Efficacy of Oncolytic Herpes Simplex Virus T-VEC Combined with BET Inhibitors as an Innovative Therapy Approach for NUT Carcinoma

Abstract

NUT carcinoma (NC) is an extremely aggressive tumor and current treatment regimens offer patients a median survival of six months only. This article reports on the first in vitro studies using immunovirotherapy as a promising therapy option for NC and its feasible combination with BET inhibitors (iBET). Using NC cell lines harboring the BRD4-NUT fusion protein, the cytotoxicity of oncolytic virus talimogene laherparepvec (T-VEC) and the iBET compounds BI894999 and GSK525762 were assessed in vitro in monotherapeutic and combinatorial approaches. Viral replication, marker gene expression, cell proliferation, and IFN-β dependence of T-VEC efficiency were monitored. T-VEC efficiently infected and replicated in NC cell lines and showed strong cytotoxic effects. This implication could be enhanced by iBET treatment following viral infection. Viral replication was not impaired by iBET treatment. In addition, it was shown that pretreatment of NC cells with IFN-β does impede the replication as well as the cytotoxicity of T-VEC. T-VEC was found to show great potential for patients suffering from NC. Of note, when applied in combination with iBETs, a reinforcing influence was observed, leading to an even stronger anti-tumor effect. These findings suggest combining virotherapy with diverse molecular therapeutics for the treatment of NC.

Keywords: BET inhibitors; NUT carcinoma; T-VEC; combination therapy; talimogene laherparepvec; virotherapy.

Figure 1

The life cycle of a herpes-simplex-virus type 1 (HSV-1)-based virotherapeutic (T-VEC) (A) and transmission electron microscopy (TEM) images of T-VEC-infected HCC2429 NUT carcinoma cells at 48 h post infection (hpi), illustrating individual steps of HSV-1 propagation (B–G): (A) (I) Glycoproteins located on the surface of HSV-1 virus particles attach to cellular receptors, followed by fusion of the viral envelope membrane with the cytoplasmic membrane of the target cell to release the virus capsid (C) into the cytoplasm. (II) The HSV-1 capsid migrates along the cytoskeleton to the cell nucleus, where it attaches; the viral DNA is released into the nucleus, leaving the empty capsid behind. (III) In the nucleus, transcription of viral genes and genome replication take place, thus enabling the assembly of progeny viral capsids. (IV) Newly formed viral capsids first attach to the nuclear lamina, are then transported through the inner nuclear membrane, and finally released into the cytoplasm. (V) Final maturation of the capsid occurs via budding into vesicles of the trans-Golgi network (TGN), which contain viral glycoproteins (dark blue spikes). (VI) Enveloped virions within cellular vesicles are transported to the cell surface. (VII) Then, vesicle and plasma membranes fuse in order to release a mature, enveloped progeny HSV-1 virion from the cell. (B) Overview of a single T-VEC-infected HCC2429 NUT carcinoma cell. Scale bar shows 1000 nm. (C) Transport of newly formed viral capsids (white arrows) through the perinuclear space (step IV in (A)). Scale bar shows 250 nm. (D) In the cytoplasm, viral capsids are wrapped into vesicles of the trans-Golgi network (TGN) for final maturation (white arrows; step V in (A)). Scale bar shows 250 nm. (E) Onset of oncolysis in a T-VEC-infected HCC2429 cell, indicated by a defect in the cellular membrane (white arrow). Scale bar shows 500 nm. (F) Multiple mature progeny T-VEC particles are released by exocytosis, lining still up on the outside of the cellular membrane (white arrows) (step VII in (A)). Scale bar shows 500 nm. (G) Completed oncolysis results in the release of a multitude of T-VEC progeny particles (white arrows). Scale bar shows 500 nm.

