Glioblastoma (GBM) is among the most aggressive human cancers. Although oncolytic virus (OV) therapy has been proposed as a potential approach to treat GBM, it frequently fails because GBM cells are usually nonpermissive to OV. Here, we describe a dual-step drug screen for identifying chemical enhancers of OV in GBM. From a high-throughput screen of 1416 FDA-approved drugs, an inhibitor of CDK4/6 was identified as the top enhancer, selectively increasing potency of two OV strains, VSVΔ51 and Zika virus. Mechanistically, CDK4/6 inhibition promoted autophagic degradation of MAVS, resulting in impaired antiviral responses and enhanced tumor-selective replication of VSVΔ51 in vitro and in vivo. CDK4/6 inhibition cooperated with VSVΔ51 to induce severe DNA damage stress and amplify oncolysis. In GBM xenograft models, combined treatment with CDK4/6 inhibitor and VSVΔ51 significantly inhibited tumor growth and prolonged the survival of tumor-bearing mice. Further investigation revealed that CDK4/6 inhibitor and VSVΔ51 synergistically induced immunogenic cell death and boosted antitumor immunity. Together, this study features a promising approach of treating aggressive GBM through the combination of CDK4/6 inhibitor with OV.

Significance:

This study proposes inhibition of cyclin-dependent kinases as a clinically translatable combinatorial strategy to enhance the efficacy of oncolytic virotherapy in GBM.

Glioblastoma (GBM) is a lethal brain tumor. Despite the current standard treatment including surgical resection, radiation, and chemotherapy (1), its median survival time is less than 2 years following diagnosis. At present, there is still no effective therapy for progressive or relapsed GBM (1). Genomic profiling has defined GBM subgroups and identified alterations in core signaling pathways, such as CDKN2A/B (50%), TP53 (50%), PTEN (33%), EGRF amplification (50%), CDK4 amplification (13%), and CDK6 amplification (1.5%; refs. 2, 3).

Oncolytic viruses (OV) are currently advancing into clinical trials (4–6). OVs are promising biotherapeutics as they are engineered to propagate within and selectively kill tumor cells (4, 7). Due to these features, OVs have the flexibility to be delivered both systemically or locally, and have the potency to act at both the primary and metastatic tumor sites (4, 6). Vesicular stomatitis virus (VSV) was introduced as a potent oncolytic candidate (8) that replicates effectively in PTEN-deficient tumor cells (9, 10). In orthotopic glioma models of the rodent brain, systemic administration of VSV showed potential in penetrating the brain blood barrier, selectively targeting and infecting not only the tumor bulk but also remote satellite glioma cell clusters (11–13). To reduce a potential neurotoxicity, researchers have engineered a VSV strain by deleting the methionine at residue 51 of the matrix protein (VSV∆51). However, in contrast to the parental strain, VSV∆51 has gained the ability to strongly induce an IFN response in cells (11–13). Evidence also suggests that Zika virus (ZIKV) has an oncolytic activity against GBM (14–16). ZIKV causes microcephaly by killing neural precursor cells but also acts as an OV against GBM. ZIKV preferentially targets GBM stem cells (GSC) through SOX2–integrin avb5 axis (16). Integrin avb5 is a GSC marker and promotes ZIKV infection; SOX2 expression promotes ZIKV replication via reducing antiviral responses (16).

As tumor cells are often defective in either producing or responding to the primary antiviral cytokine, type I IFNs (5, 17), they are generally considered more susceptible to infections of OVs. But the magnitude of the defect is quite variable and can be a barrier to effective OV replication and spread in tumor site (18–21). In these refractory tumor cells, the first line of defense, innate antiviral response is frequently activated through the recognition of pathogen-associated molecular patterns by pattern recognition receptors, culminating in the production of IFN to eliminate OV and limit its spreading within the tumor microenvironment (22).

In this study, we demonstrate that CDK4/6 inhibition can promote both tumor selective replication of OVs and cytotoxic DNA damage stress in GBM cells, leading to direct tumor cell death. CDK4/6 inhibition and VSVΔ51 also synergistically reshapes the tumor microenvironment and boosts the tumor-specific immunity in vivo. CDK4/6 inhibition enhances oncolytic therapy by potentiating oncolysis and antitumor immunity in GBM.

Cell lines and virus

The human GBM cell lines T98, SHG44, A172, U138, U118, LN229, M059K, LN18, M059J, DBTRG-05, U87, SF767, and U251 were purchased from the ATCC. The mouse glioma cell line GL261, primary patient-derived GSC1 and GSC11, two patient-derived primary GBM cells (GBM01 and GBM10) were provided by Dr. Guangmei Yan (Sun Yat-sen University, Guangzhou, China). The GBM cell lines and GBM cells were maintained at 37°C under 5% CO2 in high glucose DMEM (Gibco) supplemented with 10% Australian FBS (Gibco) and penicillin/streptomycin. GL261 cells were cultured in DMEM medium supplemented with 10% FBS (Gibco). GSC1 and GSC11 cells were cultured in DMEM/F12 medium (Gibco) supplemented with 2% B27 (Gibco), 0.5% penicillin G–streptomycin sulfate–amphotericin B complex (Corning), recombinant human EGF (20 ng/mL; R&D Systems), and recombinant human FGF-basic (20 ng/mL; PeproTech). Spheres were dissociated with Accutase (Innovative Cell Technologies) for passaging. The human astrocyte (HA) cell line was cultured in Astrocyte Medium (ScienCell, 1801). The human normal endothelial cell lines human immortal keratinocyte (HACAT) was cultured in high glucose DMEM supplemented with 10% FBS (Gibco) and penicillin/streptomycin. The vero and BHK21 cell lines was cultured in high glucose DMEM supplemented with 10% FBS (Gibco) and penicillin/streptomycin.

VSVΔ51-GFP virus was kindly provided by Dr. Tao Sun (Shanghai Jiao Tong University, Shanghi, China). VSVΔ51 was grown in BHK21. ZIKV was produced in vero cells. Virus titer was tested by plaque assay in vero cell lines and convert the PFU.

Lentivirus-mediated gene knockdown and gene transfer and stable cell lines construction

The phage-luciferase empty vector was purchased from OBiO Technology Company. The core vector and packing plasmids psPAX2 and the envelope plasmid pMD2.G were transfected into HEK293T cells using polyethylenimine. The medium was changed 8 hours after transfection. Forty-eight hours post transfection, cell supernatants containing lentivirus were collected and passed through a 0.45 μmol/L filter. GL261 cells were introduced with lentivirus for 8 hours. Forty-eight hours after introduction, the cells were selected with 2 μg/mL puromycin for 7 to 10 days to establish stably expressing cell lines. For animal models, GL261-luc showed stable express the luciferase gene and were selected to establish xenograft model in mice.

Antibodies and reagents

β-tubulin (Arigo, ARG62347, IB, 1:2,000), phosphorylated histone H2AX (2577, Cell Signaling Technology, 1:1,000 for IB, 1:100 for IHC), Ki-67 (9449s, Cell Signaling Technology, 1:400 for IHC), H2AX (7631, Cell Signaling Technology, 1:1,000), IRF3 (HUABIO, HM0923, 1:1,000), phosphorylated IRF3 (HUABIO, HML1224,1:1,000), CDK4 (ProteinTech, 11026–1-AP, 1:1,000), CDK6 (ProteinTech, 14052–1-AP, 1:1,000), RB (Cell Signaling Technology, 9309T, 1:1,000), phosphorylated–Rb (Cell Signaling Technology, 8516T, 1:1,000), MAVS (Cell Signaling Technology, 24930S, 1:1,000), MDA5 (Cell Signaling Technology, 5321S, 1:1,000), 53BP1 (Nouves, NB100–304, 1:500 for IF), VSV-G (Kerafast, EB0010, 1:1,000), RIG-I (Cell Signaling Technology, 3743S, 1:1,000), anti-rabbit IgG (H+L), Alexa Fluor 647 conjugate (Cell Signaling Technology, 4414, 1:500), Pierce goat anti-mouse IgG, (H+L), peroxidase conjugated (Thermo, 31430,1:5,000), Pierce goat anti-rabbit IgG, (H+L), peroxidase conjugated (Thermo, 31460, 1:5,000), granzyme B (granzB) monoclonal antibody (GB11), PE, eBioscience (Invitrogen, 12–8899–41), Alexa Fluor 647 anti-human FOXP3 (BioLegend, 320114), Alexa Fluor 700 anti-mouse CD8a (BioLegend, 100730), PE/Cyanine7 anti-mouse CD4 (BioLegend, 100528).

Cell viability assays

Cells were seeded in 96-well plates at 3,000 cells per well in 0.1 mL medium. After treatment, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added to the cells (1 mg/mL final concentration), and the cells were allowed to grow at 37°C for another 3 hours. MTT-containing medium was removed, and the MTT precipitate was dissolved in 100 μL DMSO. The optical absorbance was determined at 490 nm using a microplate reader (Biotech).

Drugs

In the first screening, the A549-IFNβ-GFP cells were seeded in 24-well plates at 4 × 105 per well in 0.5 medium and then pretreated with single doses (10 μmol/L) of candidate agents (1,416 FDA drugs bought from Selleck) for 1 hour, 1 multiplicity of infection (MOI) SeV for every well later for 10 hours, set the negative control (DMSO only) and positive control (SeV only) at the same time. The GFP fluorescence was analyzed by flow cytometry.

