Abstract P4-08-05: NOS inhibition reverses epithelial-to-mesenchymal transition and synergizes with alpelisib in metaplastic breast cancer
Authors: Reddy Tejaswini , Puri Akshjot , Guzman Liliana , Qian Wei , Zhou Jianying , Rosato Roberto , Zhao Hong , Thomas Christoforos , Li Xiaoxian , Mahboubi Bijan , Oo Adrian , Cho Young-Jae , Kim Baek , Thaiparambil Jose , Ayerbe Camila , Giese Noah , Moulder Stacy , Piwnica-Worms Helen , Meric-Bernstam Funda , Chang Jenny 
Published: March 2023

Abstract
Background: Metaplastic breast cancer (MpBC) is a rare, lethal, and highly chemoresistant breast cancer subtype, with no FDA-approved therapeutic options. Most MpBCs are triple-negative, yet have a worse prognosis than non-metaplastic triple-negative breast cancer (non-MpTNBC). MpBC tumors are enriched for markers of epithelial-to-mesenchymal transition (EMT)/cancer stem cells (CSC), produce high nitric oxide (NO) levels, and have a hyperactive phosphoinositide 3-kinase (PI3K) signaling pathway. Increased PI3K and inducible nitric oxide synthase (iNOS) activity are poor prognostic indicators in MpBC. NO can activate multiple oncogenic pathways spatially and temporally, such as PI3K and transforming growth factor beta (TGFβ), a critical regulator of EMT. Therefore, our study evaluates 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 (SUM159, BT549, Hs578T, HCC1806) and Patient-Derived Xenograft (PDX) models were used in our studies. Droplet digital polymerase chain reaction (ddPCR) was conducted to evaluate the iNOS-associated mutation (RPL39 A14V) and PIK3CA hotspot mutation rates in PDX models. Cell viability (SRB/Cell Titer-Glo), combination index (CI), immunoblotting, and immunofluorescence of treated MpBC cell lines and tumor tissues were evaluated. Results: Immunostaining analysis revealed that MpBC PDX tumors had elevated co-expression of iNOS and pAkt (60% vs 23%, p=0.0495) relative to non-MpTNBC PDX tumors. MpBC PDX tumors had higher RPL39 A14V (66% vs 4.7%, p&lt; 0.0006) and PIK3CA hotspot mutation rates (50% vs 19.1%, p=0.1247) than non-MpTNBC PDX tumors. Combining L-NMMA with alpelisib was synergistic in MpBC cell lines harboring PIK3CA/PIK3R1 mutations (CI&lt; 1) and antagonistic in PIK3CA-wild type and PTEN-deleted models (CI&gt;1). In vivo evaluation using MpBC PDX tumors found that L-NMMA significantly 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 (Padj = 0.0254) and keratinization pathway (Padj = 0.048) were enriched pathways in MpBC PDX tumors that responded to combination therapy. Pharmacological and genomic inhibition of iNOS reversed EMT in MpBC cells, as shown 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 an associated decrease in aldehyde dehydrogenase (ALDH1), a CSC marker. L-NMMA and alpelisib therapy also resulted in the loss of tumor-initiating ability, enhanced chemosensitivity, and improved overall survival in MpBC PDX models. These studies paralleled results from a phase 1b/2 clinical trial with L-NMMA combined with taxane chemotherapy in a cohort of anthracycline-refractory MpBC patients (n=15, NCT02834403). The clinical benefit rate was 40% (6/15), the 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 enhanced expression of E-cadherin, and decreased expression of iNOS, Zeb1, and ALDH1. Conclusion: Our findings suggest that combining L-NMMA and alpelisib is a novel therapeutic strategy to treat MpBC by reversing EMT and decreasing CSCs, rendering MpBC tumors more chemosensitive. This combination therapy is being tested in a first multicenter phase 2 study targeting this orphan disease.
Citation Format: Tejaswini Reddy, Akshjot Puri, Liliana Guzman, Wei Qian, Jianying Zhou, Roberto Rosato, Hong Zhao, Christoforos Thomas, Xiaoxian Li, Bijan Mahboubi, Adrian Oo, Young-Jae Cho, Baek Kim, Jose Thaiparambil, Camila Ayerbe, Noah Giese, Stacy Moulder, Helen Piwnica-Worms, Funda Meric-Bernstam, Jenny Chang. NOS inhibition reverses epithelial-to-mesenchymal transition and synergizes with alpelisib in metaplastic breast cancer. [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 P4-08-05.