The TrkAIII splice variant oncoprotein enhances PD-L1 expression in human SH-SY5Y neuroblastoma cells

BACKGROUND-AIM Neurotrophin receptor TrkA alternative splicing, resulting in TrkAIII variant expression, positively correlates with posttherapeutic relapse and advanced stage metastatic disease in pediatric neuroblastomas, MCPyV positive Merkel cell carcinomas and cutaneous malignant melanomas. The TrkAIII variant is characterized by exons 6 and 7 skipping, which encode regulatory domains, the absence of which result in intracellular accumulation, and ligand-independent cell cycle and stress-regulated activation. TrkAIII activation signals through PI3K-AKT but not Ras/MAPK, exhibits oncogenic activity in NB and melanoma models, and enhances both stress and chemotherapeutic resistance. Immune checkpoint inhibition mediated by programmed death ligand PD-L1 and its receptor PD-1 plays a significant role in NB pathogenesis but the regulation of this important immune-evasion pathway is poorly understood in NB. Here, we have investigated whether TrkAIII regulates PD-L1 expression and function in TrkAIII expressing SH-SY5Y NB cells. METHODS Real Time qPCR, RT-PCR and Western blot comparisons of PD-L1 mRNA and protein expression in control, TrkA and TrkAIII transfected SH-SY5Y cells, under untreated conditions and following treatment with entrectinib Trk, LY-294002 PI3K and PD98059 MEK inhibitors. PD-L1 function was assayed by IL-2 ELISA in co-cultures of control, TrkA and TrkAIII SH-SY5Y cells incubated for 48 hours with PHA and TPA-activated Jurkat T cells. RESULTS TrkAIII SH-SY5Y cells express significantly higher levels of PD-L1 than control or TrkA SH-SY5Y counterparts. Constitutive PD-L1 mRNA expression in TrkAIII but not control or TrkA SH-SY5Y cells was significantly inhibited by entrectinib and LY-294002 but not PD98059. Jurkat co-culture with control, TrkA and TrkAIII SH-SY5Y cells significantly reduced IL-2 production, confirming PD-L1 function. CONCLUSIONS The data implicate TrkAIII in promoting PD-L1 expression via PI3K in human SH-SY5Y NB cells, supporting a rational for using clinically approved therapeutic Trk inhibitors in combination with PD1/PD-L1 inhibiters in TrkAIII expressing NB.