Date Available

8-20-2023

Year of Publication

2023

Document Type

Master's Thesis

Degree Name

Doctor of Philosophy (PhD)

College

Medicine

Department/School/Program

Toxicology and Cancer Biology

Advisor

Christine Brainson

Co-Director of Graduate Studies

Jill Kolesar

Abstract

Only 20% of patients diagnosed with lung squamous cell carcinoma (LSCC) respond to immunotherapy. Anti-PD1 immunotherapy is most commonly prescribed to these patients; however, most will become refractory. It is important to understand the mechanisms underlying this problem to increase durability and survival. Building upon the work of other groups, our lab has demonstrated that the inhibition of the histone methyltransferase, EZH2, is crucial to maintaining an immunologically responsive microenvironment. Based on our data, we hypothesize that combining EZH2 inhibitors with anti-PD1 therapy will increase response and durability. To study non-small cell lung cancers (NSLC) our lab uses a variety of 2D and 3D in vitro models derived from mouse and human tissues. The experiments performed combine interferon-γ (IFNγ) with pharmacological EZH2 inhibitors to determine the combinatorial effects on gene expression of major histocompatibility complex I and II (MHC I/II). Following treatment, assays such as qPCR and flow cytometry were performed, which revealed an increase in both gene and protein expression of MHC I and II. Expression of MHC I and II is critical to antigen presentation and T cell activation; therefore, it is important to study in the context of immunotherapy. Moreover, it has been demonstrated by other groups that high EZH2 expression correlates to a decrease in MHC I and II expression in different models of cancer, and that expression of MHC II predicted response to immunotherapy by increasing progression free survival. Additionally, two in vivo models of LSCC were used to define the effects of combining an EZH2 inhibitor with anti-PD1 immunotherapy. The mouse models employed in our studies are generated through the biallelic deletion of the two tumor suppressors Lkb1 and Pten (LP) in an autochthonous manner or through a syngeneic subcutaneous graft. Both models recapitulate the histology seen in human disease as evidenced by the presence of large pockets of infiltrating neutrophils. These tumor-associated neutrophils (TANs) demonstrate immunosuppressive effects on the microenvironment within the tumor milieu through the production of enzymes and immune modulating cytokines that are detrimental to T cell activation. Following positive identification of tumor burden by MRI or palpation, the mice are placed on treatment regimen that combines anti-PD1 immunotherapy with EZH2 inhibitors. Upon subsequent MRI analysis and tumor volume calculation, we determined that the combination therapy was superior to each single agent in the autochthonous model. Furthermore, we observed that the drug EPZ-6438 contains anti-cancer activity as a single agent in the syngeneic graft model. This finding is exciting as a therapeutic option for patients who are not eligible for immunotherapy or in the case of a patient having severe adverse reactions to immunotherapy. Lastly, we performed scRNAseq on tumors from our in vivo study and identified increases in gene sets involved with IFNγ signaling and immune activation. We also identified different populations of neutrophil within tumors, demonstrated by increases of tumor eliminating phenotypes and decreases in tumor promoting populations.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2023.399

Funding Information

This study was supported by National Institutes of Health R01 CA237643, National Institutes of Health T32 ES007266-30, National Institutes of Health P30 CA177558, and V foundation V2017-010

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