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Glioblastoma Multiforme is one of the most aggressive form of malignancy of the central nervous system. To date, the treatment of this malignancy does not significantly improve the poor overall survival of the patients. The dramatic outcomes of immunotherapy in the treatment of several solid and hematologic tumors are giving new hopes for the treatment of glioblastoma. A series of clinical studies are currently ongoing, encouraged by the promising pre-clinical results achieved. PD-L1 is one of the main immunotherapy molecular targets expressed on glioma cell surface. PD-L1 interacts with the cognate receptor PD1, expressed on the lymphocytes, and conveys an immune-inhibitory signalling. Increasing evidence suggests that PD-L1, in addition to its well-known immunomodulatory function, exerts tumor intrinsic functions that sustain self-renewal and growth. Currently, the overall mechanism that regulates the expression of PD-L1 in tumor cells is still unclear. Glioma tumors express high levels of the immunophilin and co-chaperone FKBP51. Our research group have identified a splicing isoform of this immunophilin, FKBP51s, in melanoma patients and provided elements in support of a role for this isoform as a foldase that serves to PD-L1 protein maturation. The aim of my thesis work was to address the role of FKBP51s in regulation of PD-L1 expression in glioblastoma. Moreover, I also attempted to investigate whether PD-L1 could affect the tumor intrinsic pro-oncogenic properties. To analyse the role of FKBP51s on PD-L1 expression and function, I used D54 and U251 glioblastoma cell lines that constitutively express PD-L1 and FKBP51s. By immunoblot and flow cytometry, I measured the expression of FKBP51s and PD-L1. By sub-cellular fractionation and co-immunoprecipitation assays, I investigated FKBP51s localization and interaction with PD-L1. By co-cultures with PBMCs, I measured the tumor capacity to kill lymphocytes through PD-L1. By spheroids assay, immunofluorescence for Ki67 and cell counting, I measured the effect of PD-L1 and FKBP51s on glioma cell self-renewal and growth capacities. Using a murine model of glioblastoma, made by injection of GFP+luc+ U87 cells, I addressed the effect of a selective inhibitor of FKBP51, called SAFit2, on tumor growth and aggressiveness. Results obtained showed that FKBP51s is abundantly expressed in glioma cells. FKBP51s co-localized with PD-L1 in the ER. Silencing of FKBP51s expression or inhibiting its function by SAFit2 decreased glycosylated PD-L1 levels and its expression on plasma membrane. Moreover, PD-L1 silencing decreased spheroids formation especially in a subset of tumor cells which is isolated by the subventricular zone (SVZ) and has a high tumorigenic capacity. The same effect was obtained with SAFit2 that also decreased Ki67 expression and proliferation capacity of glioblastoma cells. Daily treatment with SAFit2 reduced the volumes of formed tumors, in the in vivo glioblastoma model, and reduced the vimentin expression, in comparison with tumors from mice treated with the drug vehicle. In conclusion, FKBP51s is involved in a post-transcriptional mechanism that regulates PD-L1 maturation and consequent expression on glioblastoma plasma membrane. Glioblastoma cells with a high tumorigenic capacity, identified in the SVZ, expressed higher levels of PD-L1 compared with cells from the tumor mass grown in the nigrostriatal region. Inhibiting FKBP51s function decreases PD-L1 expression and reduces self-renewal and growth capacities of glioblastoma cells in vitro and in vivo.

