Indocyanine green clearance test in liver transplantation: defining cut-off levels for graft viability assessment during organ retrieval and for the prediction of post-transplant graft function recovery - the Liver Indocyanine Green (LivInG) Trial Study Protocol

Introduction Viability assessment of the graft is essential to lower the risk of liver transplantation (LT) failure and need for emergency retransplantation, however, this still relies mainly on surgeon's experience. Post-LT graft function recovery assessment is also essential to aid physicians in the management of LT recipients and guide them through challenging decision making. This study aims to trial the use of indocyanine green clearance test (IGT) in the donor as an objective tool to assess graft viability and in the recipient to assess graft function recovery after LT.Methods and analysis This is an observational prospective single-centre study on consecutive liver transplant donors and recipients.Primary objective To determine the capability of IGT of predicting graft viability at the time of organ retrieval. Indocyanine green will be administered to the donor and the plasma disappearance rate (PDR) measured using the pulsidensitometric method. Some 162 IGT donor procedures will be required (alpha, 5%; beta, 20%) using an IGT-PDR cut-off value of 13% to achieve a significant discrimination between viable and non-viable grafts.Secondary objective IGT-PDR will be measured at different time-points in the LT recipient: during the anhepatic phase, after graft reperfusion, at 24 hours, on day 3 and day 7 after LT. The slope of IGT values from the donor to the recipient will be evaluated for correlation with the development of early allograft dysfunction.Ethics and dissemination This research protocol was approved by Fondazione Policlinico Universitario Agostino Gemelli IRCCS Ethics Committee (reference number: 0048466/20, study ID: 3656) and by the Italian National Transplant Center (CNT) (reference number: Prot.11/ CNT2021). Liver recipients will be required to provide written informed consent. Results will be published in international peer-reviewed scientific journals and presented in congresses

Synergistic inhibition of the Hedgehog pathway by newly designed Smo and Gli antagonists bearing the isoflavone scaffold

Aberrant activation of the Hedgehog (Hh) pathway is responsible for the onset and progression of several malignancies. Small molecules able to block the pathway at the upstream receptor Smoothened (Smo) or the downstream effector Gli1 have thus emerged recently as valuable anticancer agents. Here, we have designed, synthesized, and tested new Hh inhibitors taking advantage by the highly versatile and privileged isoflavone scaffold. The introduction of specific substitutions on the isoflavone's ring B allowed the identification of molecules targeting preferentially Smo or Gli1. Biological assays coupled with molecular modeling corroborated the design strategy, and provided new insights into the mechanism of action of these molecules. The combined administration of two different isoflavones behaving as Smo and Gli antagonists, respectively, in primary medulloblastoma (MB) cells highlighted the synergistic effects of these agents, thus paving the way to further and innovative strategies for the pharmacological inhibition of Hh signaling

A Smo/Gli multitarget hedgehog pathway inhibitor impairs tumor growth

Pharmacological Hedgehog (Hh) pathway inhibition has emerged as a valuable anticancer strategy. A number of small molecules able to block the pathway at the upstream receptor Smoothened (Smo) or the downstream effector glioma-associated oncogene 1 (Gli1) has been designed and developed. In a recent study, we exploited the high versatility of the natural isoflavone scaffold for targeting the Hh signaling pathway at multiple levels showing that the simultaneous targeting of Smo and Gli1 provided synergistic Hh pathway inhibition stronger than single administration. This approach seems to effectively overcome the drug resistance, particularly at the level of Smo. Here, we combined the pharmacophores targeting Smo and Gli1 into a single and individual isoflavone, compound 22, which inhibits the Hh pathway at both upstream and downstream level. We demonstrate that this multitarget agent suppresses medulloblastoma growth in vitro and in vivo through antagonism of Smo and Gli1, which is a novel mechanism of action in Hh inhibition

Mitogen-activated kinase kinase kinase 1 inhibits hedgehog signaling and medulloblastoma growth through GLI1 phosphorylation

The aberrant activation of hedgehog (HH) signaling is a leading cause of the development of medulloblastoma, a pediatric tumor of the cerebellum. The FDA‑approved HH inhibitor, Vismodegib, which targets the transmembrane transducer SMO, has shown limited efficacy in patients with medulloblastoma, due to compensatory mechanisms that maintain an active HH‑GLI signaling status. Thus, the identification of novel actionable mechanisms, directly affecting the activity of the HH‑regulated GLI transcription factors is an important goal for these malignancies. In this study, using gene expression and reporter assays, combined with biochemical and cellular analyses, we demonstrate that mitogen‑activated kinase kinase kinase 1 (MEKK1), the most upstream kinase of the mitogen‑activated protein kinase (MAPK) phosphorylation modules, suppresses HH signaling by associating and phosphorylating GLI1, the most potent HH‑regulated transcription factor. Phosphorylation occurred at multiple residues in the C‑terminal region of GLI1 and was followed by an increased association with the cytoplasmic proteins 14‑3‑3. Of note, the enforced expression of MEKK1 or the exposure of medulloblastoma cells to the MEKK1 activator, Nocodazole, resulted in a marked inhibitory effect on GLI1 activity and tumor cell proliferation and viability. Taken together, the results of this study shed light on a novel regulatory mechanism of HH signaling, with potentially relevant implications in cancer therapy

ERAP1 promotes Hedgehog-dependent tumorigenesis by controlling USP47-mediated degradation of βTrCP.