In the second screening, T98 cells were seeded the 24-well plates at 1 × 105 per well in 0.5 medium and then pretreated with the increased doses for 0.1 μmol/L, 0.3 μmol/L, 1 μmol/L, 3 μmol/L, 10 μmol/L, with or without the VSVΔ51 (MOI = 0.001) for 36 hours. Cell viability was determined by an MTT assay. AUCs were calculated using GraphPad Prism 8. Differences in the AUC are indicated by [area (a) − area (b)] area (b). Abemaciclib (LY2835219; S5716), Palbocilib (S4482), Ribocilib (S7440) were purchased from Selleck.

RNA interference

Specific and scramble siRNAs were obtained from Ribobio. Cell medium was replaced with 10% FBS in DMEM (without penicillin/streptomycin). SiRNAs were transfected using Lipofectamine RNAi MAX (13778–150, Thermo Fisher) with OPTI-MEM (31985070, Thermo Fisher).

Calreticulin exposure

The exposure of calreticulin was detected by the antibody of calreticulin for Flow cytometry. T98 cells was treated by abemaciclib (0.5 μmol/L), VSVΔ51 (0.01 MOI) or the combination of them for 24 hours, then harvest the cells, resuspended the cells for precooling of PBS. Then dyed the T98 cells for 30 minutes on the ice by the antibody of calreticulin. After wash with PBS, cells were stained with Alexa647 conjugated donkey anti-rabbit IgG (H + L) secondary antibody (1:200 dilution) on ice for 30 minutes. After wash with PBS, all samples were kept on ice and analyzed by flow cytometry within 30 minutes.

ATP secretion

T98 and GL261 cells were exposed to vehicle, abemaciclib (0.5 μmol/L), VSVΔ51 (0.01 MOI/0.1 MOI) or the combination of them for 24 hours or 36 hours. After that, supernatant was collected and spun at 2,000 × rpm, 5 minutes at 4°C to remove the inclusion of cells or debris. ATP amounts were measured via ENLITEN ATP Assay System (Promega) according to the kit instruction. Briefly, samples and standards (20 μL/well) are added into the plate on ice. Reconstituted rLuciferase/Luciferin Reagent (20 μL/well) are then added into each well rapidly. ATP-driven chemiluminescence signals are recorded with a luminescence microplate reader. ATP concentrations in samples are calculated with standard curves.

Transmission electron microscopy

T98 cells were infected with VSVΔ51 (0.001 MOI) in the presence or absence of abemaciclib (0.5 μmol/L) for 36 hours. Cells were collected by centrifugation at 1,000 × g for 5 minutes at room temperature. Cell pellets were then resuspended, washed once with PBS, pelleted at 1,500 × g for 5 minutes and fixed on ice for 4 hours in 0.1 mol/L PBS (pH 7.4) containing 2.5% glutaraldehyde and 2% paraformaldehyde. Samples were then submitted to the Servicebio Company. Electron Microscopy Facility for standard transmission electron microscopy (TEM) ultrastructural analysis.

Annexin V/7-AAD analysis

T98, HACAT and HA cells were treated with abemaciclib (0.5 μmol/L) and VSVΔ51 (0.001 MOI) for 36 hours. Then cells were centrifuged, counted, resuspended in FACS buffer (2% inactivated FCS in PBS), incubated with Annexin V-APC and 7-AAD (MultiSciences, 70-AP105–100) for 15 minutes, washed and resuspended in FACS buffer, and then analyzed using Cytoflex (Beckman Coulter) and CytExpert.

Multicolor flow cytometry analysis

Brain tumor quadrants were harvested, minced, incubated with a Brain Tumor Dissociation Kit (Miltenyi, 130–095–942), triturated, passed through a 70-mm screen, resuspended in FACS buffer, and stained with fluorochrome-conjugated anti-mouse antibodies from BioLegend or eBioscience, as well as appropriate isotype control antibodies. A Zombie Red Fixable Viability Kit (BioLegend) was used to stain dead cells. We followed a ‘no-wash’ sequential staining protocol (BioLegend) to stain dead cells and for surface staining. Intracellular FoxP3 staining was performed following the FoxP3 intracellular staining protocol (BioLegend). For single-color compensation controls, UltraComp eBeads (eBioscience) were used and stained with each of eight fluorescently conjugated antibodies according to the manufacturer's instructions. For the Zombie Red assay, cells from the non-tumor and tumor quadrants, respectively, were used as single-color compensation controls. All samples were run in a Cytoflex flow cytometer. Data were analyzed with CytExpert software. Technicians acquiring and gating the data were blinded to the treatments.

IHC assay

The expression of γH2AX, 53BP1, Ki-67 in the tumors was assessed by IHC. Briefly, tumor sections (4 μmol/L) were dewaxed in xylene, hydrated in decreasing concentrations of ethanol, immersed in 0.3% H2O2-methanol for 30 minutes, washed with PBS, and probed with monoclonal antibodies or isotype controls at 4°C overnight. After being washed, the sections were incubated with biotinylated goat anti-rabbit or anti-mouse IgG at room temperature for 2 hours. Immunostaining was visualized with streptavidin/peroxidase complex and diaminobenzidine, and sections were counterstained with hematoxylin. The IHC assay was made in a blinded manner for pathologists.

ELISA experiment

The production and secretion of human TNFα and IFNγ in splenic lymphocytes supernatants was measured with the mouse TNFα (catalog no. 1217202) and IFNγ (catalog no. 1210002) precoated ELISA kit (DAKEWE).

Drugs for animal experiment

The CDK4/6 inhibitor abemaciclib was purchased from Selleck. Both drugs were dissolved in DMSO to make a 5-mmol/L stock solution for in vitro studies. The highest DMSO concentration (0.001% DMSO) used for in vitro studies was nontoxic to the cells. For in vivo studies, abemaciclib powder (25 mg/kg/day) was dissolved in Edible alcohol and water for cells to make a homogenous suspension and was administered by oral gavage.

Animal models

This study was approved by the Institutional Animal Care and Use Committee, Sun Yat-sen University. For the subcutaneous xenograft model, dissociated GSC1 cells (3 × 106) in 100 μL of PBS were inoculated subcutaneously into the hind flanks of 5-week-old female BALB/c-nu/nu mice. After 5 days, palpable tumors had developed (∼100 mm3), and the mice were divided into four groups at random. The mice were treated with abemaciclib (25 mg/kg/day) by oral gavage on days 6 to 9 and days 12 to 15. VSVΔ51 (3×107 PFU/day) was administered by tail vein injection on days 7 to 9 and days 13 to 15. Tumor length and width were measured every 2 days, and the volume was calculated according to the formula, (length × width2)/2.

The orthotopic intracranial xenograft model were implanted stereotactically into the striatum (2.2 mm lateral from the bregma and 2.5 mm deep) to generate intracranial tumors. The GL261 cells stably expressing luciferase (3 × 105) were dissociated in 5 μL of PBS. Five days after tumor implantation, the mice were randomly divided into groups and treated with drugs. Mice were treated with abemaciclib (25 mg/kg, once per day) on days 6, 7, 8, 9, 12, 13, 14, and 15 by oral gavage; and VSVΔ51 (3×107 PFU, once per day) by tail vein injection on days 7 to 9 and days 13 to 15.

Mouse bioluminescence imaging

Mice implanted with GBM cells expressing luciferase were injected intraperitoneally with a luciferin solution (15 mg/mL in DPBS, dose of 150 mg/kg). The bioluminescence images were acquired using the IVIS Lumina system and analyzed by Living Image software. Imaging experiments were conducted at the Animal Imaging platform of the Experimental Animal Center of Sun Yat-sen University.

Quantitative RT-PCR

Total RNA was extracted with TRIzol reagent (Invitrogen). A reverse transcription system (TAKARA) was used to synthesize cDNA. SuperReal PreMix SYBR Green (AG) and an ABI Q5 Detection System were used for qRT-PCR. The mRNA results were normalized to GAPDH expression. The sequence of prime showed in the Supplementary Table S1.

Immunoblot analysis

For immunoblot analysis, whole-cell extracts were collected and lysed in RIPA lysis buffer containing 50-mmol/L Tris-HCl, pH 8.0, 150-mmol/L NaCl, 1.0% (v/v) Triton X-100, 1.0% sodium deoxycholate, and 0.1% SDS. The lysates were subjected to SDS-PAGE, transferred onto 0.45-μmol/L polyvinylidene difluoride membranes and then blotted with the indicated antibodies.

For GL261-specific antibodies measurement, the blood of tumor-bearing mice was collected and then centrifugated 3,000 r/min for 15 minutes to separate the serum. Then the serum was diluted at the ratio of 1:1,000 for immunoblotting to detect the antigen from GL261 cell lysis.

