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Purpose:Oncolytic herpes simplex virus-1 (oHSV) infection of brain tumors activates NOTCH, however the consequences of NOTCH on oHSV-induced immunotherapy is largely unknown. Here we evaluated the impact of NOTCH blockade on virus-induced immunotherapy.Experimental Design:RNA sequencing (RNA-seq), TCGA data analysis, flow cytometry, Luminex- and ELISA-based assays, brain tumor animal models, and serum analysis of patients with recurrent glioblastoma (GBM) treated with oHSV was used to evaluate the effect of NOTCH signaling on virus-induced immunotherapy.Results:TCGA data analysis of patients with grade IV glioma and oHSV treatment of experimental brain tumors in mice showed that NOTCH signaling significantly correlated with a higher myeloid cell infiltration. Immunofluorescence staining and RNA-seq uncovered a significant induction of Jag1 (NOTCH ligand) expression in infiltrating myeloid cells upon oHSV infection. Jag1-expressing macrophages further spread NOTCH activation in the tumor microenvironment (TME). NOTCH-activated macrophages increased the secretion of CCL2, which further amplified myeloid-derived suppressor cells. CCL2 and IL10 induction was also observed in serum of patients with recurrent GBM treated with oHSV (rQnestin34.5; NCT03152318). Pharmacologic blockade of NOTCH signaling rescued the oHSV-induced immunosuppressive TME and activated a CD8-dependent antitumor memory response, resulting in a therapeutic benefit.Conclusions:NOTCH-induced immunosuppressive myeloid cell recruitment limited antitumor immunity. Translationally, these findings support the use of NOTCH inhibition in conjunction with oHSV therapy.

Supplementary Figure from NOTCH-Induced MDSC Recruitment after oHSV Virotherapy in CNS Cancer Models Modulates Antitumor Immunotherapy

Supplementary Data from NOTCH-Induced MDSC Recruitment after oHSV Virotherapy in CNS Cancer Models Modulates Antitumor Immunotherapy

INTRODUCTION The median survival of Glioblastoma (GBM) patients is less than two years with the standard of care of maximal surgical resection, radiation, and temozolomide (TMZ) chemotherapy. Protein Arginine Methyltransferase 5 (PRMT5), which regulates cellular functions by symmetrically di-methylating arginine residues, is overexpressed in GBM. Inhibiting PRMT5 induces apoptosis in differentiated and senescence in stem-like GBM tumor cells. We inhibited PRMT5 in GBM neurospheres to determine if PRMT5 inhibition would enhance TMZ’s antitumor effect. METHODS We depleted PRMT5 activity, in vitro, using target-specific siRNA or LLY-283 and combined these with TMZ treatment. We evaluated the antitumor effect of this combination using cell viability assay, cell cycle analysis, apoptosis assay, and western blot. RESULTS TMZ reduced the viability of GBMNS with PRMT5 knockdown significantly more than the viability of PRMT5 intact GBMNS. The combination of TMZ and PRMT5 knockdown elevated the expression of cleaved caspase 3 and caspase3/7 indicating that PRMT5 knockdown enhanced the apoptotic effects of TMZ. Cell cycle analysis showed that depleting PRMT5 abrogated TMZ-induced G2/M cell cycle arrest. Further, treatment of PRMT5-depleted GBMNS with TMZ increased ɣ-H2AX expression compared PRMT5 intact GBMNS treated with TMZ, suggesting that PRMT5 depletion enhanced TMZ-induced DNA damage. PRMT5 knockdown also inhibited the symmetric di-methylation of RUVBL1 that is required for homologous recombination repair of TMZ treatment-related DNA damage. CONCLUSION Overall, PRMT5 inhibition sensitized GBMNS to TMZ and enhanced TMZ-related DNA damage and cytotoxicity. These findings support further development of this potential therapeutic combination.

The beta-1 subunit of integrins (CD29) is important for cell-extracellular matrix interactions. CD29 expression is increased in breast cancer cells, and it promotes their invasion, migration, and intravasation, all of which can result in breast cancer brain metastasis (BCBM). While CD29 expression can be correlated to poor prognosis of breast cancer patients, the median survival of BCBM patients predicted to have better prognosis is 36 months. Despite current BCBM treatments such as craniotomy, reaching the metastatic site is often challenging. Modified viral vectors, such as oncolytic herpes simplex virus (oHSV), have been clinically approved for the treatment of malignant melanoma and brain tumors, as they infect and selectively kill tumor cells. Previously, we identified that combination of oHSV and humanized CD29 blocking antibody (OS2966, aCD29) enhanced viral replication by reducing antiviral immune response, increased tumor cell killing, and rendered survival benefit to tumor bearing mice with intra-tumoral, but not systemic, oHSV administration. These findings suggested that the antitumor efficacy of the therapeutic combination required intratumoral delivery of anti-CD29, since the antibody could not penetrate the blood brain barrier and reach intracranial tumors. To overcome this, we report the generation of an oHSV encoding the scFv-Fc fragment of this antibody (HSV-aCD29), which retains its oncolytic function, and evaluate the impact of HSV-aCD29 on the CD29 downstream signaling. We will further characterize the safety, antitumor efficacy, and assess the immune response to HSV-aCD29 in vitro and in vivo. We anticipate that compared to our control virus, HSV-aCD29 will block CD29 activity which will reduce antiviral immune responses and enhance tumor cell lysis, thus improving oHSV efficacy. Together, this study proposes the blockade of CD29 with the targeted delivery of a CD29 blocking antibody mediated by a novel oHSV as a potential synergistic therapy against BCBM.

