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

项目总结 重组干扰素-α对骨髓增生性疾病仍是一种高效疗法 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)，但通常不显示出肿瘤负担的减少(即分子 反应)，为破译干扰素治疗耐药的机制提供了一个信息丰富的模型。 询问干扰素对人MPN干细胞的分子影响可能揭示关键的机制 治疗抵抗力。因此，我们应用了我们创新的转录本基因分型(GOT)平台-- 捕获同一细胞内的突变状态和单细胞完整转录体(scRNA-seq)-CD34+ 经干扰素治疗的CALR突变MPN患者的骨髓(BM)细胞。令人惊讶的是， 我们观察到，干扰素引起了分化景观的重大变化，在突变型和野生型中尤为明显。 祖细胞：干扰素暴露在野生型细胞上会导致淋巴祖细胞大量扩增，而 相比之下，突变的细胞表现出粒-单核细胞(GM)祖细胞的扩张(不那么显著 淋巴小室扩张)。我们的初步数据表明：(1)GM的分化偏向 CALR突变的干细胞可能是耐药的基础，(2)CALR突变诱导的UPR可能 使突变的干细胞向GM血统转化，并在治疗抵抗中发挥作用。审问这些人 假设，我们将确定控制干扰素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来实现转变，该平台捕获RNA-seq、染色质可及性和 同一干扰素治疗队列中相同数千个单细胞(GOT-ATAC)的体细胞基因分型(AIM 1a)，并通过在小鼠模型中靶向这些转铁蛋白网络(目标1b)。我们将定义UPR在治疗中的作用- 通过GOT-ATAC和染色质结合分析(Aim 2a)和通过 评估小鼠模型中UPR通路的扰动(目标2b)。最后，我们将确定影响 CALR突变MPN中DNMT3A或ASXL1共突变对干扰素耐药的研究 临床标本的单细胞多组学平台(AIM 3a)和干扰素对新小鼠的作用 双突变模型(目标3b)。该项目的中心是一个概念创新的框架，在这个框架中 我们在同一个人体内叠加肿瘤和正常的造血发育，以定义如何 作为突变状态和细胞特性的函数，治疗可以重塑分化结构。这一概念 创新是由应用于引人注目的临床的单细胞多组学平台的技术创新推动的 队列，加上对新的小鼠模型的功能评估。这些研究有可能 发现MPN对干扰素分子耐药机制的新见解，导致新的治疗方法 接近了。

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.