Abstract P2-20-08: Expression of ACVRL1 in a subset of recruited tumor-associated macrophages drives resistance to PD-1 therapy in human breast cancer
Authors: Talkhoncheh Mehrnaz Safaee , Sjölund Jonas , Balbas Paulina Bolivar , Kurzejamska Ewa , Larsson Sara , Cordero Eugenia , Oudenaarden Clara R. , Pantaleo Jessica , Jonsson Göran , Rolny Charlotte , Bocci Matteo , Pietras Kristian 
Published: March 2023

Abstract
The activin-like kinase receptor 1 (ALK1, encoded by ACVRL1) is a member of the transforming growth factor β (TGF-β) superfamily. Expression of ALK1 has been historically associated with endothelial cells, thus serving as a potential target for antiangiogenic therapy in cancer. Despite promising preclinical results with a ligand trap (RAP-041/dalantercept), clinical trials did not show additional benefit of dalantercept when combined or contrasted with the standard of care. This outcome raises the question about patient selection and optimal combination partner in the clinical setting. Moreover, from the biological perspective, how ALK1 signaling shapes the features of a tumor is still largely uncharted. Based on our previous observations, we confirmed that the genetic network correlated with ACVRL1 expression is associated with immune processes in human breast cancer. Next, we validated that inhibition of ALK1 altered the immune landscape in experimental primary breast cancer (MMTV-PyMT model), providing a rationale for a combined regimen with immunotherapy. After developing and validating a murine model of adjuvant therapy with RAP-041 (syngeneic E0771 and 4T1 cell lines), we discovered that ALK1 blockade potentiated immune checkpoint inhibitors. Based on bulk RNA-sequencing and a multicolor FACS approach, addition of RAP-041 to immunotherapy significantly reduced the number of intratumoral macrophages compared with either treatment alone. Notably, these changes were coupled with systemic alterations in peripheral blood cell composition, with the combined administration suppressing circulating monocytes. Interestingly, ALK1 signaling appears to be a pivotal contributor to this phenotype, as fewer circulating monocytes were also detected in the RAP-041 cohort. Based on our in vitro characterization, we revealed that ALK1 expression is not limited to endothelial cells, as different myeloid populations readily expressed Acvrl1, suggesting a direct functional role of ALK1. Stimulation of ALK1 did not alter macrophage polarization, even though different levels of mediators downstream of ALK1 characterized the M1/M2 states. Furthermore, apoptosis and necrosis did not seem to be affected upon activation of ALK1, while the effect on the cell cycle awaits confirmation. In vitro genetic modulation of ALK1 activity is ongoing, with co-culture systems, transendothelial adhesion and migration being currently assessed. Inspection of several human sc-RNA-sequencing datasets confirmed the existence of ACVRL1-positive monocytes in human PBMCs from healthy individuals as well as in human solid cancers, including breast cancer. Focusing on the immune compartment, ACVRL1 expression characterized recruited tumor-associated macrophages (TAMs) and their cycling counterpart. In light of this feature, longitudinal follow-up of patients exposed to chemotherapy and/or anti-PD-1/PD-L1 revealed that expression of ACVRL1 decreased following exposure to chemotherapy. Notably, high ACVRL1 expression and an increased proportion of ACVRL1-positive TAMs were found at progression with anti-PD-1 treatment. In agreement with our preclinical data, ACVRL1-positive cells were depleted from circulation upon recurrence, indicating a migration from blood to tumors. Finally, survival analysis determined that ACVRL1hi signature in macrophages correlated with a significantly lower survival in a 5-year follow-up in breast cancer. In conclusion, our work sheds light on the value of ALK1 as a promising dual antiangiogenic and immunomodulatory target for precision medicine in cancer.
Citation Format: Mehrnaz Safaee Talkhoncheh, Jonas Sjölund, Paulina Bolivar Balbas, Ewa Kurzejamska, Sara Larsson, Eugenia Cordero, Clara R. Oudenaarden, Jessica Pantaleo, Göran Jonsson, Charlotte Rolny, Matteo Bocci, Kristian Pietras. Expression of ACVRL1 in a subset of recruited tumor-associated macrophages drives resistance to PD-1 therapy in human 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 P2-20-08.