CALR突变的MPN患者经常表现出正常化， 血细胞计数（即临床反应），但通常不表现出肿瘤负荷（即分子水平）的降低。 反应），提供了一个信息模型，以破译IFN的治疗抗性的机制。 探讨干扰素对人MPN干细胞的分子影响可能揭示其机制的重要见解 治疗抵抗。因此，我们应用了我们创新的转录组基因分型（GoT）平台， 捕获相同细胞内的突变状态和单细胞全转录组（scRNA-seq）-CD 34 + 来自用IFN治疗的CALR突变的MPN患者的连续骨髓（BM）抽吸物的细胞。引人注目的是， 我们观察到IFN引起了分化景观的重大变化，在突变型和野生型中明显 祖细胞：野生型细胞上的IFN暴露导致淋巴祖细胞的大量扩增，而 相比之下，突变的细胞显示出颗粒单核细胞（GM）祖细胞的扩增（具有不太显著的 淋巴区室的扩张）。我们的初步数据表明，（1）转基因分化的偏见， CALR突变的干细胞可能是治疗抗性的基础，并且（2）CALR突变诱导的UPR可能 使突变的干细胞向GM谱系发展，并在治疗抵抗中发挥作用。审问这些 假设，我们将确定控制IFN诱导分化的转录因子（TF）网络 通过应用一种新的单细胞多组学平台，捕获RNA-seq，染色质可及性和 在相同的数千个单细胞（GoT-ATAC）中对相同的IFN处理的队列进行体细胞基因分型（Aim 1a），并通过在小鼠模型中靶向这些TF网络（Aim 1b）。我们将定义普遍定期审议在治疗中的作用- 通过GoT-ATAC和染色质结合试验（Aim 2a）以及通过 评估小鼠模型中UPR途径的扰动（目标2b）。最后，我们将确定影响 在CALR突变的MPN中，通过应用 临床样品的单细胞多组学平台（Aim 3a）和IFN对新小鼠的作用的询问 双突变模型（目标3b）。该项目以一个概念创新的框架为中心， 我们在同一个人中研究了肿瘤和正常造血发育，以确定如何 作为突变状态和细胞身份的函数，治疗重塑分化地形。这个概念 创新是通过单细胞多组学平台的技术创新实现的， 队列，再加上新的小鼠模型中的功能评估。这些研究有可能 发现新的见解的分子耐药机制干扰素在MPN，从而产生新的治疗 接近。