Abstract 5603: Melanoma PD1 expression is upregulated by MAPK inhibitors leading to an immune-independent synergism with anti-PD1 antibodies
Authors: Sanlorenzo Martina , Vujic Igor , Floris Arianna , Novelli Mauro , Donini Chiara , Gammaitoni Loretta , Giraudo Lidia , Macagno Marco , Quaglino Pietro , Fierro Maria Teresa , Giordano Silvia , Carnevale-Schianca Fabrizio , Aglietta Massimo , Sangiolo Dario 
Published: July 2017

Abstract
Background Antibodies blocking the inhibitory checkpoint PD1, expressed by T lymphocytes, and kinase inhibitors of BRAF and MEK (MAPKi) are mainstays of current treatment for patients with metastatic melanoma. Mouse models point towards a synergistic antitumor activity of anti-PD1-antibodies and MAPKi, explaining the findings by an enhanced lymphocytic activity opposing tumor adaptive resistance. Recently PD1 was found expressed by a subpopulation of melanoma cells where it could mediate tumorigenic effects, but no data exist showing how MAPKi could modulate its expression. We hypothesized that MAPKi may upregulate PD1 expression and enhance the direct, immune-cell independent antitumor action of anti-PD1 antibodies. We first explore the exact prevalence and magnitude of PD1 expression in patients and cell lines. Then we test the effect of MAPKi on its expression and explore possible immune-independent synergisms with anti-PD1-antibodies.
Methodology and results. Prevalence of PD1 expression by melanoma cells was explored in the Cancer Cell Line Encyclopedia (CCLE) and The Cancer Genome Atlas (TCGA) datasets. CCLE included 61 melanoma cell lines, which all expressed PD1 (Affymetrix mRNA 4.20 [3.81- 4.65]) with values comparable to PDL1 (4.63, range 3.73 -7.84) and PDL2 (3.73, range 3.97-8.50). The TCGA analysis included 114 melanomas, excluding those with evidence of immune infiltrate (n=256) or stromal cells (n=100) potentially interfering with PD1 assessment. PD1 mRNA was expressed by all samples (median FPKM values: 64.7, range 0.4-1461.3). Next we assessed by flow-cytometry the membrane expression of PD1 in 4 melanoma cell lines and 3 patient-derived melanoma cultures, harboring BRAF and NRAS mutations We confirmed that low rates of PD1 were expressed by all melanoma (median 1.2%, range 0.7- 4.3). The median rate of viable PD1+ melanoma cells significantly increased (12.9% range 5.9-20, p=0.0180, n =7) following treatment (96h) with MAPKi (dabrafenib and trametinib) but not with chemotherapy (fotemustine). Longer treatments (8 days) further increased the rate of PD1+ melanoma cells (31.8% range 15.0-50.3, n=3) and the effect was reversed by drug withdrawal. The MAPKi modulatory effect on PD1 expression was therapeutically exploited in vitro. The addition of PD1 blocking antibody enhanced the inhibitory activity of MAPKi on melanoma proliferation (mean inhibition rate: 66% vs 40%, n=2) in the absence of any immune cells.
Conclusions We report a new expression pattern of PD1 receptor on melanoma and its potential therapeutic perspective. PD1 is directly expressed by a small but consistent rate of melanoma cells. Treatment with MAPKi significantly enhanced the rate of PD1+ melanoma cells and direct synergism with PD1 blockade was reported without the involvement of immune response. Our findings may be clinical relevant in settings of patients treated with MAPKi.
Citation Format: Martina Sanlorenzo, Igor Vujic, Arianna Floris, Mauro Novelli, Chiara Donini, Loretta Gammaitoni, Lidia Giraudo, Marco Macagno, Pietro Quaglino, Maria Teresa Fierro, Silvia Giordano, Fabrizio Carnevale-Schianca, Massimo Aglietta, Dario Sangiolo. Melanoma PD1 expression is upregulated by MAPK inhibitors leading to an immune-independent synergism with anti-PD1 antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5603. doi:10.1158/1538-7445.AM2017-5603