Graduation Year

2023

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Medical Sciences

Major Professor

Subhra Mohapatra, Ph.D.

Committee Member

Andreas Seyfang, Ph.D.

Committee Member

Bala Chandran, Ph.D.

Committee Member

Gloria Ferreira, Ph.D.

Committee Member

Srinivas Bharadwaj, Ph.D.

Keywords

Combination, Immunotherapy, Tumor Immunology, Immunoresistance

Abstract

Lung cancer is the first cause of cancer-related deaths in both men and women with an overall five-year survival rate of 28%. Although immune checkpoint blockers (ICBs) are currently FDA-approved for the treatment of non-small cell lung cancer (NSCLC), only 17-20% of patients achieve durable responses by the induction of immunologic memory. The lack of response in most patients can be attributed to the tumor-intrinsic or tumor-extrinsic immune resistance mechanisms. A biomarker of importance is the Programmed Death Ligand-1 (PD-L1), as higher PD-L1 expression is usually associated with a better response to ICBs. Although studies have attempted to combine ICBs with chemotherapeutic agents to improve treatment outcomes, many of these combinations are not clinically attainable due to the additive adverse events. Therefore, there is an unmet need to find treatments that improve patients’ responses to ICBs without additive toxicity. Withaferin A (WFA) is a steroidal lactone isolated from the herb Withania somnifera with a pleiotropic anti-cancer activity and selective cytotoxicity in cancer cells. Although WFA has been extensively studied in preclinical cancer models, the immunomodulatory effects of WFA in NSCLC remain unexplored. This study aimed to investigate the immunomodulatory effects of WFA in NSCLC and whether it can enhance the effectiveness of ICBs by inducing anti-tumor immunity or targeting tumor immunosuppression. Our findings show that WFA-induced immunogenic cell death (ICD) is characterized by the release of danger-associated molecular patterns (DAMPs) and subsequent activation of dendritic cells (DCs) in NSCLC cells. Additionally, WFA increased the expression of the immune checkpoint molecule PD-L1 which may sensitize tumor cells to ICBs. Mechanistically, we found that WFA increased PD-L1 through the production of reactive oxygen species (ROS) as ROS scavengers (N-acetyl cysteine (NAC) or glutathione) completely abrogated WFA-mediated PD-L1 increase. In vivo testing confirmed these findings, as WFA treatment sensitized the immunoresistant LLC syngeneic mouse model to anti-PD-L1 ICB as the combination of WFA+anti-PD-L1 showed a significant reduction in tumor growth compared to the control mice and individual treatments. Moreover, the combination treatment significantly increased CD8 T-cell infiltration and activation, while reducing immunosuppressive cell populations in the tumor microenvironment (TME). Moreover, the combination treatment did not alter the body weight of the mice, highlighting the safety of WFA+anti-PD-L1 combination. Consistent with our in vitro findings, we found that ROS plays a partial role in the anti-cancer effectiveness of WFA+anti-PD-L1 combination in vivo as adding NAC partially reversed the tumor size reduction observed with the combination treatment. Further, NAC completely abrogated the change in tumor immune infiltration by reducing CD8 T-cell and increasing immunosuppressive cell infiltration. In summary, our study shows that WFA sensitizes NSCLC to anti-PD-L1 ICB by inducing ICD, PD-L1 upregulation and altering tumor-immune infiltration without increasing toxicity, providing an advantage over the other combinatorial strategies.

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