Relapse in T-cell acute lymphoblastic leukemia (T-ALL) may signify the persistence of leukemia-initiating cells (L-ICs). Ectopic TAL1/LMO expression defines the largest subset of T-ALL, but its role in leukemic transformation and its impact on relapse-driving L-ICs remain poorly understood. In TAL1/LMO mouse models, double negative-3 (DN3; CD4⁻CD8⁻CD25⁺CD44⁻) thymic progenitors harbored L-ICs. However, only a subset of DN3 leukemic cells exhibited L-IC activity, and studies linking L-ICs and chemotolerance are needed. To investigate L-IC heterogeneity, we used mouse models and applied single-cell RNA-sequencing and nucleosome labeling techniques in vivo. We identified a DN3 subpopulation with a cell cycle–restricted profile and heightened TAL1/LMO2 activity, that expressed genes associated with stemness and quiescence. This dormant DN3 subset progressively expanded throughout leukemogenesis, displaying intrinsic chemotolerance and enrichment in genes linked to minimal residual disease. Examination of TAL/LMO patient samples revealed a similar pattern in CD7⁺CD1a⁻ thymic progenitors, previously recognized for their L-IC activity, demonstrating cell cycle restriction and chemotolerance. Our findings substantiate the emergence of dormant, chemotolerant L-ICs during leukemogenesis, and demonstrate that Tal1 and Lmo2 cooperate to promote DN3 quiescence during the transformation process. This study provides a deeper understanding of TAL1/LMO-induced T-ALL and its clinical implications in therapy failure.

T 细胞急性淋巴细胞白血病 (T-ALL) 的复发可能意味着白血病起始细胞 (L-IC) 的持续存在。异位 TAL1/LMO 表达定义了 T-ALL 的最大子集，但其在白血病转化中的作用及其对复发驱动 L-IC 的影响仍知之甚少。在TAL1 / LMO小鼠模型中，双阴性 3 (DN3; CD4 ⁻ CD8 ⁻ CD25 ⁺ CD44 ⁻ ) 胸腺祖细胞含有 L-IC。然而，只有一部分 DN3 白血病细胞表现出 L-IC 活性，需要研究将 L-IC 与化学耐受性联系起来。为了研究 L-IC 异质性，我们使用小鼠模型并在体内应用单细胞 RNA 测序和核小体标记技术。我们鉴定了一个具有细胞周期限制和TAL1/LMO2活性增强的 DN3 亚群，该亚群表达与干性和静止相关的基因。这种休眠的 DN3 子集在整个白血病发生过程中逐渐扩展，表现出内在的化学耐受性和与微小残留病相关的基因的富集。对TAL / LMO患者样本的检查揭示了 CD7 ⁺ CD1a ^-胸腺祖细胞中的类似模式，先前因其 L-IC 活性而被识别，证明了细胞周期限制和化学耐受性。我们的研究结果证实了在白血病发生过程中休眠、化疗耐受的 L-IC 的出现，并证明Tal1和Lmo2在转化过程中合作促进 DN3 静止。这项研究提供了对TAL1/LMO诱导的 T-ALL 及其治疗失败的临床意义的更深入的了解。