Single-Cell Multi-Omics Analysis Reveals Cellular Subpopulations Associated with Relapse in High-Risk B-ALL Following Intensified Chemotherapy

Li Liu¹Xiaoyan Mao²Chunhui Yang³Na Li³Yan Zhou³Li Zhang⁴Nanjing Jiang⁵Yu Huang⁵Shigang Yin⁶Huangfan Xie⁶Xin Tian^5*

• ¹Qujing Medical College, Qujing, China
• ²The Affiliated Hospital of Southwest Medical University, Luzhou, China
• ³Kunming Children's Hospital, Kunming, China
• ⁴Dali University, Dali, China
• ⁵Chengdu Women’s and Children’s Central Hospital, Chengdu, China
• ⁶Southwest Medical University, Luzhou, China

Acute lymphoblastic leukemia (ALL) is the most prevalent malignant tumor in children, with B-cell ALL (B-ALL) accounting for 85% of cases. Despite advancements in chemotherapy and supportive care, a subset of high-risk B-ALL patients still experience relapse post-treatment. The molecular mechanisms underlying the relapses after intensified chemotherapy remain poorly understood. To address this, we utilized single-cell RNA sequencing (scRNA-seq) and single-cell Assay for Transposase Accessible Chromatin sequencing (scATAC-seq) to investigate the molecular landscape of peripheral blood mononuclear cells (PBMCs) from high-risk B-ALL patients following early intensified chemotherapy. Significant differences in cellular composition were observed between the remission and non-remission groups, with the non-remission group exhibiting a notable increase in HSC/MPP and Pro-B cells. Copy number variation (CNV) analysis also revealed that the CNV levels in HSC/MPP and Pro-B cells were higher in the non-remission group compared to other cell types. We subsequently identified a subcluster associated with resistance to intensified therapy within both the HSC/MPP and Pro-B cell groups. The drug-resistant subcluster of HSC/MPP cells was characterized by high expression of TCF4, EBF1, ERG, AL589693.1, and CRIM1, as well as enrichment of the allograft rejection pathway and the Notch signaling pathway. The drug-resistant subcluster of Pro-B cells was characterized by high expression of RPS29, B2M, RPL41, RPS21, NEIL1, AC007384.1, and CRIM1, as well as enrichment of the B cell receptor signaling pathway. Our study identified distinct cellular subpopulations associated with treatment failure, provide insights into the molecular mechanisms underlying treatment resistance in B-ALL and may inform the development of targeted therapies for high-risk patients.