Dissecting the role of the Cullin3-RENKCTD11 E3 ubiquitin ligase complex in the control of the Hedgehog pathway and medulloblastoma tumorigenesis

Medulloblastoma (MB) is the most common malignant pediatric brain tumor arising from alterations in cerebellum development. The Sonic Hedgehog variant (SHH-MB) is the best genetically characterized, however the molecular mechanisms responsible for its pathogenesis are not fully understood and therapeutic benefits are still limited. Mutations in key components of the SHH pathway and cytogenetic alterations (such as chromosome 17p deletion) lay the pathogenetic foundation for SHH-MB subgroup. The laboratory where I conducted my PhD previously identified the Cullin3-RING (CRL3) BTB-containing adaptor RENKCTD11 (here REN) as a tumor suppressor that maps on chromosome 17p, involved in neural progenitor development. REN acts as a negative regulator of the SHH pathway by promoting the ubiquitylation and degradation of HDAC1, a well-known SHH pathway activator. This project aims to expand the knowledge of roles of the CRL3REN complex to unveil novel molecular circuitries whose deregulation could contribute to SHH-MB onset and therapeutic targets for the treatment of SHH-MB. Affinity purification coupled to mass spectrometry was performed to identify new REN interactors. Among them, we focused on the Spalt-like transcriptional factor 4 (SALL4), a stemness regulator and oncofetal protein. SALL4, frequently overexpressed in various tumors, is closely associated with poor prognosis and reduced survival rates. Notably, elevated SALL4 levels are significantly correlated with worse overall survival in a large cohort of SHH-MB patients. Here, we found that REN induces SALL4 polyubiquitylation and proteasome-mediated degradation. Functional transcriptional assays demonstrated that SALL4 is a positive regulator of the SHH pathway working through GLI1 (the final effector of the signaling). Specifically, SALL4, GLI1, and HDAC1 are assembled in a ternary complex that promotes GLI1 protein deacetylation and its transcriptional activity. Noteworthy, genetic depletion of SALL4 inhibits SHH-dependent tumor growth both in vitro and in vivo, an effect that correlates with the reduction of SHH signature genes. Interestingly, the pharmacological inhibition of SALL4 mediated by the immunomodulatory imide drug (IMiD) thalidomide (Thal) strongly inhibits SHH-MB tumor growth in Patient-Derived Xenograft (PDX) models. Accordingly, high SALL4 expression levels correlate with worse overall survival in a large cohort of SHH-MB patients. Overall, data obtained from this project identify SALL4 as a novel CRL3REN substrate and a promising therapeutic target for SHH-dependent MBs.