Metaplastic breast cancer (MpBC) is a rare subset accounting for less than 1% of all breast cancers. MpBC exhibits the worst prognosis in comparison to triple-negative breast cancer (TNBC) with a dismal poor survival rate in patients with metastatic disease. The main therapeutic options for MpBC remain surgery and systemic chemotherapy, despite known resistance to most systemic chemotherapy. A common molecular alteration in MpBC is hyperactivation of the phosphoinositide 3-kinase (PI3K) pathway. Recently, we published that MpBC displays a gain-of-function oncogenic mutation in ribosomal protein L39 (RPL39), which is responsible for treatment resistance, stem cell self-renewal, and lung metastasis. The mechanistic function of RPL39 is mediated through inducible nitric oxide synthase (iNOS)-mediated nitric oxide production. In addition, we demonstrated that inhibiting this nitric oxide synthase (NOS) pathway using pan-NOS inhibitor NG-methyl-L-arginine acetate (L-NMMA) may represent a highly effective therapeutic option for MBC patients. Therefore, we hypothesize that the combinatorial approach of inhibiting the two major pathways implicated in MpBC, namely PI3K/Akt/mTOR and NOS pathways would lead to significant tumor regression. Alpelisib, an FDA-approved, isoform-specific PI3K inhibitor, is currently used with the antiestrogen, fulvestrant, to treat hormone receptor (HR)-positive, PIK3CA-mutated breast cancer patients. We used MpBC cell lines Hs578t and BT549 and MpBC patient-derived xenograft (PDX) models in our preliminary studies. Using flow cytometry to detect Annexin V+/DAPI+ cells, we found increased cell death in MpBC cell lines treated with L-NMMA+alpelisib combination treatment in comparison to monotherapy. Immunoblotting of samples from single (L-NMMA or alpelisib) or combination treated cell lines and PDXs showed increased PARP degradation and cleaved caspase 3/9 with combination treatment. In vivo data using PDX models showed that combination treatment of L-NMMA+alpelisib was most effective at reducing tumor volume in comparison to monotherapy. We performed preliminary bulk-RNA sequencing analysis of tumor samples collected from PDX BCM-4664 treated with vehicle control, monotherapy of L-NMMA or alpelisib, and combination therapy. Gene set enrichment analysis found that E2F and Hedgehog signaling pathways were the top two enriched pathways in BCM-4664 tumors treated with combination therapy. Pathway-focused RT-PCR of MpBC PDXs confirmed GSEA results and showed significant gene expression alterations involving E2F signaling and cell cycle regulation. Further studies are currently ongoing to elucidate the molecular mechanisms involved in enhanced cell death and decreased tumorigenesis with L-NMMA and alpelisib dual therapy. Our results support the concept that LNMMA and alpelisib combination therapy has therapeutic potential in the treatment of MpBC, which may enable rapid translational into clinical trials, and impact the exceeding poor prognosis of women with MpBC.

Citation Format: Tejaswini Parlapalle Reddy, Roberto R Rosato, Liliana Guzman, Wei Qian, Jianying Zhou, Hongbin Wang, Helen Piwnica-Worms, Stacy Moulder, Jenny C Chang. Combination PI3K and NOS targeted therapy for metaplastic breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS11-17.