A brain organoid/ALL co-culture model reveals the AP-1 pathway as critically associated with CNS involvement of BCP-ALL

Central nervous system (CNS) involvement remains a clinical hurdle in treating childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). The disease mechanisms of CNS leukemia are primarily investigated using 2D cell culture and mouse models. Given the variations in cellular identity and architecture between the human and murine CNS, it becomes imperative to seek complementary models to study CNS leukemia. Here, we present a first-of-its-kind 3D co-culture model combining human brain organoids and BCP-ALL-cells. We noticed significantly higher engraftment of BCP-ALL cell lines and patient-derived xenograft (PDX) cells in cerebral organoids as compared to non-ALL-cells. To validate translatability between organoid co-culture and in vivo murine models, we confirmed that targeting CNS leukemia relevant pathways like CD79a/Igα or CXCR4- SDF1 reduced the invasion of BCP-ALL-cells into organoids. RNA sequencing and functional validations of organoid-invading leukemia cells compared to the non-invaded fraction revealed significant upregulation of AP-1 transcription factor-complex members in organoid-invading cells. Moreover, we detected a significant enrichment of AP-1 pathway genes in ALL-PDX-cells recovered from the CNS compared to spleen blasts of mice transplanted with TCF3::PBX1+ PDX-cells, substantiating the role of AP-1 signaling in CNS disease. Accordingly, we found significantly higher levels of the AP-1-gene JUN in patients initially diagnosed as CNS-positive compared to CNS negative cases as well as CNS-relapse vs non-CNS-relapse cases in a cohort of 100 BCP-ALL-patients. Our results suggest CNS-organoids as a novel model to investigate CNS-involvement and identify the AP-1 pathway as a critical driver of CNS-disease in BCP-ALL. 1*10^5 cells of the two B cell precursor leukemia cell lines 697 and Kasumi 2 were cococultured with iPSC derived human cerebral organoids over the time course of 14 days. The cells that remainied in suspension and did not invade were used as controlls against the invading cells. The orgainoids were disolved via strainers and the leukemia cells were isolated via CD19+ Selection. The library preparation was performed according to the manufacturer's protocol using the 'Illumina Stranded Total RNA Prep, Ligation with Ribo-Zero Plus' (Illumina Inc.) kit, after purity and viability controlls. Bead-purified libraries were normalized and sequenced on the NextSeq2000 system.