Novel Synthetic Promoter Armed Oncolytic Herpes Simplex Virus For Treatment of PAX3-FOXO1 Positive Rhabdomyosarcoma
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Background
Due to the difficulty in targeting oncogenic transcription factors, there are no targeted treatments available for pediatric patients afflicted with PAX3-FOXO1 fusion positive alveolar rhabdomyosarcoma. Additionally, alveolar rhabdomyosarcomas are immunologically cold tumors inherently resistant to immune checkpoint blockade. Viral vectors present a unique opportunity to address this gap in treatment strategies as their presence causes immune infiltration, and a virus can be engineered to rely on tumor specific transcriptional networks for its own replication.
Methodology
We engineered a synthetic promoter which relies on PAX3-FOXO1 to drive expression of a downstream gene. We then placed the HSV-1 RL1 gene encoding ICP34.5 under this promoter within the G47Δ oncolytic herpes simplex virus (oHSV), generating a novel oHSV targeting cells harboring the PAX3-FOXO1 fusion gene, which we name oRP3Fus (“Orpheus;” oncolytic recombinant G47Δ targeting PAX3-FOXO1 virus). PAX3-FOXO1 specificity was determined by qRT-PCR, protein expression, and chromatin immunoprecipitation. Antitumor efficacy of oncolytic virus and anti-PD1 antibody was determined using immuno-competent mice harboring syngeneic PAX3-FOXO1 expressing rhabdomyosarcoma tumors. Bulk RNA sequencing was performed on infected and mock infected fusion positive and negative cells for differential gene expression analysis.
Results
oRP3Fus kills PAX3-FOXO1 positive human rhabdomyosarcoma lines, while remaining non-toxic to normal human skeletal muscle cells in vitro . In immunocompetent mouse models, both novel and parental viruses improved tumor control when combined with anti-PD1, while oRP3Fus treatment increased T cell infiltration within tumors. RNA and DNA extraction from tumors demonstrate viral expansion within oRP3Fus treated tumors up to three weeks post treatment. Meanwhile oRP3Fus treated mice displayed decreased amounts of necroptosis-associated gene expression within normal organs as compared to groups treated with parental virus. Bulk RNA sequencing of fusion positive and negative human rhabdomyosarcoma lines demonstrated PAX3-FOXO1 increases expression of genes involved in the TNFa-NFκB signaling axis, as well as innate and adaptive immune signaling. Additionally, ICP34.5 expression from oRP3Fus reduces RIPK1 activation.
Conclusions
ICP34.5-armed oHSV (oRP3Fus) combined with anti-PD1 provides effective antitumor activity against PAX3-FOXO1-positive alveolar rhabdomyosarcoma in vivo and should be further explored for fusion oncogene-driven tumors.
What is already known on this topic?
PAX3-FOXO1 fusion-positive and negative rhabdomyosarcoma (RMS) is highly resistant to immune checkpoint blockade. Previously, a PAX3-FOXO1 driven oncolytic adenovirus showed efficacy in an immuno-compromised fusion-positive RMS model. In fusion-negative, immuno-competent RMS models, oncolytic herpes viruses have been shown to improve survival when combined with anti-PD1 therapy, but combinational efficacy was only observed with sex-mismatched models and was never tested on fusion-positive RMS tumors in immunocompetent mice.
What this study adds?
We improved the specificity of the synthetic PAX3-FOXO1 targeted promoter. We also show, for the first time, efficacy of oncolytic herpes simplex virus-1 in immunocompetent mice harboring orthotopic PAX3-FOXO1-expressing RMS tumors. Our study also demonstrates improvement in survival efficacy in sex-matched tumors, whereas previous reports did not observe complete responses except when using sex-mismatched tumor models.
How this study might effect research, practice, or policy?
The synthetic promoter generated here can be used for future gene therapy or targeting efforts to study PAX3-FOXO1 or target PAX3-FOXO1 tumors. We observe anti-PD1 therapy benefits survival when paired with an oHSV, demonstrating oncolytic virus can sensitize PAX3-FOXO1 expressing RMS tumors to immunotherapy.
