SALL4 is a CRL3REN/KCTD11 substrate that drives Sonic Hedgehog-dependent medulloblastoma.

The Sonic Hedgehog (SHH) pathway is crucial regulator of embryonic development and stemness. Its alteration leads to medulloblastoma (MB), the most common malignant pediatric brain tumor. The SHH-MB subgroup is the best genetically characterized, however the molecular mechanisms responsible for its pathogenesis are not fully understood and therapeutic benefits are still limited. Here, we show that the pro-oncogenic stemness regulator Spalt-like transcriptional factor 4 (SALL4) is re-expressed in mouse SHH-MB models, and its high levels correlate with worse overall survival in SHH-MB patients. Proteomic analysis revealed that SALL4 interacts with REN/KCTD11 (here REN), a substrate receptor subunit of the Cullin3-RING ubiquitin ligase complex (CRL3REN) and a tumor suppressor lost in ~30% of human SHH-MBs. We demonstrate that CRL3REN induces polyubiquitylation and degradation of wild type SALL4, but not of a SALL4 mutant lacking zinc finger cluster 1 domain (ΔZFC1). Interestingly, SALL4 binds GLI1 and cooperates with HDAC1 to potentiate GLI1 deacetylation and transcriptional activity. Notably, inhibition of SALL4 suppresses SHH-MB growth both in murine and patient-derived xenograft models. Our findings identify SALL4 as a CRL3REN substrate and a promising therapeutic target in SHH-dependent cancers.

The RIG-I agonist M8 triggers cell death and natural killer cell activation in human papillomavirus-associated cancer and potentiates cisplatin cytotoxicity.

Although the activation of innate immunity to treat a wide variety of cancers is gaining increasing attention, it has been poorly investigated in human papillomavirus (HPV)-associated malignancies. Because these tumors harbor a severely impaired cGAS-STING axis, but they still retain a largely functional RIG-I pathway, another critical mediator of adaptive and innate immune responses, we asked whether RIG-I activation by the 5'ppp-RNA RIG-I agonist M8 would represent a therapeutically viable option to treat HPV+ cancers. Here, we show that M8 transfection of two cervical carcinoma-derived cell lines, CaSki and HeLa, both expressing a functional RIG-I, triggers intrinsic apoptotic cell death, which is significantly reduced in RIG-I KO cells. We also demonstrate that M8 stimulation potentiates cisplatin-mediated cell killing of HPV+ cells in a RIG-I dependent manner. This combination treatment is equally effective in reducing tumor growth in a syngeneic pre-clinical mouse model of HPV16-driven cancer, where enhanced expression of lymphocyte-recruiting chemokines and cytokines correlated with an increased number of activated natural killer (NK) cells in the tumor microenvironment. Consistent with a role of RIG-I signaling in immunogenic cell killing, stimulation of NK cells with conditioned medium from M8-transfected CaSki boosted NK cell proliferation, activation, and migration in a RIG-I-dependent tumor cell-intrinsic manner. Given the highly conserved molecular mechanisms of carcinogenesis and genomic features of HPV-driven cancers and the remarkably improved prognosis for HPV+ oropharyngeal cancer, targeting RIG-I may represent an effective immunotherapeutic strategy in this setting, favoring the development of de-escalating strategies.

Proteolysis-Targeting Chimera (PROTAC): Is the Technology Looking at the Treatment of Brain Tumors?

Post-translational modifications, such as ubiquitylation, need to be tightly controlled to guarantee the accurate localization and activity of proteins. Ubiquitylation is a dynamic process primarily responsible for proteasome-mediated degradation of substrate proteins and crucial for both normal homeostasis and disease. Alterations in ubiquitylation lead to the upregulation of oncoproteins and/or downregulation of tumor suppressors, thus concurring in tumorigenesis. PROteolysis-TArgeting Chimera (PROTAC) is an innovative strategy that takes advantage by the cell's own Ubiquitin-Proteasome System (UPS). Each PROTAC molecule is composed by a ligand that recruits the target protein of interest (POI), a ligand specific for an E3 ubiquitin ligase enzyme, and a linker that connects these units. Upon binding to the POI, the PROTAC recruits the E3 inducing ubiquitylation-dependent proteasome degradation of the POI. To date, PROTAC technology has entered in clinical trials for several human cancers. Here, we will discuss the advantages and limitations of PROTACs development and safety considerations for their clinical application. Furthermore, we will review the potential of PROTAC strategy as therapeutic option in brain tumor, focusing on glioblastoma.

