Overcoming cancer cell resistance to VSV oncolysis with JAK1/2 inhibitors

Abstract

Oncolytic vesicular stomatitis virus (VSV) has potent antitumor activity but some cancer cells are resistant to VSV killing, either constitutively or due to type I interferon (IFN) inducing an antiviral state in the cells. Here, we evaluated VSV oncolysis of a panel of human head and neck cancer cells and showed that VSV resistance in SCC25 and SCC15 cells could be reversed with Janus kinase (JAK) 1/2 inhibitors (JAK inhibitor I and ruxolitinib). Pre-treatment of cells with JAK1/2 inhibitors before or in conjunction with VSV enhanced viral infection, spread and progeny yield (100- to 1000-fold increase). In contrast, inhibitors of histone deacetylase (LBH589), phosphatidylinositol 3-kinase (GDC-0941, LY294002), mammalian target of rapamycin (rapamycin) or signal transducer and activator of transcription 3 (STAT3 inhibitor VII) were ineffective. Compared with VSV-sensitive SW579 cells, IFNα/β responsive antiviral genes (IRF-9, IRF-7, OAS1 but not MxA) are constitutively expressed in SCC25 cells. Pretreatment with JAK inhibitors reduced mRNA levels of these genes, increasing VSV expression in the cells. Interestingly, 1 h of drug exposure was sufficient to reverse SCC25 resistance to VSV and was still effective if virus was added 24 h later. Overall, we show here that JAK inhibitor I and ruxolitinib (Jakafi) can reverse resistance to VSV, supporting the rationale to incorporate JAK1/2 inhibitors in future VSV virotherapy trials.

INTRODUCTION

Vesicular stomatitis virus (VSV) has a broad tropism and uses the recently identified receptor from the low-density lipoprotein family to infect cells.¹ Vesicular stomatitis virus has several features that make it attractive as an oncolytic virotherapy agent for cancer.² The virus is highly lytic, has fast replication kinetics and a large burst size. Importantly, the majority of the population does not have pre-existing antibodies to VSV that could otherwise reduce the therapeutic activity of systemic VSV administration.³ The natural hosts of VSV include cattle, horses and pigs.^4,5 The animals recover from the infection but VSV infection of livestock is associated with significant disease, including vesicular lesions around the mouth, hoofs and teats, and loss of beef and milk production.⁶ Human infections with VSV through laboratory work or working with affected animals have occurred, and typically results in a mild fever from which the infected individual recovers with no incidents. When injected experimentally into the brains of research rodents, wild-type VSV is neurovirulent and causes acute infection of the central nervous system, resulting in neurological signs including ataxia and hind limb paralysis.⁷

To attenuate VSV (Indiana strain) for therapeutic use as a vaccine or an oncolytic agent in cancer therapy, additional transcription units, truncations in the cytoplasmic tail of the glycoprotein, microRNA targeting and insertion of picornaviral internal ribosome entry sites have been used successfully to attenuate the neurotoxicity of recombinant VSV.^8–11 Furthermore, the virus is exquisitely sensitive to the antiviral effects of type I interferon (IFN) and two popular engineering platforms involve IFN production by the infected cell. These strategies include mutation or deletion of the methionine residue 51 in the matrix (ΔM51) protein of VSV such that M is no longer able to inhibit the nuclear export of cellular mRNA (for example, IFN) or insertion of an additional transcription unit encoding IFN-β (VSV-IFNβ).^12–14 Both VSV-ΔM51 and VSV-hIFNβ have been used successfully to treat a variety of murine and human cancers.^2,3 In particular, VSV-hIFNβ is being developed for clinical testing against hepatocellular carcinoma and myeloma.^15,16 The safety of the virus was evaluated in mice, rats and rhesus macaques toxicology and pharmacology studies. When injected directly into the liver of rhesus macaques under CT guidance, the maximal feasible dose of 10¹⁰ TCID₅₀ was well tolerated with no adverse clinical symptoms and no significant hematological or chemical changes.¹⁵ VSV-hIFNβ is currently undergoing testing in a Phase I dose escalation trial (seven dose levels, 10⁵–10⁸ TCID₅₀) where patients with sorafenib refractory or intolerant hepatocellular carcinoma will receive a single intratumoral injection of VSV-hIFNβ into a target lesion (principal investigator: Mitesh Borad, Mayo Clinic Arizona, Clinical Trials.gov identifier: NCT01628640).

