Determining the Microenvironmental Contribution to Acute Myeloid Leukemia Chemoresistance

急性髓系白血病(AML)是成人最致命的血液系统恶性肿瘤，主要是由于高发病率 化疗后复发。残留疾病，免疫表型的持久性，形态上， 或一线化疗后基因定义的白血病细胞，是AML预后较差的因素。 在小鼠模型中，残留的AML细胞被证明定位于骨内骨髓区域，其中 他们可能会利用专门用于支持长期静止的造血干细胞的显微解剖学利基来 处于休眠状态，对以细胞分裂为目标的化疗产生抵抗力。然而，这些利基市场是 在人类骨髓中定义不明确，目前尚不清楚人类AML细胞是否真的与 作为一种逃避化疗的机制的典型干细胞利基。我们的中心假设是急性髓系白血病细胞 通过过表达干细胞壁龛与骨髓内保护壁龛中的细胞相互作用 受体，这使他们获得化疗耐药。在目标1中，我们将使用表位联合索引 (CODEX)，这是一种最近开发的方法，可以在单细胞水平上可视化来自 固定人体组织，解剖人类骨髓壁龛的确切组成和空间组织。 我们将第一次能够确定化疗耐药细胞在临床骨髓中的定位。 急性髓系白血病患者的活组织检查。我们将评估未分裂的残留AML细胞是否局部化 对化疗的反应优先于骨髓中的骨内干细胞壁龛。在目标2中，我们将 确定残留AML细胞上干细胞利基滞留受体是否过度表达并促进 化疗耐药。为了实现这一点，在Aim 2a中，我们将首先对AML骨骼进行单细胞RNA测序 骨髓抽吸物样本的诊断和残留病时间的测定，以及健康 控件，提供AML细胞、AML相关微环境细胞及其 健康的同行。我们将确定介导生态位保留的受体是否过度表达。在……里面 目的2B，我们将使用CRISPR-CAS9评估它们在化疗耐药中是否在功能上重要 介导AML-间质相互作用的过表达受体-配体对的介导敲除 细胞共培养系统，将用细胞毒化疗治疗。如果AML细胞确实定位于这些 干细胞中的骨内生态位与通过受体-配体介导的相互作用获得化疗耐药 利基，这些受体/配体对将是化疗增敏的一个有吸引力的靶点，这可能有助于 降低急性髓系白血病复发风险。总而言之，这个项目准备评估白血病细胞劫持是否健康 干细胞的利基可以逃避标准护理化疗，这项工作有可能揭示新的 更好地治疗这一致命疾病的治疗机会。

Acute Myeloid Leukemia (AML) is the most fatal hematologic malignancy in adults, largely due to high rates of relapse following chemotherapy. Residual disease, the persistence of immunophenotypically, morphologically, or genetically defined leukemia cells following frontline chemotherapy, is a poor prognostic factor for AML. Residual AML cells have been shown to localize to endosteal bone marrow regions in murine models, where they may utilize microanatomical niches specialized to support long-term quiescent hematopoietic stem cells to become dormant and gain resistance to chemotherapy, which targets dividing cells. However, these niches are ill-defined in human bone marrow, and it remains unclear whether human AML cells actually interact with canonical stem cell niches as a mechanism to evade chemotherapy. Our central hypothesis is that AML cells interact with cells in protective niches within bone marrow by overexpressing stem cell niche retention receptors, which allows them to acquire chemoresistance. In Aim 1, we will employ Co-Indexing by Epitopes (CODEX), a recently developed method to visualize dozens of antibody markers on a single-cell level from fixed human tissue, to dissect the exact composition and spatial organization of human bone marrow niches. For the first time, we will be able to determine where chemoresistant cells localize in clinical bone marrow biopsies from AML patients. We will assess whether or not non-dividing residual AML cells localize preferentially to endosteal stem cell niches in the bone marrow in response to chemotherapy. In Aim 2, we will determine whether stem cell niche retention receptors are overexpressed on residual AML cells and promote chemoresistance. To achieve this, in Aim 2A we will first perform single-cell RNA sequencing of AML bone marrow aspirate samples from diagnosis and time of residual disease determination, as well as healthy controls, providing an expression profile of AML cells, AML-associated microenvironmental cells, and their healthy counterparts. We will determine whether receptors mediating niche retention are overexpressed. In Aim 2B, we will assess whether they are functionally important for chemoresistance using CRISPR-Cas9 mediated knockout of overexpressed receptor-ligand pairs which mediate AML-stromal interaction in an AML- stormal co-culture system that will be treated with cytotoxic chemotherapy. If AML cells indeed localize to these endosteal niches and gain chemoresistance through receptor-ligand mediated interaction in the stem cell niche, these receptor/ligand pairs would represent an attractive target for chemosensitization, which may help reduce AML relapse risk. Together, this project is poised to evaluate whether leukemic cells hijack healthy stem cell niches to evade standard-of-care chemotherapy, and this work has the potential to reveal new therapeutic opportunities to better treat this deadly disease.