Cell lines
DB, SU-DHL4, SU-DHL2, and SU-DHL10 cells were obtained from the American Type Cell Collection (ATCC) and grown in RPMI-1640 (Sigma, USA) supplemented with 10% heat-inactivated fetal bovine serum (PAN, Germany), 100 units/mL penicillin, and 100 µg/mL streptomycin (Gibco, USA). Cell lines were maintained in 5% CO2 at 37 °C. MYC and BCL2 rearrangements were confirmed by fluorescence in situ hybridization (FISH).
Reagents
Brequinar (HY-108,325) and venetoclax (HY-15,531) were purchased from MCE (USA). DHODH antibody (ab174288) was purchased from Abcam (England). Antibodies targeting P53 (SC6243), P21 (SC817), and BAX (SC20067) were purchased from Santa Cruz. BCL2 (4223), BIM (C34C5), c-MYC (5605), p-c-MYC (46,650 and 13,748), MCL-1 (D35A5), BCL-xL (2762), α-tubulin (3873), caspase family antibody kit (9929), NFκB antibody kit (55,764), JAK/STAT antibody kit (9799), and PI3K/AKT antibody kit (9655) were purchased from CST (USA). Antibodies targeting GAPDH (10,494) and histones (19,649) were purchased from Proteintech (USA). Antibodies targeting RPL26 (102,758), RPS27 (138,642), and MRPS-6 (118,709) were purchased from Absin (China). Secondary anti-rabbit and anti-mouse polyclonal antibodies were purchased from Absin (China). Z-VAD-FMK was purchased from Selleck (America).
Cell proliferation assay
Cell proliferation was measured by the Cell Counting Kit-8 (CCK8, Dojindo Laboratories, Japan) assay according to the manufacturer’s instructions. The cell viability was calculated by the formula: Cell viability (%) = [OD (drug+) – OD (Blank)] / [OD (drug-) – OD (Blank)]×100%. The IC50 value or CI value was calculated using GraphPad Prism 7.0 (GraphPad Software, San Diego, CA, USA).
Cell cycle and apoptosis assay
For the apoptosis experiment, the percentage of viable cells was determined with an Annexin V-PE apoptosis detection kit I (BD Pharmingen, San Diego, CA) according to the manufacturer’s instructions using a FACSCanto II cytometer (BD). Cells negative for staining were considered viable, and all results were normalized to the dimethyl sulfoxide (DMSO)-treated group, which was set as 100% viable cells (0% dead cells). Data were analyzed with FlowJo software V7.6.1 (FlowJo LLC, Ashland, OR). For the cell cycle experiment, cells were harvested and fixed at 4 °C with 70% ethanol in phosphate-buffered saline (PBS) overnight. Cell suspensions were then treated with 50 µg/mL RNase A (Sigma; St Louis, MO) for 2 h before being stained with 50 µg/mL propidium iodide. The percentages of cells in the G0/G1, S, and G2/M phases were determined by flow cytometric analysis (FACSCalibur, BD). The cell cycle distribution was analyzed using BD ModFitTM LT software (BD Biosciences, San Diego, CA). Each experiment was repeated in triplicate.
RT-PCR
RNA was extracted from relevant cells, and cDNA was generated using SuperScript® III RT (Thermo Fisher) with oligo-dT primers. qRT-PCR was performed using Power SYBR® Green PCR Master Mix (Applied Biosystems) according to the manufacturer’s instructions. GAPDH expression was used as an internal control. PCR was performed in duplicate wells on an ABI 9700 thermocycler (Applied Biosystems, Foster City, CA) under the following cycling conditions: 95 °C for 10 min and 35 cycles of 95 °C for 15 s and 60 °C for 1 min. The results were analyzed with GraphPad Prism 7 and are expressed as N-fold differences according to the ΔΔCq method as follows: relative expression = 2–∆ΔCt, where ΔCt = Ct (target gene) – Ct (control gene) [16]. The primer sequences are listed in Supplementary Table 1.
DHODH knockout
We utilized the CRISPR-Cas9 design site (crispr.mit.edu) to identify sgRNAs targeting the protein-coding region of the human DHODH gene. We chose a sgRNA (5’-CAGTCACGGGCTTTCAGTGG-3’) based on the efficiency and low frequency of off-target sites. The lentiviral plasmids CRISPRV2-Cas9-Puro and CRISPRV2-Cas9-sgRNA-Puro were purchased from Miaoling Plasmid Biomart, China. We infected SU-DHL4 cells using the lentivirus packaging method.
C-MYC overexpression cell construction
The c-MYC-overexpressing lentivirus pLV-hef1a-Puro-WPRE-CMV-MYC (human, NM_002467-3Xflag) and empty vector pLV-hef1a-Puro-WPRE-CMV-MCS-3Xflag were purchased from Hesheng Gene Company (China). DB and SU-DHL4 cells were transfected with the virus according to the instructions.
Assessment of brequinar and venetoclax synergy
Combination indexes (CIs) for combinations of brequinar and venetoclax were calculated using Compusyn (CombosynInc, Paramus, NJ) according to the Chou-Talalay algorithm. The median CIs for all assessed combinations are shown.
