PI3K inhibitor significantly enhances the antitumor activity of oncolytic virus in osteosarcoma

Background: Osteosarcoma (OS) is a highly aggressive malignancy with less than 30% 5-year survival rate among patients with metastatic or recurrent cancer. However, the treatment for osteosarcoma has not been modified in the last three decades. Oncolytic viruses have shown encouraging results in pre-clinical trials, but have failed to translate into high therapeutic efficacy in clinical trials. In this study, we will determine the therapeutic effect of combining PI3K inhibitor with an oncolytic virus against osteosarcoma. Material and Methods: Osteosarcoma cell lines and xenograft model were treated with ZSTK474 and/or VSVΔ51, the tumor suppressive ability was verified by in vitro cytotoxicity experiments and in vivo antitumor activity experiments, and the antitumor mechanism was explored through the study of apoptosis-related signaling pathways. Results: ZSTK474 sensitized the osteosarcoma cells to VSVΔ51, and augmented apoptosis via endoplasmic reticulum stress. The combination treatment also showed greater in vivo tumor inhibition compared to either ZSTK474 or VSVΔ51 alone, and significantly enhanced the tumor infiltration of immune cells. Conclusion: PI3K inhibitors combined with oncolytic virus is a promising strategy against osteosarcoma.


Background
Osteosarcoma (OS) is the most common form of bone cancer in children and adolescents, and belongs to a large family of mesenchymal-derived solid tumors with considerable histological, genetic and molecular heterogeneity [1][2][3]. It is initiated by somatic TP53 and/or RB1 mutations that lead to chromosomal instability and malignant transformation [4]. The 5-year survival rate of patients diagnosed with metastatic or 3 recurrent osteosarcoma is less than 30% [5]. A recent multi-center clinical trial showed that increasing the dose of chemotherapy improved prognosis of patients but did not prolong survival in high-risk populations, thus underscoring the need for novel treatment strategies [6].
Oncolytic viruses are a novel immunotherapeutic tool that has shown encouraging results in various cancer models [7]. The oncolytic herpes virus Talimogene Laherparepvec (T-VEC) was recently approved by FDA for treating metastatic melanoma [8]. Vesicular stomatitis virus (VSV), an enveloped negative sense strand RNA virus of the Rhabdoviridae family, has shown strong oncolytic activity in preclinical models and is currently undergoing clinical trials [9,10]. Site-directed mutagenesis of VSV allows it to preferentially target tumors without infecting the healthy cells [11]. For example, VSVΔ51 can only replicate in tumor cells and not in normal cells due to the methionine deletion at position 51 [12].
Studies show that oncolytic viruses infect cancer cells through a dual mechanism of selective replication and lysis, and induce an anti-tumor immune response in the host [13].
However, these viruses cannot exert a potent "oncolytic" effect on their own, and the ensuing anti-tumor immune response is also limited [14]. To improve their therapeutic effects against solid tumors, combination therapy or genetically engineered viruses are currently being explored [15,16].
The PI3K/AKT/mTOR signaling pathway is constitutively active in many cancers, and promotes excessive proliferation of tumor cells while inhibiting apoptosis [17,18]. Blocking the PI3K-Akt pathway not only mitigates tumor progression but also induces endoplasmic reticulum stress (ERS) in the tumor cells [19]. Interestingly, oncolytic virus-induced apoptosis can be significantly enhanced by aggravating ERS in the host cells through pharmacological agents [20,21].
In this study, we determined whether the PI3K inhibitor ZSTK474 enhanced the oncolytic 4 activity of VSVΔ51 in osteosarcoma cells, and found that the former acted as a potent adjuvant and significantly increased viral cytotoxicity in vitro. In addition, the combination therapy also increased the infiltration of immune effector cells in the osteosarcoma xenografts in vivo, resulting in greater inhibition of tumor growth. Therefore, our findings present a novel immunotherapeutic strategy against osteosarcoma using oncolytic virus and pharmacological induction of ERS.