Immunofluorescence

T98, U251 cells (2×105 per dish) were seeded into glass bottom Culture dishes and incubated with abemaciclib (0.5 μmol/L), VSVΔ51 (MOI = 0.01) single or combination for 24 hours. Four percent paraformaldehyde (Polysciences 18814) was used to fix tumor cells on cover slips for 15 minutes at room temperature. Dishes were then rinsed in 1×PBS and blocked with 0.3% Triton X-100 and 5% BSA (Bovine Serum Albumin, Solarbio-9048–46–8) in PBS for 30 minutes at 37°C. Dishes were incubated at 37°C with γH2AX (Ser139, 20E3) or 53BP1 antibody at 1:500 or 1:1,000 dilution for 1 hour. Then incubation at room temperature in anti-rabbit antibody Alexa Fluor 555 at 1:1,000 dilution in PBS with 1% BSA at 37°C. The dishes were rinsed in PBS three times and nuclei were stained by incubating the coverslips at room temperature in the dark with 4′,6-diamidino-2-phenylindole (DAPI, Thermol Fisher) diluted at 1:1,000 in PBS, followed by additional rinses in PBS and sterile water. The dishes were analyzed with a Laser Scanning Confocal Microscope (Zeiss, LSM780, Germany). Foci were counted in 100 cells per treatment condition.

RNA sequencing and transcriptome data analysis

Total RNA was extracted from T98 cells using the TRIzol reagent (Life Technologies). Samples were sent to Beijing Genomics Institute for RNA sequencing (RNA-seq) analysis. Functional analysis of differentially expressed genes was performed by gene ontology (GO). The data generated in this study are publicly available in Gene Expression Omnibus (GEO) at GSE166914.

Lymphocytotoxicity test

Splenic lymphocytes were isolated from the spleen with lymphocyte separation fluid (Dakewe). Lymphocytes were cocultured with pre-seeded GL261 cells for 2 days. The cells were washed with PBS twice to remove suspended lymphocytes, and the viability of adherent tumor cells was detected by MTT assays as described above. Lymphocytotoxicity was calculated with the following formula: lymphocytotoxicity  =  (Atumor cells − Atumor cells+lymphocytes)/(Atumor cells − Ablank) × 100%.

Lactate dehydrogenase cytotoxicity assay

The cocultured supernatant was harvested without cells, then used 50 μL of sample and 50 μL of lactate dehydrogenase (LDH) Detection Reagent (J2308) in a 96-well plate, incubated for 60 minutes at room temperature, then recorded luminescence by GloMax instrument.

Plague assay

Briefly, after infection, supernatants or sera containing virus were collected and diluted to infect Vero cells plated on 24-well plates at 90% confluence. Two hours post infection, the supernatants were removed, the cells were washed several times with PBS, and methylcellulose (0.8%) was added. Forty-eight hours later, the cells were stained with crystal violet (0.2%) for 12 hours. Plaques (pfu/mL) were counted.

Serum chemistry parameters measurement

Briefly, the blood of tumor-bearing mice was collected and then centrifugated 3,000 r/min for 15 minutes to separate the serum. Then the biochemical parameters of the serum were measured by the Servicebio Company (China, Hubei).

Statistical analysis

All experiments were repeated at least two times, and the data are shown as the mean ± SD. Statistical significance between different groups was calculated by two-tailed unpaired Student t test or two-way ANOVA. For the survival curve of mice, the log-rank (Mantel-Cox) test was used for the comparison. A P value < 0.05 was considered to be significant. Data were analyzed with GraphPad Prism 8.0 Software.

Data availability

The RNA-seq raw data have been deposited in GEO at GSE166914. All relevant data and materials that are included in this study are available from corresponding author upon reasonable request.

Dual-step screening of FDA-approved drugs identified CDK4/6 inhibitor as an enhancer of OVs

To evaluate the sensitivity of GBM to OVs, a variety of widely used glioma cell lines (GL261, T98, SHG44, A172, U138, U118, LN229, M059K, LN18, M059J, DBTRG-05, U87, SF767, and U251) and two patient-derived primary GBM cells (GBM01 and GBM10) were treated with VSVΔ51 (MOI = 0.001, 0.01, 0.1, 1) for 48 hours (Fig. 1A). The measurement of cell viability revealed that glioma cell lines showed different response to VSVΔ51 treatment. It was observed that 7 of 16 glioma cell lines were relatively refractory to VSVΔ51 infection, suggesting that it is necessary to improve the oncolytic activity in refractory glioma cells to promote the clinical application of OVs.

Figure 1.

A dual-step screening of FDA-approved drugs identified CDK4/6 inhibitor as top enhancer for OVs. A, Relative cell viability in 14 glioma cell lines and 2 primary patient-derived glioma cells (GBM-01, GBM-10) treated with VSVΔ51 (MOI = 1, 0.1, 0.01, 0.001). The percent cell inhibition is color-coded by quartile. Data represents means (n = 3). B, A flow diagram of the drug-screening protocol. The first step of drug screening was to look for drugs inhibiting IFNβ expression. A549-IFNβ-GFP cells were pretreated with the 10 μmol/L of FDA drugs for 1 hour, then infected by SeV (MOI = 1) for 10 hours to stimulate IFNβ expression. The ratio of GFP+ cell was measured by the flow cytometry. The first screening identified 70 drugs that could reduce the proportion of virus-induced GFP-positive cells to less than 30%. The second screening was designed to look for drugs that could increase the oncolytic effect of OVs. Dose–response curves were generated for each drug in the absence or presence of OV VSVΔ51, and the DAUC (fold) was calculated according to the formula (AUCsingle−AUCcombined)/AUCcombined; the orange areas represent DAUC. The biologic replicate of the first screening is one (n = 1). Data represents means± SD (n = 3) for second screening. C, Representative DAUC for drugs identified in the second round of screening. In this case, the drug A significantly increased the oncolytic effect of VSVΔ51.The top 10 drugs identified in the second round of screening according to DAUC values. D, The expression of CDK4 and CDK6 in the primary glioma and normal tissue in the TCGA database.

Figure 1.

A dual-step screening of FDA-approved drugs identified CDK4/6 inhibitor as top enhancer for OVs. A, Relative cell viability in 14 glioma cell lines and 2 primary patient-derived glioma cells (GBM-01, GBM-10) treated with VSVΔ51 (MOI = 1, 0.1, 0.01, 0.001). The percent cell inhibition is color-coded by quartile. Data represents means (n = 3). B, A flow diagram of the drug-screening protocol. The first step of drug screening was to look for drugs inhibiting IFNβ expression. A549-IFNβ-GFP cells were pretreated with the 10 μmol/L of FDA drugs for 1 hour, then infected by SeV (MOI = 1) for 10 hours to stimulate IFNβ expression. The ratio of GFP+ cell was measured by the flow cytometry. The first screening identified 70 drugs that could reduce the proportion of virus-induced GFP-positive cells to less than 30%. The second screening was designed to look for drugs that could increase the oncolytic effect of OVs. Dose–response curves were generated for each drug in the absence or presence of OV VSVΔ51, and the DAUC (fold) was calculated according to the formula (AUCsingle−AUCcombined)/AUCcombined; the orange areas represent DAUC. The biologic replicate of the first screening is one (n = 1). Data represents means± SD (n = 3) for second screening. C, Representative DAUC for drugs identified in the second round of screening. In this case, the drug A significantly increased the oncolytic effect of VSVΔ51.The top 10 drugs identified in the second round of screening according to DAUC values. D, The expression of CDK4 and CDK6 in the primary glioma and normal tissue in the TCGA database.

Close modal

Meanwhile, we detected the IFNB1 expression following VSVΔ51 infection in above cell lines and found that IFNB1 expression was negatively correlated with the sensitivity to VSVΔ51 infection (Supplementary Fig. S1A and S1B), indicating that the IFN pathway could provide molecular targets for improving the efficacy of OVs in refractory glioma cells.

Next, we performed a dual-step screening of 1416 FDA-approved drugs (Supplementary Table S2) for screening the chemical enhancers of OV therapy. The first step of screening aimed to identify candidates for inhibiting the IFN response (Fig. 1B). We have previously constructed a fluorescence reporting system for type I IFN expression in A549 cells (A549-IFNβ-GFP; ref. 23), which was more efficient and laborsaving than constructing new IFNβ reporter glioma cell lines. Through flow cytometry analysis of A549-IFNβ-GFP cells, we identified 70 drug candidates (Supplementary Table S3) that had the potential for inhibiting the IFN response and reduced the ratio of GFP+ cells (cut off : GFP+ < 30%; Fig. 1B; Supplementary Fig. S1C).

The second screening step aimed to identify candidates for enhancing the oncolytic effect of OVs (Fig. 1B). We conducted the combinatory screening using VSVΔ51 in refractory T98 cells. Cell viability was measured after treatment with increasing doses of candidate drugs in the presence or absence of VSVΔ51. Differences in the area under the curve (DAUC) with or without VSVΔ51 virus for each agent were calculated. The agents were ranked according to the DAUC values (Fig. 1C).

After screening the FDA drugs library, we identified multiple compounds targeting different pathways that can cooperate with VSVΔ51, the top 10 of which are listed (Fig. 1C). Our screening also identified several previously reported chemical enhancers of OVs, including STAT inhibitor (24), JAK inhibitor (25, 26), and microtubule disruption agent (21), indicating the efficiency of our screening system. A CDK4/6 inhibitor, abemaciclib, was identified as the best enhancer for OV VSVΔ51 in our screening (DAUC = 1.10 for T98; Fig. 1C).

From the Cancer Genome Atlas Program (TCGA) database, CDK4 and CDK6 are found to be significantly upregulated in glioma compared with normal issue (Fig. 1D), indicating that CDK4 and CDK6 may represent a selective target for combining treatment with OV in glioma.