In general, cancer cells have defective IFN signaling pathways and are susceptible to oncolysis by the IFN-inducing viruses, VSV-ΔM51 or VSV-hIFNβ.^17,18 In contrast, non-transformed cells have intact IFN signaling pathways and respond to VSV infection with production of type I IFN, and induction of an antiviral state that inhibits viral amplification in the infected cell and viral spread into neighboring cells. Each IFN receptor (IFNAR1 and IFNAR2) subunit binds constitutively to a single specific member of the Janus kinase (JAK) family: IFNAR1 to tyrosine kinase 2 (TYK2) and IFNAR2 to JAK1.¹⁹ Type I IFN binding induces the phosphorylation of JAK1, TYK2, intracellular tyrosine residues of each receptor chain and signal transducers and activators of transcription (STATs). Activated STATs dimerize, dissociate from the receptor and together with IRF-9 (ISGF3 complex) translocate to the nucleus to induce the expression of more than 300 IFN-stimulated genes (ISGs).^20–22 The four main effector pathways of IFN-mediated antiviral response are the Mx GTPase pathway, the 2′5′-oligoadenylate-synthetase-directed ribonuclease L pathway, the protein kinase R (PKR) pathway and the ISG15 ubiquitin-lie pathway.¹⁹ These pathways serve to inhibit viral transcription, degrade viral RNA, inhibit translation and modify protein function to limit the spread and infection of the virus.

In this report, we evaluated the oncolytic activities of VSV-green fluorescent protein (GFP) and VSV-ΔM51-GFP in a panel of human head and neck squamous cell carcinoma (HNSCC) cell lines and showed that some cell lines were highly sensitive to VSV oncolysis, whereas others were resistant to VSV. Previous studies have investigated pharmacological strategies to improve VSV oncolysis in resistant cells by incorporating drugs and inhibitors of IFN-associated signaling or apoptotic pathways. For example, histone deacetylase (HDAC) inhibitors and JAK inhibitors increased VSV replication in otherwise resistant human prostate PC3 cancer cells.^23,24 Rapamycin, a highly specific inhibitor of the mammalian target of rapamycin (mTOR), increased VSV oncolysis of human and rat glioblastoma cells.²⁵ To overcome VSV resistance and oncolysis in HNSCC, we evaluated a panel of inhibitors of JAK1/2, HDAC, phosphatidylinositol 3-kinase (PI3K), mTOR or signal transducer and activator of transcription 3 (STAT3) and showed that JAK inhibitors and not the others can reverse VSV resistance in HNSCC cells. Importantly, Jakafi (ruxolitinib), which was recently approved for clinical use in myleoproliferative disorders, was effective, making it feasible to consider incorporating this drug into VSV virotherapy.

MATERIALS AND METHODS

Cell lines, drugs and viruses

HNSCC cell lines SW579, FaDu, SCC15 and SCC25 (American Type Culture Collection, Manassas, VA, USA) were maintained in medium as recommended by American Type Culture Collection. TG101348 (SAR302503) and Ruxolitinib (INCB018424) were purchased from Selleckchem (Houston, TX, USA), JAK Inhibitor I was from EMD Millipore (Billerica, MA, USA). VSV-GFP (VSV-expressing GFP; Indiana strain) and VSV-ΔM51-GFP were kindly provided, respectively, by Glen Barber (University of Miami) and John Bell (Ottawa Hospital Research Institute). Viruses were propagated on Vero African green monkey kidney cells and titered by TCID₅₀ titrations on Vero cells as previously described.²⁶

Virus infection and replication assay

Cells were plated and treated with or without inhibitors. After 24 h of incubation (or otherwise stated), cells were infected with VSV-GFP or VSV-ΔM51-GFP at multiplicity of infection (MOI) of 0.5. The supernatants were harvested 24, 48 and 72 h post infection and stored at −80 °C until the time of analysis. Viral progeny was determined by TCID₅₀ assay on Vero cells.

