P050 | Single-cell Sequencing Reveals Pathways and Perturbations Predictive of Central Nervous System Dissemination in B-cell Acute Lymphoblastic Leukemia
Year: 2025
Type: article
Abstract: 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.
Authors: L. V. Cappelli, F. Iannelli, D. Cardinali, L. Fumagalli, A. Natoni, M. Di Trani, S. Chiaretti, R. Chiarle, S. Pileri, R. Foà
Language: English
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W7093122908
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