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Background FKBP51 immunophilin is abundantly expressed by immune cells. Co-inhibitory immune receptor signalling generates the splicing isoform FKBP51s. Tregs stained by FKBP51s are increased in melanoma patients and their counts are associated with anti-CTLA-4 response. An expansion of FKBP51s+PD-L1+ monocytes was measured in a group of non-responding patients to anti-CTLA-4. The aim of this work was to confirm the predictive value of response of FKBP51s+Tregs in a cohort of patients undergoing anti-PD1 treatment and shed light on a monocyte subset co-expressing PD-L1/FKBP51s. Methods Co-cultures of organoids and autologous lymphocytes were used to confirm that tumour T-cell interaction can induce FKBP51s. PBMC immunophenotype and flow cytometry served to assess and monitor FKBP51s+Treg and FKBP51s+PD-L1+ monocytes in 22 advanced melanoma patients treated with anti-PD1. Silencing and overexpression of FKBP51s in human macrophages served to address the protein role in the tolerant macrophages’ behaviour. Results FKBP51s+Tregs count was increased in responders and had a prognostic value. Non-responders showed an early increase in FKBP51s+ PD-L1+ monocytes during anti-PD1 treatment. Manipulation of FKBP51s modulated the macrophage–phenotype, with forced protein expression promoting aspects associated with tolerance. Conclusions FKBP51s may guide in the selection and monitoring of melanoma patient candidates to immune-checkpoint-targeted therapy. Manipulation of FKBP51s may overcome resistance.

Recent advances in tumor immunology, fostered by dramatic outcomes with cancer immunotherapy, have opened new scenarios in cancer metastasis. The cancer stemness/mesenchymal phenotype and an excess of immune suppressive signals are emerging as Intertwined aspects of human tumors. This review examines recent studies that explored the mechanistic links between cancer cell stemness and immunoevasion, and the evidence points to these key events in cancer metastasis as two sides of the same coin. This review also covers the mechanisms involved in tumor expression of programmed cell death ligand 1 (PD-L1), a major factor exploited by human neoplasias to suppress immune control. We highlight the convergence of mesenchymal traits and PD-L1 expression and examine the functions of this immune inhibitory molecule, which confers cancer cell resistance and aggressiveness.

Despite Glioblastoma (GBM) frequently expressing programmed cell death ligand-1 (PD-L1), treatment with anti-programmed cell death-1 (PD1) has not yielded brilliant results. Intratumor variability of PD-L1 can impact determination accuracy. A previous study on mouse embryonic fibroblasts (MEFs) reported a role for cyclin-D in control of PD-L1 expression. Because tumor-cell growth within a cancer is highly heterogeneous, we looked at whether PD-L1 and its cochaperone FKBP51s were influenced by cell proliferation, using U251 and SF767 GBM-cell-lines. PD-L1 was measured by Western blot, flow cytometry, confocal-microscopy, quantitative PCR (qPCR), CCND1 by qPCR, FKBP51s by Western blot and confocal-microscopy. Chromatin-Immunoprecipitation assay (xChIp) served to assess the DNA-binding of FKBP51 isoforms. In the course of cell culture, PD-L1 appeared to increase concomitantly to cyclin-D on G1/S transition, to decrease during exponential cell growth progressively. We calculated a correlation between CCND1 and PD-L1 gene expression levels. In the temporal window of PD-L1 and CCND1 peak, FKBP51s localized in ER. When cyclin-D declined, FKBP51s went nuclear. XChIp showed that FKBP51s binds CCND1 gene in a closed-chromatin configuration. Our finding suggests that the dynamism of PD-L1 expression in GBM follows cyclin-D fluctuation and raises the hypothesis that FKBP51s might participate in the events that govern cyclin-D oscillation.

Thrombocytopenia after TAVI is common and clinically detrimental. Retrospectively, we observed Portico recipients had a more profound platelet drop than Evolut recipients. We thus investigated periprocedural platelet damage and/orpro-inflammatory state in 64 TAVI recipients at baseline and after implantation. Platelet damage was assessed by annexin V staining and monocyte-phagocytic phenotype was assessed according to CD14/CD36 expression. Serum cytokines were measured in 20 patients. The formaldehyde-based storage solution altered platelets. When, before being loaded onto the delivery system, Portico underwent one additional flushing to those recommended, the receiving patients showed thrombocytopenia, platelet damage, and CD36-monocyte count were mitigated. A general increase in IL-6 was recorded in overall TAVI recipients, but a high serum level of IL-8, a potent thrombocytopenia inducer, was measured in Portico recipients only, including those with extra-rinsed valve. Our study suggests a platelet-injury effect by storage-solution and generates the hypothesis of a role for the biomaterial in stimulating innate-immunity. Larger prospective studies are needed. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1007/s12265-021-10117-9.

