Regulatory Mechanisms and Therapeutic Targeting of PD-L1 Trafficking and Stability in Cancer Immunotherapy

Abstract

The PD-L1/PD-1 signaling axis is a pivotal regulator of T-cell activity and a key mechanism by which tumors evade immune surveillance. Inhibiting this pathway has resulted in significant anti-tumor responses, establishing immune checkpoint blockade (ICB) as a crucial component of modern cancer therapy. However, many patients with high PD-L1 expression do not respond to PD-1/PD-L1 blockade, underscoring the necessity for a deeper investigation into the mechanisms underlying this resistance. Recent studies have identified DRG2 as a critical modulator of anti-PD-1 therapeutic efficacy. While DRG2 depletion enhances IFN-γ signaling and increases the overall PD-L1 levels, it disrupts the recycling of endosomal PD-L1, resulting in reduced surface expression and impaired PD-1 interaction, ultimately compromising therapeutic outcomes. Furthermore, TRAPPC4, HIP1R, and CMTM6 help stabilize PD-L1 by preventing lysosome degradation. When depleted, these proteins have been shown to boost the body's immune response against tumors. Research into the complex regulatory mechanisms of PD-L1 suggests that targeting DRG2, TRAPPC4, HIP1R, and CMTM6 could enhance the effectiveness of PD-1/PD-L1 blockade therapies. This strategy could create exciting new possibilities for cancer immunotherapy and improve patient outcomes.

Keywords: CMTM6; HIP1R; PD-1 pathway; PD-L1 trafficking; Ras-associated binding proteins (Rab); TRAPPC4; immune checkpoint blockade; immune checkpoint blockade DRG2; post-translational modifications; tumor immune evasion.

Figure 1

Post-translational modifications (PTMs) regulate PD-L1 trafficking, stability, and function. This schematic illustrates the structural domains of PD-L1 and highlights key post-translational modifications that modulate its intracellular trafficking and stability. PD-L1 consists of a signal peptide (SIG), IgV-like domain (residues 19–127), IgC-like domain (128–239), transmembrane domain (TM), and intracellular domain (ICD). N-linked glycosylation at asparagine residues N192 and N200, mediated by B3GNT3, and at N219 (enzyme unknown, indicated by “?”), stabilizes PD-L1 and enhances its interaction with PD-1. Phosphorylation at threonine and serine residues (T180/S184 by GSK3β and S195 by AMPK) influences PD-L1 degradation and trafficking. Palmitoylation at cysteine residue C272, catalyzed by the DHHC family of palmitoyltransferases, protects PD-L1 from lysosomal degradation and supports its membrane localization. Acetylation of PD-L1 at lysine 263 (K263) by p300 promotes its retention on the cell membrane, while acetylation at lysine 270 (K270) by the HBXIP/p300 complex leads to its accumulation in the cytoplasm. Together, these PTMs finely tune PD-L1’s availability at the cell surface and contribute to immune evasion mechanisms in cancer cells.

Figure 2

Role of post-translational modifications (PTMs) and their regulatory enzymes in PD-L1 stability. This circular diagram summarizes the major PTMs that modulate PD-L1 turnover and subcellular localization, along with the key enzymes responsible for these modifications. Glycosylation, mediated by STT3A and B3GNT3, stabilizes PD-L1 and enhances its interaction with PD-1. Ubiquitination by E3 ligases such as Cullin-3-SPOP and STUB1 targets PD-L1 for proteasomal degradation. In contrast, deubiquitination by CSN5, USP9X, OTUB1, and others counteracts degradation and prolongs PD-L1 stability. Phosphorylation by kinases, including JAK1, AMPK, and GSK3β, influences both the glycosylation and degradation pathways. Palmitoylation by ZDHHC9 and DHHC3 enhances PD-L1 membrane localization and prevents lysosomal degradation. These PTMs dynamically regulate PD-L1 surface levels and contribute to tumor immune evasion.

Figure 3

DRG2 regulates key signaling pathways involved in PD-L1 stability and expression. This circular schematic summarizes the role of developmentally regulated GTP-binding protein 2 (DRG2) in modulating upstream regulators that influence PD-L1 trafficking, degradation, and surface expression. DRG2 affects the EGFR and VEGF pathways, both of which modulate PD-L1 expression and trafficking. It also regulates hypoxia-inducible factor 1α (HIF-1α), which contributes to PD-L1 stabilization under hypoxic conditions. DRG2-mediated suppression of GSK3β prevents the phosphorylation-dependent degradation of PD-L1, enhancing its stability. Additionally, DRG2 modulates NF-κB signaling, which promotes PD-L1 transcription and prevents its degradation. These interactions position DRG2 as a central node in controlling PD-L1 availability and immune evasion in tumors.

Figure 4

Intracellular trafficking and regulation of PD-L1 by DRG2, TRAPPC4, HIP1R, and CMTM6 in cancer cells. This schematic illustrates the endocytic trafficking route of PD-L1 and the roles of regulatory proteins in its intracellular localization and stability. Upon internalization from the plasma membrane, PD-L1 enters early endosomes marked by Rab5. DRG2 facilitates the stabilization of endosomal tubules, promoting PD-L1 sorting and trafficking. PD-L1 may be directed toward late endosomes marked by Rab7 for lysosomal degradation or recycled back to the plasma membrane via Rab11-positive recycling endosomes. TRAPPC4 and CMTM6 promote the recycling of PD-L1 to the cell surface, supporting immune evasion by maintaining PD-L1 availability for interaction with PD-1 on T cells. In contrast, HIP1R directs PD-L1 to lysosomes for degradation. CMTM6 also stabilizes PD-L1 on the plasma membrane by preventing its degradation. Together, these pathways tightly regulate PD-L1 localization and surface expression, which influence the tumor’s ability to suppress T-cell-mediated immune responses.