MEK inhibition mediates immunomodulatory factors and cell populations in advanced biliary tract cancer Open Access

Joyce, Amanda Nicole Ruggieri (Fall 2022)

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Biliary tract cancer (BTC) is a rare group of aggressive gastrointestinal malignancies with a five-year survival rate of less than 10%. Patients are often diagnosed at late stages when tumors are refractory to treatment and resection is not possible. The Ras/Raf/MEK/ERK signaling pathway has an important role in the development and progression of this disease, but inhibitors of MEK have had limited success in patients with BTC. Immunotherapy has also produced modest efficacy in advanced BTC and is largely limited by a lack of tumor-infiltrating lymphocytes characteristic of these tumors. However, MEK inhibitors have been shown to increase infiltration of CD8+ T cells in various other tumor models and combine with immune checkpoint blockade to improve anti-tumor activity. A recent clinical trial demonstrated that combining the MEK inhibitor cobimetinib with the PD-L1 blocking antibody atezolizumab leads to improved progression-free survival in patients with advanced, metastatic BTC. In this dissertation, we investigate the effects of systemic MEK inhibition (MEKi) combined with PD-L1 blockade in these patients and seek to understand the mechanism by which MEK inhibition leads to increased T cell infiltration and improved anti-tumor activity. We show that dual MEK/PD-L1 blockade alters the concentrations of growth factors and populations of immune cells in peripheral blood, correlating with improved clinical outcomes. We also demonstrate that MEK inhibition does not limit cell viability in BTC cell lines in vitro, but does alter the production of immunomodulatory cytokines and chemokines, suggesting that MEKi elicits anti-tumor activity on the tumor microenvironment rather that on tumor cells directly. Finally, we highlight that MEKi limits CD8+ T cell activation in patients with advanced BTC and discuss the role of T cell costimulatory agonists in enhancing the efficacy of dual MEK/PD-L1 blockade. Overall, the data presented here provides a foundation for future investigation to identify potential therapeutic strategies to enhance the efficacy of MEK inhibitors with immune checkpoint blockade in this aggressive disease.

Table of Contents

Table of Contents



Table of Contents

List of Figures

List of Tables

Chapter 1: Introduction

1.1. Introduction

1.2. Fundamentals of the human immunological response

1.3. Inflammation-mediated tumorigenesis and evasion of the immune response

1.3.2. Immunosuppressive cells facilitate tumor progression

1.4. Immune checkpoint blockade therapy and resistance mechanisms in cancer

1.4.1. Fundamentals of immune checkpoint blockade for the treatment of cancer

1.4.2. Mechanisms of resistance to immune checkpoint blockade

1.5. Biliary Tract Cancers – Epidemiology, molecular basis, tumor microenvironment

1.5.1 Physiological classification and risk factors

1.5.2. Tumor immune microenvironment of BTC

1.6. Ras/Raf/MEK/ERK Pathway in cancer development, progression, and anti-tumor immune responses

1.6.1. Functions of oncogenic Ras signaling in tumor development

1.6.2. Immunological implications of MEK/ERK signaling

1.7. Therapeutic development in BTC – clinical trials and roadblocks to overcome

1.7.1. Molecular targeted treatment strategies for BTC

1.7.2. Progress in immune checkpoint blockade development for advanced BTC

1.8. Summary, scope, and goals for dissertation

1.9. Tables

Chapter 2: Combined MEK/PD-L1 inhibition alters peripheral cytokines and lymphocyte populations correlating with improved clinical outcomes in advanced biliary tract cancer

2.1. Author’s Contribution and Acknowledgements of Reproduction

2.2 Abstract

2.3. Introduction

2.4. Results

2.4.1. Differential production of cytokines, chemokines, and growth factors in the blood of BTC patients compared to healthy donors

2.4.2. PD-1 and BTLA-expressing T cells are elevated in BTC patients

2.4.3. MEK inhibition significantly alters growth factor levels when combined with anti-PD-L1 therapy in advanced BTC that correlate with improved clinical outcomes

2.4.4. Regulation of T lymphocyte populations with an exhausted phenotype by dual MEK/PD-L1 blockade correlates with improved clinical outcomes

2.4.5. High baseline CD8+ T cells correlate with improved overall survival following dual MEK/PD-L1 blockade

2.5. Discussion

2.6. Materials and Methods

2.6.1. Patients and Treatment

2.6.2. Cytokine, chemokine, and growth factor analysis

2.6.3. Flow cytometry

2.6.4. Statistical analyses

2.7. Acknowledgements

2.8. Tables

Chapter 3: MEK inhibition alters immunomodulatory factor production in biliary tract cancer cell lines, modulates immune cell phenotypes, and impairs T cell activation in biliary tract cancer patients.

3.1. Author Contributions and Acknowledgement of Reproduction

3.2 Introduction

3.3 Results

3.3.1 Cobimetinib inhibits ERK phosphorylation in BTC cell lines but does not interrupt cell viability

3.3.2 MEK inhibition modulates the production of soluble factors by BTC cells

3.3.3. Comprehensive immune profiling of advanced BTC patients receiving atezolizumab with or without cobimetinib revealed relationships between immune checkpoint expression and clinical outcomes.

3.3.4. Addition of cobimetinib impairs T cell activation in patients receiving concurrent PD-L1 inhibition

3.4 Discussion

3.5 Methods

3.5.1. Cell culture

3.5.2. Immunoblot analysis

3.5.3. Cell viability assay

3.5.4. Cytokine, chemokine, and growth factor analysis

3.5.5. Flow cytometry

3.5.6. Patient sample processing

3.5.7. Statistical Analysis

3.6. Tables

Chapter 4: Conclusions, Future Directions, and Closing Remarks

4.1 Introduction

4.2. MEK inhibition mediates the production of soluble factors in the tumor microenvironment

4.3. Peripheral factors altered by MEK inhibition in advanced BTC patients

4.4. Roles for other immune checkpoint molecules in advanced BTC and immunotherapeutic development

4.5. Restoring MEK-mediated inhibition of T cell activation for improved clinical outcomes in advanced BTC

4.6. Modulation of the tumor microenvironment as a means to regulate antitumor T cell activity

4.7. Future studies and concluding remarks

Chapter 5: References

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