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XRCC1 inactivation does not restore the sensitivity to TMZ in MSH6-deficient cells

Western blot analysis of p-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), p-MEK1/2 (Ser217/221), p-p90RSK (Ser380), p-MSK1 (Thr581) in JIMT-1 BR-3, MDA-MB-361, MDA-MB-231 BrM2 and BS-004 cell lines treated with GDC-0084 in increasing concentrations for a total of 6 hours

Purpose:Previous studies have shown that the PI3K/Akt/mTOR pathway is activated in up to 70% of breast cancer brain metastases, but there are no approved agents for affected patients. GDC-0084 is a brain penetrant, dual PI3K/mTOR inhibitor that has shown promising activity in a preclinical model of glioblastoma. The aim of this study was to analyze the efficacy of PI3K/mTOR blockade in breast cancer brain metastases models.Experimental Design: The efficacy of GDC-0084 was evaluated in PIK3CA-mutant and PIK3CA wild-type breast cancer cell lines and the isogenic pairs of PIK3CA wild-type and mutant (H1047R/+) MCF10A cells in vitro. In vitro studies included cell viability and apoptosis assays, cell-cycle analysis, and Western blots. In vivo, the effect of GDC-0084 was investigated in breast cancer brain metastasis xenograft mouse models and assessed by bioluminescent imaging and IHC.Results:In vitro, GDC-0084 considerably decreased cell viability, induced apoptosis, and inhibited phosphorylation of Akt and p70 S6 kinase in a dose-dependent manner in PIK3CA-mutant breast cancer brain metastatic cell lines. In contrast, GDC-0084 led only to growth inhibition in PIK3CA wild-type cell lines in vitro. In vivo, treatment with GDC-0084 markedly inhibited the growth of PIK3CA-mutant, with accompanying signaling changes, and not PIK3CA wild-type brain tumors.Conclusions:The results of this study suggest that the brain-penetrant PI3K/mTOR targeting GDC-0084 is a promising treatment option for breast cancer brain metastases with dysregulated PI3K/mTOR signaling pathway conferred by activating PIK3CA mutations. A national clinical trial is planned to further investigate the role of this compound in patients with brain metastases.

Purpose:Emergence of mismatch repair (MMR) deficiency is a frequent mechanism of acquired resistance to the alkylating chemotherapeutic temozolomide (TMZ) in gliomas. Poly(ADP-ribose) polymerase inhibitors (PARPi) have been shown to potentiate TMZ cytotoxicity in several cancer types, including gliomas. We tested whether PARP inhibition could re-sensitize MSH6-null MMR-deficient gliomas to TMZ, and assessed the role of the base excision repair (BER) DNA damage repair pathway in PARPi-mediated effects.Experimental Design:Isogenic pairs of MSH6 wild-type and MSH6-inactivated human glioblastoma (GBM) cells (including both IDH1/2 wild-type and IDH1 mutant), as well as MSH6-null cells derived from a patient with recurrent GBM were treated with TMZ, the PARPi veliparib or olaparib, and combination thereof. Efficacy of PARPi combined with TMZ was assessed in vivo. We used genetic and pharmacological approaches to dissect the contribution of BER.Results:While having no detectable effect in MSH6 wild-type GBMs, PARPi selectively restored TMZ sensitivity in MSH6-deficient GBM cells. This genotype-specific restoration of activity translated in vivo, where combination treatment of veliparib and TMZ showed potent suppression of tumor growth of MSH6-inactivated orthotopic xenografts, compared with TMZ monotherapy. Unlike PARPi, genetic and pharmacological blockage of BER pathway did not re-sensitize MSH6-inactivated GBM cells to TMZ. Similarly, CRISPR PARP1 knockout did not re-sensitize MSH6-inactivated GBM cells to TMZ.Conclusions:PARPi restoration of TMZ chemosensitivity in MSH6-inactivated glioma represents a promising strategy to overcome acquired chemoresistance caused by MMR deficiency. Mechanistically, this PARPi-mediated synthetic phenotype was independent of BER blockage and was not recapitulated by loss of PARP1.

Supplementary Figure Legend

Photomicrographs illustrating p-MEK1/2 (Ser221) and p-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) expression in mouse brains harboring either JIMT-1 BR-3 or MDA-MB-231 BrM2 intracranial tumors, stratified by treatment with sham or GDC-0084.

PARP inhibitors restore sensitivity to temozolomide in MSH6-inactivated, temozolomide resistant glioblastoma cells

GDC-0084-induced changes in cell cycle in the PIK3CA-mutant cell line MDA-MB-361 and in the PIK3CA-wildtype cell line BS-004