VSV cell killing assay

HNSCC (3 × 10⁴) cells were plated in 96-well plates and treated with or without inhibitors diluted in reduced serum media, Opti-MEM (Life Technologies, Carlsbad, CA, USA). Following 24 h of incubation with drugs or media, cells were infected with VSV-GFP or VSV-ΔM51-GFP at MOI of 0.5. Forty-eight hours post infection, media were removed and the cells were washed with PBS, and 100 μl of fresh media with 20 μl per well of Cell Titer 96 AQueous One Solution Cell Proliferation Assay (MTS; Promega, Madison, WI, USA) were added. The plates were read 1 h later at 490 nm using an ELISA plate reader (TECAN Infinite M200Pro, Tecan, San Jose, CA, USA).

RNA isolation and reverse transcription-PCR

Total RNA was extracted from 10⁶ cells of each treatment using RNeasy Plus mini kit (QIAGEN, Valencia, CA, USA). One microgram of total RNA was used as template for reverse transcription using Superscript III 1st strand synthesis supermix (Life Technologies). Semi-quantitative PCR analysis was performed using the following primer sets: IRF-9 sense: 5′-CAAGCAGGACTTCCGGGAGG-3′ and IRF-9 antisense: 5′-CTTCCTGTGGCTCAGGGCTG-3′;²⁷ IRF-7 sense: 5′-TGGTCCTGGTGAAGCTGGAA-3′ and IRF-7 antisense: 5′-GATGTCGTCATAGAGGCTGTTGG-3′;²⁸ OAS1 sense: 5′-TCGGACGGTCTTGGAATTAGTC-3′ and OAS1 antisense: 5′-TAATTCAGCCAGGCCTCAGC-3′;²⁹ MxA sense: 5′-GCTACACACCGTGACGGATATGG-3′ and MxA antisense: 5′-CGAGCTGGATTGGAAAGCCC-3′;²⁷ PKR sense: 5′-GCCTTTTCATCCAAATGGAATTC-3′ and PKR antisense: 5′-GAAATCTGTTCTGGGCTCATG-3′; VSV-N sense: 5′-TGATAGTACCGGAGGATTGACGAC-3′,³⁰ VSV-N antisense: 5′-CCTTGCAGTGACATGACTGCTCTT-3′;¹⁵ GAPDH sense: 5′-GGTATCGTGGAAGGACTCATGAC-3′ and GAPDH antisense: 5′-ATGCCAGTGAGCTTCCCGTTCAGC′3′.³¹ Semi-quantitative PCR conditions were optimized to obtain reproducible and reliable amplification within the logarithmic phase of the reaction. Amplification for IRF9, 2′–5′ OAS, PKR and GAPDH, involved denaturation 94 °C for 3 min, primer annealing at 55 °C for 45 s, and extension at 72 °C for 45 min, 27 cycles; for IRF7, denaturation at 94 °C for 3 min, primer annealing 58 °C for 45 s, an extension at 72 °C, 29 cycles; for VSV-N, denaturation at 94 °C for 3 min, primer annealing at 60 °C for 30 s, an extension at 72 °C for 30 s, 29 cycles; for MxA, denaturation at 94 °C for 3 min, primer annealing at 50 °C for 45 s, an extension at 72 °C for 45 s, 29 cycles.

