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Abstract 3581: Multi-omic analysis identifies mechanisms of resistance to CD19 CAR T-cell therapy in children with acute lymphoblastic leukemia


 Background: Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. Despite the survival rate of 90% for newly diagnosed children with ALL, the outcome for relapsed patients is historically poor with a less than 30% survival. CD19 CAR T-cell therapy (CART19) has shown remarkable response rates, between 80-90% in relapsed/refractory disease. Little is known about antigen-independent factors that predict initial resistance to CART19. We hypothesized that leukemias that are resistant to CART19 are distinct from sensitive leukemias and that these differences can be detected prior to therapy.
 Methods: To interrogate differences between resistant and sensitive leukemias, we obtained pre-treatment bone marrow aspirates (BMAs) from patients enrolled in a clinical trial at Seattle Children’s Hospital (PLAT-02). Samples were categorized based on patient response, with non-response defined as not achieving and maintaining minimal residual disease negativity at Day +63. Our study included 7 resistant and 7 sensitive leukemias as controls. We performed whole exome sequencing, bulk RNA-seq, PacBio-seq of the CD19 locus, array-based methylation, ATAC-seq, scRNA-seq, and CyTOF.
 Results: We found that non-response to CART19 is independent of leukemic subtype. Despite blasts being CD19+ in all patients by flow cytometry, we identified alternative splicing of CD19 in one non-responder, while the remaining non-responders expressed high levels of wildtype CD19. We discovered a distinctive DNA methylation pattern in the non-responders characterized by hypermethylation of PRC2 targets in embryonic and cancer stem cells (p = 8.15E-25) Furthermore, using gene set enrichment analysis of ATAC-seq data, we found increased accessibility of chromatin at regions associated with stem cell proliferation (NES = 2.31; p < 0.0001) and cell cycling (NES = 2.27; p < 0.0001). We found a greater similarity between accessibility patterns of non-responders to hematopoietic progenitors, including hematopoietic stem cells (p = 0.037) and common myeloid progenitors (p = 0.047). These findings were supported by an increased frequency of cell subpopulations expressing a multi-lineage phenotype (CD19, CD20, CD33, CD34; p = 0.009). Moreover, we find decreased expression of antigen presentation and processing pathways across all leukemic cells relative to responders (p = 0.0001).
 Conclusions: This study, one of the most comprehensive multi-omic analyses of samples from patients treated with CAR T-cells, identified resistance mechanisms that can be detected prior to treatment. We report the novel association of a stem cell phenotype, lineage plasticity, and decreased antigen presentation with resistance. These results support further refinement of eligibility for CART19 for children with leukemia and highlights the need for alternative of complimentary approaches for these patients.
 Citation Format: Katherine E. Masih, Rebecca Gardner, Hsien-Chao Chou, Abdalla Abdelmaksoud, Young K. Song, Luca Mariani, Vineela Gangalapudi, Berkley E. Gryder, Ashley Wilson, Serifat O. Adebola, Benjamin Z. Stanton, Chaoyu Wang, Xinyu Wen, Gregoire Altan-Bonnet, Michael C. Kelly, Jun S. Wei, Martha L. Bulyk, Michael C. Jensen, Rimas J. Orentas, Javed Khan. Multi-omic analysis identifies mechanisms of resistance to CD19 CAR T-cell therapy in children with acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3581.