Enhancing Immunotherapy Potential Through PD-L1 Upregulation—Combination of Anti-PD-L1 and mTOR Inhibitors

Background Introduction

In recent years, immune checkpoint inhibitors (ICIs) have made significant progress in cancer treatment, particularly in the treatment of urothelial cancer (UC). PD-1/PD-L1 inhibitors restore T cell anti-tumor activity by blocking the binding of PD-1 and PD-L1, significantly improving survival in some patients. However, despite the durable efficacy of these drugs in certain patients, the overall response rate remains low, with only 20-23% of patients benefiting. Therefore, improving the efficacy of immunotherapy has become a key focus of current research.

The mTOR (mechanistic target of rapamycin) signaling pathway plays an important role in various cancers, especially in bladder cancer (BC), where mTOR pathway mutations are relatively common. Previous studies have suggested that mTOR inhibitors may enhance the efficacy of immunotherapy by modulating PD-L1 expression in the tumor microenvironment. However, the specific relationship between mTOR inhibitors and PD-L1 expression has not been fully explored. To address this, a research team from IMIM (Hospital del Mar Research Institute) and Dana-Farber Cancer Institute conducted a study to investigate the effects of mTOR inhibitors on PD-L1 expression and evaluate the potential of their combination with anti-PD-L1 antibodies.

Source of the Paper

This paper was co-authored by Anna Hernández-Prat, Joaquim Bellmunt, and other researchers from IMIM, Dana-Farber Cancer Institute, Harvard Medical School, and other institutions. The paper was accepted on June 25, 2024, and published online on September 11, 2024, in the journal Molecular Oncology, with the DOI 10.¹⁰⁰²⁄₁₈₇₈-0261.13699.

Research Process and Results

1. Research Process

The research team first evaluated the effects of three PI3K/AKT/mTOR pathway inhibitors (TAK-228, Everolimus, and TAK-117) on PD-L1 expression in bladder cancer cell models. The specific process was as follows:

a) Cell Culture and Treatment

The study used seven bladder cancer cell lines (T24, HT-1197, TCCSUP, UM-UC-3, J82, RT4, and CAL-29), representing tumor models with high and low PD-L1 expression. Cells were cultured under standard conditions and treated with TAK-228, Everolimus, and TAK-117 for 48 hours.

b) Detection of PD-L1 and HLA-I Expression

Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were used to detect PD-L1 mRNA and protein expression levels. Additionally, flow cytometry was used to detect cell surface PD-L1 and HLA-I expression.

c) Mechanism Investigation

To explore the mechanism by which TAK-228 upregulates PD-L1, the research team analyzed the effects of mTOR inhibitors on S6 and GSK3β phosphorylation and evaluated the role of EGF (epidermal growth factor) and IFN-β (interferon-β) in PD-L1 expression. By blocking the EGF receptor (EGFR) and IFN-β receptor (IFNAR2), the role of these factors in PD-L1 upregulation was further validated.

d) Immune Cell Co-Culture Experiments

The research team co-cultured bladder cancer cells with peripheral blood mononuclear cells (PBMCs) or CD8+ T cells from healthy donors to assess the impact of TAK-228 on the ability of immune cells to kill tumor cells. Additionally, the role of the anti-PD-L1 antibody (Atezolizumab) in enhancing immune cell killing was studied.

e) Patient-Derived Tumor Sample Studies

The research team also used patient-derived tumor samples (Patient-Derived Explants, PDE), treated them ex vivo with TAK-228, and detected changes in PD-L1 expression through immunohistochemistry (IHC).

2. Main Results

a) Baseline Expression of PD-L1 and HLA-I

The study found significant differences in PD-L1 expression among different bladder cancer cell lines under basal conditions. T24, TCCSUP, and HT-1197 cell lines exhibited high PD-L1 expression, while RT4, UM-UC-3, CAL-29, and J82 cell lines exhibited low PD-L1 expression. Additionally, HLA-I expression levels also varied, with CAL-29, TCCSUP, and J82 cell lines showing high HLA-I expression, while T24, RT4, UM-UC-3, and HT-1197 cell lines showed low HLA-I expression.

b) Effects of mTOR Inhibitors on PD-L1 Expression

TAK-228 and Everolimus significantly increased PD-L1 mRNA and protein expression levels, particularly in cell lines with high PD-L1 expression. TAK-228 also enhanced PD-L1 stability by inhibiting S6 phosphorylation and activating GSK3β. Furthermore, TAK-228 promoted PD-L1 expression by increasing the secretion of EGF and IFN-β.

c) Immune Cell Co-Culture Experiments

The study found that bladder cancer cells treated with TAK-228 exhibited some resistance to the killing effects of PBMCs and CD8+ T cells. However, the addition of the anti-PD-L1 antibody (Atezolizumab) partially reversed this resistance, particularly in T24 cells.

d) Patient-Derived Tumor Sample Studies

In patient-derived tumor samples, TAK-228 significantly increased PD-L1 expression, especially in high-grade tumors. This result was consistent with in vitro findings, further supporting the potential of TAK-228 in enhancing PD-L1 expression.

3. Conclusions and Significance

This study reveals that mTOR inhibitors, particularly TAK-228, may enhance the efficacy of anti-PD-L1 immunotherapy by upregulating PD-L1 expression. This finding provides a theoretical basis for the combination of mTOR inhibitors and immune checkpoint inhibitors, especially in tumors with low PD-L1 expression, where mTOR inhibitors may sensitize tumors to immunotherapy by inducing PD-L1 expression.

4. Research Highlights

• Mechanism of PD-L1 Upregulation: The study is the first to detail the molecular mechanism by which mTOR inhibitors upregulate PD-L1 expression through EGF- and IFN-β-dependent pathways.
• Immune Cell Co-Culture Experiments: Through PBMC and CD8+ T cell co-culture experiments, the study validated the impact of TAK-228 on immune cell killing of tumor cells and demonstrated the potential enhancing effect of anti-PD-L1 antibodies.
• Validation with Patient-Derived Tumor Samples: The study not only validated the role of TAK-228 in cell lines but also further confirmed its clinical potential using patient-derived tumor samples.

Summary

This study provides important experimental evidence for the combination of mTOR inhibitors and immune checkpoint inhibitors, particularly in tumors with low PD-L1 expression, such as bladder cancer. mTOR inhibitors may enhance the efficacy of immunotherapy by inducing PD-L1 expression. This discovery offers new insights for the design of future clinical trials and holds promise for bringing benefits to more cancer patients.