Western blot analysis

Total protein was extracted from 10⁶ cells using 200 μl RIPA buffer (25 mm Tris-HCL (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) complemented with protease/phosphatase inhibitors cocktail (Cell Signaling Technology, Danvers, MA, USA). Total protein was quantified with Pierce BCA protein assay kit (Thermo Scientific, Waltham, MA, USA) using bovine serum albumin as standard. Protein extracts representing 10 μg of the total protein were separated on 10% SDS-polyacrylamide gel electrophoresis and electrophoretically transferred onto nitrocellulose membranes. The membranes were blocked in 5% of bovine serum albumin in Tris-buffered saline-Tween overnight, incubated with primary antibodies polyclonal rabbit α-JAK1 (sc-277, Santa Cruz, Dallas, TX, USA), rabbit polyclonal α-STAT1 (ab2415, Abcam, Cambridge, MA, USA), rabbit monoclonal α-STAT1 phospho, which binds to residues surrounding serine 727 (ps727, Epitomics, Burlingame, CA, USA), polyclonal rabbit α-VSV¹⁵ or rabbit polyclonal α-Actin (sc-1616, Santa Cruz, Dallas, TX, USA) washed three times with TBS-Tween, incubated with secondary antibody goat α-rabbit IgG (H + L) conjugated with horse radish peroxidase (PI1000, Vector Labs, Burlingame, CA, USA). Signal was developed using Pierce ECL western blotting substrate kit (Thermo Scientific).

RESULTS

VSV resistance of HNSCC can be overcame by JAK1/2 inhibitors Human HNSCC cancer cell lines, SW579, FaDu, SCC15 and SCC25, were infected with VSV-GFP at MOI of 0.01, 0.1 or 1.0. Forty-eight hours post infection, cell viability was measured by MTS cell proliferation assay. The HNSCC cell lines were highly sensitive to VSV oncolysis (80% cell death at MOI of 0.01) except for SCC25 that was constitutively resistant to VSV-GFP killing, showing less than 10% cell death at MOI of 1.0 (Figure 1a). Despite increasing the MOI to 10, only 40% of SCC25 cell killing was achieved (data not shown).

Figure 1.

Janus kinase (JAK) 1/2 inhibitors increase vesicular stomatitis virus-green fluorescent protein (VSV-GFP) susceptibility in SCC25 cells. (a) SW579, FaDu, SCC15, SCC25 cell lines were analyzed for VSV-GFP-induced cytotoxicity by MTS cell viability assay. (b) SCC25 was treated with JAK1/2 inhibitors 24 h before VSV. Cells were infected with VSV-GFP and cell viability was determined 48 h later. (c) Representative photographs of VSV infection in treated cells with and without JAK1/2 inhibitors. (d) Viral progeny from b was measured at different times post infection by TCID₅₀ assay. Average of three replicates (mean ± s.d.). *P ≤ 0.05, **P ≤ 0.001, ***P ≤ 0.0001.

To identify drugs that could increase the effectiveness of VSV killing, SCC25 cells were pretreated for 24 h with a panel of inhibitors of various signaling pathways at three different concentrations (Table 1). The concentrations were chosen so that drug alone did not induce significant cell death (data not shown). Cells were then infected with VSV-GFP (MOI of 0.1 and 1.0) and cell viability was measured 48 h later. Broad-spectrum HDAC inhibitor (LBH-589), mTOR inhibitor (rapamycin), PI3K inhibitors (GDC-0941, LY294002) and STAT 3 inhibitor (STAT3 inhibitor VII) did not improve VSV infection or oncolysis in the SCC25 cell line (Table 1). In contrast, JAK2 inhibitor (TG101348), JAK1/2 inhibitor (ruxolitinib) and JAK family inhibitor (JAK Inhibitor I) significantly increased the cytotoxicity and infectivity of VSV-GFP on SCC25 (Figures 1b and c). At the highest drug and VSV concentrations tested, the amount of cell killing increased from 10 to 70% with TG101348, to 90% with ruxolitinib and to more than 99% with JAK Inhibitor I (Figure 1b). There was minimal cell killing with drug alone in the absence of VSV at the concentrations used.

Table 1.

