Abstract P2-20-09: Enhancement of anti-tumor immunity in ER+ breast cancer through dual inhibition of MCL1 and caspases
Authors: Yi Jae Kyo , Spektor Alexander 
Published: March 2023

Abstract
Breast cancer is one of the most commonly diagnosed cancer types and a leading cause of cancer death in women in the United States. However, it is difficult to treat with immunotherapy because of its immunological “cold” status. This underscores the importance of a novel clinical approach to increase efficacy of immunotherapy including acute upregulation of Type I Interferon (IFN) response. One strategy to enhance Type I IFN response is by release of damage-associated molecular patterns (DAMPs) from mitochondria, including mitochondrial DNA, RNA and oxidized proteins that are recognized by pattern recognition receptors (PRRs) in cytoplasm and activate downstream IFN signaling. Because many cancers including breast cancer experience increased and dysregulated mitochondrial biogenesis at baseline, they contain significantly higher levels of DAMPs compared to normal cells, but these substrates are sequestered in mitochondria and not accessible to PRRs. We set out to develop a strategy to exploit increased levels of DAMPs in cancer cell mitochondria to enhance Type I IFN response. While DAMPs are released from mitochondria upon apoptosis caused by mitochondrial outer membrane permeabilization (MOMP), Type I IFN response is still silenced due to cleavage of PRRs by activated caspases. One way to induce MOMP is by inhibition of Myeloid Cell Leukemia-1 (Mcl-1), an anti-apoptotic family protein that is frequently upregulated in breast cancer. Interestingly, breast cancer cell lines showed the highest dependency on Mcl-1 among solid cancers and inhibitors of Mcl-1 have shown efficacy in breast cancer early clinical trials. Based on our preliminary observations showing that Mcl-1 inhibition activates a robust Type I IFN response in MCF7 breast cancer cells deficient for caspase 3, we hypothesized that combining Mcl-1 inhibitors with caspase inhibitors would greatly enhance Type I IFN response in a robust and specific manner by facilitating release on DAMPs into the cytoplasm while preventing inactivation of PRRs. Strikingly, in contrast with Mcl-1 inhibitor treatment alone, combination treatment led to dramatic increase in expression levels of IFN-β and interferon-stimulated genes (ISGs) in MCF7 and ZR-75-1 ER+ breast cancer cell lines with minimal effects on cytotoxicity. In addition, T47d, a breast cancer line highly resistant to Mcl-1 inhibition due to compensation by other Bcl-2 family proteins, showed greatly enhanced Type I IFN response upon combined Mcl-1, Bcl-2 and caspase inhibition. Because reduced expression of MHC class I molecules on cell surface is one of the most important immune escape mechanisms in tumors including breast cancer, we examined whether the proposed combinatorial treatment would overcome this by enhancing expression of MHC class I. Immunofluorescence (IF) staining confirmed that the combination treatment dramatically increased total HLA-ABC protein expression on plasma membrane of ZR-75-1. Furthermore, combinatorial treatment upregulated transcription of most genes responsible for antigen presentation, a multistep process consisting of antigen peptide generation and loading of MHC class I molecules. Lastly, we sought to examine how elevated chemokine expression may impact patients with breast cancer. We examined association of cytokines highly upregulated by the combination treatment with immune cell infiltration using a TCGA dataset that included over a thousand breast cancer samples. This analysis was performed by xCell algorithm and demonstrated strong association of that cytokine expression with CD8+ T, B and DC infiltrate. Our results demonstrate that the combination of Mcl-1 and caspase inhibitors greatly enhances Type I IFN response and may improve anti-tumor immunity. We plan to further test this drug combination in immunocompetent mouse models, laying the foundation for a clinical trial assessing the combination treatment in patients with metastatic ER+ breast cancer.
Citation Format: Jae Kyo Yi, Alexander Spektor. Enhancement of anti-tumor immunity in ER+ breast cancer through dual inhibition of MCL1 and caspases. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-20-09.