P064 | XPO1 mutations drive BCR hyperactivation and early disease progression in chronic lymphocytic leukemia

Introduction. XPO1 encodes exportin-1, a key shuttle protein regulating nuclear–cytoplasmic transport. The recurrent hotspot mutation E571K has been linked to early disease progression in chronic lymphocytic leukemia (CLL). ATAC- and RNA-seq analyses revealed that XPO1-mutated CLL displays a distinct chromatin landscape with increased accessibility at transcription factor binding sites downstream B-cell receptor (BCR) and upregulation of MIR155HG. We hypothesize that mutant XPO1 enhances nuclear export of DUSP1, a nuclear phosphatase inhibiting MAPKs, leading to MIR155HG overexpression, increased miR-155 and reduced SHIP1, with consequent BCR amplification. This mechanism may be linked to the aggressive clinical behavior and shorter time to first treatment (TTFT) observed in XPO1-mutated CLL, although the precise molecular mechanisms have not been fully understood.Methods. To dissect the downstream effects of the E571K, HG3 CLL cell line was engineered using CRISPR-Cas9. A control was generated in parallel. Clones were validated by next generation sequencing and analyzed by flow cytometry for immunophenotype, phosphoflow and calcium flux upon anti-IgM/IgD stimulation. XPO1–target protein colocalization was assessed by ImageStream and Western blot.Results. HG3 CLL cell line was engineered though CRISPR-Cas9 to introduce E571K mutation together with two additional silent mutations disrupting the Cas9 recognition site, preventing further cleavage after repair. In parallel, a control cell line harboring only the two silent mutations was generated. All CRISPR-edited clones retained a CLL-like immunophenotype with lambda light chain restriction and surface IgM/IgD positivity. Both the XPO1E571K and control clones carried homozygous del13q and wild-type TP53. To evaluate BCR reactiveness, calcium flux was measured. The E571K clone displayed stronger calcium flux upon anti-IgM stimulation (p=0.009) compared to the control (Figure 1A) and a similar trend was observed upon anti-IgD stimulation. This is possibly explained by higher phosphorylated levels of p-SYK (p