BackgroundFKBP51 is a co-chaperone with isomerase activity, abundantly expressed in glioma. We previously identified a spliced isoform (FKBP51s) and highlighted a role for this protein in the upregulation of Programmed Death Ligand 1 (PD-L1) expression in melanoma. Because gliomas can express PD-L1 causing a defective host anti-tumoral immunity, we investigated whether FKBP51s was expressed in glioma and played a role in PD-L1 regulation in this tumour.MethodsWe used D54 and U251 glioblastoma cell lines that constitutively expressed PD-L1. FKBP51s was measured by immunoblot, flow cytometry and microscopy. In patient tumours, IHC and qPCR were used to measure protein and mRNA levels respectively. FKBP51s depletion was achieved by siRNAs, and its enzymatic function was inhibited using selective inhibitors (SAFit). We investigated protein maturation using N-glycosidase and cell fractionation approaches.ResultsFKBP51s was expressed at high levels in glioma cells. Glycosylated-PD-L1 was increased and reduced by FKBP51s overexpression or silencing, respectively. Naïve PD-L1 was found in the endoplasmic reticulum (ER) of glioma cells complexed with FKBP51s, whereas the glycosylated form was measured in the Golgi apparatus. SAFit reduced PD-L1 levels (constitutively expressed and ionizing radiation-induced). SAFit reduced cell death of PBMC co-cultured with glioma.ConclusionsHere we addressed the mechanism of post-translational regulation of PD-L1 protein in glioma. FKBP51s upregulated PD-L1 expression on the plasma membrane by catalysing the protein folding required for subsequent glycosylation. Inhibition of FKBP51s isomerase activity by SAFit decreased PD-L1 levels. These findings suggest that FKBP51s is a potential target of immunomodulatory strategies for glioblastoma treatment.

Gliomas aberrantly express programmed cell death ligand-1 (PD-L1), which has a pivotal role in immunoevasion. The splicing isoform of FKBP5, termed FKBP51s, is a PD-L1 foldase, assisting the immune checkpoint molecule in maturation and expression on the plasma membrane. The concept that PD-L1 supports tumor-intrinsic properties is increasingly emerging. The aim of the present work was to confirm the pro-tumoral effect of PD-L1 on human glioma cell survival, stemness capacity and resistance, and to address the issue of whether, by targeting its foldase either chemically or by silencing, the aggressive tumor features could be attenuated. PD-L1-depleted glioma cells have a reduced threshold for apoptosis, while PD-L1 forced expression increases resistance. Similar results were obtained with FKBP51s modulation. The ability of PD-L1 to counteract cell death was hampered by FKBP51s silencing. PD-L1 expression was particularly high in glioma cells with a cancer-stem-cell profile. Moreover, PD-L1 sustained the spheroid formation capability of glioma cells. Targeting of FKBP51s by small-interfering RNA (siRNA) or the specific inhibitor SAFit2, reduced the number of formed spheroids, along with PD-L1 expression. Finally, in an orthotopic mouse model of glioblastoma, daily treatment with SAFit2 significantly reduced tumor PD-L1 expression, and tumor growth. In treated mice, caspase-3 activation and reduced vimentin expression were observed in excised tumors. In conclusion, targeting of FKBP51s hampers PD-L1 and its pro-tumoral properties, thereby affecting the self-renewal and growth capacities of glioblastoma cells in vitro and in vivo.

