原发性对酪氨酸激酶抑制剂（TKI）的抵抗是慢性髓细胞白血病最佳治疗结果的重要障碍，但对于造成反应异质性的因素知之甚少。通过单细胞RNA测序，我们确定了预处理骨髓单个核细胞中的八个统计学显著特征，与敏感性（主要分子反应或MMR）或伊马替尼极端抵抗力（最终爆发危机转化）相关。通过机器学习，我们还确定了LSC和NK基因表达谱预测伊马替尼反应的>80％准确性，包括零误报预测BC。一个典型的红细胞指定（TAL1/KLF1/GATA1）调控子是患者骨髓造血干细胞（LSCs）中MMR的标志，并与体内红细胞祖细胞（ERP）扩增有关（p<0.05），在体外表现出明显的2-10倍（在A组中为6.3倍，在C组中为1.09倍）的红细胞过多的偏见。值得注意的是，与骨髓祖细胞相比，ERP表现出极高的TKI敏感性（p<0.001）。这些LSC特征随着渐进性抵抗而丧失，在转化的患者中出现MYC和IRF1驱动的炎症调控子。MMR患者还表现出一种通常罕见的高功能自适应样NK细胞亚型（CD57+NKG2C+）的56倍扩增（p<0.01），随着渐进性抵抗而减少，而注定发展为BC的患者则积累了优先NK细胞耐受的抑制性NKG2A+ NK细胞（通过HLA-E结合目标细胞）。最后，我们开发了一组简明的抗体以验证我们的单细胞RNA测序结果。该组抗体将有助于原发性抵抗的前瞻性研究，并评估TKI反应异质性中预定因素与获得性因素的贡献。Copyright © 2023 American Society of Hematology.

Primary resistance to tyrosine kinase inhibitors (TKI) is a significant barrier to optimal outcomes in chronic myeloid leukemia, but little is known about the factors contributing to response heterogeneity. Using scRNA-sequencing, we identified eight statistically significant features in pretreatment bone marrow mononuclear cells which correlated with either sensitivity (major molecular response or MMR) or extreme resistance to imatinib (eventual blast crisis transformation). Employing machine-learning, we also identified LSC and NK gene expression profiles predicting imatinib response with >80% accuracy, including zero false positives for predicting BC. A canonical erythroid-specifying (TAL1/KLF1/GATA1) regulon was a hallmark of LSCs from patients with MMR and was associated with erythroid progenitor (ERP) expansion in vivo (p<0.05), and a marked 2-10-fold (6.3-fold in Group A vs 1.09-fold in Group C) erythroid over myeloid bias in vitro. Notably, ERPs demonstrated exquisite TKI sensitivity compared to myeloid progenitors (p<0.001). These LSC features were lost with progressive resistance, and in patients who transformed, MYC- and IRF1-driven inflammatory regulons became evident. Patients with MMR also exhibited a 56-fold expansion (p<0.01) of a normally rare subset of hyperfunctional adaptive-like NK cells (CD57+NKG2C+) which diminished with progressive resistance, while patients destined for BC accumulated inhibitory NKG2A+ NK cells favoring NK cell tolerance (through HLA-E binding on target cells). Finally, we developed a parsimonious set of antibodies to validate our scRNA-seq findings. This panel will be useful in prospective studies of primary resistance, and assessing the contribution of predetermined versus acquired factors in TKI response heterogeneity.Copyright © 2023 American Society of Hematology.