Panel of drugs tested in conjunction with VSV infection in SCC25 cells

Drugs	Concentrations used in this study	Targets	Enhance VSV oncolysis?
LBH589 (Panobinostat, NVP-LBH589)	5 nM, 50 nM, 250 nM	Broad-spectrum HDAC inhibitor41,42	No
GDC-0941	0.2 μM, 1μM, 5μM	PI3K inhibitor against p110α43	No
LY294002	2μM, 10μM, 50μM	PI3K inhibitor: competitive inhibitor for ATP-binding site44,45	No
STAT3 Inhibitor VII	20 nM, 0.1 μM, 0.5 μM	Cellular JAK1/JAK2/TYK2/STAT3 activation46	No
Rapamycin	25 nM, 0.25 μM, 2.5 μM	Binds to immunophilin FKBP12 to inhibit the activity of FRAP/mTOR,47	No
TG101348 (SAR302503)	0.01 nM, 0.1 nM, 1 nM	ATP-competitive JAK2 inhibitor48,49	Yes, + (70% kill)
Jakafi (INCB018424)	250 nM, 2.5 μM, 25 μM	JAK family inhibitor for JAK1, JAK2 and JAK350	Yes, + + (90% kill)
JAK inhibitor I (P6, DBI, 2-(1,1-dimethylethyl)-9-fluoro-3,6-dihydro-7H-bens51-imiaz [4,5-f]isoquinolin-7-one, Pyridone 6)	1 μM, 10 μM, 50 μM	JAK family inhibitor for JAK1, JAK2 and JAK3, and TYK232,33	Yes, + + + (>99% kill)

Abbreviations: FRAP, FKBP-12-rapamycin associated protein; HDAC, histone deacetylase; mTOR, mammalian target of rapamycin; PI3K, phosphatidylinositol-3-kinase; STAT, signal transducer and activator of transcription; TYK2, tyrosine kinase 2; VSV, vesicular stomatitis virus.

Cell killing at multiplicity of infection 1.0 with the highest drug concentration tested is denoted for drugs showing positive effect on VSV oncolysis.

To further understand the effect of JAK inhibitors on VSV-induced killing of SCC25, cells were treated with the respective inhibitors at indicated concentrations for 24 h before infection by VSV-GFP (MOI of 0.5). Viral spread, as indicated by GFP expression, was significantly increased in treated cells compared with cells that did not receive drug treatment (Figure 1c). By 72 h, more than 90% of cells were GFP positive (infected) or were non-viable (loss of GFP expression). The impact of JAK inhibitors treatment on VSV-GFP progeny production in SCC25 cells was also analyzed (Figure 1d). Conditioned media from VSV-infected cells were harvested at 24, 48 and 72 h post infection and viral progeny was measured by TCID₅₀ assay on Vero cells. Virus progeny production was increased significantly when cells were pretreated with JAK1/2 inhibitors (Figure 1d). JAK inhibitor I was the most potent and significantly increased viral progeny yield by 100- to 1000-fold.

JAK1/2 inhibitors increases cancer cell killing by VSV-ΔM51-GFP

In contrast to VSV-GFP, VSV-ΔM51-GFP is attenuated by deletion of methionine 51 in the matrix protein and induces production of IFN in infected cells, limiting viral spread in cells with functional IFN signaling pathways. As such, the virus is tolerated to higher levels when injected intranasally into Balb/c mice¹² VSV-ΔM51-GFP efficiently infects FaDu and SW579, whereas SCC25 is highly resistant and SCC15 show intermediate resistance. Resistance of SCC25 and SCC15 to VSV-ΔM51-GFP infection and oncolysis can be overcome by pretreatment with JAK inhibitor I or ruxolitinib (Figures 2b and c). Pre-treatment of VSV-ΔM51-GFP-infected SCC25 and SCC15 cells with JAK inhibitors also resulted in a significant 100- to 500-fold increase (P < 0.05) in production of viral progeny compared with infected cells that received no inhibitors (Figure 2d).

Figure 2.

