Malasomma, Chiara (2025) PD-L1 BEYOND IMMUNE EVASION: NUCLEAR FUNCTIONS SUPPORTING GLIOBLASTOMA PROLIFERATION. [Tesi di dottorato]

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Abstract

Glioblastoma (GBM) is the most aggressive primary malignancy of the central nervous system, marked by high mortality and frequent recurrence. Outcomes remain poor despite standard care, highlighting the need for new treatment approaches. A hallmark of GBM is the high expression of programmed cell death ligand-1 (PD-L1), which promotes immune evasion. Nevertheless, clinical trials of anti-PD-1/PD-L1 antibodies have yielded limited benefit in GBM. The aim of this study was to determine whether PD-L1 exerts functions beyond its canonical role in immune evasion of GBM, particularly, whether it promotes GBM proliferation and growth. To this end, PD-L1 expression in glioblastoma cells was modulated by silencing or over-expression. Proliferation was quantified by flow-cytometric Ki67 immunostaining and by RT-qPCR of proliferation-associated transcripts (CCND1, PCNA, MYC). These experiments demonstrated that glioblastoma cell proliferation scales with PD-L1 abundance. Cellular fractionation demonstrated that PD-L1 was both cytosolic and nuclear in GBM cells. Moreover, subnuclear fractionation showed glycosylated PD-L1 (~50kDa, MW) in the soluble nucleoplasmic fraction, while a lower MW band (~37kDa), potentially a PD-L1 splice isoform, was detected in the chromatinbound fraction. Because a previous study in breast cancer showed that the nuclear entry of PD-L1 is regulated by acetylation dynamics, and that trichostatin A (TSA) reduces PD-L1 nuclear localization, we investigated in GBM cells the effect of TSA on nuclear PD-L1 level and cell proliferation. We found that TSA reduced GBM proliferation; however, unexpectedly, analysis of nuclear fractions revealed an increase, rather than a decrease, in nuclear PD-L1 levels. Since this variation in nuclear PD-L1 was accompanied by increased levels of the transcriptional co-regulator YY1, we next explored a possible interaction between PD-L1 and YY1. To this end, we immunoprecipitated nucleoplasmic YY1 in GBM cells with or without PD-L1 overexpression. Although our results apparently excluded a direct interaction between the two proteins, they revealed a markedly reduced acetylation pattern of YY1 in PDL1-overexpressing GBM cells compared with controls. Notably, acetylated YY1 often functions as a transcriptional co-repressor. Consistent with this notion, preventing YY1 deacetylation with TSA counteracted the PD-L1-induced upregulation of CCND1 expression. In conclusion, our study identifies a tumor-intrinsic role for PD-L1 in promoting GBM proliferation and raises the hypothesis that YY1 acts as an inhibitory co-transcriptional regulator downstream of PD-L1. These findings support a model in which PD-L1 contributes to glioblastoma malignancy through acetylation-sensitive, tumor-intrinsic mechanisms that extend beyond immune evasion, and point to epigenetic modifiers as potential therapeutic targets.

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