The Hedgehog (Hh) pathway is essential for embryonic development and tissue homeostasis. Aberrant Hh signaling may occur in a wide range of human cancers, such as medulloblastoma, the most common brain malignancy in childhood. Here, we identify endoplasmic reticulum aminopeptidase 1 (ERAP1), a key regulator of innate and adaptive antitumor immune responses, as a previously unknown player in the Hh signaling pathway. We demonstrate that ERAP1 binds the deubiquitylase enzyme USP47, displaces the USP47-associated βTrCP, the substrate-receptor subunit of the SCFβTrCP ubiquitin ligase, and promotes βTrCP degradation. These events result in the modulation of Gli transcription factors, the final effectors of the Hh pathway, and the enhancement of Hh activity. Remarkably, genetic or pharmacological inhibition of ERAP1 suppresses Hh-dependent tumor growth in vitro and in vivo. Our findings unveil an unexpected role for ERAP1 in cancer and indicate ERAP1 as a promising therapeutic target for Hh-driven tumors

Synergistic inhibition of the Hedgehog pathway by newly designed Smo and Gli antagonists bearing the isoflavone scaffold

Hedgehog (Hh) signaling pathway inhibition has emerged in recent years as a druggable target for anticancer therapy. [1] We previously identified Glabrescione B (GlaB), an isoflavone naturally found in the seeds of Derris glabrescens (Leguminosae), as a novel small molecule that proved to interfere with Gli1/DNA interaction. [2] We provided the total synthesis of GlaB based on the deoxybenzoin route, allowing the structure activity relationship elucidation through the preparation of a small-size focused library of isoflavones. Target preference has been achieved by the introduction of specific bulky substitutions at meta position (targeting Gli1) or para position (targeting Smo) of the isoflavone’s ring B that is able to block the pathway respectively at the downstream effector Gli1 or the upstream receptor Smo. [3] Interestingly, the combined administration of two different isoflavones behaving as Smo and Gli1 antagonists in primary medulloblastoma cells has shown synergistic Hh inhibition at doses that are around 20-fold lower than single administration, thus leading the way to further and innovative combination therapy for the treatment of Hh-dipendent tumors. This approach seems to effectively overcome the drug resistance, particularly at the level of Smo. Thus, we combined the pharmacophores targeting Smo and Gli1 into a single and individual isoflavone, compound 22, which inhibits the Hh pathway at both upstream and downstream level and suppresses medulloblastoma growth in vitro and in vivo through antagonism of Smo and Gli1 providing a novel mechanism of action in Hh inhibition. Our study encourages the use of a multitargeting approach for the treatment of Hh-driven tumors and provides significant support in oncology research for the development of new clinically relevant Hh inhibitors. References: [1]. Ghirga F et al. (2018). Bioorganic Med. Chem. Lett. 28: 3131-3140. [2]. Infante P et al. (2015). The EMBO Journal 34: 200-217. [3]. Berardozzi S et al. (2018). Eur. J. Med. Chem. 156: 554-562. [4]. Lospinoso Severini et al. (2019). Cancers 11. 1518

ERAP1 promotes Hedgehog-dependent tumorigenesis by controlling USP47-mediated degradation of βTrCP.

The Hedgehog (Hh) pathway is essential for embryonic development and tissue homeostasis. Aberrant Hh signaling may occur in a wide range of human cancers, such as medulloblastoma, the most common brain malignancy in childhood. Here, we identify endoplasmic reticulum aminopeptidase 1 (ERAP1), a key regulator of innate and adaptive antitumor immune responses, as a previously unknown player in the Hh signaling pathway. We demonstrate that ERAP1 binds the deubiquitylase enzyme USP47, displaces the USP47-associated βTrCP, the substrate-receptor subunit of the SCFβTrCP ubiquitin ligase, and promotes βTrCP degradation. These events result in the modulation of Gli transcription factors, the final effectors of the Hh pathway, and the enhancement of Hh activity. Remarkably, genetic or pharmacological inhibition of ERAP1 suppresses Hh-dependent tumor growth in vitro and in vivo. Our findings unveil an unexpected role for ERAP1 in cancer and indicate ERAP1 as a promising therapeutic target for Hh-driven tumors