Abstract PS1-12-26: Neurofibromin loss as a driver of bone metastasis in estrogen receptor-positive breast cancer

Abstract Approximately 80% of breast cancers (BCa) expresses estrogen receptor-α (ER), and ER+ BCa most frequently metastasizes to the bone. The driver of bone metastasis remains undefined. To address this un-met need, we focus on a key tumor suppressor, neurofibromin (NF1), which is both a GTPase-activating protein that represses RAS signaling and a direct transcriptional corepressor that represses ER. NF1 inactivation, often observed as somatic mutations, has been shown to be enriched in metastatic vs primary ER+ breast cancer. In our analysis, NF1 copy number loss (CNL) was identified in 62% of ER+ breast cancer patients in a cohort who subsequently developed metastases. Notably, NF1-CNL was especially prevalent in bone metastases as compared to metastases in other tissues/organs. To functionally assess the metastatic potentials of NF1 inactivation, luciferase-tagged human NF1 KO ER+ BCa cells were orthotopically transplanted into mouse mammary fat pads. After primary tumor resection, bioluminescence imaging revealed that NF1 KO cells disseminated more extensively than NF1 + controls, with marked enrichment in bone and skeletal tissues. Furthermore, when directly delivered into bone via intra-iliac artery injection, NF1 KO ER+ BCa cells demonstrated significantly accelerated growth within the bone microenvironment. Bone metastasis in ER+ BCa is frequently accompanied by osteoclast (OC)-induced bone loss, which releases stored growth factors that fuel tumor progression, thus creating a vicious cycle. We analyzed previously published RNA-seq data and found that “ossification” and “bone-remodeling” are key functional categories enriched among genes whose expression is affected by NF1-depletion in ER+ BCa cells. Bone abnormalities are common in neurofibromatosis type 1 patients caused by germline loss of NF1. It is possible that bone remodeling is a core function controlled by NF1 but hijacked by cancer cells. To test this, two ER+ BCa cell lines were each co-cultured with the OC progenitor cell line RAW264.7 in the presence of RNAKL. OC formation, assessed by TRAP staining, microscopy, and qPCR, was more efficiently induced by NF1-depleted cells than by controls. To identify factors mediating this effect, we performed mass-spectrometry on the culture medium from the co-cultures, and RNA-seq on mRNA extracted from the cells. Both analyses revealed upregulation of ICAM1, a molecule that facilitates fusion of pre-OC and promotes OC formation. Pharmacological inhibition of ICAM1 confirmed its functional necessity for OC induction in this setting, as expected. Immune suppression is a critical driver of tumor progression. When co-cultured with T-cells derived from human PMBCs, NF1 KO ER+ BCa cells more efficiently suppressed proliferation and IFN-γ production than NF1 + cells. We further developed a CAR-T cell line that exhibits cytotoxic effect on HER2-expressing cells, such as MCF7 cells. NF1 KO MCF7 cells were more resistant to killing by these CAR-T cells, while concurrently induce apoptosis in these CAR-T cells. Collectively, these data support the model whereby NF1 loss in ER+ BCa cells induce OC formation and T cell suppression, increasing tumor burden in the bone. Proper targeting of RAS and ER pathways for treatments may help mitigate bone metastasis in NF1-deficient ER+ BCa. Citation Format: Z. Zhao, Z. Zheng, J. T. Lei, M. J. Baik, Y. Wu, L. K. Somes, O. A. Harb, J. Wang, U. Rasaily, O. A. Chang, H. Wang, I. Bado, Z. Gugala, M. Anurag, B. Zhang, A. Sreekumar, V. Hoyos, X. H. Zhang, E. C. Chang. Neurofibromin loss as a driver of bone metastasis in estrogen receptor-positive breast cancer [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS1-12-26.