The immune system actively counteracts the tumorigenesis process; a breakout of the immune system function, or its ability to recognize transformed cells, can favor cancer development. Cancer becomes able to escape from immune system control by using multiple mechanisms, which are only in part known at a cellular and molecular level. Among these mechanisms, in the last decade, the role played by the so-called “inhibitory immune checkpoints” is emerging as pivotal in preventing the tumor attack by the immune system. Physiologically, the inhibitory immune checkpoints work to maintain the self-tolerance and attenuate the tissue injury caused by pathogenic infections. Cancer cell exploits such immune-inhibitory molecules to contrast the immune intervention and induce tumor tolerance. Molecular agents that target these checkpoints represent the new frontier for cancer treatment. Despite the heterogeneity and multiplicity of molecular alterations among the tumors, the immune checkpoint targeted therapy has been shown to be helpful in selected and even histologically different types of cancer, and are currently being adopted against an increasing variety of tumors. The most frequently used is the moAb-based immunotherapy that targets the Programmed Cell Death 1 protein (PD-1), the PD-1 Ligand (PD-L1) or the cytotoxic T lymphocyte antigen-4 (CTLA4). However, new therapeutic approaches are currently in development, along with the discovery of new immune checkpoints exploited by the cancer cell. This article aims to review the inhibitory checkpoints, which are known up to now, along with the mechanisms of cancer immunoediting. An outline of the immune checkpoint targeting approaches, also including combined immunotherapies and the existing trials, is also provided. Notwithstanding the great efforts devoted by researchers in the field of biomarkers of response, to date, no validated FDA-approved immunological biomarkers exist for cancer patients. We highlight relevant studies on predictive biomarkers and attempt to discuss the challenges in this field, due to the complex and largely unknown dynamic mechanisms that drive the tumor immune tolerance.

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

Tumor necrosis factor-α (TNF-α) is a pleiotropic cytokine, whose role in melanoma is controversial. Although high-dose TNF-α is approved for the treatment of patients with in transit-metastatic melanoma confined to the limb, diverse preclinical models of melanoma have shown that TNF-α can induce cell invasion. Biomarkers that can differentiate between the dual role of TNF-α are needed. TRAF2 is critical to TNF receptor-induced activation of nuclear factor-κB (NF-κB), allowing shifting from death to survival-signaling cascades. The large immunophilin FKBP51 acts as a scaffold and catalyst in the IκB kinase complex assembly and activation. Here, using microscopy and an electrophoretic mobility-shift assay, we provide further evidence in support of the essential role of FKBP51 in sustaining the TNF-α NF-κB signaling in melanoma. Through the cross-linking reaction with the chemical linker disuccinimidyl glutarate, we show that a direct interaction occurs between FKBP51 and TRAF2 in melanoma cells. Immunohistochemistry of tumor samples from 24 patients with cutaneous melanomas showed a correlation between the expressions of the two proteins. Given the association of FKBP51 and TRAF2 with TNF-α-induced NF-κB signaling and their correlation in tumor samples, we propose that the two proteins can be exploited as useful markers for the identification of those melanoma tumors that can benefit from TNF-α inhibition. Future studies will address this hypothesis.

(Duh et al. 1994; Holthuis et al. 1994). The sequence analyses revealed that fascin proteins form a unique family of actin-bundling proteins, sharing no apparent homology with nonfascin actin-bundling proteins including alpha-actinin, villin, and fimbrin.Vertebrates have three fascin genes (fascin-1 through À3): fascin-1 shows widespread expression in a variety of tissues, whereas expression of fascin-2 is restricted to retina and hair cell stereocilia, whereas fascin-3 is restricted to testis. Fascin is absent in Dictyostelium discoideum, C. elegans, or yeasts. As expected from the actin-bundling activity, fascin is mainly localized in filopodia of mammalian cultured cells, microvilli of sea urchin eggs, and drosophila bristles. Consistent with highly motile structure of filopodia, fascin is shown to promote cell motility and metastasis of tumor cells. Intriguingly, a fascin homologue is found in microvilli and filopodia of choanoflagellates. Because this unicellular organism is considered as the last ancestor of multicellular animals, fascin1 is suggested to be an ancestral component for filopodial assembly machinery (Sebe-Pedros et al. 2013). Structure and Function of FascinX-ray structural analyses revealed that fascin has a b-trefoil structure (Sedeh et al. 2010), showing Fascin1 plays an important role in the formation of filopodia in mammalian cells. In vitro, fascin1 makes parallel actin bundles with uniform polarity, which is consistent with its localization in filopodia. Upregulation of fascin1 induces membrane protrusions and increases cell motility of epithelial cells, as well as colonic epithelial and carcinoma cells. Conversely, Fascin1 knock down has been reported to block filopodia assembly of B16F1 mouse melanoma cells, as well as colon carcinoma cells and mature antigen-presenting dendritic cells (DCs) (Ross et al. 1998).In addition to binding to actin filaments, fascin1 has been recently reported to bind directly to microtubules (Villari et al. 2015). Fascin1-microtubule binding occurred independently of fascin1-actin binding. The association was shown to increase the dynamics of focal adhesions, as well as that of microtubules, thereby controlling cell motility. This regulation of focal adhesion dynamics may be controlled via FAK because fascin1 was found to bind to FAK. Genetic AnalysesAnalyses of Drosophila singed mutations indicate that fascin1 is involved in female sterility, in addition to the gnarled bristle phenotype. In Drosophila oogenesis, each developing oocyte is surrounded by and connected to 15 nurse cells via intercellular bridges. Nurse cell cytoplasmic contents flow into the oocyte along actin filaments traversing these cytoplasmic bridges. A singed allele affects the microfilament structure required for this nurse cell cytoplasmic flow, resulting in female sterility (Cant et al. 1994). Interestingly, fascin1 is involved in prostaglandin-mediated actin remodeling (Groen et al. 2012). Prostaglandin is synthesized in Drosophila by peroxidase (Pxt), a cyclooxygenase (COX)-like...

