1. YF-PRJ8-1011 tested against CDK kinases
CDK4 and CDK6 kinase activity was measured by using GST-Rb rotein as a general substrate. Compounds were tested in 10-dose IC50 duplicate mode starting at 5 µM with a 3-fold serial dilution. The control compound, Staurosporine was tested in 10-dose IC50 mode starting at 20 µM with 4-fold serial dilution. Reactions were carried out at Km ATP according to the RBC Km binning structure: CDK4/cyclin D1 (100 µM ATP), CDK4/cyclin D3 (5 µM ATP), CDK6/cyclin D1 (100 µM ATP), CDK6/cyclin D3 (100 µM ATP). The ADP-Glo kinase assay kit (Promega) was adopted to measure IC50 values of YF-PRJ8-1011. When the enzyme activities at the highest concentration of compounds were less than 65%, curve fits were performed.
2. Cell Establishment And Culture Conditions
The patient-derived DIPG cells (including TT150630, TT150714, TT150728 and DIPG17) and primary pontine neural progenitor cells (PPCs) were kind gifts from Dr. Yu Sun. TT190326 was established as other DIPG cells. The cell culture method was performed as previously reported by Sun et al[8]. DIPG cells and PPCs were cultured in matrigel (356,234, BD, 1%, 4–12 h at 37°C) coated plates with serum-free medium containing DMEM (Invitrogen), B27 (Gibco, 1:50), N2 (Gibco, 1:100), Insulin-Transferrin-Selenium (ITS, Macgene, 20 µg/ml), bFGF (PeproTech, 20 ng/ml), EGF (PeproTech, 20 ng/ml) and penicillin streptomycin (Hyclone, 1:100). All the cells were cultured at 37°C in a 5% CO2 atmosphere.
3. Western Blot Analyses
3. Western Blot Analyses
The total protein was extracted with RIPA lysis buffer (Solarbio) according to the manufacturer's protocol. Whole cell lysates of DIPG cells and PPCs were generated by lysing 2×10^6 washed cells in 200 µL RIPA lysis buffer plus 1% phenylmethylsulfonyl fluoride (PMSF), followed by incubation on ice for 30 min. The lysed sample was centrifuged at 10000-14000g for 3–5 min, and western blots were then performed on the supernatant of centrifuged samples. After the determination of protein concentration with BCA Protein Assay Kit (Thermo), the samples were denatured by boiling them with 5×loading buffer (Scintol) at 100°C for 5 min. A 50 µg protein sample was separated by 10% SDS-polyacrylamide gels at 120V for 1.5 h and then transferred to PVDF Transfer Membranes (Millipore) at 300mA for 1h. After they were blocked with 5% skimmed milk powder in PBST buffer (including 3.2 mM Na2HPO4, 0.5 mM KH2PO4, 1.3 mM KCl, 135 mM NaCl and 0.05% Tween-20, pH 7.4) for at least 1 h, the membranes were incubated with primary antibodies, including anti-pRb (ab173289, 1:5000), anti-Rb (ab181616, 1:2000), anti-CDK4 (CST,12790, 1:1000), anti-CDK6 (CST, 13331, 1:1000), anti-CyclinD1 (CST, 2922, 1:1000), anti-P16 (CST, 18769, 1:1000) and anti-β-Actin (Easybio, 1:3000), at 4°C overnight. The membranes were washed 3 times with PBST buffer for 10 min, and then incubated with secondary antibodies Goat anti-mouse- IgG(H + L)-HRP-conjugated (Easybio, 1:3000), and Goat anti-rabbit IgG(H + L) (Easybio, 1:3000) for approximately 1 h at room temperature. After washing 3 times for 10 min in PBST, the membranes were scanned by the Tanon 5200 Chemiluminescent Imaging System.
4. Cell Viability Assays
DIPG cells were first counted, and approximately 10^4 cells were seeded onto each well in 96-well plates. The cells were treated with YF-PRJ8-1011 at concentrations of 6.25 nM, 25 nM, 100 nM, 400 nM, 1600 nM, 6400 nM, and 25600 nM, respectively. Cell viability was determined by CellTiter-Glo assays (G7572, Promega) according to the manufacturer's protocol. In brief, CellTiter-Glo® Buffer and CellTiter-Glo® Substrate were thoroughly mixed to form CellTiter-Glo® Reagent. 100ul CellTiter-Glo® Reagent was added to each well in 96-well plates and the contents were mixed on an orbital shaker to induce cell lysis. The plates were incubated at room temperature for 10 min to stabilize the luminescent signal. The luminescence was recorded to measure cell viability. Cell viability was assessed 48 h after treatment (T48) and calculated as T48h. The value for control-treated cells was set to 100%. Triplicate wells were used for each concentration. IC50 values were calculated by Graphpad prism 8.