CDK4/6 inhibition increased the oncolytic effect of VSVΔ51 and ZIKV in GBM cells

To investigate whether CDK4/6 inhibition enhances the oncolysis, we chose more refractory glioma cell lines to measure the synergistic activity of the combination treatment (Fig. 2A). The treatment of abemaciclib enhanced the oncolytic effect of VSVΔ51 in these cell lines (DAUC = 1.10 for T98, 0.91 for LN229, 0.94 for GL261; Fig. 2A). We further quantify the sensitization by measuring the half-inhibitory concentration (IC50) shift. The treatment of abemaciclib sensitized the glioma cells toward VSVΔ51 at various degrees (IC50 shift of 1185-fold for T98, 635-fold for GL261, and 809-fold for LN229; Fig. 2B).

Figure 2.

CDK4/6 inhibition increased the oncolytic effect of VSVΔ51 and ZIKV in refractory GBM cells. A, T98, LN229, and GL261 cells were treated with increasing doses of CDK4/6 inhibitor abemaciclib in the absence or presence of VSVΔ51 virus (MOI = 0.001) for 48 hours. Cell viability was measured and DAUC was calculated. B, EC50 shifts were determined for T98, LN229, and GL261 cells treated with escalating titers of VSVΔ51 with or without abemaciclib (0.5 μmol/L) for 48 hours. C, T98 cells were treated with increasing doses of different CDK4/6 inhibitors (palbociclib and ribociclib) in the absence or presence of VSVΔ51 for 48 hours. Then cell viability was measured and DAUC was calculated. D and E, Western blot analysis of cells transfected with siRNAs targeting the CDK4 and CDK6. Cells were transfected with siRNA for 24 hours, then treated with VSVΔ51 (MOI = 0.001) for another 48 hours. The cell viability was measured by MTT staining. siNC, negative control siRNA. F, GSC1 and GSC11 cells were infected by ZIKV (MOI = 10) with different concentrations of abemaciclib. Cell viability was measured and DAUC was calculated. Data represents the mean ± SD (n = 3) in A–F. *, P < 0.05; **, P < 0.01.

Figure 2.

CDK4/6 inhibition increased the oncolytic effect of VSVΔ51 and ZIKV in refractory GBM cells. A, T98, LN229, and GL261 cells were treated with increasing doses of CDK4/6 inhibitor abemaciclib in the absence or presence of VSVΔ51 virus (MOI = 0.001) for 48 hours. Cell viability was measured and DAUC was calculated. B, EC50 shifts were determined for T98, LN229, and GL261 cells treated with escalating titers of VSVΔ51 with or without abemaciclib (0.5 μmol/L) for 48 hours. C, T98 cells were treated with increasing doses of different CDK4/6 inhibitors (palbociclib and ribociclib) in the absence or presence of VSVΔ51 for 48 hours. Then cell viability was measured and DAUC was calculated. D and E, Western blot analysis of cells transfected with siRNAs targeting the CDK4 and CDK6. Cells were transfected with siRNA for 24 hours, then treated with VSVΔ51 (MOI = 0.001) for another 48 hours. The cell viability was measured by MTT staining. siNC, negative control siRNA. F, GSC1 and GSC11 cells were infected by ZIKV (MOI = 10) with different concentrations of abemaciclib. Cell viability was measured and DAUC was calculated. Data represents the mean ± SD (n = 3) in A–F. *, P < 0.05; **, P < 0.01.

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To exclude the off-target effects of abemaciclib, we chose two other CDK4/6 inhibitors, palbociclib and ribociclib (27), to measure the synergistic activity of the combination treatment. DAUC values of two CDK4/6 inhibitors were calculated in the above cell lines (DAUC = 0.75 for palbociclib, 0.80 for ribociclib in T98 cells; Fig. 2C; Supplementary Fig. S2A–S2D). In addition, knockdown of CDK4 and CDK6 by siRNA also enhanced oncolysis of VSVΔ51 in T98 cells and GL261 cells (Fig. 2D and E). These results indicated that CDK4/6 inhibition could sensitize the refractory glioma cells to OV infection.

It is worthwhile to evaluate the sensitization of CDK4/6 inhibitors across various OVs. ZIKV has been reported to be an emerging OV strain for gliomas (14–16), with specificity for targeting glioma stem cells (GSC). Therefore, we measured the synergistic effect of abemaciclib and ZIKV in two patient-derived GSCs lines (GSC-1 and GSC-11). We found that abemaciclib treatment enhanced the oncolytic effect of ZIKV in both GSC-1 and GSC-11 cells (DAUC = 0.68 for GSC1, 0.53 for GSC11, Fig. 2F). Collectively, CDK4/6 inhibition broadly enhanced oncolysis across different glioma cells and various OV strains.

CDK4/6 inhibitor promoted the tumor-selective viral replication

We next evaluated the effect of CDK4/6 inhibition on viral replication. Fluorescence imaging showed that abemaciclib treatment or siCDK4&6 enhanced viral spread in glioma cells (Fig. 3A and B; Supplementary Fig. S3A and S3B). The abemaciclib treatment also improved the expression of VSV-G at mRNA and protein level (Fig. 3C and D). Viral titer measurement confirmed that abemaciclib treatment enhanced viral production in T98, Gl261, and primary GBM10 cells (Fig. 3E; Supplementary Fig. S3C). We further evaluated the effect of treatment sequence on viral replication. The abemaciclib was given sequential or concomitantly with VSVΔ51(Supplementary Fig. S3D). We found that only concomitant treatment of abemaciclib could increase the VSV-G expression and viral replication in T98 cells, but not the sequential treatment (Supplementary Fig. S3D). These data suggest that the CDK4/6 inhibitors should be given concomitantly with the OV.

Figure 3.

CDK4/6 inhibitor promoted the tumor-selective viral replication. A and B, Abemaciclib treatment promoted the spread of VSVΔ51 in T98 (A) and GL261 (B). Cells were infected with VSVΔ51 (0.001 for T98 cells; 0.1 MOI for GL261 cells) in the presence or absence of abemaciclib (0.5 μmol/L). Viral reporter GFP was monitored after 24 hours of VSVΔ51 infection. The ratio of infected cells (GFP+) was measured by flow cytometry. Scale bar, 100 μm. C, Relative level of viral gene VSV-G by qPCR. T98 cells were treated with VSVΔ51 (0.001) and abemaciclib (0.5 μmol/L) for 24 hours or 36 hours. D, Immunoblot analysis of viral G protein (VSV-G). T98 and GL261 cells were treated the same as A and B for 36 hours. E, Plague assay to test the titer of VSVΔ51. The T98, GL261, and GBM10 cells were treated with VSVΔ51 for 48 hours (0.001 for T98 and GBM10 cells; 0.1 MOI for GL261 cells). Then the cellular supernatant was harvested for the plague assay. F and G, Abemaciclib treatment did not promote the viral spread of VSVΔ51 in normal cells HACAT (F) and HA (G). Cells were infected with VSVΔ51 (0.001 MOI) in the presence or absence of abemaciclib (0.5 μmol/L). Viral reporter GFP was monitored after 24 hours of VSVΔ51 infection. The ratio of infected cells (GFP+) to uninfected cells was measured by flow cytometry. Scale bar, 100 μm. H, Relative levels of viral mRNA VSV-G by qPCR. HACAT and HA cells were treated the same as F and G for 24 hours. I, Immunoblot analysis of viral G protein (VSV-G). HACAT and HA cells were treated the same as F and G for 24 hours. J, Plague assay to test the titer of VSVΔ51. The HACAT and HA cells were treated with VSVΔ51 (0.001 MOI) and abemaciclib at the indicated concentration. Then the cellular supernatant was harvested for the plague assay. K, Relative level of viral gene VSV-G by qPCR for C57 animal tissue. GL261 subcutaneous xenografts in C57 BL/6J were treated with VSVΔ51 (3×107 PFU/day, intravenous injection) and abemaciclib (25 mg/kg/day, oral gavage; n = 7 or 4 from two independent experiments). The mRNA of VSV-G was measured by qPCR in tumor and different tissue. Data represents the mean ± SD (n = 3) in A, B, C, E, H and J; n = 7 or 4 in K. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., nonsignificant.

Figure 3.