Glioblastoma (GBM) is the most aggressive brain malignancy, which despite continuing worldwide efforts to develop new therapies, remains a deadly disease with limited treatment options. The asparagine hydroxylase factor inhibiting HIF (FIH1) is located in chromosome 10q24, a region often deleted in GBM, suggesting a tumor protective role. FIH1 has been implicated in GBM as an inhibitor of HIF1α-mediated transcription of VEGF-A, leading to decreased angiogenesis. We previously showed that oncolytic HSV (oHSV) infection reduces the expression FIH1 via a virus encoded miRNA. Furthermore, we have found that FIH1 also negatively regulates NOTCH signaling, which is elevated following oHSV treatment. Thus, we hypothesize that FIH1 reconstitution in tumors will block pro-tumorigenic NOTCH and HIF1α signaling. Using FIH1-overexpressing GBM cells we observed a reduction in vascular permeability, measured by intravital imaging, and decreased expression of angiogenesis markers. In vivo, combination of FIH1 overexpression with oHSV treatment resulted in enhanced survival in mice bearing intracranial GBM tumors. Furthermore, FIH1 overexpression correlated with the enrichment of genes associated with oxidative phosphorylation and ROS pathways, suggesting the potential for multi-modality therapeutic strategies.

Purpose: Oncolytic herpes simplex virus-1 (oHSV) infection of brain tumors activates NOTCH, however the consequences of NOTCH on oHSV-induced immunotherapy is largely unknown. Here we evaluated the impact of NOTCH blockade on virus-induced immunotherapy. Experimental Design: RNA sequencing (RNA-seq), TCGA data analysis, flow cytometry, Luminex- and ELISA-based assays, brain tumor animal models, and serum analysis of patients with recurrent glioblastoma (GBM) treated with oHSV was used to evaluate the effect of NOTCH signaling on virus-induced immunotherapy. Results: TCGA data analysis of patients with grade IV glioma and oHSV treatment of experimental brain tumors in mice showed that NOTCH signaling significantly correlated with a higher myeloid cell infiltration. Immunofluorescence staining and RNA-seq uncovered a significant induction of Jag1 (NOTCH ligand) expression in infiltrating myeloid cells upon oHSV infection. Jag1-expressing macrophages further spread NOTCH activation in the tumor microenvironment (TME). NOTCH-activated macrophages increased the secretion of CCL2, which further amplified myeloid-derived suppressor cells. CCL2 and IL10 induction was also observed in serum of patients with recurrent GBM treated with oHSV (rQnestin34.5; NCT03152318). Pharmacologic blockade of NOTCH signaling rescued the oHSV-induced immunosuppressive TME and activated a CD8-dependent antitumor memory response, resulting in a therapeutic benefit. Conclusions: NOTCH-induced immunosuppressive myeloid cell recruitment limited antitumor immunity. Translationally, these findings support the use of NOTCH inhibition in conjunction with oHSV therapy.

NOTCH signaling is a method of cell-cell communication where membrane bound NOTCH ligands on signal-sending cells can bind to and initiate cleavage of the NOTCH receptor, releasing NICD which can initiate signal transduction in adjacent “signal-receiving” cells. We have recently shown that oHSV treatment of GBM cells induces NICD cleavage and NOTCH activation in adjacent uninfected glioma cells. RNA sequencing of GBM cells post-infection also uncovered Gene Ontology NOTCH signaling pathway to be significantly upregulated. This activation was induced by viral miRNA-H16, which represses FIH-1 expression. FIH-1 was found to be a negative regulator of Mib1, a ubiquitin ligase, which activates NOTCH ligand-mediated activation of adjacent signal-receiving cells bearing the NOTCH receptor (Otani et al Clin. Can. Res. 2020). Here we have investigated the impact of oHSV-induced NOTCH signaling on the tumor microenvironment. Treatment of brain tumors in immune competent mice with oHSV and NOTCH blocking gamma secretase inhibitor (GSI) induced an anti-tumor memory immune response. Long term survivors in mice treated with the combination also completely rejected subsequent tumor re-challenge in the other hemisphere. UMAP of flow cytometry of tumor-bearing hemispheres and functional analysis of isolated cellular fractions from treated mice showed a significant influx of MDSC cells after oHSV treatment that was rescued in mice treated with oHSV and GSI. Ongoing mechanistic studies are uncovering a significant induction of NOTCH in tumor associated macrophages that aids in recruitment of MDSC cells. Overall these studies have uncovered a significant impact of oHSV therapy on GBM tumor microenvironment and presents opportunities for combination therapies that can help improve therapeutic benefit and anti-tumor immunity.