5. Cell Proliferation Analysis
Flow cytometric analysis of Ki-67 was adopted to evaluate cell proliferation. Foxp3 Transcription Factor Staining Buffer Set (eBioscience) was used for Ki67 staining, according to its protocol. In brief, 1 portion of Foxp3 Fixation/Permeabilization Concentrate was mixed with 3 portions of Foxp3 Fixation/Permeabilization Diluent to generate fresh Foxp3 Fixation/Permeabilization working solution. 1 portion of 10X Permeabilization Buffer was mixed with 9 portions of distilled water to generate 1X working solution of Permeabilization Buffer. The cells were washed three times, followed by the addition of 1 mL of Foxp3 Fixation/Permeabilization working solution to each tube, and incubated for 30–60 min at room temperature with foil. 2 mL 1X Permeabilization Buffer was added to each tube, followed by the centrifugation of samples at 500 g for 4 min. After the supernatant was discarded, the pellet was resuspended in residual volume of 1X Permeabilization Buffer, followed by the addition of Ki-67 (Biolegend) to incubate with foil at room temperature for 30 min. The cells were washed with 2 mL 1X Permeabilization, followed by the resuspension of stained cells in PBS. The samples and the data were analyzed by flow cytometry (BD FACS Calibur) and FlowJo7.6, respectively.
6. Rna-seq Library Preparation And Sequencing
RNA integrity was evaluated by the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). Total RNA was used as input materials for RNA sample preparations. In brief, poly-T oligo-attached magnetic beads were adopted to purify mRNA from total RNA. Divalent cations were used to perform fragmentation under elevated temperature in First Strand Synthesis Reaction Buffer(5X). First-strand cDNA was synthesized by using random hexamer primers and M-MuLV Reverse Transcriptase (RNase H-). Subsequently, second-strand cDNA synthesis was performed by using DNA Polymerase I and RNase H. Remaining overhangs were converted to blunt ends through exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, the Adaptor with a hairpin loop structure was ligated to prepare hybridization. In order to preferentially select cDNA fragments with a length of 370 ~ 420 bp, the RNA-seq library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then, PCR was performed by using Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. Finally, PCR products were purified with AMPure XP system, followed by the evaluation of library quality with the Agilent Bioanalyzer 2100 system. The clustering of the index-coded samples was performed on a cBot Cluster Generation System by TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the manufacturer’s instructions. After cluster generation, library preparations were sequenced on an Illumina Novaseq platform, followed by the generation of 150 bp paired-end reads.
7. Rna-seq Analysis
Gene Ontology (GO) enrichment analysis of differentially expressed genes was performed by the cluster Profiler R package, in which gene length bias was adjusted. GO terms with adjusted P value (< 0.05) were considered to be significantly enriched by differential expressed genes. KEGG is a database resource for understanding advanced functions and utilities of the biological system, such as cells, organisms and the ecosystem, from the information at the molecular-level, especially large-scale molecular datasets generated by genome sequencing and other high-throughput experimental technologies (http://www.genome.jp/kegg/). Cluster Profiler R package was adopted to detect the statistical enrichment of differentially expressed genes in KEGG pathways. Gene Set Enrichment Analysis (GSEA) is a computational method to determine whether a pre-defined gene set can show a significantly consistent difference between two biological states. The genes in the gene set were ranked according to the degree of differential expression in the two samples, and then the predefined gene set was detected to see whether it was enriched at the top or bottom of the list. In gene set enrichment analysis, expression changes in sets of genes rather than individual ones are detected and thus subtle expression changes in them can also be included. The local version of the GSEA analysis tool (http://www.broadinstitute.org/gsea/index.jsp) as well as GO and KEGG datasets were independently used for GSEA analysis.
8. Bbb Analysis Of Yf-prj8-1011
Plasma, cerebrospinal fluid (CSF) and brain sample concentrations were measured by a LC/MS/MS method. Briefly, 50 µL aliquots of plasma or brain samples and 3 µL aliquots of CSF ones were processed according to a protein precipitation procedure, with a mixture of methanol, acetonitrile, and Terfenadine used as internal standards. The 5-fold diluted supernatant was injected onto an ACE 5 C4 (50 mm * 2.1 mm) column, and the YF-PRJ8-1011 and internal standards were eluted and directed into Sciex API5500 mass spectrometer for detection with 5 mM NH4OAc (0.05% FA) – ACN (0.1% FA). The acquisition was performed for quantitation in positive ionization mode with multiple reaction monitoring (MRM), and the MRM channels for YF-PRJ8-1011 and the internal standard (Terfenadine) were 379.20/293.20 and 472.33/436.20, respectively. Both Data collection and linear regression with weighting (1/X2) were performed by Analyst Software version 1.6.3. Non-compartmental analysis was carried out to determine the PK parameters of YF-PRJ8-1011 by WinNonlin Version 8.0.
