P050 | SINGLE-CELL SEQUENCING REVEALS PATHWAYS AND PERTURBATIONS PREDICTIVE OF CENTRAL NERVOUS SYSTEM DISSEMINATION IN B-CELL ACUTE LYMPHOBLASTIC LEUKEMIA

BACKGROUND. Prognosis of B-ALL has improved particularly through targeted treatment and immunotherapy. CNS dissemination remains a life-threatening and poorly understood complication. METHODS. We performed single-cell RNA sequencing (scRNA-seq) on 7 B-ALL diagnostic bone marrow (BM) samples - 2 with hematologic and CNS relapse, 2 with isolated CNS relapse, 2 with hematologic relapse and 1 in continuous complete remission (n=15-20.000 cells/sample) - to characterize involved cell types, and explore differential clusters and their related transcriptomic fingerprints. RESULTS. Reference-based cell identity assignment by SingleR coupled with cell type-specific markers showed a prevalence of B cells at differentiation stages compatible with leukemic blasts (Fig. 1A). After unsupervised clustering, we verified the expression of a previously published 5-gene signature associated with CNS dissemination (Sapienza MR et al, Hematol Oncol. 2023). CNS+ ALL samples significantly enriched this signature, also verified at cluster resolution (Fig. 1B and C). We interrogated differentially-expressed genes (DEGs) between CNS+ only and BM relapse only samples (Fig. 1D). In CNS+ samples we found a significant enrichment of pathways associated with survival and proliferation including: NOTCH, PI3K-AKT, MAPK and JAK-STAT. Additionally, TGF-β pathway upregulation (SMAD3, TGFBR1/2, EP300) may promote a quiescent, immune-evasive and stem-like phenotype, while overexpression of THBS1 and TGFBRs could enhance leukemic adhesion to or remodeling of cerebral microcirculation. Leukemic cells from CNS+ samples also overexpressed key adhesion genes (ITGB1, ITGA5, LAMB2, THBS1), supporting extra-cellular matrix (ECM) interaction and CNS homing, along with CD36 and SDC1, which favor infiltration, immune evasion and niche adaptation. Finally, CNS+ samples significantly upregulated genes modulating tight junction dynamics (PRKCB, PRKCD, PRKCE, RHOA, RRAS), promoting junction disassembly and migration, and neuroactive ligand-receptor genes as NGFR (a key enhancer of CNS migration), P2RY14, P2RY13 and GAB1 (responsible for CNS chemotaxis). CONCLUSIONS. Leveraging on scRNA-seq, in patients with CNS+ ALL leukemic blasts are able to activate a coordinated program involving survival signaling (via TGF-β, NOTCH, PI3K/AKT and MAPK pathways), ECM interaction and junction remodeling (Fig. 1E). This enables them to adhere to, migrate across, and adapt to the CNS microenvironment, supporting persistence and therapy resistance.