Abstract 2261: Deciphering cross-resistance mechanisms associated to targeted therapies and immunotherapy in BRAFmutmelanoma: The role of tumor-associated macrophages and galectin-1
Authors: Veigas Florencia , Mahmoud Yamil Damian , Merlo Joaquin P. , Cocco Montana Manselle , Massaro Mora , Gatto Sabrina , Morales Rosa , Girotti Maria R. , Perez-Saez Juan M. , Rabinovich Gabriel A. 
Published: April 2023

Abstract
Both targeted therapies (TT) and immunotherapies have revolutionized the treatment of metastatic melanoma patients. Particularly, TT with BRAFi or BRAFi/MEKi leads to prompt but not durable responses in most patients due to the development of resistance within 1 year. Tumors refractory to TT derive little benefit from subsequent immunotherapy. Here we aim to elucidate how previous treatments with TT shape tumor-immune landscape and how it could be overcome therapeutically. Firstly, we analyzed the gene expression profile of a single-cell RNA-Seq dataset of 48 biopsies (16,291 cells) from melanoma patients before and during treatment with anti-PD1/anti-CTLA4. Our single-cell data analysis showed that macrophages and CD8+ T-cells of non-responders to immunotherapy upregulate a specific glycosylation-related signature unraveling a novel potential mechanism of resistance. Interestingly, we found a new subset of macrophages associated with resistance that arises during treatment. Macrophages of non-responders express high levels of galectin-1 (Gal-1), an immunosuppressive lectin implicated in tumor progression and immune escape, and display an M2-like phenotype regulated by the activation of specific transcription factors and signaling pathways including TGF-b, hypoxia, and VEGF. This observation was validated on bulk RNA-Seq datasets, where we observed that melanoma patients refractory to immunotherapy have a higher M2-macrophage infiltrate at baseline by MIXTURE (p-value&lt;0.05). To study cross-resistance, we first analyzed by MIXTURE the immune microenvironment of melanoma patients treated with BRAFi or BRAFi/MEKi using 6 published transcriptomic datasets. We identified the prevalence of an immunosuppressive microenvironment significantly enriched in M2-macrophages in relapsed biopsies (p-value &lt; 0.05) accompanied by low levels of T cells activation markers and dysregulation of the TGF-b/Gal-1 axis. Interestingly, we observed that the expression of Gal-1 is increased in human and mouse melanoma cell lines resistant to TT (p-value&lt;0.05). To further elucidate the mechanisms underlying this effect and identify potential therapeutic targets, we developed an immunocompetent mouse model of resistance to BRAFi/MEKi in BRAFmut melanoma which mimics the M2 macrophage-centered immune profile observed in melanoma patients and the low response rates to immunotherapy at progression in TT-refractory tumors. Our preliminary findings suggest that Gal-1-glycan interactions could promote an immune evasive microenvironment in BRAFi/MEKi-resistant tumors, preventing subsequent response to immunotherapy. Hence, the Gal1-glycan axis arises as a potential actionable target for the development of combination therapies in non-responding melanoma patients.
Citation Format: Florencia Veigas, Yamil Damian Mahmoud, Joaquin P. Merlo, Montana Manselle Cocco, Mora Massaro, Sabrina Gatto, Rosa Morales, Maria R. Girotti, Juan M. Perez-Saez, Gabriel A. Rabinovich. Deciphering cross-resistance mechanisms associated to targeted therapies and immunotherapy in BRAFmutmelanoma: The role of tumor-associated macrophages and galectin-1 [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 2261.