Glioblastoma can avoid immune surveillance and induce tumor tolerance, through inhibitory molecules, e.g. PD-L1. Ionizing radiation (IR), used to treat this tumor, is known to increase tumor expression of PD-L1, thus inducing resistance mechanisms. Finding molecular determinants involved in IR-induced PD-L1 may provide a target for preventing such an effect and improve radiotherapy outcomes. We demonstrated that the short isoform of the cochaperone FKBP51 (FKBP51s) regulated PD-L1 expression in melanoma. In glioma, FKBP51s was expressed at high levels, together with PD-L1 and its silencing reduced PD-L1 levels. Conversely, overexpression of FKBP51s increased PD-L1. Different PD-L1 isoforms were observed by immunoblot. A lower band (~37 kDa) corresponding to the naïve protein and two upper bands (~50, ~68 kDa) ascribable to post-translationally modified isoforms. FKBP51s was found mainly bound to the heaviest bands of PD-L1, reasonably mature protein, while the canonical isoform FKBP51 appeared to bind only the naïve protein. Mature PD-L1 protein consists in carbohydrates addition, the principal chemical modification to most plasma membrane proteins, and, particularly, N-glycosylation. Treatment of immunoprecipitated PD-L1 protein with PNGaseF produced a decrease of the highest band and the appearance of a lower band, corresponding to the naïve PD-L1, in accordance with the concept that the heaviest band of PD-L1 is a glycosylated form. Moreover, following subcellular fractionation to obtain extracts from ER and Golgi compartments, we found that naïve 37 kDa PD-L1 was detectable in the ER, but not in the Golgi. The PD-L1 glycosylated band was expressed in ER in a small proportion and mostly in the Golgi. FKBP51s, but not the canonical FKBP51, was found in ER. Co-IP of FKBP51s and PD-L1 from ER extract confirmed the two proteins interacted each other in ER. Our results show that naïve PD-L1 colocalized in the ER of glioma cell complexed with FKBP51s, while the PD-L1 glycosylated form was measured in the Golgi apparatus. Treatment of glioma cell with increasing doses of IR upregulated PD-L1 expression, in a dose-response manner. Particularly, we found a significant increase in PD-L1 expression at 4 and 8 Gy, in comparison with unirradiated glioma cell. Moreover, IR induction of mature PD-L1 was efficiently counteracted by FKBP51s silencing. Subcellular fractionation of glioma cell subjected to IR in kinetics showed an early and transitory decrease in FKBP51s ER levels at 3hrs, in line with a reduction of the glycosylated band in the whole lysate. After 8 hrs from IR, FKBP51s rose up again in the ER inducing a full maturation of PD-L1. These findings suggested that FKBP51s has a role in catalyzing PD-L1 folding, an essential step to glycosylation, through which it controls the affinity for PD1. This study identifies FKBP51s as an essential element that regulates PD-L1 expression on glioma cell, which is exploited by the tumor to resist to IR. Citation Format: Paolo D'Arrigo, Michele Russo, Elia Guadagno, Roberto Pacelli, Maria Laura Del Basso De caro, Anna Rea, Martina Tufano, Stefania Staibano, Gennaro Ilardi, Maria Fiammetta Romano, Simona Romano. Ionizing radiation-induced PD-L1 upregulation in glioma: a crucial role for the molecular chaperone FKBP5 [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 586. doi:10.1158/1538-7445.AM2017-586

Background Long‐term data on durability of currently available transcatheter heart valves are sparse. We sought to assess the incidence of long‐term (8‐year) structural valve dysfunction and bioprosthetic valve failure in a cohort of patients with transcatheter aortic valve replacement ( TAVR ) who reached at least 5‐year follow‐up. Methods and Results Consecutive patients with at least 5‐year follow‐up available undergoing TAVR from June 4, 2007 to March 30, 2012 were included. Structural valve dysfunction and bioprosthetic valve failure were defined according to newly standardized European Association of Percutaneous Cardiovascular Interventions/ European Society of Cardiology/European Association for Cardio‐Thoracic Surgery criteria and reported as cumulative incidence function to account for the competing risk of death. A total of 288 consecutive patients with a mean age of 80.7±5.3 years and with a mean Society of Thoracic Surgery mortality score of 8.1±5.1% were analyzed. Survival rate at 8 years was 29.8%. Mean pressure gradients decreased from 53.3±15.9 mm Hg (pre‐ TAVR ) to 10.5±4.5 mm Hg (in‐hospital post‐ TAVR ) ( P Bioprosthetic valve failure was observed in a total of 11 patients (8‐year cumulative incidence function: 4.51%; 95% confidence interval , 1.95%–8.76%). Severe and moderate structural valve dysfunctions were reported in 7 patients (8‐year cumulative incidence function: 2.39%; 95% confidence interval, 0.77%–5.71%) and 13 patients (8‐year cumulative incidence function: 5.87%; 95% confidence interval , 3.06%–9.96%), respectively. Aortic valve reintervention (redo TAVR ) was successfully performed in 2 patients (0.7%) presenting with symptomatic severe restenosis and intraprosthetic regurgitation subsequent to endocarditis. Conclusions In an aged population of patients with symptomatic severe aortic stenosis treated with first‐generation bioprostheses, TAVR was associated with a survival rate of 30% but low rates of bioprosthetic valve failure and structural valve dysfunction at 8 years.