The inclusion of customary justice mechanisms is increasingly being invoked as an answer to the top-down, externally-driven approach to transitional justice. But the practice of engaging with customary justice systems proves complicated. The approach of governments and the international community has been criticized as 'ethnojustice', where a male-elderly version of customary justice is invented and imposed, based on a myth of community consensus. In Somalia, the government and international community are currently considering a role for customary justice systems in the reintegration of low-risk disengaged Al-Shabaab (AS) combatants. In this article we combine unique data on local perceptions regarding the return of ex-combatants in Somalia with insights from the literature, to critically examine the prospects of engaging customary justice mechanisms in South-Central Somalia in the reintegration of disengaged Al Shabaab combatants and of a supporting role for the international donor community and the government.

Melanoma often exploits Treg to avoid immune attack. Treg is a heterogeneous population with respect to immunosuppressive capability. Lymphocytes are particularly rich in FKBP51 (encoded by FKBP5 gene), known as the receptor for FK506. Melanoma aberrantly expresses this protein, which sustains resistance and invasion. Melanoma/immune cell interaction, through PD-L1/PD1, bidirectionally generates FKBP5 splicing inducing a lower molecular weight form termed FKBP51s. In 64 advanced melanoma patient PBMCs, we found that FKBP51s marked a Treg subset that correlated to anti-CTLA4 response. More precisely, a Treg FKBP51s+ count <1% was associated with unresponsiveness (normal donor-range 0.1-0.7%). Aim of the present study was to assess the role of Treg FKBP51s+ as potential biomarker of response in a different cohort of patients treated with anti-PD1. In addition, the suppressive potential of Treg FKBP51s+ in comparison with that of Treg FKBP51s- is investigated. To date, we have outcomes of 11 patients. For each patient, we have collected up to 10 blood samples, at T0 and before each treatment, to monitor Tregs. In 5 responder patients, Treg FKBP51s+ was >1.2 and <4.8; in 5 non responder patients, the count was >0.04 and <0.8 After a transient increase following the first administration, the count decreased to 0.3+0.2% in responder patients. Interestingly, a patient with count =0.7% developed autoimmune side effects that led to therapy discontinuation. Resolution of side effects was accompanied by a value increase to 9.9%; anti-PD1 re-administration was then successful. In vitro iTreg generation showed that FKBP51s was upregulated in Treg CD25high, Ki67high and p70S6khigh, corresponding to a highly metabolically active profile with strong suppressive capability. In conclusion, melanoma patients that benefit from immune checkpoint targeted therapy are recognizable by an expansion of a Treg subset which is marked by FKBP51s, a splicing protein isoform generated by triggering of surface antigens (PD-L1, PD1), abundantly expressed on highly suppressive Tregs. Citation Format: Teresa Troiani, Simona Romano, Paolo D'Arrigo, Anna Rea, Martina Tufano, Emilio F. Giunta, Giuseppe Matarrese, Claudio Procaccini, Nunzia Novizio, Vincenza Vigorito, Deriggio Faicchia, Giuseppe Argenziano, Fortunato Ciardiello, Maria F. Romano. Identification of a highly suppressive Treg subset associated with immunotherapy response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5712.