CDK4/6 inhibitor promoted the tumor-selective viral replication. A and B, Abemaciclib treatment promoted the spread of VSVΔ51 in T98 (A) and GL261 (B). Cells were infected with VSVΔ51 (0.001 for T98 cells; 0.1 MOI for GL261 cells) in the presence or absence of abemaciclib (0.5 μmol/L). Viral reporter GFP was monitored after 24 hours of VSVΔ51 infection. The ratio of infected cells (GFP+) was measured by flow cytometry. Scale bar, 100 μm. C, Relative level of viral gene VSV-G by qPCR. T98 cells were treated with VSVΔ51 (0.001) and abemaciclib (0.5 μmol/L) for 24 hours or 36 hours. D, Immunoblot analysis of viral G protein (VSV-G). T98 and GL261 cells were treated the same as A and B for 36 hours. E, Plague assay to test the titer of VSVΔ51. The T98, GL261, and GBM10 cells were treated with VSVΔ51 for 48 hours (0.001 for T98 and GBM10 cells; 0.1 MOI for GL261 cells). Then the cellular supernatant was harvested for the plague assay. F and G, Abemaciclib treatment did not promote the viral spread of VSVΔ51 in normal cells HACAT (F) and HA (G). Cells were infected with VSVΔ51 (0.001 MOI) in the presence or absence of abemaciclib (0.5 μmol/L). Viral reporter GFP was monitored after 24 hours of VSVΔ51 infection. The ratio of infected cells (GFP+) to uninfected cells was measured by flow cytometry. Scale bar, 100 μm. H, Relative levels of viral mRNA VSV-G by qPCR. HACAT and HA cells were treated the same as F and G for 24 hours. I, Immunoblot analysis of viral G protein (VSV-G). HACAT and HA cells were treated the same as F and G for 24 hours. J, Plague assay to test the titer of VSVΔ51. The HACAT and HA cells were treated with VSVΔ51 (0.001 MOI) and abemaciclib at the indicated concentration. Then the cellular supernatant was harvested for the plague assay. K, Relative level of viral gene VSV-G by qPCR for C57 animal tissue. GL261 subcutaneous xenografts in C57 BL/6J were treated with VSVΔ51 (3×107 PFU/day, intravenous injection) and abemaciclib (25 mg/kg/day, oral gavage; n = 7 or 4 from two independent experiments). The mRNA of VSV-G was measured by qPCR in tumor and different tissue. Data represents the mean ± SD (n = 3) in A, B, C, E, H and J; n = 7 or 4 in K. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., nonsignificant.

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In contrast, abemaciclib treatment did not influence the viral spread and replication in the normal cells, including primary HAs and HACATs (Fig. 3F and G). It also did not improve the expression of VSV-G in HA and HACAT cells (Fig. 3H and I). Virus titer measurement revealed that different doses of abemaciclib also did not enhance viral production in non-tumor cells (Fig. 3J). Combination treatment with abemaciclib and VSVΔ51 showed no synergistic effect on cell death in HA and HACAT cells (Supplementary Fig. S3E–S3H).

We further analyzed the effect of CDK4/6 inhibitor on viral replication in vivo. We established a subcutaneous tumor model using GL261 cells in C57 mice and intravenously injected VSVΔ51. Abemaciclib treatment significantly increased the VSV-G expression in the tumor tissues, but not in the kidney, liver, lung, brain, heart, and lung (Fig. 3K). These data suggested that the enhancement of viral replication by CDK4/6 inhibition was tumor-specific both in vitro and in vivo.

CDK4/6 inhibition suppressed the antiviral response by autophagic degradation of MAVS

To understand how CDK4/6 inhibition promote the replication of OV, we first characterized the innate immune response to viral infection. We found that the CDK4/6 inhibitor reversed the induction of IFNB1 and IFN-stimulated genes (ISG) by VSVΔ51 infection in different glioma cell lines and the primary GBM10 cells (Fig. 4A; Supplementary Fig. S4A and S4B). Consistently, dual knockdown of CDK4 and CDK6 also abolished the VSVΔ51-activated IFNB1 and ISGs expression (Supplementary Fig. S4C).

Figure 4.

CDK4/6 inhibitor suppressed the type I IFN pathway by promoting autophagic degradation of MAVS. A, Abemaciclib treatment inhibited IFN signaling stimulated by VSVΔ51 infection. Relative mRNA expression of IFNB1 and CXCL10 in T98, GL261, and GBM10 cells by qPCR. Cells were treated with abemaciclib (0.5 μmol/L) and VSVΔ51 (MOI = 0.001 for T98 and GBM10 cells; MOI = 0.1 for GL261 cells) for 12 hours. GAPDH was used as the housekeeping gene. B, The immunoblot analysis of key molecules in the IFN pathway. T98 and GBM10 cells were treated with abemaciclib (0.5 μmol/L), VSVΔ51 (MOI = 0.01), or the combination for 24 hours, then the expression of proteins in IFN pathway were measured by immunoblot. C, The mRNA levels of MAVS in T98 cells. T98 cells were treated the same as in B. GAPDH was used as the housekeeping gene. D, Immunoblot analysis of lysates from T98 cells infected with VSVΔ51 in the presence or absence of abemaciclib (0.5 μmol/L) for 16 hours and then treated with cycloheximide for the indicated times to inhibit protein translation. E, Immunoblot analysis of lysates from T98 cells infected with VSVΔ51 in the presence or absence of abemaciclib (0.5 μmol/L) for 18 hours, then the cells were treated with DMSO, MG132 (25 μmol/L), NH4Cl (10 mmol/L), chloroquine (20 μmol/L), or BafA1 (200 nmol/L) for 6 hours. F, TEM images of T98 cells treated with abemaciclib (0.5 μmol/L), VSVΔ51 (MOI = 0.001), or the combination for 36 hours. Higher magnification images are highlighted by the red box. Red arrows, lysosome in the tumor cells. Scale bar, 2 μm. G, The immunofluorescence analysis of LysoTracker following different treatments with abemaciclib (0.5 μmol/L), VSVΔ51 (MOI = 0.001), or the combination for 12 hours. Scale bar, 10 μm. H–K, The MAVS rescue experiment via knockdown of LAMP2A or LIPA. T98 cells were transfected with siRNA targeting LAMP2A or LIPA for 24 hours. Then cells were treated with abemaciclib (0.5 μmol/L), VSVΔ51 (MOI = 0.01 for 24 hours; MOI = 0.001 for 48 hours) or the combination for another 24 hours (H, I, and J) or 48 hours (K). The expression of MAVS was measured by immunoblot (H). The mRNA expression of IFNB1 (I) and VSVG (J) was measured by qPCR. The viral titer was measured by plague assay (K). Data represents the mean ± SD (n = 3) in A, C, I, J and K. *, P < 0.05; **, P < 0.01; ***, P < 0.001. n.s., nonsignificant

Figure 4.

CDK4/6 inhibitor suppressed the type I IFN pathway by promoting autophagic degradation of MAVS. A, Abemaciclib treatment inhibited IFN signaling stimulated by VSVΔ51 infection. Relative mRNA expression of IFNB1 and CXCL10 in T98, GL261, and GBM10 cells by qPCR. Cells were treated with abemaciclib (0.5 μmol/L) and VSVΔ51 (MOI = 0.001 for T98 and GBM10 cells; MOI = 0.1 for GL261 cells) for 12 hours. GAPDH was used as the housekeeping gene. B, The immunoblot analysis of key molecules in the IFN pathway. T98 and GBM10 cells were treated with abemaciclib (0.5 μmol/L), VSVΔ51 (MOI = 0.01), or the combination for 24 hours, then the expression of proteins in IFN pathway were measured by immunoblot. C, The mRNA levels of MAVS in T98 cells. T98 cells were treated the same as in B. GAPDH was used as the housekeeping gene. D, Immunoblot analysis of lysates from T98 cells infected with VSVΔ51 in the presence or absence of abemaciclib (0.5 μmol/L) for 16 hours and then treated with cycloheximide for the indicated times to inhibit protein translation. E, Immunoblot analysis of lysates from T98 cells infected with VSVΔ51 in the presence or absence of abemaciclib (0.5 μmol/L) for 18 hours, then the cells were treated with DMSO, MG132 (25 μmol/L), NH4Cl (10 mmol/L), chloroquine (20 μmol/L), or BafA1 (200 nmol/L) for 6 hours. F, TEM images of T98 cells treated with abemaciclib (0.5 μmol/L), VSVΔ51 (MOI = 0.001), or the combination for 36 hours. Higher magnification images are highlighted by the red box. Red arrows, lysosome in the tumor cells. Scale bar, 2 μm. G, The immunofluorescence analysis of LysoTracker following different treatments with abemaciclib (0.5 μmol/L), VSVΔ51 (MOI = 0.001), or the combination for 12 hours. Scale bar, 10 μm. H–K, The MAVS rescue experiment via knockdown of LAMP2A or LIPA. T98 cells were transfected with siRNA targeting LAMP2A or LIPA for 24 hours. Then cells were treated with abemaciclib (0.5 μmol/L), VSVΔ51 (MOI = 0.01 for 24 hours; MOI = 0.001 for 48 hours) or the combination for another 24 hours (H, I, and J) or 48 hours (K). The expression of MAVS was measured by immunoblot (H). The mRNA expression of IFNB1 (I) and VSVG (J) was measured by qPCR. The viral titer was measured by plague assay (K). Data represents the mean ± SD (n = 3) in A, C, I, J and K. *, P < 0.05; **, P < 0.01; ***, P < 0.001. n.s., nonsignificant

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As shown in Fig. 4B, CDK4/6 inhibitor reduced the level of MAVS and the phosphorylation of TBK-1 and IRF-3 after VSVΔ51 infection in T98 and primary GBM10 cells, indicating that CDK4/6 inhibition blocked the signal transduction of IFN pathway via modulating MAVS expression. However, the mRNA expression of MAVS was not decreased by CDK4/6 inhibitor (Fig. 4C). CDK4/6 inhibition significantly enhanced the rate of MAVS degradation in cycloheximide chase experiments (Fig. 4D). These data indicated that CDK4/6 inhibition caused the proteomic degradation of MAVS, led to the blockade of IFN pathway.