Among the high-grade malignant gliomas (III-IV), grade IV glioma, or glioblastoma (GBM), is the most common and destructive form of brain cancer. Despite rigorous molecular, preclinical, and clinical research these tumors remain a challenge to treat and 70% of patients diagnosed with GBM will succumb to the disease within 2 years. New immune-based cancer treatments are currently in development, among which, anti-PD-1 is in a phase II clinical trial for recurrent glioblastoma. Tumor cells exploit PDL-1/PD1 pathway to induce T cell exhaustion and evade host antitumour immunity. PDL-1 expression on glioblastoma has been shown to correlate with a bad prognosis. There is strong demand for knowledge about the mechanisms regulating PDL-1 expression in glioblastoma. Our research group, has previously identified a relevant role for FKBP51, and its spliced isoform FKBP51s, in PDL-1 expression and melanoma resistance to the therapy. FKBP51 is an immunophilin capable of immunosuppression, originally cloned in lymphocytes. Because FKBP51 was identified as highly expressed in glioblastoma, correlating with tumor aggressiveness, we investigated whether this protein and/or its spliced isoform FKBP51s could be involved in regulation of PDL-1 expression in glioblastoma. In addition, we looked at the role of these protein-isoforms in glioblastoma resistance to chemotherapy. To this aim, we used two different glioblastoma cell lines termed D54 and U251. Our results suggest that, differently from melanoma, in which PDL-1 is inducible and associated with FKBP51 splicing, glioblastoma cell lines express high basal levels of PDL-1. Interestingly, both the canonical and the spliced FKBP51 isoforms were highly expressed in the glioblastoma cell lines. Expression of PDL-1 was regulated by FKBP51s knockin and knockout and the levels changed in accordance with FKBP51s levels. Moreover, in glioblastoma cells silenced for FKBP51s, the decrease in PDL-1 levels was associated with a significant increase in apoptosis, either spontaneous apoptosis and the one stimulated by etoposide or temozolomide. Two recently developed compounds, selective inhibitor of FKBP51, termed SAFit1 and SAFit2 were able to reduce expression of PDL-1 on glioblastoma cell lines. Particularly, SAFit 2 was found to be more effective, in accordance with the notion that it is more brain-permeable than SAFit1. SaFit 2 was also shown to increase sensitivity to etoposide of glioblastoma cell, although to a lesser extent when compared to FKBP51siRNAs. In conclusion, our preliminary results suggest that FKBP51s plays a relevant role in glioblastoma resistance and PDL-1 expression. Future studies are needed to shed light on the mechanisms underlying FKBP51s-induced PDL-1 expression and signaling pathway downstream to FKBP51s/PDL-1 that sustain glioblastoma resistance.

Constitutive activation of NF-κB occurs in a significant percentage of human cancers, including melanoma. Aberrant NF-κB activation sustains cancer survival, contributes to tumor invasion and accounts for therapy resistance. Several studies support a role for FKBP51 in NF-κB activation, however, mechanisms by which FKBP51 controls NF- κB pathway are still not known. FKBP51 has been previously found to interact with IKKα. We studied FKBP51 interactions with a wide number of-κB proteins

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.

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