SMA-miRs (miR-181a-5p, -324-5p, and -451a) are overexpressed in spinal muscular atrophy skeletal muscle and serum samples.

Background: Spinal muscular atrophy (SMA) is a neuromuscular disorder characterized by the degeneration of the second motor neuron. The phenotype ranges from very severe to very mild forms. All patients have the homozygous loss of the SMN1 gene and a variable number of SMN2 (generally 2-4 copies), inversely related to the severity. The amazing results of the available treatments have made compelling the need of prognostic biomarkers to predict the progression trajectories of patients. Besides the SMN2 products, few other biomarkers have been evaluated so far, including some miRs. Methods: We performed whole miRNome analysis of muscle samples of patients and controls (14 biopsies and 9 cultures). The levels of muscle differentially expressed miRs were evaluated in serum samples (51 patients and 37 controls) and integrated with SMN2 copies, SMN2 full-length transcript levels in blood and age (SMA-score). Results: Over 100 miRs were differentially expressed in SMA muscle; 3 of them (hsa-miR-181a-5p, -324-5p, -451a; SMA-miRs) were significantly upregulated in the serum of patients. The severity predicted by the SMA-score was related to that of the clinical classification at a correlation coefficient of 0.87 (p<10-5). Conclusions: miRNome analyses suggest the primary involvement of skeletal muscle in SMA pathogenesis. The SMA-miRs are likely actively released in the blood flow; their function and target cells require to be elucidated. The accuracy of the SMA-score needs to be verified in replicative studies: if confirmed, its use could be crucial for the routine prognostic assessment, also in presymptomatic patients. Funding: Telethon Italia (grant #GGP12116).

Drug Delivery Systems for Hedgehog Inhibitors in the Treatment of SHH-Medulloblastoma.

Medulloblastoma (MB) is a highly aggressive pediatric tumor of the cerebellum. Hyperactivation of the Hedgehog (HH) pathway is observed in about 30% of all MB diagnoses, thereby bringing out its pharmacological blockade as a promising therapeutic strategy for the clinical management of this malignancy. Two main classes of HH inhibitors have been developed: upstream antagonists of Smoothened (SMO) receptor and downstream inhibitors of GLI transcription factors. Unfortunately, the poor pharmacological properties of many of these molecules have limited their investigation in clinical trials for MB. In this minireview, we focus on the drug delivery systems engineered for SMO and GLI inhibitors as a valuable approach to improve their bioavailability and efficiency to cross the blood-brain barrier (BBB), one of the main challenges in the treatment of MB.

Specific Protein 1 and p53 Interplay Modulates the Expression of the KCTD-Containing Cullin3 Adaptor Suppressor of Hedgehog 2.

The Hedgehog (Hh) signaling pathway plays a crucial role in normal embryonic development and adult tissue homeostasis. On the other end, dysregulated Hh signaling triggers a prolonged mitogenic response that may prompt abnormal cell proliferation, favoring tumorigenesis. Indeed, about 30% of medulloblastomas (MBs), the most common malignant childhood cerebellar tumors, exhibit improper activation of the Hh signaling. The oncosuppressor KCASH2 has been described as a suppressor of the Hh signaling pathway, and low KCASH2 expression was observed in Hh-dependent MB tumor. Therefore, the study of the modulation of KCASH2 expression may provide fundamental information for the development of new therapeutic approaches, aimed to restore physiological KCASH2 levels and Hh inhibition. To this end, we have analyzed the TATA-less KCASH2 proximal promoter and identified key transcriptional regulators of this gene: Sp1, a TF frequently overexpressed in tumors, and the tumor suppressor p53. Here, we show that in WT cells, Sp1 binds KCASH2 promoter on several putative binding sites, leading to increase in KCASH2 expression. On the other hand, p53 is involved in negative regulation of KCASH2. In this context, the balance between p53 and Sp1 expression, and the interplay between these two proteins determine whether Sp1 acts as an activator or a repressor of KCASH2 transcription. Indeed, in p53-/- MEF and p53 mutated tumor cells, we hypothesize that Sp1 drives promoter methylation through increased expression of the DNA methyltransferase 1 (DNMT1) and reduces KCASH2 transcription, which can be reversed by Sp1 inhibition or use of demethylating agents. We suggest therefore that downregulation of KCASH2 expression in tumors could be mediated by gain of Sp1 activity and epigenetic silencing events in cells where p53 functionality is lost. This work may open new venues for novel therapeutic multidrug approaches in the treatment of Hh-dependent tumors carrying p53 deficiency.

