Author ORCID Identifier

https://orcid.org/0000-0002-4112-0450

Date of Award

Summer 8-29-2024

Document Type

Thesis (Master's)

Department or Program

Microbiology and Immunology

First Advisor

Tyler J. Curiel, M.D., M.P.H

Second Advisor

Yina H. Huang, Ph.D.

Third Advisor

Lucas A. Salas, M.D., M.P.H., Ph.D.

Abstract

Cutaneous melanoma is the most aggressive form of skin cancer even though it takes only about 1% of all skin cancers. Even among all cutaneous melanomas, NRAS-mutant melanoma is more aggressive than any other, and about 10-25% of all cutaneous melanoma cases have mutations in NRAS. NRAS is a member of the RAS family of proto-oncogenic GTPase proteins, and it plays a key role in signal transduction pathways responsible for cellular survival, proliferation, and metabolism. Immunotherapy is the first line of defense against NRAS-mutant melanoma because, despite tremendous efforts made for decades, it has not been successful to develop small molecules targeting mutant forms of NRAS for melanoma therapy. Even then, patients with NRAS-mutant melanoma often develop resistance to the currently available immunotherapy. Thus, we are in urgent need of developing novel therapeutic strategies against NRAS-mutant melanoma. PDL1 is an extensively studied immune checkpoint molecule for its ability to inhibit T-cell-mediated immunity. PDL1 is often overexpressed in cancers as a result of malignant degeneration of tumor cells-of-origin, promoting tumor immune-evasiveness. Besides suppressing T-cell-mediated immune responses, it has been reported that PDL1 is involved in mediating numerous cellular processes and responses as a signaling molecule or by controlling transcription of specific genes, and tumor-cell-intrinsic PDL1 signaling can promote immunopathogenic effects. IFN-I is a family of cytokines that are capable of controlling gene expression of thousands of genes. IFN-I can be both protective and pathogenic. It has been reported that IFN-I can induce immunopathogenic and pro-tumor effects by promoting resistance to chemo- and radiotherapy and by triggering epigenetic changes in cancer cells in a way that they remember and become resistant to antitumor immune responses made by host immune cells. A couple of genes that can also be transcribed in response to IFN-I signaling are IDO1 and TDO2, which are the key enzymes of Kyn pathway of the Trp metabolism. Trp metabolism plays a significant role in regulating immune responses. In normal and healthy conditions, proper Trp metabolism restricts inflammation and prevents autoimmunity by metabolizing Trp, an essential amino acid, and triggering anergy and apoptosis of T cells and inhibiting their proliferation and differentiation. IDO1 and TDO2 are the key enzymes in this Trp metabolism. Many have reported overexpression of IDO1 and/or TDO2 in cancer models both in animals and humans, and inhibition of IDO1 in preclinical studies and early-stage clinical studies yielded promising results with therapeutic benefits. However, with a failure of a randomized Phase III clinical trial that could not demonstrate added benefits of IDO1 compared to ICB monotherapy, none of the available IDO1 inhibitors has been approved by FDA for cancer therapy. I questioned if the aggressiveness of NRAS-mutant melanoma is rooted from IDO1/TDO2 enzymatic activity. Modulation of IDO1/TDO2 expression by mutated NRAS has been suggested with a study that used MEKi for a cancer study. Co-expression of PDL1 and IDO1 or TDO2, separately, in various cancer types has been reported by many studies. UVB, a primary environmental risk factor for melanomagenesis, triggers IFN-I signaling which is now gaining attention for its immunopathogenic traits. Thus, I decided to test IDO1/TDO2 inducibility in NRAS-mutant melanoma with UVB and proinflammatory cytokines, tumor-PDL1-effects on IDO1/TDO2 expression, and tumor-PDL1-dependent treatment effects of IDO1 inhibition. I used NRASQ61R mouse melanoma cell lines derived from our lab’s novel autochthonous tamoxifen-inducible NRASQ61R mouse models to test the IDO1/TDO2 inducibility and melanoma PDL1 effects on IDO1/TDO2 expression. With the same cell lines, with tremendous help from my colleagues, I tested PDL1-dependent treatment effects of IDO1 inhibition in vivo. Additionally, I speculated that there are biological processes and functional pathways that are responsible for promoting therapy responsiveness and resistance to IDO inhibition. Thus, I performed bioinformatic analyses with publicly available and personally-granted RNA-seq data of therapy responders and non-responders to identify gene sets that govern responsiveness and resistance. The gene sets were enriched to infer the responsible biological processes and functional pathways. This work tackles the complicated relationship between NRAS mutant, melanoma PDL1, and immunometabolism with a focus on Trp metabolism. I bring new insights into antitumor immunity mediated by IDO1 inhibition, and potentially TDO2 inhibition, by demonstrating changes in immune cell populations and potential novel therapeutic strategies.

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