Figure 2

Viability of human NC cell lines after monotherapeutic treatment with T-VEC: 143100 (A), HCC2429 (B), Ty-82 (C), 10-15 (D), 14169 (E), and JCM (F) NC tumor cells were infected with T-VEC at different multiplicities of infection (MOIs) ranging from 0.0001 to 1 or remained uninfected (MOCK). At 96 h post infection (hpi), the remaining NC tumor cells were determined by SRB viability assay. T-VEC-mediated oncolysis was calculated relative to MOCK control. The mean ± SD of at least two independent experiments performed in quadruplicates is shown. *** p < 0.001, **** p < 0.0001.

Figure 3

Influence of IFN-β pretreatment on the oncolytic efficacy of T-VEC in 143100 NC cells: (A) 143100 NC cells were pretreated with 2 ng/mL IFN-β for 16 h before infection with T-VEC at two different tumor cell line-adjusted multiplicities of infection (MOIs) or remained uninfected (MOCK + IFN-β). At 96 h post infection (hpi), the remaining tumor cells were determined by SRB viability assays. The anti-tumor effect of each treatment modality was calculated relative to untreated control (MOCK). The mean ± SD of at least two independent experiments performed in triplicates is shown. ** p < 0.01, *** p < 0.001, **** p < 0.0001. (B) 143100 NC cells were pretreated with IFN-β analogue to (A) and infected with T-VEC at MOIs 0.0001 and 0.001 for the timepoints 16 and 24 hpi and at MOIs 0.00005 and 0.0001 for the timepoints 72 and 96 hpi, respectively. At 16, 24, 72, and 96 hpi supernatants were harvested and T-VEC-mediated expression of the marker protein GM-CSF was measured via ELISA. (C) Immunofluorescence images of 143100 NC cells pretreated with IFN-β and infected with T-VEC (MOI 0.1) for 8 or 16 h. ++, moderate HSV-1 staining; +++, strong HSV-1 staining; Ø, no HSV-1 staining. Scale bar shows 5 µm.

Figure 4

Viability of human NC cell lines after combinatorial treatment with T-VEC and either BET inhibitor (iBET) BI894999 (BI) or GSK525762 (GSK): 143100 (A), HCC2429 (B), Ty-82 (C), 10-15 (D), 14169 (E), and JCM (F) NC cells were treated with T-VEC, BI, and GSK at indicated multiplicities of infection (MOIs) or concentrations alone or with the appropriate combinations or remained untreated (MOCK). MOIs and iBET concentrations were adjusted to the appropriate cell lines. The remaining tumor cells were determined by SRB viability assay at 96 h post infection (hpi). The anti-tumor effect of each treatment modality is calculated relative to MOCK control. The mean ± SD of at least two independent experiments performed in triplicates is shown. Calculated significances always refer to the most potent monotherapy. * p < 0.05; ** p < 0.01, *** p < 0.001, **** p < 0.0001, n.s. not significant.

Figure 5

Real-time analysis of 143100 NC cells treated with T-VEC alone or after combinatorial treatment with the BET inhibitors BI894999 (BI) (A) or GSK525762 (GSK) (B). 24 h after seeding, 143100 NC cells were infected with T-VEC (MOI 0.0001) alone or in combination with 5 nM BI (A) or 0.5 µM GSK (B) or remained untreated (MOCK). Triton X-100 was added as a negative control inducing maximum lysis of tumor cells. Real-time cell proliferation was monitored using the xCELLigence^® RTCA SP system. Measured electrode impedance is expressed as cell index. One representative of two independent experiments performed in triplicates is shown. Vertical dashed lines indicate the time point of T-VEC infection.

Figure 6

Viral replication of T-VEC in NC cell lines alone and after combination with either BET inhibitor (iBET) BI894999 (BI) or GSK525762 (GSK): 143100 (A), HCC2429 (B), Ty-82 (C), 10-15 (D), 14169 (E), and JCM (F) tumor cells were infected with T-VEC at a multiplicity of infection (MOI) of 0.0001 alone or in combination with BI or GSK at cell line-adjusted concentrations. Viral replication was analyzed via plaque assay at 1, 24, 48, 72, and 96 h post infection (hpi).