A stem cell epigenome is associated with primary nonresponse to CD19 CAR T-cells in pediatric acute lymphoblastic leukemia.

CD19 CAR T-cell therapy (CD19-CAR) has changed the treatment landscape and outcomes for patients with pre-B cell acute lymphoblastic leukemia (B-ALL). Unfortunately, primary non-response (PNR), sustained CD19+ disease and concurrent expansion of CD19-CAR, occurs in 20% of patients and is associated with adverse outcomes. Although some failures may be attributable to CD19 loss, mechanisms of CD19-independent, leukemia intrinsic resistance to CD19-CAR remain poorly understood. We hypothesized that PNR leukemias are distinct compared to primary sensitive (PS) leukemias and that these differences are present prior to treatment. We utilized a multi-omic approach to investigate this in 14 patients (7 PNR and 7 PS) enrolled in the PLAT-02 trial at Seattle Children's Hospital. Long-read PacBio sequencing identified one PNR where 47% of CD19 transcripts had exon 2 skipping, but other samples lacked CD19 transcript abnormalities. Epigenetic profiling discovered DNA hypermethylation at genes targeted by polycomb repressive complex 2 (PRC2) in embryonic stem cells. Similarly, ATAC-seq revealed reduced accessibility at these PRC2 target genes with a gain in accessibility of regions characteristic of hematopoietic stem cells and multi-lineage progenitors in PNR. Single cell RNA-seq and CyTOF analyses identified leukemic subpopulations expressing multi-lineage markers and decreased antigen presentation in PNR. We thus describe the association of a Stem Cell Epigenome (SCE) with primary resistance to CD19-CAR therapy. Future trials incorporating these biomarkers, with addition of multi-specific CAR T-cells targeting against leukemic stem cell or myeloid antigens, and/or combined epigenetic therapy to disrupt this distinct stem cell epigenome may improve outcomes of patients with B-ALL.