Glabrescione B delivery by self-assembling micelles efficiently inhibits tumor growth in preclinical models of Hedgehog-dependent medulloblastoma.

Aberrant activation of the Hedgehog (Hh) pathway leads to the development of several tumors, including medulloblastoma (MB), the most common pediatric brain malignancy. Hh inhibitors acting on GLI1, the final effector of Hh signaling, offer a valuable opportunity to overcome the pitfalls of the existing therapies to treat Hh-driven cancers. In this study, the toxicity, delivery, biodistribution, and anticancer efficacy of Glabrescione B (GlaB), a selective GLI1 inhibitor, were investigated in preclinical models of Hh-dependent MB. To overcome its poor water solubility, GlaB was formulated with a self-assembling amphiphilic polymer forming micelles, called mPEG5kDa-cholane. mPEG5kDa-cholane/GlaB showed high drug loading and stability, low cytotoxicity, and long permanence in the bloodstream. We found that mPEG5kDa-cholane efficiently enhanced the solubility of GlaB, thus avoiding the use of organic solvents. mPEG5kDa-cholane/GlaB possesses favorable pharmacokinetics and negligible toxicity. Remarkably, GlaB encapsulated in mPEG5kDa-cholane micelles was delivered through the blood-brain barrier and drastically inhibited tumor growth in both allograft and orthotopic models of Hh-dependent MB. Our findings reveal that mPEG5kDa-cholane/GlaB is a good candidate for the treatment of Hh-driven tumors and provide relevant implications for the translation of GlaB into clinical practice.

The SHH/GLI signaling pathway: a therapeutic target for medulloblastoma.

INTRODUCTION: Medulloblastoma (MB) is a heterogeneous tumor of the cerebellum that is divided into four main subgroups with distinct molecular and clinical features. Sonic Hedgehog MB (SHH-MB) is the most genetically understood and occurs predominantly in childhood. Current therapies consist of aggressive and non-targeted multimodal approaches that are often ineffective and cause long-term complications. These problems intensify the need to develop molecularly targeted therapies to improve outcome and reduce treatment-related morbidities. In this scenario, Hedgehog (HH) signaling, a developmental pathway whose deregulation is involved in the pathogenesis of several malignancies, has emerged as an attractive druggable pathway for SHH-MB therapy. AREAS COVERED: This review provides an overview of the advancements in the HH antagonist research field. We place an emphasis on Smoothened (SMO) and glioma-associated oncogene homolog (GLI) inhibitors and immunotherapy approaches that are validated in preclinical SHH-MB models and that have therapeutic potential for MB patients. Literature from Pubmed and data reported on ClinicalTrial.gov up to August 2020 were considered. EXPERT OPINION: Extensive-omics analysis has enhanced our knowledge and has transformed the way that MB is studied and managed. The clinical use of SMO antagonists has yet to be determined, however, future GLI inhibitors and multitargeting approaches are promising.

DUBs Activating the Hedgehog Signaling Pathway: A Promising Therapeutic Target in Cancer.

The Hedgehog (HH) pathway governs cell proliferation and patterning during embryonic development and is involved in regeneration, homeostasis and stem cell maintenance in adult tissues. The activity of this signaling is finely modulated at multiple levels and its dysregulation contributes to the onset of several human cancers. Ubiquitylation is a coordinated post-translational modification that controls a wide range of cellular functions and signaling transduction pathways. It is mediated by a sequential enzymatic network, in which ubiquitin ligases (E3) and deubiquitylase (DUBs) proteins are the main actors. The dynamic balance of the activity of these enzymes dictates the abundance and the fate of cellular proteins, thus affecting both physiological and pathological processes. Several E3 ligases regulating the stability and activity of the key components of the HH pathway have been identified. Further, DUBs have emerged as novel players in HH signaling transduction, resulting as attractive and promising drug targets. Here, we review the HH-associated DUBs, discussing the consequences of deubiquitylation on the maintenance of the HH pathway activity and its implication in tumorigenesis. We also report the recent progress in the development of selective inhibitors for the DUBs here reviewed, with potential applications for the treatment of HH-related tumors.

The RNA-Binding Ubiquitin Ligase MEX3A Affects Glioblastoma Tumorigenesis by Inducing Ubiquitylation and Degradation of RIG-I.

