Targeting FOXP3 mRNA splicing for breast cancer immunotherapy

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Project Abstract The development of cancer immunotherapy including immune checkpoint inhibitors and adoptive cell transfer (ACT)-based therapies revolutionized cancer treatment. The success, however, is limited to a relatively small subset of patients and cancer types. Regulatory T cells (Tregs) play a central role in maintaining immune system homeostasis and negatively regulate immune-mediated inflammation such as autoimmune diseases, asthma and allergy. However, tumor infiltrating Tregs also suppress effective anti-tumor immunity. FOXP3 is a master regulator of Treg development and function. Human FOXP3 gene encodes two major isoforms through mRNA alternative splicing – a long full-length isoform (FOXP3L) and a shorter isoform lacking exon 2 region (FOXP3S). To study the function of FOXP3S in Tregs, we generated a Foxp3S mouse line expressing only the Foxp3S isoform. Our preliminary studies with orthotopic mouse breast tumor models demonstrate that the Foxp3S mice are completely resistant to tumor development, while the littermate wildtype mice expressing Foxp3L exhibit significant tumor growth. Intratumoral injection of Foxp3S-promoting morpholino drug (MO) into the breast tumors in wildtype mice markedly increases IFN-γ+ CD4 and CD8 T cells in the tumors and thus suppresses tumor growth. Interestingly, bioinformatics analysis of transcriptomic data from The Cancer Genome Atlas (TCGA) reveals that FOXP3S mRNA expression in human breast cancer is positively correlated with clinical outcomes. Given that over 60% of tumor-infiltrating CD4 T cells are Tregs and the most dominant TCRs from intratumoral Tregs were tumor-reactive and recognize specific tumor neoantigens, we hypothesize that promoting FOXP3S isoform expression will reprogram tumor-reactive Tregs and convert these immune suppressive Tregs to tumor-specific T helpers thus promoting antitumor immunity. We propose to use unique mouse lines, FOXP3S-promoting morpholinos and patient-derived organoids to test our hypothesis. We will determine the mechanisms for FOXP3S-mediated antitumor immunity and further define the efficacy of FOXP3S- promoting morpholinos in breast cancer treatment. Successful completion of this study will identify a novel therapeutic strategy for breast cancer immunotherapy.