Chloride intracellular channel 1 activity is not required for glioblastoma development but its inhibition dictates glioma stem cell responsivity to novel biguanide derivatives

Abstract

Background Chloride intracellular channel-1 (CLIC1) activity controls glioblastoma proliferation. Metformin exerts antitumor effects in glioblastoma stem cells (GSCs) inhibiting CLIC1 activity, but its low potency hampers its translation in clinical settings. Methods We synthesized a small library of novel biguanide-based compounds that were tested as antiproliferative agents for GSCs derived from human glioblastomas, in vitro using 2D and 3D cultures and in vivo in the zebrafish model. Compounds were compared to metformin for both potency and efficacy in the inhibition of GSC proliferation in vitro (MTT, Trypan blue exclusion assays, and EdU labeling) and in vivo (zebrafish model), migration (Boyden chamber assay), invasiveness (Matrigel invasion assay), self-renewal (spherogenesis assay), and CLIC1 activity (electrophysiology recordings), as well as for the absence of off-target toxicity (effects on normal stem cells and toxicity for zebrafish and chick embryos). Results We identified Q48 and Q54 as two novel CLIC1 blockers, characterized by higher antiproliferative potency than metformin in vitro, in both GSC 2D cultures and 3D spheroids. Q48 and Q54 also impaired GSC self-renewal, migration and invasion, and displayed low systemic in vivo toxicity. Q54 reduced in vivo proliferation of GSCs xenotransplanted in zebrafish hindbrain. Target specificity was confirmed by recombinant CLIC1 binding experiments using microscale thermophoresis approach. Finally, we characterized GSCs from GBMs spontaneously expressing low CLIC1 protein, demonstrating their ability to grow in vivo and to retain stem-like phenotype and functional features in vitro. In these GSCs, Q48 and Q54 displayed reduced potency and efficacy as antiproliferative agents as compared to high CLIC1-expressing tumors. However, in 3D cultures, metformin and Q48 (but not Q54) inhibited proliferation, which was dependent on the inhibition dihydrofolate reductase activity. Conclusions These data highlight that, while CLIC1 is dispensable for the development of a subset of glioblastomas, it acts as a booster of proliferation in the majority of these tumors and its functional expression is required for biguanide antitumor class-effects. In particular, the biguanide-based derivatives Q48 and Q54, represent the leads to develop novel compounds endowed with better pharmacological profiles than metformin, to act as CLIC1-blockers for the treatment of CLIC1-expressing glioblastomas, in a precision medicine approach.

Metformin antiproliferative activity is exclusively mediated by the membrane functional expression of the Chloride Intracellular Channel 1 in glioblastoma stem cells

“…In a recent publication, we demonstrated the existence of GBM subsets whose aggressiveness is unrelated to CLIC1 expression whereas displaying resistance towards metformin treatment 42 . This introduces the possibility to establish novel eligibility criteria for discriminating patients' cohorts in a personalized trial setting based on CLIC1 expression.…”

Disruption of metazoan gene regulatory networks in cancer alters the balance of co-expression between genes of unicellular and multicellular origins

“…3 G). The most highly connected gene in this module is CLIC1, a chloride ion channel with high expression and potential therapeutic and prognostic value in adult glioma [ 39 , 40 ]. Our work links CLIC1 expression with EGFR activity in LGG.…”

Mitochondrial gene expression signature predicts prognosis of pediatric acute myeloid leukemia patients

“…Furthermore, several novel SDH inhibitors have been identified using in silico library design which can be potentially utilised in future studies (60). Interestingly, CLIC1 inhibitors has also been explored in glioblastoma cells and found that inhibition of CLIC1 sensitizes glioblastoma stem cells by inhibiting proliferation, migration, invasiveness and self-renewable in vitro and in vivo (61)(62)(63). Along with this, using transcriptomic profile of patients with high and low risk score, drug sensitivity assay using FDA approved drugs can be performed to identify drugs precise targeted therapy for patients with high-risk score in future studies (64)(65)(66).…”