Abstract 2658: NOS inhibition augments PI3K inhibitor-induced DNA damage and mesenchymal-to-epithelial transition in metaplastic breast cancer
Authors: Reddy Tejaswini P. , Puri Akshjot , Guzman-Rojas Liliana , Mahboubi Bijan , Qian Wei , Zhou Jianying , Kim Baek , Moulder Stacy , Piwnica-Worms Helen , Rosato Roberto , Chang Jenny C. 
Published: June 2022

Purpose: Metaplastic breast cancer (MpBC) is a highly chemoresistant and aggressive breast cancer variant with limited therapeutic options. Most MpBCs harbor a triple-negative breast cancer (TNBC) phenotype, yet have a worse prognosis compared to TNBC. We and others discovered two key molecular alterations in MpBC associated with poor overall survival: 1) hyperactivation of the phosphoinositide 3-kinase (PI3K) signaling pathway and 2) enhanced inducible nitric oxide synthase (iNOS) signaling. Nitric oxide produced from iNOS is able to activate various oncogenic pathways, such as PI3K signaling. In MpBC, both PI3K and NOS signaling pathways may synergistically work to enhance chemoresistance. We propose to evaluate whether pan-NOS inhibitor NG-monomethyl-l-arginine (L-NMMA) augments the efficacy of alpha isoform-specific PI3K inhibitor alpelisib in MpBC in vitro and in vivo models.

Methods: MpBC cell lines (Hs578T, BT549, SUM159) and MpBC Patient-Derived Xenograft (PDX) models were used in this study. Cell viability was determined with SRB Assay and the combination index was evaluated using Chou-Talalay Method. Flow cytometry was conducted to evaluate cellular apoptosis and cell cycle distribution analysis. In vivo efficacy of NOS and PI3K inhibition was assessed using MpBC PDX models. Transcriptional analysis of PDX tumors treated with vehicle control, single-agent (L-NMMA or alpelisib), or combination therapy (L-NMMA+alpelisib) was conducted to reveal top enriched pathways. Immunoblotting and immunofluorescence analyses of treated MpBC cell lines and PDX tissues were performed.

Results: Combination of L-NMMA and alpelisib synergistically reduced cellular proliferation (CI&lt;1), enhanced apoptosis, depleted nucleotide pools, and arrested MpBC cell lines in G2/M phase of cell cycle relative to single-agent treatment. Furthermore, NOS+PI3K inhibition significantly suppressed tumor volume in MpBC PDX models with PIK3CA mutations, although less significantly in MpBC PIK3CA-wild type PDX models, augmented efficacy of taxane chemotherapy, and improved overall survival. Transcriptional analysis of MpBC PDX tumors treated with combination therapy versus vehicle control revealed that epithelial-to-mesenchymal transition, DNA repair, and E2F targets were among the top enriched pathways, suggesting their plausible involvement in the mechanism of response. Immunoblotting/immunofluorescence analyses of cell lines/PDX models confirmed that combination therapy enhances DNA damage, reduces expression of homologous recombination-mediated DNA repair proteins, and induces mesenchymal-to-epithelial transition, rendering MpBC cells potentially more chemosensitive.

Conclusion: Combined NOS and PI3K inhibition is a novel therapeutic strategy that may improve the efficacy of chemotherapy in patients with MpBC.

Citation Format: Tejaswini P. Reddy, Akshjot Puri, Liliana Guzman-Rojas, Bijan Mahboubi, Wei Qian, Jianying Zhou, Baek Kim, Stacy Moulder, Helen Piwnica-Worms, Roberto Rosato, Jenny C. Chang. NOS inhibition augments PI3K inhibitor-induced DNA damage and mesenchymal-to-epithelial transition in metaplastic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2658.