Understanding the molecular basis of cancer immunotherapy for melanoma

Abstract

In recent years, cancer immunotherapy has emerged as a highly promising treatment option, aiming to harness the power of the immune system to combat cancer, especially for melanoma. However, the intricate nature of the tumor microenvironment and its various components present significant challenges in cancer immunotherapy. Furthermore, intrinsic and therapy-induced resistance are also clinical challenges that restrict the effectiveness of cancer immunotherapy. Mechanisms such as the suppressive tumor microenvironment, tumor heterogeneity and immune dysregulation, have been demonstrated to contribute to immune resistance. To overcome these limitations, the development of next-generation checkpoint inhibitors is necessary. This thesis focused on the interplay between melanoma and T cells (Chapter 2), as well as the melanoma side (Chapter 3), and the T cell side (Chapter 4) to gain deeper insights and identify novel targets of immunotherapy for melanoma. It is essential to understand the molecular basis of cancer immunotherapy for melanoma. In Chapter 2, we showed that IDO1-dependent tryptophan depletion is a critical mechanism for cytotoxic CD8 T cell-derived IFN antitumor activity. Thus, tryptophan replenishment by epacadostat adversely protects melanoma cells to immune elimination. Ribosome profiling revealed that IFN causes general protein translation stalling, which is reversed by IDO1 inhibition. Impaired translation is accompanied by an amino acid deprivation-dependent stress response carrying ATF4high and MITFlow transcriptomic signatures, which was also observed in human melanoma specimens. Further corroborating this clinically, the ability to downregulate MITF is predictive of improved outcome of checkpoint blockade treated melanoma patients. Our results show that anti-tumor activity by T cells and IFN is partially triggered by translation stress mediated by IDO1-dependent tryptophan depletion, which is rescued by epacadostat, thereby adversely protecting tumor cells to T cell attack. In Chapter 3, we identified TAF6L as a novel regulator of PD-L1 using an unbiased FACS-based epigenomic remodeler shRNA library screen in melanoma cells. TAF6L knockdown or depletion by CRISPR/Cas9 leads to elevated PD-L1 RNA and protein expression. Furthermore, TAF6L-deficient melanoma cells enhance PD-L1 inhibitory functions when encounter T cell interactions. Consistent with these findings, PD-L1 is inversely correlated with TAF6L expression in a number of clinical human cancer samples. Together, our study provides a novel transcriptional regulatory mechanism of PD-L1, contributing to our understanding the regulation of PD-L1 expression in tumor cells. Chapter 4 describes an unbiased FACS-based loss-of-function genome-wide CRISPR/Cas9 sgRNA screen that was conducted to identify novel regulators of Lag-3 on mouse T cells. Several candidates were analyzed and selected, which were enriched in FACS-sorted Lag-3 low population and potentially clinically more relevant. Most of the hits are Golgi apparatus and endoplasmic reticulum-associated proteins, or involved with glycosphingolipids biosynthesis. It was demonstrated that the surface expression of Lag-3 is reduced by the depletion of B4galt5, B3galt4 and B4galnt1, although their knockout efficiency has yet to be analyzed. Collectively, our study could identify novel modulators of Lag-3, which may uncover regulatory mechanisms of Lag-3 transportation or post-translational modifications.