Abstract 3268: Biomarkers of drug-tolerant persistent melanoma cells in human, correlation with tumor microenvironment and response to treatment
Authors: Chen Hongxu , Shen Shensi , Faouzi Sara , Alfaro Alexia , Baille Dorothee , Roy Séverine , Benannoune Naima , Hermann Hugues , Muhammadnejad Samad , Vagner Stephan , Robert Caroline 
Published: June 2023

Abstract
Introduction: Drug tolerant cancer persister cells can be silent for years but constitute a reservoir of genetic resistance and can eventually result in tumor relapse. We showed that targeted therapy (TT)-tolerant BRAFV600 melanoma persister cells (MPC) have a reduced global mRNA translation activity but an increased translation efficiency of a subset of mRNAs encoding proteins involved in transcription, chromatin remodeling and epigenetic changes. Here, we identify protein markers of MPC in patient biopsies treated either with TT or with immune checkpoint inhibitors (ICI). Since persister cells are not destroyed by the immune system, we explored the interactions of MPC with their immune microenvironment. Single cell expression of these candidate MPC markers, as well markers of the tumor microenvironment (31 lymphoid and myeloid markers), were studied in sequential biopsies from melanoma patients treated with TT or ICI by CyTOF. Spatial localization of MPC markers and various types of immune cells were studied by multiplex immunohistochemistry (Akoya Codex). In addition, the in vitro effect of the cell supernatants of MPC or naïve parental cells on immune cells was studied.
Results: Biomarkers of persistence included 7 protein markers that are regulated at the translational level (MLL3, HDAC6, CCSER2, MT-ND5, ANK2, HIPK2, CBP) and 4 that are transcriptionally regulated (MITF, NGFR, CD36 and SLIT2). Some MPC markers were expressed at baseline in melanoma patients’ biopsies (n = 27) and their baseline expression tended to be associated with progressive disease (to TT or ICI). Paired biopsies from 18 patients showed that an increase in HDAC6, ANK2, HIPK2, CBP5 and MITF was statistically correlated with a short survival regardless of the type of treatment. Baseline lymphocytic infiltrate, especially CD4+ T cells, was associated with favorable clinical outcome. In addition, we found that the supernatants derived from naïve parental and persister cells have distinct effects on human CD4+, CD8+ T cells and macrophages. MPC supernatant had a significant inhibitory effect on the proliferation and activation of CD4+, CD8+ T cells as well as the expression of immune checkpoint markers (PD-1, PD-L1, CTLA4, LAG3, Tim3) compared to supernatant of naïve parental cells. We also found that supernatants of MPC induced M2-like polarization of macrophages. One major candidate to convey this effect is MIF (Macrophage migration inhibitory factor) that is specifically increased in MPC supernatant.
Conclusion: Melanoma persister cells can be identified in patients’ biopsies during TT as well as ICI therapy. The expression of MPC protein markers is associated with poor clinical outcome regardless of the type of treatment. Their expression is inversely correlated with a hot immune environment that could result from the effect of immunosuppressive cytokines secreted by persister cells.
Citation Format: Hongxu Chen, Shensi Shen, Sara Faouzi, Alexia Alfaro, Dorothee Baille, Séverine Roy, Naima Benannoune, Hugues Hermann, Samad Muhammadnejad, Stephan Vagner, Caroline Robert. Biomarkers of drug-tolerant persistent melanoma cells in human, correlation with tumor microenvironment and response to treatment [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 3268.