9. Maximum Tolerated Dose (Mtd)
In order to help determine the optimal concentration of YF-PRJ8-1011 for further in vivo experiments, the MTD values of the compound for female B6 mice were evaluated. After administration of 50mg/kg, 100 mg/kg, 250 mg/kg and 500 mg/kg, respectively, weight changes were measured every 3 or 4 days for 2 consecutive weeks. Heart, brain, kidney, spleen, lung and liver were all removed for HE staining to observe whether there was related organ damage for them 24h after administration of 500mg/kg.
10. Dipg Xenograft Mouse Models And Administration Of Yf-prj8-1011
6-week female NOD/ShiLtJGpt-Prkdcem26Cd52Il2rgem26Cd22/Gpt mice (GemPharmatech) were used to establish Orthotopic xenograft mouse models. The method for establishing the animal model was as described previously. Briefly, NCG mice were anesthetized with 2.5% avertin i.p. and positioned in a stereotaxic instrument (KDS Legato 130). The posterior skull skin of the mice was incised and lambdoid suture was located as a mark. A skull hole was made by an electric drill (RWD). Luciferase engineered DIPG cells were re-suspended in 5 µl PBS at a density of 10^5 cells/µl and implanted into the brainstem of NCG mice. The tumor burden was measured by bioluminescence imaging 2 weeks after the implantation. D-luciferin (Goldbio) suspended in PBS (15 mg/mL) was injected (150 mg/kg, i.p.) 10 min before acquisitions. Bioluminescence images were taken by Xenogen IVIS Spectrum Imaging System (Caliper Life Sciences). The ROI tool was adopted to measure Bioluminescence imaging data by Living Image software Version 3.0 (Caliper Life Sciences). Once the luminescence signal was detected, the tumor-bearing mice were randomly assigned to the control, YF-PRJ8-1011 or palbociclib treated groups. Then, the two drugs or vehicle was orally administered at a dose of 100 mg/kg/day for 14 consecutive days.
11. Radiation And Yf-prj8-1011 Treatment For Dipg Xenograft Mouse Models
DIPG xenograft mouse models were randomized into groups (treatment and control groups) before treatment. Treatment groups included YF-PRJ8-1011 alone, 4Gy whole brain XRT alone and 4Gy whole brain XRT with YF-PRJ8-1011 groups. YF-PRJ8-1011 was administered orally at 100 mg/kg/day for 21 consecutive days.
12. Hematoxylin And Eosin (H&e) Staining And Immunohistochemistry (Ihc)
Mouse brainstem tissue was fixed in 4% paraformaldehyde (PFA) for 48 h. PFA-fixed brainstem tissue was paraffin-embedded after dehydration in ethanol gradients. The sections were cut to 5µm-thickness by a Paraffin microtome (Leica RM2235). After the slides were placed in a 56–60°C oven for 45 min, the sections were deparaffinized with xylene three times, for 5 min each. The slides were transferred to fresh absolute ethanol and then successively to 95%, 70%, and 50% alcohols for 5 min each. The slides were stained with H&E and IHC. The methods for H&E and immunohistochemical (IHC) staining were described as previously reported[7].
13. Plasmids And Reagents
Luciferase-GFP were cloned into pLEX based lentivirus vector, which was used to establish DIPG cells with luciferase-GFP. The human antibodies for western blot test in this study included anti-pRb (ab173289, 1:5000), anti-Rb (ab181616, 1:2000) and anti-β-Actin (Easybio, 1:3000). Secondary antibodies included Goat anti-mouse- IgG(H + L)-HRP-conjugated (Easybio, 1:3000), and Goat anti-rabbit IgG(H + L) (Easybio, 1:3000).
14. Statistical Analysis
All the experiments were performed three times. Graphpad prism 8 was adopted for statistical analysis. Statistical significance was calculated by an unpaired two-tailed t-test between groups. P < 0.05 indicates statistical significance: P < 0.05 (∗), P < 0.01 (∗∗), P < 0.001 (∗∗∗), and P < 0.0001 (∗∗∗∗).
15. Ethics Approval
This study was approved by Animal Welfare Ethics Committee of Beijing Neurosurgical Institute.