Janus kinase (JAK1/2) inhibitors increased vesicular stomatitis virus-ΔM51-green fluorescent protein (VSV-ΔM51-GFP) infection in SCC25 and SCC15 cells. (a) SW579, FaDu, SCC15, SCC25 were analyzed for VSV-ΔM51-GFP-induced cytotoxicity by MTS cell viability assay. (b) Cells were treated with JAK1/2 inhibitors 24 h before VSV and cell viability was determined 48 h later. (c) Representative photographs of VSV infection in treated cells. (d) Viral progeny produced by infected SCC15 and SCC25 cells (TCID₅₀ assay). Average of three replicates (mean ± s.d.). *P ≤ 0.05, **P ≤ 0.001, ***P ≤ 0.0001.

JAK1/2 inhibitors decrease IFN-α/β inducible genes and increase VSV expression

To understand why SCC25 is constitutively resistant to VSV and how JAK inhibitors facilitate VSV infection in the HNSCC cells, reverse transcription-PCR for IFN responsive genes was performed. VSV-permissive SW579 and VSV-resistant SCC25 were treated with ruxolitinib (25 μm) or JAK inhibitor I (10 μM) for 24 h after which the cells were infected with VSVs (MOI of 0.5) or mock infected. Twenty-four hours later, cellular RNA was harvested and semi-quantitative reverse transcription-PCR for representative genes that have pivotal roles in virus detection (IFN regulatory factor, IRF-7), activation of type I IFN production (IRF-9) and limiting viral replication (IFN-α/β inducible antiviral genes MxA, OAS1, PKR). As shown in Figure 3a, IRF7, IRF9, OAS1 but not MxA, are constitutively expressed in SCC25 and not in SW579. Exposure of SCC25 to IFNα did not further increase transcription of these genes. Treatment of SCC25 cells with ruxolitinib and JAK inhibitor I for 24 h reduced constitutive expression of IRF7, IRF9 and OAS1 (Figure 3a). PKR mRNA level in SCC25 was not affected by inhibitor treatment (Figure 3a). Importantly, mRNA levels of VSV nucleocapsid were higher in SCC25 cells pretreated with JAK inhibitors at 24 h post infection than cells without drug treatment (Figure 3a). In contrast, IRF-7, IRF-9 or OAS1 is not constitutively expressed in VSV-permissive SW579 cells but IRF9 and OAS1 were upregulated by IFNα treatment or exposure to VSV-ΔM51-GFP. Ruxolitinib and JAK inhibitor I reduced the activation level of IRF-9 and OAS1 in IFNα-treated SW579.

Figure 3.

Treatment with Janus kinase (JAK) 1/2 inhibitors decreased interferon (IFN)-α/β inducible genes and increased vesicular stomatitis virus (VSV) gene expression. VSV-resistant SCC25 and VSV-permissive SW579 cells were treated with JAK1/2 inhibitors 24 h before VSV-green fluorescent protein (GFP) and VSV-ΔM51-GFP infection. (a) Semi-quantitative reverse transcription-PCR for VSV-N, IRF7, IRF9, PKR, MxA and OAS1. Immunoblot showing (b) expression of JAK1, signal transducer and activator of transcription 1 (STAT1) and phosphorated-STAT1 or (c) VSV proteins (glycoprotein G, nucleocapsid N, phosphoprotein P, and matrix M) under different treatment conditions.

JAK1/2 inhibitors decreased JAK1 and STAT1 protein expression and increased VSV expression in SCC25

JAK1/2 inhibitors bind to the ATP-binding site of JAK1 and JAK2 thus preventing activation of JAKs and the subsequent activation of proteins in the JAK1/STAT1 signaling pathway.^32,33 To determine the effect of JAK inhibitors on JAK1 and STAT1 protein expression in SCC25 cells, immunoblotting was performed. The cells were treated with IFNα (500 U) or with ruxolitinib (25 μM) or JAK inhibitor I (10 μm) for 24 h. As shown in Figure 3b, SCC25 cells express JAK1 and STAT1 proteins and STAT1 is constitutively active (phosphorylated pSTAT1). IFN treatment did not further increase the expression of JAK, STAT1 and pSTAT1 proteins (Figure 3b). Treatment of cells with JAK1/2 inhibitors reduced the constitutive expression of JAK1, STAT1 and pSTAT1 proteins. Another cohort of SCC25 cells was infected with VSV-GFP or VSV-ΔM51-GFP. It is apparent from the immunoblot that JAK inhibitor I was more potent than ruxolitinib at improving VSV expression in the infected SCC25 cells (Figure 3c). As expected, VSV expression was robust in the permissive SW579 cells.