RNA-sequencing (RNA-SEQ) analysis
RNA extraction
A total of 5*106 cells were suspended in 1000 µL TRIzol Reagent (Life Technologies). Then, RNA was extracted using an RNeasy Micro Kit (Qiagen, Germany) according to the manufacturer’s protocol.
Illumina RNA sequencing
A total amount of 1 µg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using the NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following the manufacturer’s recommendations, and index codes were added to attribute sequences to each sample. See “Supplementary Materials and Methods” for further information.
Sequencing data analysis
Differential expression analysis of two groups (two biological replicates per condition) was performed using the DESeq2 R package (1.16.1). The resulting P-values were adjusted using Benjamini and Hochberg’s approach for controlling the false discovery rate. Gene Ontology (GO) enrichment analysis of differentially expressed genes was implemented using the cluster Profiler R package, in which gene length bias was corrected. The GO terms/KEGG pathways analysis screen criteria were evaluated by Padj < 0.05. The “significant genes” between venetoclax and control groups were limited, so we used |log Foldchange|>0 as a threshold for further analysis.
Western blot analysis
Proteins were prepared according to the handbook of Protein Extraction Kit from KeyGEN BioTECH (China). A 40 µg protein sample from each group was separated by SDS-PAGE and transferred onto a nitrocellulose membrane (Millipore, USA). The protein expression was detected by specific antibodies combined with HRP-conjugated secondary antibodies (goat anti-rabbit or goat anti-mouse)(Abison, China). Images were taken using an Olympus IX-71 microscope controlled by DeltaVision SoftWoRx. The immunoblots were quantified using ImageJ software (National Institute of Mental Health, Bethesda, MD, USA). For some molecules with molecular weight very close to that of the loading control, we first adjust our sample loading volume based on loading control. Then we loaded each sample (the same sample we used to adjust the loading volume) with recorded volumes to test different molecules.
Co-immunoprecipitation assay
Lysis buffer was prepared with NP40 and PMSF (100:1) (Solarbio, China). Cell lysates were precleared with 20 µl of Protein A + G Agarose (SC-2003, Santa Cruz, USA) for 3 h at 4 °C before incubating with anti-BCL2, anti-BAX, or normal IgG antibodies (all from CST) overnight at 4 °C. Then, the proteins bound to each antibody were precipitated with protein agarose A + G for 4 h at 4 °C before they were resolved and analyzed by western blotting.
In vivo studies
Four- to six-week-old SCID- NOD mice (SPF Biotechnology, Beijing, China) were bred under specific pathogen-free conditions. SU-DHL-4 cells (5 × 106 cells) were injected subcutaneously into the right flank of the mice. Treatment started when the tumors were about 50mm3, and the mice were divided randomly into four groups, receiving vehicles(control), venetoclax, brequinar, or a combination of venetoclax and brequinar, respectively. Diameters of the tumors were measured, and the volume was calculated according to the equation: Tumor volume(mm3) =[Length(mm) × Width2(mm)]/2. Venetoclax (VEN) was administered at 50 mg/kg once daily by oral gavage in 60% phosal-50PG, 30% polyethylene glycol-400, and 10% ethanol [17]. Brequinar (BRQ) was given 15 mg/kg intraperitoneally (IP) every three days, dissolved in 10% DMSO and 5% Tween-80 in PBS. When imaging, 1% pentobarbital sodium was used at 80 mg/kg via intraperitoneal injection. Mice were euthanized when the tumor volume reached 2000 mm3. In addition to tumor volume, any mouse that showed symptoms of tumor ulceration, cachexia, dehydration, loss of body weight of 20%, inability to get food and water, or paralysis was regarded to reach a humane endpoint and would be euthanized. Mice were euthanized by carbon dioxide asphyxiation at the flow rate of 30–70% of the chamber volume per minute. Death was confirmed by continuous exposure to CO2 for at least 15 min after respiratory arrest.
Histology and immunohistochemistry
Fresh tumor tissues were fixed in 10% neutral buffered formalin overnight, washed once with PBS, and stored in 70% ethanol at 4 °C. The tissues were dehydrated and embedded in paraffin according to standard protocols. Embedded tissues were sectioned at a thickness of 3 μm for H&E or immunohistochemistry analysis. IHC analysis was performed according to the antibodies protocol. The primary antibodies, including c-myc (1:200, 5605, Cell Signaling Technology), RPL26L1 (1:200, ABS146832, Absin), RPS27 (1:200, ABS138642, Absin) MRPS6(1:200, ABS118709, Absin) were incubated overnight at 4 °C. Staining was performed using the Images were randomly taken from the renal cortex (three images per tumor) at × 400 magnification using an Olympus microscope.
Statistical analysis
The unpaired Student’s t-test was used to compare the two groups’ differences One-way ANOVA followed by Dunnett’s or Turkey’s test was used to compare the difference between more than two groups. Two-way ANOVA followed by Bonferroni post-hoc analysis was used to compare the difference between groups in two factors. The specific statistical method was described in the graph’s legend. Statistical analysis was performed using GraphPad Prism version 7.0 (GraphPad Software, Inc.). Data from three independent experiments are shown as the mean ± standard error (SE). P values < 0.05 were considered significant.
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