Abstract 3447: NOS inhibition reverses epithelial-to-mesenchymal transition and synergizes with alpelisib in metaplastic breast cancer
Authors: Reddy Tejaswini P. , Puri Akshjot , Guzman-Rojas Liliana , Thomas Christoforos , Qian Wei , Zhou Jianying , Zhao Hong , Li Xiaoxian , Mahboubi Bijan , Oo Adrian , Young-Jae Cho , Kim Baek , Thaiparambil Jose , Chervo Maria Florencia , Rosato Roberto , Ayerbe Camila , Giese Noah , Moulder Stacy , Piwnica-Worms Helen , Meric-Bernstam Funda , Chang Jenny C. 
Published: April 2023

Abstract
Metaplastic breast cancer (MpBC) is a rare and highly chemoresistant breast cancer subtype, with a median survival of 8 months after metastatic disease, and no standard therapeutic options. MpBCs are enriched for epithelial-to-mesenchymal transition (EMT)/cancer stem cell (CSC) markers, produce high levels of nitric oxide (NO), and have a hyperactive phosphoinositide 3-kinase (PI3K) signaling pathway. NO activates multiple oncogenic pathways, such as PI3K and transforming growth factor beta (TGFβ), a regulator of EMT. Therefore, we hypothesized that pan-NOS inhibitor NG-monomethyl-l-arginine (L-NMMA) could augment the efficacy of α-specific PI3K inhibitor alpelisib in MpBC in vitro and in vivo models.
Immunostaining analysis revealed that MpBC PDX tumors had elevated co-expression of iNOS and pAkt (60% vs 23%, p=0.04) relative to triple-negative breast cancer (TNBC) PDX tumors. MpBC PDX tumors had higher RPL39 A14V (66% vs 4.7%, p&lt; 0.00) and PIK3CA hotspot mutation rates (50% vs 19.1%, p=0.31) than TNBC PDX tumors. L-NMMA was synergistic with alpelisib in MpBC cell lines with PIK3CA/PIK3R1 mutations and antagonistic in PIK3CA-wild type and PTEN-deleted models. In vivo evaluation using MpBC PDX tumors found that L-NMMA augmented the efficacy of alpelisib in reducing tumor volume in PIK3CA-mutated MpBC PDX models. Transcriptomic analysis found gene sets associated with EMT reversal, such as the formation of cornified envelope (NES = 2.04 Nom p&lt;0.00) and keratinization pathway (NES = 2.06, Nom p&lt;0.00), were enriched pathways in MpBC PDX tumors that responded to combination therapy. Pharmacological/genomic inhibition of iNOS reversed EMT in MpBC cells, by decreased expression of Zeb1, TGFβ, Snail, Vimentin, and increased expression of E-cadherin and ZO-1 in immunoblotting analysis. MpBC cells with NOS2 knockout acquired an epithelial-like cellular morphology, and this reversal of EMT rendered MpBC cells more sensitive to alpelisib and taxane-chemotherapy. MpBC PDX tumors that responded to combination therapy also exhibited a reversal in EMT, with a decrease in aldehyde dehydrogenase (ALDH1), a CSC marker. Combination therapy also reduced tumor-initiating ability, enhanced chemosensitivity, and improved overall survival in MpBC PDX models.
These studies paralleled a phase 1b/2 clinical trial with L-NMMA+taxane chemotherapy in a cohort of anthracycline-refractory MpBC patients (NCT02834403). The clinical benefit rate was 40% (6/15), overall response rate was 20% (3/15), and one patient achieved a pathologic complete response. Relative to baseline tumors, the responder end-of-treatment tumors had undergone reversal of EMT, with decreased expression of iNOS and ALDH1.
We find that combining L-NMMA and alpelisib is a novel therapeutic strategy to treat MpBC, and combination therapy is being tested in a first multicenter phase 2 study for patients with MpBC.
Citation Format: Tejaswini P. Reddy, Akshjot Puri, Liliana Guzman-Rojas, Christoforos Thomas, Wei Qian, Jianying Zhou, Hong Zhao, Xiaoxian Li, Bijan Mahboubi, Adrian Oo, Cho Young-Jae, Baek Kim, Jose Thaiparambil, Maria Florencia Chervo, Roberto Rosato, Camila Ayerbe, Noah Giese, Stacy Moulder, Helen Piwnica-Worms, Funda Meric-Bernstam, Jenny C. Chang. NOS inhibition reverses epithelial-to-mesenchymal transition and synergizes with alpelisib in metaplastic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3447.