The leukemia stem cell (LSC) compartment is a complex reservoir fueling disease progression in acute myeloid leukemia (AML). The existence of heterogeneity within this compartment is well documented but prior studies have focused on genetic heterogeneity without being able to address functional heterogeneity. Understanding this heterogeneity is critical for the informed design of therapies targeting LSC, but has been hampered by LSC scarcity and the lack of reliable cell surface markers for viable LSC isolation. To overcome these challenges, we turned to the patient-derived OCI-AML22 cell model. This model includes functionally, transcriptionally and epigenetically characterized LSC broadly representative of LSC found in primary AML samples. Focusing on the pool of LSC, we used an integrated approach combining xenograft assays with single-cell analysis to identify two LSC subtypes with distinct transcriptional, epigenetic and functional properties. These LSC subtypes differed in depth of quiescence, differentiation potential, repopulation capacity, sensitivity to chemotherapy and could be isolated based on CD112 expression. A majority of AML patient samples had transcriptional signatures reflective of either LSC subtype, and some even showed coexistence within an individual sample. This work provides a framework for investigating the LSC compartment and designing combinatorial therapeutic strategies in AML.

白血病干细胞（LSC）区室是一个复杂的储存库，可促进急性髓系白血病（AML）疾病的进展。该区室中异质性的存在已有充分记录，但先前的研究主要集中在遗传异质性上，而无法解决功能异质性。了解这种异质性对于针对 LSC 的疗法的知情设计至关重要，但由于 LSC 稀缺性和缺乏用于可行 LSC 分离的可靠细胞表面标记物而受到阻碍。为了克服这些挑战，我们转向患者来源的 OCI-AML22 细胞模型。该模型包括具有功能、转录和表观遗传学特征的 LSC，广泛代表了原发性 AML 样本中发现的 LSC。着眼于 LSC 库，我们采用了将异种移植测定与单细胞分析相结合的综合方法，以确定具有不同转录、表观遗传和功能特性的两种 LSC 亚型。这些 LSC 亚型在静止深度、分化潜力、增殖能力、化疗敏感性方面存在差异，并且可以根据 CD112 表达进行分离。大多数 AML 患者样本具有反映任一 LSC 亚型的转录特征，有些甚至在单个样本中表现出共存。这项工作为研究 LSC 区室和设计 AML 组合治疗策略提供了一个框架。