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.

Magnetic resonance imaging (MRI) is the gold standard for glioblastoma (GBM) patient evaluation. Additional non-invasive diagnostic modalities are needed. GBM is heavily infiltrated with tumor-associated macrophages (TAMs) that can be found in peripheral blood. FKBP51s supports alternative-macrophage polarization. Herein, we assessed FKBP51s expression in circulating monocytes from 14 GBM patients. The M2 monocyte phenotype was investigated by qPCR and flow cytometry using antibodies against PD-L1, CD163, FKBP51s, and CD14. MRI assessed morphologic features of the tumors that were aligned to flow cytometry data. PD-L1 expression on circulating monocytes correlated with MRI tumor necrosis score. A wider expansion in circulating CD163/monocytes was measured. These monocytes resulted in a dramatic decrease in patients with an MRI diagnosis of complete but not partial surgical removal of the tumor. Importantly, in patients with residual tumor, most of the peripheral monocytes that in the preoperative stage were CD163/FKBP51s− had turned into CD163/FKBP51s+. After Stupp therapy, CD163/FKBP51s+ monocytes were almost absent in a case of pseudoprogression, while two patients with stable or true disease progression showed sustained levels in such circulating monocytes. Our work provides preliminary but meaningful and novel results that deserve to be confirmed in a larger patient cohort, in support of potential usefulness in GBM monitoring of CD163/FKBP51s/CD14 immunophenotype in adjunct to MRI.

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.

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.

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 1.2 and 0.04 and 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.

In the 70s, after a decade from the purification of cyclosporine, a selective immunosuppressant agent and potent tool in transplantation medicine, a novel molecule was purified from bacteria Streptomyces tsukubaensis. This molecule, called FK506, showed the same selective immunosuppressant action as cyclosporine but was 10 to 100 fold more potent. In an attempt to clarify the molecular mechanism through which the new drug exerted such a selective effect on T-cells activation, two laboratories identified the cytosolic receptor for FK506. This so-called FK506 binding protein (FKBP) was purified from bovine thymus, human spleen, and Jurkat T-cell line. The isolated FKBP had an approximate molecular mass of 14 kDa and showed an isomerase activity similar to the recently purified cyclosporine-binding protein, cyclophilin, but, it was inhibited by FK506 and rapamycin but not cyclosporine. The subsequent cloning of FKBP gene revealed that FKBP and cyclophilin had dissimilar sequences in spite of their common enzymatic activity. The identified FKBP gene encoded for a protein of 108 aminoacids with a relative molecular mass of 11,819. For this reason, the progenitor of this nascent class of proteins was later known as FKBP12. The subsequent studies showed that FKBP12 was just a member of a ubiquitous and evolutionarily conserved sub-family of proteins which differ from each other in their molecular weight and structure. All FKBPs share a highly conserved domain, termed “FK-12 like domain”, capable of binding to FK506 and exerting isomerase properties, i.e. interconversion from cis-to-trans and trans-to-cis of peptide bonds involving proline, on protein substrates. A schematic historical background of the 17 FKBPs so far identified is shown. A general overview of FKBP structure, function and eventually associated disease is given in this monograph, with the order of proteins following the chronology of discovery.