One hour of drug pretreatment is sufficient to improve VSV oncolysis

All the above studies used 24 h of inhibitor treatment before exposure of cells to VSV. Here, the timing required for drug exposure was further investigated. SCC25 cells were incubated with JAK1/2 inhibitors for 1, 3 or 24 h, after which the drug was removed and VSV-GFP or VSV-ΔM51-GFP was added (MOI of 0.5). As shown in Figure 4a, a relatively short exposure time of 1 or 3 h to the inhibitors rendered SCC25 cells susceptible to VSV oncolysis. There was no significant difference in cell killing between the 1, 3 or 24 h treatment groups at the respective drug and virus concentrations.

Figure 4.

The effect of drug exposure times on vesicular stomatitis virus (VSV) infection and oncolysis. (a) SCC25 cells were incubated with Janus kinase (JAK) inhibitor I or ruxolitinib for 1, 3 or 24 h, after which the inhibitor was removed and cell infected with VSV-green fluorescent protein (GFP) or VSV-ΔM51-GFP. Cell viability was determined 48 h post virus infection. (b) SCC25 cells were incubated with inhibitors for 1 h after which the drug was removed and virus added 1, 3 or 24 h later. Representative photographs of cells are shown and (c) cell viability was determined at 48 h post virus infection. Average of three replicates (mean ± s.d.). *P ≤ 0.05, **P ≤ 0.001.

To determine the durability of the reversal of VSV resistance post inhibitor treatment, SCC25 cells were exposed to JAK Inhibitors for 1 h, after which the drug was removed and VSV was added 1, 3 or 24 h later (Figure 4b). Comparable GFP expression and cytotoxicity were observed regardless if the cells were infected with viruses 1, 3 or 24 h post inhibitor removal (Figure 4b). Cell proliferation assays showed that SCC25 cells could be infected and killed more than 99% by VSV-GFP and VSV-ΔM51-GFP even after the drugs had been removed 24 h ago (Figure 4c).

DISCUSSION

In this study, we demonstrate that there is variability in the susceptibility of human HNSCC cells to oncolysis by VSV. Although FaDu and SW579 cells are highly permissive to VSV infection, SCC25 is highly resistant and SCC15 has intermediate resistance. Combining virus therapy with JAK inhibitor I or ruxolitinib (Jakafi) enhanced VSV infection, spread and replication in the resistant cells, resulting in significant cell death. JAK inhibitor I is an ATP competitive inhibitor of JAKs (http://www.emdmillipore.com) and displays potent inhibitory activity against JAK1 (IC₅₀ = 15 nM for murine JAK1), JAK2 (IC₅₀ = 1 nM), JAK3 (Ki = 5 nM) and TYK2 (IC₅₀ = 1 nM). The IC₅₀ of ruxolitinib is 3.3 nM for JAK1 and 2.8 nM for JAK2.³³ In our study, JAK inhibitor I was more potent than ruxolitinib at blocking the IFN-inducible JAK1/STAT1 signaling pathway in HNSCC cells to yield a higher level of VSV expression, progeny yield and oncolysis of the infected SCC25 and SCC15 cells. Based on results from other drug/VSV combination studies, we also evaluated inhibitors of other pathways important in IFN or antiviral responses, including HDAC, PI3K, mTOR and STAT3 inhibitors. The roles of these proteins in antiviral responses are varied. STAT3 has an important role in IFN signaling,³⁴ whereas HDAC activity, commonly correlated with transcriptional repression, is essential for transcriptional induction of ISGs.³⁵ HDAC function is also required for gene induction driven by virus activation of IRF3, and essential for establishment of an antiviral response against VSV.³⁵ Pretreatment of human prostate PC3 cancer cells with HDAC inhibitors, MS-275 or SAHA (Vorinostat), enhanced VSV oncolysis by blunting IFN response and inhibited the increase in IRF-7 and MxA mRNA levels post virus infection.²³ The PI3K/mTOR/Akt pathway is important in coordinating innate immune defense and is an important regulator of type I IFN production via activation of IRF7.³⁶ Combination therapy of VSV-ΔM51 with rapamycin (inhibitor of mTOR) reduced IFN production and significantly improved survival of rats bearing malignant glioma.²⁵ However, our study shows that none of these inhibitors were effective at reversing VSV resistance in the HNSCC SCC15 or SCC25 cells.