To examine the detailed mechanism of MAVS degradation, different inhibitors, including the proteasome inhibitor MG132, lysosome inhibitor NH4Cl and chloroquine and late-phase autophagy inhibitor bafilomycin A1 (BafA1) were used to block different degradation pathways. The results showed that CDK4/6 inhibitor–mediated degradation of MAVS was completely blocked by chloroquine and BafA1, but not by MG132, indicating that CDK4/6 inhibitor–mediated MAVS degradation took place via autophagic processes (Fig. 4E). TEM analysis showed that VSVΔ51/abemaciclib combination induced the formation of autolysosomes (Fig. 4F). Lysotracker Red assays also confirmed that VSVΔ51/abemaciclib combination increased lysosome quantity (Fig. 4G). Knockdown of two key autolysosome formation regulatory genes, lysosome-associated membrane protein 2 (LAMP2A; ref. 28) and lysosomal acid lipase (LIPA; ref. 29), could rescue MAVS expression during VSVΔ51/abemaciclib combination treatment (Fig. 4H; Supplementary Fig. S4D). Further experiments showed that siLAMP2A or siLIPA restored the IFNB1 expression under VSVΔ51/abemaciclib combination treatment (Fig. 4I). The enhancement of viral replication was also reversed by siLAMP2A or siLIPA (Fig. 4J and K). These results indicated that CDK4/6 inhibition abolished IFN signal transduction and the antiviral response by promoting autophagic degradation of MAVS.

Combination of CDK4/6 inhibitor and VSVΔ51 induced cytotoxic DNA damage stress in GBM cells

To address how CDK4/6 inhibition facilitates oncolysis, we performed RNA-seq to gain insight into the possible mechanisms. As expected, GO analysis revealed that VSVΔ51/abemaciclib combination treatment downregulated the genes relating to “defense response to virus” and “type I interferon signaling”, compared with single treatment with VSVΔ51 (Fig. 5A). Strikingly, DNA damage related GO process terms were also significantly enriched by the treatment of VSVΔ51/abemaciclib combination, including “response to DNA damage stimulus,” “chromatin organization,” and “DNA repair” (Fig. 5A). Gene set enrichment analysis (GSEA) also revealed that VSVΔ51/abemaciclib combination suppressed the DNA damage repair (DDR) pathway, including DNA mismatch repair and homologous recombination (Fig. 5B and C; Supplementary Fig. S5A). These data indicated that VSVΔ51/abemaciclib combination treatment triggered the DNA damage response.

Figure 5.

Combination of CDK4/6 inhibitor and VSVΔ51 induced cytotoxic DNA damage stress in GBM cells. A, GO analysis of RNA-seq data showed the downregulated biological process pathway by VSVΔ51/abemaciclib combination treatment, compared with single treatment with VSVΔ51. The T98 cells were treated with abemaciclib (0.5 μmol/L) and VSVΔ51 (MOI = 0.01) for 24 hours, and then the total RNA of cells was isolated for the RNA-seq (n = 3). B and C, GSEA analysis of KEGG gene sets. Plots show an enrichment of gene signatures associated with mismatch repair pathway (B) and homologous recombination (C) in VSVΔ51/abemaciclib combination group. NES, normalized enrichment score. D, The immunofluorescence analysis of DNA damage signal in T98 cells and U251 cells. Cells were treated with abemaciclib (0.5 μmol/L) and OV VSVΔ51 (MOI = 0.01) for 24 hours, then cells were fixed and stained with anti-53BP1 (red) and anti-γH2AX antibody (purple). Nuclei were stained with DAPI (blue). Scale bar, 10 μm. Left, the quantification analysis of γH2AX and 53BP1 foci was performed. E, Relative mRNA level of DDR-related genes. The T98 cells were treated with abemaciclib (0.5 μmol/L) and VSVΔ51 (MOI = 0.01) for 24 hours, then qRT-PCR was performed to detect the mRNA expression of the genes. F, Immunoblot analysis of the expression of p-H2AX, RAD51, XRCC1, RAD54, BLM, and POLD1. T98 cells were treated with abemaciclib (0.5 μmol/L), VSVΔ51 (MOI = 0.01), or the combination for 24 hours. G, The flow cytometric analysis of cell death in T98 cells. Cells were infected with VSVΔ51 (MOI = 0.001) with or without abemaciclib (0.5 μmol/L) for 36 hours, the cells were stained with Annexin-V/7-AAD, then flow cytometric analysis was performed. Data represents the mean ± SD (n = 6) in D; (n = 3) in E and G. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 5.

Combination of CDK4/6 inhibitor and VSVΔ51 induced cytotoxic DNA damage stress in GBM cells. A, GO analysis of RNA-seq data showed the downregulated biological process pathway by VSVΔ51/abemaciclib combination treatment, compared with single treatment with VSVΔ51. The T98 cells were treated with abemaciclib (0.5 μmol/L) and VSVΔ51 (MOI = 0.01) for 24 hours, and then the total RNA of cells was isolated for the RNA-seq (n = 3). B and C, GSEA analysis of KEGG gene sets. Plots show an enrichment of gene signatures associated with mismatch repair pathway (B) and homologous recombination (C) in VSVΔ51/abemaciclib combination group. NES, normalized enrichment score. D, The immunofluorescence analysis of DNA damage signal in T98 cells and U251 cells. Cells were treated with abemaciclib (0.5 μmol/L) and OV VSVΔ51 (MOI = 0.01) for 24 hours, then cells were fixed and stained with anti-53BP1 (red) and anti-γH2AX antibody (purple). Nuclei were stained with DAPI (blue). Scale bar, 10 μm. Left, the quantification analysis of γH2AX and 53BP1 foci was performed. E, Relative mRNA level of DDR-related genes. The T98 cells were treated with abemaciclib (0.5 μmol/L) and VSVΔ51 (MOI = 0.01) for 24 hours, then qRT-PCR was performed to detect the mRNA expression of the genes. F, Immunoblot analysis of the expression of p-H2AX, RAD51, XRCC1, RAD54, BLM, and POLD1. T98 cells were treated with abemaciclib (0.5 μmol/L), VSVΔ51 (MOI = 0.01), or the combination for 24 hours. G, The flow cytometric analysis of cell death in T98 cells. Cells were infected with VSVΔ51 (MOI = 0.001) with or without abemaciclib (0.5 μmol/L) for 36 hours, the cells were stained with Annexin-V/7-AAD, then flow cytometric analysis was performed. Data represents the mean ± SD (n = 6) in D; (n = 3) in E and G. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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Fluorescence immunocytochemistry analysis confirmed that the VSVΔ51/abemaciclib combination significantly intensified the DNA damage signals, 53BP1 and γH2AX foci, compared with single treatment or control (Fig. 5D; Supplementary Fig. S5B).

Previous studies have reported that viral infection can cause DNA damage in the host cells (30). We speculated that CDK4/6 inhibition could amplify virus-induced DNA damage stress. To test our hypothesis, we evaluated the role of CDK4/6 inhibitor on the expression of genes associated with DDR. Single treatment of VSVΔ51 increased a panel of DDR genes (BML, RAD51, RAD54L, BRCA1, BRCA2, PCNA, RFC3, XRCC1, FANCA, and POLD1; Fig. 5E). However, the addition of abemaciclib reversed VSVΔ51-mediated upregulation of DDR genes and amplified DNA damage stress (Fig. 5E). Western blot analysis also confirmed the accumulation of γH2AX and the downregulation of DDR-related gene expression (RAD51, XRCC1, RAD54, BLM, and POLD1) under the VSVΔ51/abemaciclib combination treatment (Fig. 5F). Furthermore, annexin-V/7-AAD staining revealed that the percentage of dead cells was remarkably higher in the combination treatment group, compared with treatment with VSVΔ51 or abemaciclib alone (Fig. 5G).

These data suggested that CDK4/6 inhibition facilitated oncolysis by amplifying cytotoxic DNA damage stress. CDK4/6 inhibition suppressed virus-induced expression of DDR genes and synergized with OV to induce damaged-DNA accumulation, further increasing the effect of oncolytic therapy.

CDK4/6 inhibitor increased VSVΔ51 therapeutic efficacy in both orthotopic and subcutaneous GBM models

To evaluate the therapeutic potential of VSVΔ51/abemaciclib combination in vivo, the nude mice with GSC1 subcutaneous tumors were divided into four group and treated with (i) vehicle, (ii) oral administration of abemaciclib, (iii) intravenous VSVΔ51 virus, (iv) the combination of abemaciclib and VSVΔ51 (Fig. 6A). The VSVΔ51/abemaciclib combination markedly reduced tumor growth and tumor burden of GSC1 xenografts in nude mice (Fig. 6B and C).

Figure 6.

