Quisinostat is a brain-penetrant radiosensitizer in glioblastoma

Histone deacetylase (HDAC) inhibitors have garnered considerable interest for the treatment of adult and pediatric malignant brain tumors. However, owing to their broad-spectrum nature and inability to effectively penetrate the blood-brain barrier, HDAC inhibitors have failed to provide substantial clinical benefit to patients with glioblastoma (GBM) to date. Moreover, global inhibition of HDACs results in widespread toxicity, highlighting the need for selective isoform targeting. Although no isoform-specific HDAC inhibitors are currently available, the second-generation hydroxamic acid–based HDAC inhibitor quisinostat possesses subnanomolar specificity for class I HDAC isoforms, particularly HDAC1 and HDAC2. It has been shown that HDAC1 is the essential HDAC in GBM. This study analyzed the neuropharmacokinetic, pharmacodynamic, and radiation-sensitizing properties of quisinostat in preclinical models of GBM. It was found that quisinostat is a well-tolerated and brain-penetrant molecule that extended survival when administered in combination with radiation in vivo. The pharmacokinetic-pharmacodynamic-efficacy relationship was established by correlating free drug concentrations and evidence of target modulation in the brain with survival benefit. Together, these data provide a strong rationale for clinical development of quisinostat as a radiosensitizer for the treatment of GBM.


Immunocytochemistry
Cells were grown as adherent cultures on laminin-coated glass coverslips (Thermo Fisher Scientific) in GSC media.Twenty-four hours after plating, the cells were treated with QST or DMSO diluted in GSC media.Seventy-two hours after treatment, cells were fixed with 4% paraformaldehyde for 13 minutes at room temperature.Cells were washed with phosphatebuffered saline (PBS) and subsequently permeabilized and blocked with 5% normal goat serum (Sigma Aldrich) and 0.2% Triton X-100 in PBS (blocking solution) for 30 minutes at room temperature.The cells were incubated with primary antibodies overnight at 4 °C in blocking solution.Primary antibodies used in this study included rabbit anti-Ki67 (1:1000; Abcam, 15580), rabbit anti-Cleaved Caspase 3 (1:400; Cell Signaling Technologies, 9661), rabbit antigamma H2AX (phospho Ser139; 1:1000, Abcam, ab11174), and mouse anti-human Nestin (1:500; Novus Biologicals, 10C2).The following day, the cells were washed with PBS 3 times, incubated with fluorophore-conjugated secondary antibodies at 1:1000 dilutions (Alexa Fluor 568 goat anti-mouse, Abcam, ab175473; Alexa Fluor 488 goat anti-rabbit, Abcam, ab150077) for 1 hour at room temperature, and washed in PBS 3 more times.Cells were mounted onto Lo Cascio C et al. 3 SuperFrost Plus microscope slides using Fluoroshield Mounting Medium containing DAPI (Abcam).Images were acquired using a confocal microscope (Leica Microsystems; TCS SP5) operated with Leica Application Suite software.The fraction of Ki67-and Cleaved Caspase 3positive cells were counted from 5 independent images from each condition.The mean and standard deviation were calculated from 3 biological replicates for all control and QST-treated experiments.

Image Acquisition
Analysis of immunostaining of cultured GSCs was performed on confocal stacks (with a step size of 0.5-1.5 μm) acquired with either a 20× water-immersion objective or a 63× oilimmersion objective on a laser-scanning confocal microscope (Leica Microsystems; TCS SP5) operated with Leica Application Suite software.All images were processed using ImageJ software (National Institutes of Health).

Flow Cytometry
For cell cycle analysis following short-term DMSO or QST treatment in GSC cultures, approximately 1 million cells were harvested, washed in cold 1× PBS once, and fixed with 3-4 mL of ice cold 70% ethanol, added dropwise while vortexing.Cells were fixed on ice for 30 minutes and washed twice with 1× PBS.Cells were then resuspended in 500 μL of propidium iodide/RNAse staining buffer (BD Biosciences, 550825) for 15 minutes at room temperature before analysis.Data from all the samples were obtained using a Fortessa flow cytometer (BD Biosciences) and analyzed using the FlowJo software (TreeStar).Cell cycle analysis was performed in triplicate for all cell lines.
For apoptosis analysis, the Dead Cell Apoptosis Kit with Annexin V FITC and Propidium Iodide for Flow Cytometry (Thermo Fisher Scientific, V13242) was followed using the manufacturer instructions, with the exception that we employed ViaDye Red Fixable Viability Dye Kit (Cytek, SKU R7-60008) instead of propidium iodide to label necrotic cells.Samples were run on the AURORA 3L 24 color spectrum cytometers (Cytek), unmixed by SpectroFlo application, and analyzed with FlowJo 10.8.1 software (Beckton Dickinson).Recommended negative controls for flow cytometry include (1) no Annexin V FITC and no ViaDye Red, (2) Annexin V FITC alone, and (3) ViaDye Red alone.The following gating strategy was used: debris population was excluded as the subset of cells with low forward scatter in the forward scatter area versus side scatter area plot.After excluding the debris, cells were gated on the Annexin versus ViaDye Red log plot with the classification as live (Annexin V negative, ViaDye Red negative), early apoptotic (Annexin V positive, ViaDye Red negative), late apoptotic (Annexin V positive, ViaDye Red positive), or necrotic (Annexin V negative, ViaDye Red positive).The frequency of the different cell subsets was reported as frequency on the total cell population, excluding debris.