Background and aim: FKBP51 is an immunophilin encoded by FKBP5 gene on chromosome 6. It is a protein resident in lymphocytes and involved in immune response. Recently, our group demonstrated that the inhibitory checkpoint PD-L1/PD1 promoted the alternative splicing of FKBP5 gene, resulting in increased expression of its variant 4, in PBMC of melanoma patients. The aim of this study was to address whether such a molecular signature could help in identifying an immune profile associated with increased probability of immunotherapy response. Experimental Design: The splicing FKBP51 isoform or FKBP51s was measured in peripheral blood T lymphocytes subsets (CD3/CD4, CD3/CD8, CD25 and PD-L1) and CD14 monocytes from a cohort of 118 patients and 77 age- and sex-matched healthy controls, by flow cytometry. Blood samples were collected before patients underwent ipilimumab treatment. Furthermore, in 64 out of 118 patients, expression of FKBP51s was also assessed in regulatory T cells. Results: Physiologically, each PBMC subset analyzed contained a fraction of an FKBP51spos component, which resulted expanded in melanoma patients. We also measured an increase in CD3/CD8 and PD-L1 lymphocytes in patients. CD4 T lymphocytes showed the FKBP51sneg fraction significantly impaired, which might reflect the condition of impaired T cell help. Treg count was increased, in accordance with previous studies. The count of FKBP51sposTregs defined a subgroup of nonresponder patients to ipilimumab, by 92.6%. A 2D hierarchical partitioning of data from FKBP51s-immunophenotype (heatmap) revealed 3 main clusters: C1 (33 pts, 51,5%), C2 (14 pts, 22%) and C3 (17 pts, 26,5%). FKBP51spos Treg subset appeared globally increased in all clusters. In C1, values of effector T cells and monocytes were not different from normal donors. C2 showed a significant increase of FKBP51spos PD-L1 monocytes and a significant probability of not responding (Chi-square=5.46; p=0.019). This finding is consistent with the hypothesis that response to ipilimumab is prevented by accessory cells exerting a negative immune regulatory control. C3 showed a significant increase of FKBP51s in overall lymphocyte subsets analyzed. Patients in this cluster showed reduced overall survival. This finding suggests that a prevalence of activation (CD25) and co-inhibitory (PD-L1) markers together with the expansion of FKBP51spos effector T cells might reflect a condition of chronic lymphocyte stimulation in some advanced melanoma patients, contributing to T cell-exhaustion. Conclusions: FKBP51s-based immunophenotype of melanoma patients revealed several profiles virtually related to a negative immune regulatory control and highlights an impairment of a Treg subset endowed with increased suppressive potential. Such FKBP51sTreg subset is likely to be associated with immunotherapy response (Chi-square=9.916, p=0.002) . Citation Format: Simona Romano, Ester Simeone, Anna D'Angelillo, Paolo D'Arrigo, Mario Capasso, Vito Alessandro Lasorsa, Martina Tufano, Anna Rea, Michele Russo, Nicola Zambrano, Paolo Antonio Ascierto, Maria Fiammetta Romano. An FKBP5-based immunophenotype for assessment of the immunosuppression status and possible prediction of immunotherapy response in melanoma patients [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 2624. doi:10.1158/1538-7445.AM2017-2624

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. Citation Format: Michele Russo, Paolo D’Arrigo, Felix Hausch, Anna Rea, Martina Tufano, Maria Fiammetta Romano, Simona Romano. Study of PDL-1 regulation and expression in glioblastoma and its role in cancer resistance. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2216.

// Paolo D’Arrigo 1, * , Michele Russo 1, * , Anna Rea 1 , Martina Tufano 1 , Elia Guadagno 2 , Maria Laura Del Basso De Caro 2 , Roberto Pacelli 2 , Felix Hausch 3 , Stefania Staibano 2 , Gennaro Ilardi 2 , Silvia Parisi 1 , Maria Fiammetta Romano 1 and Simona Romano 1 1 Department of Molecular Medicine and Medical Biotechnologies, Federico II University, Naples, Italy 2 Department of Advanced Biomedical Sciences, Federico II University, Naples, Italy 3 Technical University Darmstadt Institute of Organic Chemistry and Biochemistry, Darmstadt, Germany * These authors have contributed equally to this work Correspondence to: Maria Fiammetta Romano, email: mariafiammetta.romano@unina.it , romano@dbbm.unina.it Keywords: PD-L1, glioblastoma multiforme, FKBP51, ionizing radiation, SAFit Received: March 10, 2017 Accepted: June 11, 2017 Published: July 17, 2017 ABSTRACT Background: FKBP51 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. Methods: We 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. Results: FKBP51s was expressed at high levels in glioma cells. Glycosylated-PD-L1 was increased and reduced by FKBP51s overexpression or silencing, respectively. Naive 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. Conclusions: Here 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.