CDK4/6 inhibitor increases VSVΔ51 therapeutic efficacy in both orthotopic and subcutaneous GBM models. A and B, Nude mice with GSC-1 subcutaneous xenografts were treated with abemaciclib (25 mg/kg/day, oral gavage), VSVΔ51 (3×107 PFU/day, intravenous injection) or combination treatment. Tumor volume and weight presented as the mean ± SD (n = 5). C, Representative images and weights of tumor xenografts for each group from A. D and E, GL261 subcutaneous xenografts in C57 were treated with abemaciclib (25 mg/kg/day, oral gavage), VSVΔ51 (3×107 PFU/day, intravenous injection), or combination (n = 7). Tumor size was recorded every 2 days and the tumor-free ratio was calculated at the end of the experiment. F, The tumor-free mice (n = 5) treated previously by the combination treatment (D) were rechallenged on day 90 with a 2-fold increase in the number of GL261 cells (6 × 106). Naïve mice of similar age (3 months) were implanted as controls (n = 5). Tumor volume presented as the mean ± SD (n = 5). G and H, Survival time of C57 mice bearing GL261 intracranial xenograft. The mice were treated with abemaciclib (25 mg/kg/day, oral gavage), VSVΔ51 (3×107 PFU/day, intravenously injection), or combination treatment (n = 10). I and J, Survival time of C57 mice bearing CT2A intracranial xenograft. The mice were treated with abemaciclib (25 mg/kg/day, oral gavage), VSVΔ51 (3×107 PFU/day, intravenously injection), or combination treatment (n = 10). K, Monitoring of tumor progression via bioluminescence imaging of luciferase in GL261-luc xenograft mice on day 5 and day 16. Quantitative radiance of mice was analyzed. L, Hematoxylin and eosin staining of mouse brain sections from H and J was performed. Scale bar, 1,000 μm. M, Tumor tissues from GSC-1 subcutaneous xenografts (A) or GL261 intracranial xenograft (G) were evaluated through immunofluorescence analysis and quantitative analysis of IHC of VSVΔ51 reporter and IHC for Ki-67 (a marker of proliferation), 53BP1 and γH2AX (markers of DNA damage). Scale bar, 50 μm. Data represents the mean ± SD [n = 5 in A and F; n = 7 in D; n = 10 in G and I; n = 5 (left) and 4 (right) in M]. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; n.s., nonsignificant.

Figure 6.

CDK4/6 inhibitor increases VSVΔ51 therapeutic efficacy in both orthotopic and subcutaneous GBM models. A and B, Nude mice with GSC-1 subcutaneous xenografts were treated with abemaciclib (25 mg/kg/day, oral gavage), VSVΔ51 (3×107 PFU/day, intravenous injection) or combination treatment. Tumor volume and weight presented as the mean ± SD (n = 5). C, Representative images and weights of tumor xenografts for each group from A. D and E, GL261 subcutaneous xenografts in C57 were treated with abemaciclib (25 mg/kg/day, oral gavage), VSVΔ51 (3×107 PFU/day, intravenous injection), or combination (n = 7). Tumor size was recorded every 2 days and the tumor-free ratio was calculated at the end of the experiment. F, The tumor-free mice (n = 5) treated previously by the combination treatment (D) were rechallenged on day 90 with a 2-fold increase in the number of GL261 cells (6 × 106). Naïve mice of similar age (3 months) were implanted as controls (n = 5). Tumor volume presented as the mean ± SD (n = 5). G and H, Survival time of C57 mice bearing GL261 intracranial xenograft. The mice were treated with abemaciclib (25 mg/kg/day, oral gavage), VSVΔ51 (3×107 PFU/day, intravenously injection), or combination treatment (n = 10). I and J, Survival time of C57 mice bearing CT2A intracranial xenograft. The mice were treated with abemaciclib (25 mg/kg/day, oral gavage), VSVΔ51 (3×107 PFU/day, intravenously injection), or combination treatment (n = 10). K, Monitoring of tumor progression via bioluminescence imaging of luciferase in GL261-luc xenograft mice on day 5 and day 16. Quantitative radiance of mice was analyzed. L, Hematoxylin and eosin staining of mouse brain sections from H and J was performed. Scale bar, 1,000 μm. M, Tumor tissues from GSC-1 subcutaneous xenografts (A) or GL261 intracranial xenograft (G) were evaluated through immunofluorescence analysis and quantitative analysis of IHC of VSVΔ51 reporter and IHC for Ki-67 (a marker of proliferation), 53BP1 and γH2AX (markers of DNA damage). Scale bar, 50 μm. Data represents the mean ± SD [n = 5 in A and F; n = 7 in D; n = 10 in G and I; n = 5 (left) and 4 (right) in M]. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; n.s., nonsignificant.

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We next developed the subcutaneous tumors with GL261 cells in immune-competent C57 mice (Fig. 6D). VSVΔ51/abemaciclib combination treatment induced tumor regression in all tumor-bearing mice (Fig. 6E), indicating that VSVΔ51/abemaciclib combination may boost the antitumor immunity. To determine if VSVΔ51/abemaciclib combination treatment induces a memory response in the mice, mice with completely regressed tumors after VSVΔ51/abemaciclib combination treatment were rechallenged with a 2-fold increase in the number of GL261 cells on the opposite flank. The naïve mice were injected with the same number of Gl261 cells as the control group. Remarkably, all tumor-free mice previously treated by VSVΔ51/abemaciclib combination spontaneously rejected the rechallenged GL261 tumors (Fig. 6F). In contrast, all age-matched naïve mice challenged with same amount of GL261 cells developed tumors (Fig. 6F), demonstrating the induction of antitumor immune memory after VSVΔ51/abemaciclib combination treatment.

In the orthotopic GBM xenograft model with GL261 cells or CT2A cells, Kaplan–Meier analysis demonstrated that single treatment of VSVΔ51 or abemaciclib did not improve the survival time of the mice in the two GBM xenograft model, but the VSVΔ51/abemaciclib combination treatment significantly increased survival time relative to other groups (Fig. 6GJ). Bioluminescence in vivo imaging analysis also confirmed that the VSVΔ51/abemaciclib combination increased tumor latency of intracranial GL261 xenograft, compared with the single treatment or control group (Fig. 6K). Hematoxylin and eosin staining of mouse brain revealed that VSVΔ51/abemaciclib combination treatment significantly reduced the volume of intracranial GL261 tumor or CT2A tumor (Fig. 6L).

At the end of the experiment, the levels of γH2AX, 53BP1, Ki-67, and VSVΔ51-reporter GFP were further examined in the tumor sections. We observed that Ki-67 staining was significantly decreased, while VSVΔ51-reporter (GFP) were elevated in the combination group compared with single treatment (Fig. 6M). Meanwhile, DNA damage signals γH2AX and 53BP1 were elevated by the VSVΔ51/abemaciclib combination treatment (Fig. 6M). These results indicated that CDK4/6 inhibition enhanced the replication of OV in the tumor and synergistically triggered the DNA damage accumulation in vivo.

We also performed experiments to detect the phosphorylation of Rb protein in brain tumor tissue, which is the direct target of the CDK4 and CDK6. IHC staining revealed that single treatment of abemaciclib and VSVΔ51/abemaciclib combination treatment could significantly decrease the phosphorylation of RB in intracranial GL261 xenografts (Supplementary Fig. S6A and S6B), indicating that abemaciclib has the potential to penetrate the blood-brain barrier in our models.

The body weight of mice had no significant differences (Supplementary Fig. S7A). Histologic analyses displayed no significant abnormal pathologic change in vital tissues, including heart, liver, kidney, lung, and spleen (Supplementary Fig. S7B). Serum chemistry parameter measurements also showed no abnormal changes of liver and kidney function (Supplementary Fig. S7C).

Collectively, these data indicated that the combination of CDK4/6 inhibition and OV treatment was tolerable and effective in both orthotopic and subcutaneous GBM models.

Combinational treatment of CDK4/6 inhibitor and VSVΔ51 reshaped the tumor microenvironment and elicited the antitumor immunity

The striking therapeutic effect in immune-competent model drives us to find the effect of VSVΔ51/abemaciclib combination on the antitumor immunity. We first observed that VSVΔ51/abemaciclib combination treatment promoted calreticulin exposure (Fig. 7A) and extracellular ATP secretion (Fig. 7B) compared with single treatment, indicating that VSVΔ51/abemaciclib combination enlarged the immunologic cell death of glioma cells.

Figure 7.

CDK4/6 inhibitor combined with VSVΔ51 increasing the antitumor immune response in GBM. A, Calreticulin exposure was examined by flow cytometry. T98 cells were treated with abemaciclib (0.5 μmol/L), VSVΔ51 (MOI = 0.01), or combination therapy for 24 hours. Quantification of CRT exposure. B, ATP secretion in supernatant was detected by a bioluminescence detection kit after 24 hours and 36 hours in T98 cells and GL261 cells. C, The percentage of CD4+, CD8+ T cell and Tregs (Foxp3+) among CD4+ T cells were analyzed in brain tumor tissue by flow cytometry. The activity of CD8+ T cells was evaluated by measuring granzB expression. D, Quantification of the results in C. E, The lymphocyte from spleen were cocultured with GL261 cells for 48 hours to evaluate lymphocytotoxicity. F, The release of LDH was detected by LDH Cytotoxicity Assay kit. G, TNFα and IFNγ were analyzed from the supernatants of splenic lymphocytes cocultured with GL261. H, Immunoblot analysis of circulating tumor antibodies against GL261 cells from the serum of C57 mice. Data represents the mean ± SD (n = 3) in A and B; n = 4 in G; n = 5 in D, E, and F. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., nonsignificant.

Figure 7.