The VSV-resistant SCC25 cells constitutively express high levels of IRF7, IRF9 and OAS1 mRNA. In contrast, these genes are not detectable in VSV-permissive SW579 cells where they are activated only after IFN or VSV treatment. Addition of JAK inhibitors reduced IRF7 and IRF9 mRNA only twofold in SCC25, whereas OAS1 mRNA levels showed no appreciable change. In contrast to the data on PC3 cancer cells where both HDAC and JAK inhibitors were effective at reversing VSV resistance, our data show that only JAK inhibitors were effective in the HNSCC cells.^23,24 Interestingly, Carey et al. showed that besides constitutive expression of numerous antiviral gene products (for example, MxA, OAS1), VSV resistance in PC3 cells is also compounded by delays in VSV penetration (by 10–30 min), primary transcription and viral protein synthesis (by 6–8 h) relative to the VSV-permissive LNCaP cells. Clearly, there are numerous factors in play that contribute to relative resistance of different cell types to VSV oncolysis. Paglino and van den Pol³⁷ performed a broad analysis on a panel of 13 sarcomas representing seven tumor types and showed that synovial sarcoma SW982 demonstrated remarkable resistance, even at a MOI of 100. These authors found that the basal pre-infection levels of ISGs, MxA and ISG-15 were aberrantly high in SW982 cells. JAK inhibitor I given 4 days pre-infection (but not coincident with VSV) reversed this antiviral state. Moerdyk-Schauwecker et al. showed that pancreatic cancer cells have substantial diversity in their susceptibility to VSV oncolysis, and constitutively express at least some ISGs, including the antiviral genes MxA and OAS.³⁸ Treatment of cells for 48 h with JAK inhibitor I reduced MxA and OAS protein to undetectable levels in the uninfected cells.

Contrary to the above reports showing that 2–4 days of JAK inhibitor drug pre-treatment are needed to impact the VSV susceptibility of sarcoma and prostate cancer lines, VSV resistance in HNSCC cells can be reversed by pre-treatment of cells with inhibitors for 1 h. Interestingly, this inhibitory effect persists for 24 h after removal of the drug. This sustained effect is unexpected as JAK1/2 inhibitors bind reversibly to the ATP-binding site of JAK1 and JAK2 to prevent activation of JAKs and proteins in the Jak/Stat signaling pathway.^32,33 However, the possibility that the inhibitors can be co-administered with VSV is very convenient to facilitate logistics of clinical trials. Although JAK inhibitor I is not being developed for clinical use, there are several JAK inhibitors under clinical testing for a variety of anti-cancer or anti-inflammatory applications.³⁹ Ruxolitinb and Tofacitinib (JAK3, JAK1 inhibitor) were recently approved by the Food and Drug Administration, respectively, for the treatment of patients with intermediate or high-risk myelofibrosis or rheumatoid arthritis.⁴⁰ As researchers continue to develop JAK inhibitors that are more specific, future studies could perhaps more accurately define the mechanism of virus resistance in cancer cells for improved oncolysis and therapeutic outcome.