FCRL1 promotes tonic PI3K/AKT signaling and constrains inducible BCR pathways 2260146

Abstract

Abstract Introduction Fc receptor-like 1 (FCRL1) is an understudied B cell immunoregulatory protein with tyrosine-based signaling in humans and mice that peaks on naïve and memory subsets. Among malignancies, FCRL1 is upregulated by chronic lymphocytic leukemia (CLL) and germinal center (GC)-derived B cell lymphomas that rely on tonic PI3K/AKT signals. Early studies found FCRL1 could co-activate B cell receptor (BCR) functions, but broader understanding of its regulatory roles in human B cells remains limited. Methods To interrogate its biology, we used a CRISPR/Cas9 strategy to target FCRL1 in a GC-derived B cell lymphoma cell line that highly expresses it. Single-cell sorting was performed to isolate and expand FCRL1-deficient clones. Indel profiling, mRNA quantification, and flow cytometry analyses confirmed FCRL1 knockout at both the transcript and protein levels. To comprehensively define FCRL1-dependent pathways in an unbiased manner, RNA sequencing (RNA-seq) and gene set enrichment analysis (GSEA) were used to compare FCRL1-sufficient and -deficient cells. Transcriptomic findings were validated by Western blot and phospho-flow cytometry under basal conditions and following BCR stimulation. Results Expanded FCRL1-deficient clones showed altered granularity and enhanced BCR-mediated apoptosis. RNA-seq and gene set enrichment analysis (GSEA) revealed that the loss of FCRL1 drove the upregulation of multiple hallmark pathways including mTORC1, NF-kB, apoptosis, INFg, and hypoxia, indicating a repressive role for it in resting B cells. Cells lacking FCRL1 also exhibited reduced global tyrosine phosphorylation (pY) and p-AKT (T308), but had higher Src-family kinase (SFK) and p-PLCg2 activity both at rest and upon BCR ligation. Conclusion These results implicate FCRL1 as a dual-regulator, which promotes tonic PI3K/AKT signals but limits inducible BCR activation, and expose its potential significance in B cell-related malignancies and humoral disorders. Funding Source Leukemia Lymphoma Society and the UAB O’Neal Comprehensive Cancer Center Topic Categories Immune Response Regulation: Molecular Mechanisms (IRM)