CDK4/6 inhibitor combined with VSVΔ51 increasing the antitumor immune response in GBM. A, Calreticulin exposure was examined by flow cytometry. T98 cells were treated with abemaciclib (0.5 μmol/L), VSVΔ51 (MOI = 0.01), or combination therapy for 24 hours. Quantification of CRT exposure. B, ATP secretion in supernatant was detected by a bioluminescence detection kit after 24 hours and 36 hours in T98 cells and GL261 cells. C, The percentage of CD4+, CD8+ T cell and Tregs (Foxp3+) among CD4+ T cells were analyzed in brain tumor tissue by flow cytometry. The activity of CD8+ T cells was evaluated by measuring granzB expression. D, Quantification of the results in C. E, The lymphocyte from spleen were cocultured with GL261 cells for 48 hours to evaluate lymphocytotoxicity. F, The release of LDH was detected by LDH Cytotoxicity Assay kit. G, TNFα and IFNγ were analyzed from the supernatants of splenic lymphocytes cocultured with GL261. H, Immunoblot analysis of circulating tumor antibodies against GL261 cells from the serum of C57 mice. Data represents the mean ± SD (n = 3) in A and B; n = 4 in G; n = 5 in D, E, and F. *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., nonsignificant.

Close modal

We then systematically profiled the immune cells that infiltrated into the tumor via flow cytometry analysis. The VSVΔ51/abemaciclib combination treatment significantly increased the percentage of tumor-infiltrating CD8+ effector T cells (Teff) and lowered the quantity of tumor-infiltrating regulatory T cells (Treg), which led to a marked increase in the ratio of Teffs to Tregs (Fig. 7C and D; Supplementary Fig. S8). To further assess the functional activity of infiltrating CD8+ T cells, we analyzed granzB expression. We found that VSVΔ51/abemaciclib combination significantly increased granzB-positive infiltrating CD8+ T cells (Fig. 7C and D).

We found that splenic lymphocytes from the combination group showed stronger cytotoxicity against GL261 cells than those from monotherapy or control groups (Fig. 7E and F). ELISA analysis found that splenic lymphocytes from the combination group had the highest levels of IFNγ and TNFα (Fig. 7G). Circulating tumor antibodies against GL261 cells from mice that received the combined therapy were detected at higher levels than those who received monotherapy or control treatment. (Fig. 7H). These observations indicated that VSVΔ51/abemaciclib combination treatment augmented tumor-specific immunity.

As OVs are advancing through clinical trials, preclinical studies should realistically assess the limitations of the virus as a monotherapy and explore opportunities to enhance oncolytic efficacy in OV-resistant tumors (6). Identifying small molecules that act synergistically with OVs may be an effective strategy for optimizing oncolysis. In this study, through a dual-step screening of FDA-approved drugs we discovered that CDK4/6 inhibitor could cooperate with OVs, including VSVΔ51 and ZIKV. Herein, we showed that CDK4/6 inhibition elicited two effects as a chemical enhancer of OVs in refractory glioma cells: promoting selective viral replication and magnifying cytotoxic DNA damage stress, which subsequently triggered the immunologic cell death and awakened tumor-specific immunity.

CDK4 and CDK6 are serine/threonine kinases that modulate cell-cycle entry by phosphorylation of the retinoblastoma protein (RB1), thereby inhibiting its transcriptional repression function (27). These kinases are frequently activated in human cancer and been proposed as therapeutic interventions (31). GBM is characterized by the inactivation of the CDKN2 locus (50%) encoding the CDK4/6 inhibitor p16INK4 and the amplification of CDK4 (13%) and CDK6 (1.5%; refs. 2, 3). Considering the abnormal activation of CDK4/6 in GBM cells compared with normal cells, the enhanced viral replication by CDK4/6 inhibition is relatively limited in GBM cell. The selectivity of viral replication ensures the safety of the combination of OVs and CDK4/6 inhibition for treating GBM.

The infection of OVs can induce DNA damage in host cells (30), which subsequently activates the DDR pathway to avoid cell death. Targeting CDK12/13 or CDK7 is reported to regulate the expression of core DNA damage response genes, resulting in DDR deficiencies and conferring sensitivity to DNA-damaging agents (31, 32). In this study, we demonstrated that targeting CDK 4/6 triggered the downregulation of DDR-related gene expression, impairing the recovery from cytotoxic DNA damage after infection of OV.

Eukaryotic cells possess intrinsic defense mechanisms against viral infection that directly restrict viral replication (32). It is now well established that cancer-specific aberrations in genes encoding proteins involved in the WNT signaling pathway and other cancer-related genes, such as RAS, TP53, RB1, PTEN, can predispose cancer cells to virus infection (9, 33). This is due to cross-talk between oncogenic signaling and antiviral pathways, which creates a permissive environment for viral replication. Therefore, cells that become malignant by acquiring defects in IFN signaling often unwittingly develop sensitivity to viral infection. Due to heterogeneity in tumor cells, the extent of defects is variable (19–21).

Our data demonstrated that OV infection induced the activation of antiviral factors in refractory cancer cells, restricting OV replication and oncolysis. Nevertheless, we observed that CDK4/6 inhibitors abrogated the induced expression of the genes related to the IFN cascade. In this study, we demonstrated that autophagic degradation of MAVS induced by CDK4/6 inhibitors blocked the antiviral type I IFN response. Other studies also found that several members of CDK had an impact on IFN signal transduction by regulating STAT1 phosphorylation or the translation of type I IFN production (34, 35). However, CDK4/6 inhibition also has been reported to induce endogenous retroviral genes expression and promote IFN pathway in breast carcinoma, but not in glioma (36). Therefore, how CDK family regulates IFN pathway is still controversial. We hypothesized that the heterogeneity of the genetic background of cells can influence the effect of CDK4/6 inhibition on IFN pathway.

Here we presented, for the first time, a new useful potential of CDK4/6 inhibitors to enhance selective OV replication and its oncolytic efficacy in GBM, especially resistant GBM cells. Currently, there are three approved, orally bioavailable, highly selective, small-molecule inhibitors of CDK4/6, abemaciclib, palbociclib, and ribociclib, for treating advanced hormone receptor–positive and HER2-negative metastatic breast cancer (37). Abemaciclib has been shown to cross the blood-brain barrier effectively (10-fold more than palbociclib), with cerebrospinal fluid concentrations being comparable to plasma concentrations, suggesting the potential anti-glioma effect of abemaciclib, but not palbociclib in vivo (38, 39). Currently, there are three ongoing clinical trials of using abemaciclib for treating GBM (NCT02644460, NCT04074785, NCT04391595).With regard to OVs, some candidates also have finally made their way to the clinic with the approval of talimogene laherparepvec based on herpes simplex virus 1 for the treatment of advanced melanoma (40). Therefore, the combination therapy with OV and CDK4/6 inhibitor has clear implications for cancer therapy.

Our current study does present some limitations. Although the combination therapy with OV and CDK4/6 inhibitor can induce antitumor activity in mouse glioma models and inhibit the tumor growth, detailed mechanisms need to be revealed. As the combination indeed induce the immunologic cell death and increase the infiltration of T lymphocytes, it is worthy to explore whether addition of other immune checkpoint blockades to combination of OV and CDK4/6 inhibitor, such as anti–PD-1 therapy, may induce more remarkable therapeutic effect in the clinic.

In summary, our study suggests that CDK4/6 inhibitors provide a significant therapeutic benefit when combined with the OV in the treatment of GBM.

F. Xing reports grants from National Natural Science Foundation of China, Natural Science Foundation of Guangdong Province, and grants from Shenzhen Scientific and Technological Innovation Talents Project during the conduct of the study. D. Guo reports grants from Shenzhen Municipal Science and Technology Innovation Council, Ministry of Science and Technology of the People's Republic of China, and grants from Guangdong Provincial Science and Technology Department during the conduct of the study. No disclosures were reported by the other authors.

J. Xiao: Data curation, formal analysis, validation, investigation, visualization, writing–original draft. J. Liang: Data curation, validation, investigation, visualization. J. Fan: Validation. P. Hou: Resources. X. Li: Writing–review and editing. H. Zhang: Writing–review and editing. K. Li: Writing–review and editing. L. Bu: Resources, methodology. P. Li: Resources, methodology. M. He: Methodology. Y. Zhong: Methodology. L. Guo: Software. P. Jia: Methodology. Q. Xiao: Methodology. J. Wu: Methodology. H. Peng: Methodology. C. Li: Methodology. F. Xing: Data curation, formal analysis, supervision, funding acquisition, investigation, methodology, writing–original draft, writing–review and editing. D. Guo: Data curation, formal analysis, supervision, funding acquisition, project administration, writing–review and editing.

The authors thank Prof. Tao Sun (Shanghai Jiao Tong University) for providing VSV∆51 and Prof. Guangmei Yan (Sun Yat-sen University) for providing patient-derived glioma cells (GSC-1, GSC-11, GBM01, and GBM10). The authors would like to acknowledge Dr. Zezhong He (Case Western Reserve University) for editorial work.

The work is supported by the Shenzhen Science and Technology Program (grant #JCYJ20200109142201695 to D. Guo), National Natural Science Foundation of China (grant #82173829 and #81803568 to F. Xing), Natural Science Foundation of Guangdong Province (grant #2018A030310099 to F. Xing), Shenzhen Scientific and Technological Innovation Talents Project (grant #RCBS20200714114923232 to F. Xing), Guangdong Zhujiang Talents Program (#2016LJ06Y540 to D. Guo) and National Ten-thousand Talents Program (to D. Guo).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

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Supplementary data