Preliminary exploration of the synergistic mechanism between EBV LMP1 and MYD88 L265P mutation on the efficacy of PD-1 Inhibitors in ABC-DLBCL

Background:​ Activated B-cell-like diffuse large B-cell lymphoma (ABC-DLBCL) is aggressive with poor prognosis. Epstein-Barr virus (EBV) infection and the MYD88 L265P mutation are key molecular drivers in ABC-DLBCL, typically exhibiting mutual exclusivity, potentially due to functional overlap. PD-1 inhibitors show limited efficacy in unselected relapsed/refractory DLBCL patients. However, our retrospective analysis of relapsed/refractory non-GCB DLBCL patients suggested that rare "double-positive" (EBV-positive/MYD88 L265P mutant) patients had higher response rates to PD-1 inhibitors. Objective:​ This study investigated how EBV LMP1 modulates intracellular signaling in the context of MYD88 L265P mutation (causing constitutive JAK/STAT activation) and evaluated its synergistic effect with the PD-1 inhibitor tislelizumab. Methods:​ Clinical data from 54 relapsed/refractory non-GCB DLBCL patients treated with PD-1 antibodies were analyzed. Patients were stratified by EBER ISH (EBV) and MYD88 sequencing. The HBL-1 cell line (MYD88 L265P mutant, EBV-negative) was used. LMP1 was overexpressed via transfection. Groups: control, tislelizumab alone, LMP1 overexpression, LMP1+tislelizumab. qPCR measured mRNA levels of NF-κB p65, MMP9, c-Myc, TLR3, STAT3, JAK3, LMP1, PD-1, PD-L1. Western blot detected protein expression and phosphorylation of key molecules. Results:​ Among 21 patients with complete molecular data, both "double-positive" patients achieved an objective response (100%), compared to 22.2% in EBV-negative/MYD88 mutant (n=9) and 60.0% in EBV-positive/MYD88 wild-type (n=5) groups. HBL-1 cells showed high baseline p-JAK3 and p-STAT3. LMP1 overexpression suppressed p-JAK3 and p-STAT3. HBL-1 cells expressed tumor cell-intrinsic PD-1 mRNA. Tislelizumab alone inhibited p-JAK3/p-STAT3 phosphorylation. The combination of LMP1 overexpression and tislelizumab showed the strongest inhibition, indicating synergy. Conclusion​: MYD88 L265P mutation may cause tumor cell addiction to STAT3 activation maintained by tumor cell-intrinsic PD-1 signaling. LMP1 co-existence adds inhibitory pressure, potentially enhancing this dependency. Tislelizumab blockade may yield a synthetic lethal effect. Combined EBV status and MYD88 profiling could predict PD-1 inhibitor sensitivity in DLBCL, requiring further validation.