Evaluation of nitroxoline and derivatives

1) Treatment of at least two cell lines with nitroxoline to determine sensitivity. IC50 value will be determined (3 days treatment with single increasing doses of nitroxoline). Subsequently extended growth curves (5 or more days) and colony assays will be performed using a single dose of the IC50 value. 2) Time course treatment with IC50 value of nitroxoline and subsequent RNA extraction and RNA-sequencing. Results obtained will be analyzed using standard bioinformatics procedures including heat maps, meta-plot analyses and MA plots. These data and analyses will be compared to those obtained using JQ1 (gold-standard iBET) that we already generated and analyzed (Piunti et al. Nature Medicine, 2017). 3) BET inhibitors generally have measurable activity on target in 1 hour or less from administration in cell culture. To demonstrate nitroxoline BET inhibitor activity on target, ChIP-sequencing on bromodomain proteins (i.e. BRD2 and BRD4) will be performed and analyzed using previous experimental strategies (Piunti et al. Nature Medicine, 2017). Expected results 1) I expect DIPG cells to be sensitive to micromolar concentration of nitroxoline (IC50 included between 1-10M)1 in all the experiments proposed. 2) I expect a good overlap (over 60-70% similarly regulated genes) between RNA-results obtained from nitroxoline and JQ1 time course treatment experiments. 3) I expect a reduction of Bromodomain proteins binding to chromatin measured by ChIP-seq signal upon treatment with nitroxoline. I expect this reduction to be comparable in terms of magnitude and genomic locations to the one induced by JQ1 treatment. If the experimental design in Part I generates data that closely match the expected results, nitroxoline could be consider a bona fide compound with BET inhibitor properties. This will also allow the project to move to the second part. 5-12 months goals (Part II) 1) Toxicity study to determine the maximum tolerated dose and evaluation of side effects. This will reproduce what previously reported2. 2) Pharmacokinetic studies for the bioavailability of nitroxoline in the brain (CSF/serum ratio and half-life value). 3) Treatment of DIPG xenograft mouse model with nitroxoline and assessment of therapeutic efficacy by measuring tumor growth inhibition in vivo (i.e. luciferase signal coming from the tumor) and animal survival (i.e. Kaplan-Meier curve). Similar to what reported before (Piunti et al. Nature Medicine, 2017). 4) Assessment of target inhibition in vivo will be performed by immunohistochemical analyses of slides generated from brain sections containing tumors coming from treated and untreated mice. H3K27ac mark will be used as surrogate antigen to determine target inhibition (Piunti et al. Nature Medicine).