Materials And Methods
Cell lines and reagents
Cells were cultured at 37 °C in a humidified incubator with 5% CO2. 5 VSV production, quantification, and infection GVP expressing VSVD51, a recombinant derivative of the Indiana VSV serotype, was provided by Dr. John Bell and Dr. David Stojdl (Ottawa Health Institute) [12]. VSVD51 was cultivated in the Vero cells and virus titers were quantified by the standard plaque analysis method as previously described [22].

Cell viability assay
The 143B cells were seeded in a 96-well plate at the density of 3000 cells/well. Following treatment with ZSTK474, thiazole blue (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium) was added to each well as the final concentration of 1 mg/mL, and the cells were incubated for 3 h at 37 °C. The medium was removed and the MTT crystals were dissolved in 100 µL DMSO, and the absorbance at 570 nm was measured using Pickers (iMark, Bio-Rad).

Western blotting
Cells or tissues were lysed with mammalian protein extraction reagent (M-PER; Thermo Science) and separated by SDS-PAGE. The proteins bands were transferred to immunoblot membranes and probed with antibodies against IRE1α, p-ERK, ATF6, CHOP, caspase-12 and GAPDH. The membrane was developed on a ChemiDoc XRS + system (Bio-Rad) using Immobilon Western chemiluminescence HRP substrate (Millipore), and the protein bands were quantified using Image J software.

Transmission electron microscopy
The 143B cells were infected with VSVΔ51 at 0.001 PFU/cell for 24 h in the presence or absence of ZSTK474. The infected cells were centrifuged at 1000 g for 5 min at room temperature. After washing once with PBS, the cells were pelleted at 1500 g for 5 min,

Statistical analysis
All statistical analyses were performed using GraphPad Prism software. Different groups were compared with Student's t test or analysis of variance (ANOVA) as appropriate.
Tumor volumes were calculated by repeated measurement of single factor analysis of variance. Kaplan-Meier survival curves were compared by the log-rank test. P values less than 0.05 were considered statistically significant.

ZSTK474 sensitizes osteosarcoma cells to VSVΔ51
The target cells were treated with varying doses of ZSTK474 in the presence/absence of low titer (0.001 PFU/cell) VSVΔ51 oncolytic virus (Fig. 1A). ZSTK474 decreased the viability of osteosarcoma cells in a dose-dependent manner, and also augmented the cytotoxic effect of VSVΔ51. Almost 80% of the infected cells were dead when additionally treated with 4 µM ZSTK474, indicating that the latter sensitizes osteosarcoma cells to VSVΔ51 (Fig. 1B).
The synergistic effect of ZSTK474 and VSVΔ51 depends on ERS-induced apoptosis The surge in protein synthesis during viral replication frequently overloads the endoplasmic reticulum, resulting in ERS [23] that triggers the unfolded protein response (UPR) to restore protein homeostasis and decrease aggregates of misfolded peptides.
However, persistent UPR due to severe ERS can lead to apoptosis [24]. Consistent with this, the 143B cells treated with ZSTK474 or VSVΔ51 showed markedly swollen endoplasmic reticulum within 24 h, which increased further in the dual treated cells 8 ( Fig. 2A, B). Furthermore, the ERS markers including IRE1α and p-PERK were also significantly upregulated in the cells treated with both ZSTK474 and VSVΔ51 compared to the monotherapy groups, while that of activating transcription factor 6 (ATF6) was not affected (Fig. 2C). As shown in Fig. 2D, the caspase-12 pathway was strongly activated in the combination treatment group, along with upregulation of C/EBP-homologous protein (CHOP) compared to the ZSTK474 or VSVΔ51-treated cells. Finally, Hoechst 33342 staining showed a significant increase in the number of pyknotic nuclei in the dual treated tumor cells, indicating apoptosis induction. However, karyopyknosis was not obvious in the ZSTK474 or VSVΔ51-treated cells (Fig. 2E). Taken together, ZSTK474 augmented VSVΔ51induced ERS and apoptosis in osteosarcoma cells.

