Targeting Hedgehog pathway in medulloblastoma: identification of SALL4A as a new activator and isoflavone c22 as multitarget inhibitor

Hedgehog (Hh) pathway is essential for embryonal development and tissues homeostasis; its alteration causes several human cancers, including medulloblastoma (MB), the most common brain malignancy in childhood. Because of its crucial role in tumorigenesis, the regulation of the Hh pathway and the identification of new Hh modulators emerged as a field of great interest in tumor biology. My Ph. D. project was focused on two main goals: Aim 1 | Identification of new molecular mechanisms involved in Hh signalling pathway regulation and MB tumorigenesis. To address this aim, we focused on the identification of new interactors of the known Hh negative regulator and tumor suppressor RENKCTD11. Through mass spectrometry analysis, we identified the transcription factor SALL4A. SALL4A plays a key role in maintaining pluripotency and self-renewal of embryonic stem cells, regulating different signalling pathway. SALL4A expression is inhibited in the post-natal period in many adult tissues, but it is reactivated in different tumors and is often related to worse prognosis and lower survival rate. Our data demonstrate that SALL4A is a substrate of RENKCTD11, that induces its poly-ubiquitylation and its consequent proteasome-mediated degradation. In order to investigate its biological role in Hh signalling, we demonstrated that SALL4A enhances GLI1 activity working in complex with HDAC1, a well known Hh activator. Of note, we observed that the proliferation ability of human MB cell lines increases in presence of SALL4A, whereas their migration rate is reduced after its genetic depletion; in vivo, SALL4A genetic depletion leads to a reduction of Hh-dependent tumor growth. Our findings identify SALL4A as a previously unknown regulator of Hh pathway able to promote, in complex with HDAC1, GLI1 activity and to contribute to Hh-dependent tumorigenesis. Hence, SALL4A stands as a new molecular target involved in the onset and progression of Hh-dependent tumors and represents an interesting focus in cancer research. Aim 2 | Pharmacological targeting of Hedgehog pathway. Given the primary role of Hh signalling in tumorigenesis and in the maintenance of cancer stem cell niches, this pathway is now considered an attractive therapeutic target in cancer. In recent years, many efforts have focused on the development of drugs that can block the activating effect of SMO receptor. However, several studies underlined some limitations of the inhibitors identified so far, linked in particular to the poor selectivity, the onset of drug resistance events and the activation of GLI1 mediated by other oncogenic pathways. These evidences raise the need to identify new and more effective Hh inhibitors able to overcome drug resistance and to counteract tumor growth. To this end, my research activity was aimed at identifying, characterizing and optimizing new molecules able to block the oncogenic potential of Hh. In particular, by combining the most profitable pharmacophores for targeting SMO and GLI1 by synthetic isoflavones, we designed and synthesized the isoflavone 22, a small molecule that acts as a multitarget Hh inhibitor blocking both SMO and GLI1 activity at the same time. Compound 22 is able to inhibit Hh-dependent tumor growth in human and murine MB cells at sub-micromolar concentration, as a consequence of the reduction in GLI1 expression levels. Isoflavone 22 remarkably shows a strong anti-tumor effect also in vivo by suppressing cell proliferation and promoting apoptosis. Molecular modeling further corroborated the multitarget mechanism of action of compound 22, showing that the drug is able to fit the ligand binding site in both SMO and GLI1. Overall, these results reveal a valuable form of targeted therapy to increase the efficacy and to decrease the toxicity of individual anticancer agents; our findings discover the first multitarget Hh inhibitor that impinges Hh-dependent tumor growth and stands as new potential weapons against Hh-driven tumors.