Targeting Citron Kinase catalytic activity for high grade brain tumors treatment

High-grade brain tumors (HGBTs) are among the most lethal human cancers, characterized by extensive heterogeneity and intrinsic resistance to conventional therapies. Current treatments remain largely insufficient, highlighting the need for new therapeutic approaches. HGBT cells share molecular and functional traits with cortical radial glia cells, which exhibit severe defects in proliferation and survival when genes associated with primary hereditary microcephaly (MCPH) are inactivated. MCPH genes have collectively been proposed as potential therapeutic targets for high-grade brain tumors, and proof-of-concept about their actual suitability have been obtained for ASPM, KIF14, CDK6, CENPE, and CITK. This work focused on characterizing the potential of Citron kinase (CITK) in the most prevalent HGBTs, glioblastoma (GBM) and medulloblastoma (MB). In particular, the effects of CITK inactivation were investigated using both genetic and pharmacological approaches. Based on literature data indicating high affinity between CITK and the multikinase inhibitor Lestaurtinib, biochemical and in vitro assays were performed to assess its ability to inhibit CITK catalytic activity. Treatment of MB and GBM cells with Lestaurtinib largely phenocopied CITK loss, leading to reduced proliferation, increased binucleation, DNA damage accumulation, and apoptotic cell death. To develop more effective and selective compounds, a drug discovery program was established. Starting from a library of 10,979 molecules, a high-throughput screening followed by selectivity assays identified three candidate inhibitors. When tested on tumor cells, these compounds did not fully reproduce CITK-loss phenotypes. Similar results were obtained with specific inhibitors developed by other groups. These data suggest that the dependence of cells from CITK activity may be very context-specific. Accordingly, mouse models expressing inactive CITK do not recapitulate human microcephaly phenotypes, while human forebrain organoids with similar mutations display severe defects in neuroepithelial organization, polarity, and survival. In addition, scaffold but not catalytic function of CITK is essential for full anti-proliferative effects. These data strongly suggest that to counteract the permissive role of CITK in sustaining tumor cell viability it will be necessary to abolish protein expression. To address this problem, we are generating and testing CITK-targeting PROTACs, based on the structure of the previously identified catalytic inhibitors. Finally, we have developed a bioinformatic approach to identify the genetic contexts that confer sensitivity to CITK loss across tumor types, which may guide the rational development of CITK-based therapeutic strategies not only for HGBTs but also for other aggressive cancers