ZSTK474 and VSVΔ51 synergistically inhibit tumor growth i n v i v o
The in vivo therapeutic potential of ZSTK474 and VSVΔ51 was evaluated in an osteosarcoma xenograft model, as outlined in Fig. 3A. Compared to ZSTK474 or VSVΔ51 alone, the combination treatment significantly inhibited tumor growth (P < 0.001; Fig. 3B-D). Furthermore, the tumor weight in the combination treatment group was significantly lower than that in ZSTK474 or VSVΔ51 groups (Fig. 3E). Consistent with this, in situ Ki67 expression was significantly lower and that of IRE1a, CHOP and C-caspase-12 were higher in the tumor tissues of the combination treatment group compared to the ZSTK474 or VSVΔ51-treated tumors (Fig. 4A, B). Thus, PI3K inhibition enhanced viral infection-induced ERS and apoptosis in tumor tissues, which translated to greater therapeutic effect.

ZSTK474 and VSVΔ51 enhance immune cell infiltration into tumors
Oncolytic viruses trigger the anti-tumor immune response after infecting the tumor cells, thus converting the "cold tumor" into a "hot tumor" [25]. Since the PI3K-AKT-mTOR signaling pathway regulates immune responses, we also analyzed the immune cell 9 infiltrates in the tumors following each treatment regimen. Compared to ZSTK474 or VSVΔ51 alone, their combination significantly enhanced the infiltration of NK cells into the tumors and decreased that of MDSCs (Fig. 5A). The number of tumor-infiltrating macrophages was lower in each treatment group compared to that in the untreated controls. Furthermore, the CD8+/CD4 + T cell ratio in the tumors was significantly increased in the combination treatment vs either monotherapy groups, indicating greater activation of anti-tumor immune responses in the former (Fig. 5B). Taken together, ZSTK474 also amplifies VSVΔ51-mediated anti-tumor immune response in vivo.

Discussion
Osteosarcoma is a malignant tumor characterized by formation of neoplastic or neoplasticlike bone tissues, and accounts for about 20-34% of all primary malignant bone tumors [26]. The 5-year survival rate of patients with metastatic or recurrent osteosarcoma is less than 30%, thereby requiring novel treatment strategies. Oncolytic viruses are a promising immunotherapeutic tool against cancer cells and several are currently in clinical trials. The FDA approved T-Vec developed by Angen in October 2015 for the treatment of metastatic melanoma [27]. The main focus of oncolytic virus therapy is to directly kill the tumor cells as well as activate the anti-tumor immune response [28]. Genetically engineered recombinant oncolytic viruses have also been designed in order to improve antitumor activity [29]. However, this strategy is limited by the small viral genomes and technical difficulty. In addition, excessive modification of the virus skeleton reduces its infection ability, and therefore oncolytic function [30]. Thus, recent studies are focusing on "conditionally enhancing" the efficacy of oncolytic viruses through pharmacological agents [31].

ZSTK474 increased apoptosis in the virus-infected tumor cells by enhancing ERS in vitro,
and amplified the virus-induced host immune response in the tumor-bearing mice. The therapeutic effects of this combination were significantly greater compared to either VSVΔ51 or ZSTK474 alone.

Conclusions
Our findings provide a strong experimental basis for using small molecule inhibitors of AKT and mTOR as adjuvants along with oncolytic virus to enhance therapeutic effects against solid tumors. There are however some limitations in our study that ought to be addressed.
We injected the oncolytic virus via the intravenous route to ensure tumor-specific infection of the oncolytic viruses, although studies show that intra-tumoral or peritumoral injection results in better anti-tumor effects [40]. Therefore, the optimum dosage regimen needs to be further explored in the follow-up experiments. In conclusion, the combination of VSVΔ51 and ZSTK474 is a promising new strategy for treating osteosarcoma. Availability of data and materials All data are available in the included figures.