CD19 CAR-T cell therapy in AML1-ETO positive Acute Myeloid Leukemia: A multi-omics analysis of response determinants

Abstract

Abstract Background: CD19-directed CAR-T therapy has demonstrated remarkable clinical efficacy in B-cell malignancies, leading to multiple FDA approvals for R/R B-cell lymphomas and leukemias. However, the therapeutic potential of CD19 CAR-T cells in AML remains largely unexplored. Recent studies have identified CD19 expression in a subset of AML cases harboring the AML1-ETO (RUNX1-RUNX1T1) fusion oncogene, suggesting a potential therapeutic vulnerability. This aberrant CD19 expression provides a scientific rationale for investigating CD19-targeted immunotherapy in this molecularly defined AML subgroup. This study systematically evaluated the clinical efficacy of CD19 CAR-T therapy in two R/R AML1-ETO positive patients and explored key factors associated with treatment response. Methods: Two R/R AML1-ETO positive AML patients were enrolled and received CD19 CAR-T cell therapy. A comprehensive multi-omics approach was employed, combining clinical outcomes with single-cell RNA sequencing and T-cell receptor sequencing of infused CAR-T products and paired post-infusion bone marrow samples. To further dissect the immune landscape, we performed computational integration with public databases to define immune cell subsets and characterize the bone marrow microenvironment. Results: Single-cell transcriptomic profiling revealed distinct immunological landscapes between responder and non-responder patients. Patient 1 achieved a morphological complete remission that persisted for 4 months. Phenotypic characterization of the CAR-T product demonstrated predominance of CD4+ T cells (80%) with elevated expression of the pro-inflammatory cytokine TNFα and co-stimulatory molecules ICOS, CD40LG, and OX40. Gene set enrichment analysis identified significant upregulation of immune activation and cytokine signaling pathways consistent with potent anti-leukemic activity. Longitudinal monitoring revealed that disease recurrence at month 4 coincided with profound downregulation of CD19 expression on leukemic blasts, indicating antigen escape as the primary resistance mechanism. Conversely, Patient 2 exhibited no appreciable clinical response. Transcriptional profiling of the infused product demonstrated significant upregulation of T-cell exhaustion markers LAG-3, TIGIT, and TIM-3, suggesting pre-existing functional impairment. Leukemic cells from patient 2 exhibited enrichment of oxidative phosphorylation, antigen processing, and anti-apoptotic pathways, indicating intrinsic resistance mechanisms. Furthermore, the bone marrow microenvironment was immunosuppressive, potentially exacerbating treatment resistance. TCR sequencing revealed comparable clonal diversity between both products, suggesting that TCR repertoire characteristics were not determinative of therapeutic outcome. Conclusions: CD19 CAR-T therapy exhibits preliminary efficacy in AML1-ETO positive AML, with CD4⁺ T-cell activation correlating with clinical response. Antigen escape and intrinsic resistance drive relapse, underscoring the necessity for combinatorial approaches with transplantation or next-generation CAR constructs to overcome these limitations.