Divergent roles of PKM2 in regulating PD-L1 and PD-L2 expression and their implications in human and mouse cancer models

Abstract

Cancer cells evade immune detection through checkpoint molecules like PD-L1 and PD-L2 which suppress T-cell activation. While PD-L1 is well-studied, the role of PD-L2 remains unclear. Pyruvate kinase M2 (PKM2), a metabolic enzyme, influences immune checkpoint regulation, but its role in PD-L1 and PD-L2 modulation is not well defined. Here, we investigate the role of pyruvate kinase M2 (PKM2) in modulating the immune checkpoint molecules PD-L1 and PD-L2 via GATA3 in cancer cells, with insights from both human and mouse models. We find that PKM2 enhances PD-L1 expression while inhibiting PD-L2, a dual regulatory mechanism that facilitates immune evasion. Knockdown and overexpression experiments revealed GATA3 as a key mediator. PKM2 knockout reduced GATA3 level, leading to decreased PD-L1 and increased PD-L2 expression. Chromatin immunoprecipitation (ChIP)-qPCR demonstrates that GATA3 functions as a direct transcription factor capable of binding to the promoters of PD-L1 and PD-L2. In silico analyses of 81 esophageal squamous cell carcinoma (ESCC) cases from the TCGA database demonstrate that PKM2 mRNA is unrelated to PD-L1 and PD-L2 expression but is negatively correlated with CD8+ T-cell infiltration in ESCC. To further validate these findings, we establish a xenograft model using immune-competent C57/BL6N mice, where knockdown of PKM2 results in significant downregulation of both PD-L1 and PD-L2 expression. Collectively, these findings underscore the divergent roles of PKM2 in regulating immune checkpoint expression in human and mouse cancer models and suggest that targeting the PKM2-GATA3 axis could enhance cancer immunotherapy by fine-tuning PD-L1 and PD-L2 levels.

1. Introduction

Cancer cells have developed sophisticated mechanisms to evade immune detection, enabling them to thrive and metastasize despite host immune system surveillance [1]. One of the most critical pathways involved in immune evasion is the regulation of immune checkpoint molecules, such as programmed death-ligand 1 (PD-L1) [2,3] and programmed death-ligand 2 (PD-L2) [4,5]. PD-L1, which binds to the PD-1 receptor on T cells, effectively disables the immune response against tumors, making it a prime target for cancer immunotherapies [6]. PD-L2, while less studied than PD-L1, has emerged as an important immune checkpoint molecule with unique roles in modulating the immune response in cancer. Unlike PD-L1, which is ubiquitously expressed on both tumor and immune cells, PD-L2 expression is more restricted and is predominantly found on antigen-presenting cells such as macrophages and dendritic cells. This selective expression pattern suggests that PD-L2 may play a distinct role in shaping the tumor microenvironment by influencing T-cell priming and activation. Recent studies have demonstrated that PD-L2 contributes to immune evasion by binding to the PD-1 receptor on T cells, thereby suppressing their activity. Moreover, PD-L2 has been implicated in the resistance to anti-PD-1 therapies, highlighting its potential as both a biomarker and a therapeutic target. For example, PD-L2 overexpression has been linked to poor prognosis in several cancers, including non-small cell lung cancer and gastric cancer, where its presence may indicate an alternative immune suppression pathway that is independent of PD-L1. These findings underscore the need for a deeper understanding of the functional role of PD-L2 in cancer and its implications for the development of more comprehensive immunotherapy strategies.

In addition to immune checkpoint regulation, cancer cells rely heavily on metabolic enzymes to support their rapid growth and immune evasion strategies. Pyruvate kinase M2 (PKM2), a key enzyme in glycolysis, has garnered attention not only for its metabolic role but also for its influence on various nonmetabolic functions [8,9], including gene expression and immune modulation [10]. PKM2 is known to interact with several transcription factors, including STAT3 [11] and HIF-1α [12], to regulate PD-L1 expression in cancer cells. However, the specific role of PKM2 in modulating PD-L2 and the underlying mechanisms remain poorly understood. This study aims to elucidate the divergent roles of PKM2 in regulating PD-L1 and PD-L2 expression via GATA3, providing insights into potential therapeutic targets for cancer immunotherapy.