In Vitro Irradiation Studies
For all in vitro radiosensitization experiments involving treatment with IR using the RS 2000 irradiator (Rad Source) or the X-Rad225XL irradiator (Precision X-Ray), GSCs were plated on laminin-coated tissue culture-treated 96-well plates and incubated at 37 C overnight for 24 hours.The next day, cells were pretreated with QST or an equivalent volume of DMSO for 1 hour and then irradiated with various doses of IR (cell-line dependent).Cell viability was measured as described above using the CellTiter-Glo assay (Promega) 3 to 5 days after treatment.For experiments involving protein characterization of IR-treated cells preceded by treatment with QST, whole-cell lysates were collected at 1, 2, 6, and 24 hours after IR.Radiation was delivered using a RS2000 series biological irradiator (Rad Source Technologies) or the X-Rad225XL irradiator (Precision X-Ray).

Flank Tumor Implantation
For flank implantations, cells were prepared in a 1:1 ratio with 100 uL Matrigel (Corning #356234) and 100 uL of a single-cell suspension of U87 in PBS (500,000 cells) in a 1 mL syringe fitted with a 26-gauge needle.Mice were anesthetized with isoflurane in a plastic desiccator placed in an externally vented fume hood.The U87-Matrigel cell suspension was then subcutaneously injected into the flank of the mouse on the posterior/lateral aspect of the lower rib cage.Mice were monitored daily, and growth of flank tumor area was measured with a digital caliper (ThermoFisher) twice a week.Mice were sacrificed once the tumor size grew to more than 2000 mm 3 in size.

Live Bioluminescence Imaging
Two weeks after implantation, mice were examined for tumor growth by monitoring bioluminescence every 7 days using the IVIS Xenogen Spectrum platform.D-Luciferin Potassium Salt (Gold Biotechnology) was dissolved in PBS at a final concentration of 15 mg/mL.All mice were weighed each week and administered D-Luciferin via an intraperitoneal injection (10 μl/g).Fifteen minutes after injection, the mice were sedated using gaseous isoflurane (Piramal) and placed inside an IVIS Spectrum in vivo imaging system (Perkin Elmer) Lo Cascio C et al. 6 for bioluminescence imaging.The total flux (photons/second) within the region of interest was calculated using the Living Image software 4.5 (Perkin Elmer).

Preparation of QST for In Vivo Use
For in vivo preparation, QST was dissolved in 50% polyethylene glycol (PEG) 300, 50% sterile water solution for 10 mg/kg dosing.The suspension was then sonicated for 10 minutes to allow the drug to completely dissolve.Finally, the pH of both the drug and the vehicle solutions was adjusted to 7.4 before intraperitoneal dosing.

Determination of Optimal Administration Route
Foxn1 nu nude male mice (The Jackson Laboratory) were used to determine the drug administration route that would result in the best QST bioavailability.Three cohorts of mice were treated with a single dose of 10 mg/kg QST delivered through intraperitoneal, subcutaneous, or oral gavage routes (3 mice per cohort).Following administration of the single dose, approximately 30 µL of blood was drawn from the tip of the tails at the following time points: 0.5, 1, 2, 4, 6, 8, and 24 hours.The collected blood was immediately centrifuged at 3000 rpm for 10 minutes at 4 °C to separate plasma, which was subsequently flash frozen.At the 24hour time point, following the last blood sample collection, mice were sacrificed and the whole brains from each mouse were dissected and flash-frozen for subsequent analysis.

In Vivo Irradiation Studies
Intracranial or flank tumor-bearing nude mice were sedated with gaseous isoflurane before IR.On the first week of treatment, 2 hours after treatment with QST or vehicle, the mice were treated with 2 Gy of ionizing radiation on MWF for a total 6 Gy (3 doses).Ionizing radiation was administered with the RS2000 series biological research irradiator (Rad Source Technologies).

Treatment of Flank-Implanted Mice with QST and/or Radiation
Implanted flank tumors were allowed to grow until the tumor size reached 100 mm 3 volume.Mice were randomized into groups before treatment and underwent treatment on MWF for the entire duration of the experiment until the tumor volumes exceeded 2000 mm 3 .Treatment groups included vehicle (50% PEG-300), QST alone (10 mg/kg), flank IR treatment with vehicle (2 Gy), and flank IR treatment (2 Gy) with QST (10 mg/kg).For mice receiving IR treatment with or without QST, mice were treated with 2 Gy on MWF for a total 6 Gy during the first week of treatment.QST was administered to mice through intraperitoneal injections 2 hours before flank tumor radiation treatment.Upon completion of the radiation regimen, IR-treated mice subsequently received QST or vehicle alone for the rest of the experiment.Tumor growth and treatment response were monitored by manually measuring the tumor area twice per week starting at 13 days after implantation.Upon reaching the tumor volume threshold (2000 mm 3 ), mice were sacrificed, and plasma and tissue samples were harvested for PD and PK analyses 2 hours after treatment with a final dose of QST (10 mg/kg).

