Acute myeloid and lymphoid leukemias often harbor chromosomal translocations involving the KMT2A gene, encoding the KMT2A lysine methyltransferase (also known as mixed-lineage leukemia-1), and produce in-frame fusions of KMT2A to other chromatin-regulatory proteins. Here we map fusion-specific targets across the genome for diverse KMT2A oncofusion proteins in cell lines and patient samples. By modifying CUT&Tag chromatin profiling for full automation, we identify common and tumor-subtype-specific sites of aberrant chromatin regulation induced by KMT2A oncofusion proteins. A subset of KMT2A oncofusion-binding sites are marked by bivalent (H3K4me3 and H3K27me3) chromatin signatures, and single-cell CUT&Tag profiling reveals that these sites display cell-to-cell heterogeneity suggestive of lineage plasticity. In addition, we find that aberrant enrichment of H3K4me3 in gene bodies is sensitive to Menin inhibitors, demonstrating the utility of automated chromatin profiling for identifying therapeutic vulnerabilities. Thus, integration of automated and single-cell CUT&Tag can uncover epigenomic heterogeneity within patient samples and predict sensitivity to therapeutic agents.

急性髓细胞和淋巴细胞白血病通常存在涉及KMT2A的染色体易位基因，编码 KMT2A 赖氨酸甲基转移酶（也称为混合谱系白血病-1），并产生 KMT2A 与其他染色质调节蛋白的框内融合。在这里，我们为细胞系和患者样本中的多种 KMT2A 肿瘤融合蛋白绘制了跨基因组的融合特异性靶点。通过修改 CUT&Tag 染色质分析以实现完全自动化，我们确定了 KMT2A 肿瘤融合蛋白诱导的异常染色质调节的常见和肿瘤亚型特异性位点。KMT2A 肿瘤融合结合位点的一个子集由二价（H3K4me3 和 H3K27me3）染色质特征标记，单细胞 CUT&Tag 分析显示这些位点显示细胞间异质性，提示谱系可塑性。此外，我们发现基因体中 H3K4me3 的异常富集对 Menin 抑制剂敏感，展示了自动染色质分析在识别治疗漏洞方面的效用。因此，自动化和单细胞 CUT&Tag 的整合可以揭示患者样本中的表观基因组异质性并预测对治疗药物的敏感性。