Glioblastoma multiforme (GB) is the most malignant primary brain tumor in humans, with an overall survival of approximatively 15 months. The molecular heterogeneity of GB, as well as its rapid progression, invasiveness and the occurrence of drug-resistant cancer stem cells, limits the efficacy of the current treatments. In order to develop an innovative therapeutic strategy, it is mandatory to identify and characterize new molecular players responsible for the GB malignant phenotype. In this study, the RNA-binding ubiquitin ligase MEX3A was selected from a gene expression analysis performed on publicly available datasets, to assess its biological and still-unknown activity in GB tumorigenesis. We find that MEX3A is strongly up-regulated in GB specimens, and this correlates with very low protein levels of RIG-I, a tumor suppressor involved in differentiation, apoptosis and innate immune response. We demonstrate that MEX3A binds RIG-I and induces its ubiquitylation and proteasome-dependent degradation. Further, the genetic depletion of MEX3A leads to an increase of RIG-I protein levels and results in the suppression of GB cell growth. Our findings unveil a novel molecular mechanism involved in GB tumorigenesis and suggest MEX3A and RIG-I as promising therapeutic targets in GB.

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.

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

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 ßTrCP, the substrate-receptor subunit of the SCFßTrCP ubiquitin ligase, and promotes ßTrCP 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.

Targeting Hedgehog Signalling through the Ubiquitylation Process: The Multiple Roles of the HECT-E3 Ligase Itch.

Hedgehog signalling (Hh) is a developmental conserved pathway strongly involved in cancers when deregulated. This important pathway is orchestrated by numerous regulators, transduces through distinct routes and is finely tuned at multiple levels. In this regard, ubiquitylation processes stand as essential for controlling Hh pathway output. Although this post-translational modification governs proteins turnover, it is also implicated in non-proteolytic events, thereby regulating the most important cellular functions. The HECT E3 ligase Itch, well known to control immune response, is emerging to have a pivotal role in tumorigenesis. By illustrating Itch specificities on Hh signalling key components, here we review the role of this HECT E3 ubiquitin ligase in suppressing Hh-dependent tumours and explore its potential as promising target for innovative therapeutic approaches.

Itch/ß-arrestin2-dependent non-proteolytic ubiquitylation of SuFu controls Hedgehog signalling and medulloblastoma tumorigenesis.

Suppressor of Fused (SuFu), a tumour suppressor mutated in medulloblastoma, is a central player of Hh signalling, a pathway crucial for development and deregulated in cancer. Although the control of Gli transcription factors by SuFu is critical in Hh signalling, our understanding of the mechanism regulating this key event remains limited. Here, we show that the Itch/ß-arrestin2 complex binds SuFu and induces its Lys63-linked polyubiquitylation without affecting its stability. This process increases the association of SuFu with Gli3, promoting the conversion of Gli3 into a repressor, which keeps Hh signalling off. Activation of Hh signalling antagonises the Itch-dependent polyubiquitylation of SuFu. Notably, different SuFu mutations occurring in medulloblastoma patients are insensitive to Itch activity, thus leading to deregulated Hh signalling and enhancing medulloblastoma cell growth. Our findings uncover mechanisms controlling the tumour suppressive functions of SuFu and reveal that their alterations are implicated in medulloblastoma tumorigenesis.

Chemical, computational and functional insights into the chemical stability of the Hedgehog pathway inhibitor GANT61.

This work aims at elucidating the mechanism and kinetics of hydrolysis of GANT61, the first and most-widely used inhibitor of the Hedgehog (Hh) signalling pathway that targets Glioma-associated oncogene homologue (Gli) proteins, and at confirming the chemical nature of its bioactive form. GANT61 is poorly stable under physiological conditions and rapidly hydrolyses into an aldehyde species (GANT61-A), which is devoid of the biological activity against Hh signalling, and a diamine derivative (GANT61-D), which has shown inhibition of Gli-mediated transcription. Here, we combined chemical synthesis, NMR spectroscopy, analytical studies, molecular modelling and functional cell assays to characterise the GANT61 hydrolysis pathway. Our results show that GANT61-D is the bioactive form of GANT61 in NIH3T3 Shh-Light II cells and SuFu-/- mouse embryonic fibroblasts, and clarify the structural requirements for GANT61-D binding to Gli1. This study paves the way to the design of GANT61 derivatives with improved potency and chemical stability.