Melanoma delivers immune suppressive stimuli through PDL-1. FK506 binding protein 51 (FKBP51) is an immunophilin capable of immune suppression. We, previously, demonstrated a relevant role for this protein in melanoma biology and progression. Melanoma also expresses a splicing variant of FKBP51 (isoform 2 or ISO2), whose function is unknown. Aim of this study was to generate knowledge on the mechanism regulating PDL-1 expression in melanoma, and find biomarkers predictive of response to immunotherapy. We generated melanoma cells overexpressing the canonical FKBP51 or ISO2, and measured levels of PDL-1. Flow cytometry showed that expression of PDL1 in ISO2-melanoma (41.5+4.2%) was significantly higher than expression in control cells (15.5+4.7%) or melanoma overexpressing the canonical FKBP51 (23.2+5.7%). These results suggested a role for ISO2 in regulation of PDL-1 expression, that was confirmed in another tumor cell line, namely HeLa. Western blot assay showed different bands of PDL-1 (around 70kD, 50kD and 37kD). The band at 70kD was present in the tumor, but not in lymphocytes and, reasonably, corresponded to the form exposed on the membrane. This 70kD band was particularly upregulated in ISO2-melanoma. Tumor infiltrating lymphocytes (TIL) express a strong immunohistochemistry signal for FKBP51. Since a molecular mimicry suits melanoma and lymphocytes, we investigated whether PBMCs of melanoma patients, that are a good a source of TIL, expressed the two isoforms. We measured, by QPCR, isoforms levels in PBMCs of 70 healthy donors, 92 primary melanoma and 60 metastatic melanoma patients. Our results show that expression of ISO2, but not canonical FKBP51, was significantly higher in PBMCs of patients than in PBMCs of controls (Kruskal-Wallis test, p<0.0001). In addition, ISO2 expression in PBMCs from metastatic melanoma patients (range 7.0-36.0 au) was significantly higher than expression in PBMCs from primary melanoma (range 1.0-3.5 au)(p<0-007). Healthy donors PBMCs expressed ISO2 when cocultured with melanoma and such expression changed in accordance with PDL-1 expression on melanoma, as suggested by PDL-1 knockdown experiments. Moreover, ISO2 resulted increased in PBMCs cultured with PDL-1 recombinant protein, but not BSA. These findings suggested that the increased expression of ISO2 in PBMCs of melanoma patients was stimulated by the interaction between the tumor and PBMCs, through PDL-1/PD1. In conclusion, our results show that 1) ISO2, a splicing variant of FKBP51, upregulates expression of PDL1 in melanoma; 2) PDL1-PD1 interaction between tumor and lymphocytes induces ISO2 in lymphocytes; 3) increased ISO2 levels were measured in PBMCs of 152 melanoma patients; 4) ISO2 levels were especially high in PBMCs of advanced melanoma patients. Collectively, our results suggest ISO2 is a promising melanoma biomarker related to PDL-1.
Citation Format: Maria Fiammetta Romano, Anna D'Angelillo, Simona Romano, Ester Simeone, Paolo Ascierto, Stefania Staibano, Paolo D'Arrigo, Massimiliano Scalvenzi, Gennaro Ilardi, Rita Bisogni. The isoform 2 of FKBP51 is induced by PDL-1/PD1 interaction and marks peripheral blood mononuclear cells of melanoma patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2912. doi:10.1158/1538-7445.AM2014-2912