Treatment of Mice with Intracranially Implanted QST and/or Radiation
Mice with implanted tumors were allowed to grow until the tumor bioluminescence score reached 10 8 radiance (p/s/cm 3 /sr).Mice were randomized into groups before treatment.For survival studies, mice underwent treatment on MWF for the entire duration of the experiment until moribund.For mice receiving IR treatment with or without QST, mice were treated with 2 Gy on MWF for a total of 6 Gy in the first week of treatment.Treatment groups included vehicle (50% PEG-3000), QST (10 mg/kg) alone, 6 Gy whole-brain IR treatment with vehicle, and 6 Gy whole-brain IR treatment with 10 mg/kg QST.QST was administered to mice through intraperitoneal injections two hours prior to whole-brain radiation treatment.Upon completion of the radiation regimen, IR-treated mice subsequently received QST or vehicle alone for the rest of the experiment.Tumor growth and treatment response was monitored by IVIS bioluminescence once per week.For survival studies, mice were sacrificed, and samples were collected for PD and PK analyses once moribund 2 hours after treatment with a final dose of QST (10 mg/kg).
For short-term PK-PD correlation studies, tumor-bearing mice were randomized into groups and underwent a single week of treatment with QST (10 mg/kg) on MWF.For mice receiving IR treatment with or without QST, mice were treated with 2 Gy on MWF for a total of 6 Gy.QST was administered to mice through intraperitoneal injections 2 hours before wholebrain radiation treatment.On the third and last day of treatment, mice were sacrificed and processed for PD and PK analyses 3 hours after administration of QST or vehicle.For PD analyses, the mice were euthanized with isoflurane, and the tumors were dissected out of the brain and flash-frozen for subsequent analysis through Western blotting and RNA sequencing.
Tissue from the hemisphere contralateral to the tumor was also collected as a normal brain/nontumor reference sample.For PK analyses, the mice were anesthetized with isoflurane, and at least 300 μL of blood was drawn from the right ventricle of the heart.The blood was collected in tubes containing 10 μL of 0.1 M KOH in EDTA to prevent blood coagulation.Blood samples were immediately centrifuged at 3000 rpm for 10 minutes at 4 °C to allow separation of plasma.After collection of the blood, the tumor was dissected out of the brain and flash-frozen.
Both plasma and erythrocytes were flash-frozen for subsequent analysis.

Stem Cell Media
The stability of QST was determined in BALB/c mouse plasma, male nude athymic perfused and nonperfused mouse brain homogenate (1:9 w/v of PBS; pH 7.4), human brain (obtained from VRL Eurofins) homogenate (1:4 w/v of PBS; pH 7.4), pooled human plasma (Innovative Research), and neurobasal media.QST stock solutions (1 mM) were prepared in acetonitrile, subsequently diluted in a 40% methanol mixture, and added to the matrices to make final concentrations of 100 nM or 10 nM.Then 50 or 30 µL of either plasma or brain homogenate containing QST were aliquoted into 1.5-mL microcentrifuge tubes (Eppendorf) and were incubated at either 4 °C or 37 °C for 0, 2, 4, 6, 12, or 24 hours (n = 3 at each time point).

Reverse Transcription Quantitative Polymerase Chain Reaction
Total RNA was extracted from orthotopic tumors by using the RNeasy Mini Kit (Qiagen) in accordance with the manufacturer's instructions.RNA was quantified on a NanoDrop Spectrophotometer (Tecan), and 1 μg of total RNA was used for complementary DNA synthesis by using the SuperScript VILO kit (Life Technologies).Quantitative polymerase chain reaction was performed using inventoried TaqMan assays for respective target genes and housekeeping control genes (18S) on the QuantStudio 6 Flex Real-Time PCR System (Life Technologies).
Fold change in gene expression was analyzed using the delta delta Ct method.

Supplemental Figure 4 .
Stability of QST in mouse plasma is improved upon addition of esterase inhibitors.(A) Stability of QST (100 nM) in mouse plasma and mouse brain homogenate when sample preparation is performed at 4 °C.(B) Stability of QST (500 nM) in mouse plasma spiked with 3 different esterase inhibitors: 0.25 mM bis(p-nitrophenyl) phosphate (BNPP), 1.25 mM phenylmethylsulfonyl fluoride (PMSF), and 1 mM dithiobis(2-nitrobenzoic acid) (DTNB) over the course of 24 hours at 37 °C.(C) Stability of QST (10 nM and 100 nM) in neurobasal cell culture medium over the course of 24 hours at 37 °C.