Purpose: Emergence of mismatch repair (MMR) deficiency is a frequent mechanism of acquired resistance to the alkylating chemotherapeutic temozolomide (TMZ) in gliomas. Poly(ADP-ribose) polymerase inhibitors (PARPi) have been shown to potentiate TMZ cytotoxicity in several cancer types, including gliomas. We tested whether PARP inhibition could re-sensitize MSH6-null MMR-deficient gliomas to TMZ, and assessed the role of the base excision repair (BER) DNA damage repair pathway in PARPi-mediated effects. Experimental Design: Isogenic pairs of MSH6 wild-type and MSH6-inactivated human glioblastoma (GBM) cells (including both IDH1/2 wild-type and IDH1 mutant), as well as MSH6-null cells derived from a patient with recurrent GBM were treated with TMZ, the PARPi veliparib or olaparib, and combination thereof. Efficacy of PARPi combined with TMZ was assessed in vivo. We used genetic and pharmacological approaches to dissect the contribution of BER. Results: While having no detectable effect in MSH6 wild-type GBMs, PARPi selectively restored TMZ sensitivity in MSH6-deficient GBM cells. This genotype-specific restoration of activity translated in vivo, where combination treatment of veliparib and TMZ showed potent suppression of tumor growth of MSH6-inactivated orthotopic xenografts, compared with TMZ monotherapy. Unlike PARPi, genetic and pharmacological blockage of BER pathway did not re-sensitize MSH6-inactivated GBM cells to TMZ. Similarly, CRISPR PARP1 knockout did not re-sensitize MSH6-inactivated GBM cells to TMZ. Conclusions: PARPi restoration of TMZ chemosensitivity in MSH6-inactivated glioma represents a promising strategy to overcome acquired chemoresistance caused by MMR deficiency. Mechanistically, this PARPi-mediated synthetic phenotype was independent of BER blockage and was not recapitulated by loss of PARP1.

Immunotherapy with the success of checkpoint blockade brings hope for cancer treatment with enduring or complete responses in various types of advanced malignancies, however, it has not benefited a number of so called “cold tumors”, such as glioblastoma multiforme (GBM), the most common and aggressive adult brain cancer. GBM is invariably lethal with a median survival of less than 15 months and characterized by a highly immunosuppressive tumor microenvironment, which desires new means of GBM immune-reactivation. Tissue-resident memory CD8+ T (TRM) cells are non-recirculating memory T cell subpopulation that resides permanently in peripheral tissues. TRM cells provide long-lived protective immunity against not only local pathogen infection but also tumor development through immediate effector function and recruitment of circulating immune cells upon antigen re-exposure. In this study, we found that memory T cells specific to some common human viruses that have infected almost everybody exist in clinical GBM specimens and can be activated by viral peptides, which are MHC I restricted CD8+ T cell epitopes derived from those viruses. In orthotopic GBM immunocompetent mouse models, we detected abundant virus specific TRM cells in the tumor microenvironment of GBMs intracranially established in mice that were previously exposed to the virus. Excitingly, intratumoral injection of viral peptides stimulated the reactivation of TRM cells, indicated by secretion of immuno-stimulatory cytokines, and demonstrated significant anti-tumor efficacy. This study provides proof of principle for TRM cell-based GBM therapy, which is a novel therapeutic paradigm with the translational potential for this deadly malignancy.

INTRODUCTION Lactate dehydrogenase A (LDHA) encodes an enzyme that catalyzes the inter-conversion between pyruvate and lactate in glycolysis. Here, we demonstrate that LDHA mediates a novel role in DNA repair independent of this metabolic function. METHODS siRNA screen, The Cancer Genome Atlas (TCGA) survival analysis, ionizing radiation (IR), g-H2AX, and chromatin assays, site-directed mutagenesis. RESULTS In an orthogonal siRNA-informatic screen to identify genes 1) when silenced caused IR sensitivity in patient-derived glioblastoma lines and 2) lowered expression is associated with improved survival in TCGA, LDHA surfaced as the top candidate. The survival association was validated by LDHA immunohistochemical staining in an independent collection of glioblastoma samples. In vitro and in vivo, silencing of LDHA sensitized glioblastoma lines to IR and enhanced radiation-induced g-H2AX accumulation. Such sensitization was not observed after treatment with an LDHA inhibitor, suggesting the metabolic function of LDHA is distinct from its role in DNA repair. Supporting this hypothesis, truncation mutations that suppressed the LDHA glycolysis function minimally affected its role in DNA repair. Mechanistically, cytoplasmic LDHA translocates into the nucleus in response to IR. This translocation was associated with subsequent chromatin transition into an open conformation and enhanced homologous recombination. CONCLUSION The novel LDHA function in DNA repair suggests intricate crosstalks between glycolytic metabolism and DNA repair, offering a new platform for glioblastoma therapeutic development.

Glioblastoma multiforme (GBM) is among the most aggressive, treatment-resistant cancers, and despite standard of care surgery, radiation and chemotherapy, is invariably fatal. GBM is marked by local and systemic immunosuppression, contributing to resistance to existing immunotherapies that have had success in other tumor types. Memory T cells specific for previous infections reside in tissues throughout the host and are capable of rapid and potent immune activation. Here, we show that virus-specific memory CD8 + T cells expressing tissue-resident markers populate the mouse and human glioblastoma microenvironment. Reactivating virus-specific memory T cells through intratumoral delivery of adjuvant-free virus-derived peptide triggered local immune activation. This delivery translated to antineoplastic effects, which improved survival in a murine glioblastoma model. Our results indicate that virus-specific memory T cells are a significant part of the glioblastoma immune microenvironment and may be leveraged to promote anti-tumoral immunity.

Glioblastoma multiforme (GBM) is among the most aggressive, treatment resistant cancers, and despite standard of care surgery, radiation and chemotherapy, is invariably fatal. GBM is marked by local and systemic immunosuppression, contributing to resistance to existing immunotherapies that have had success in other tumor types. Memory T cells specific for previous infections reside in tissues throughout the host and these tissue resident memory T cells (TRM) are capable of rapid and potent immune activation. Here, we show that virus-specific memory CD8+ T cells expressing tissue resident markers populate mouse and human glioblastoma microenvironment. Reactivating virus-specific TRM through intra-tumoral delivery of adjuvant-free virus-derived peptide triggered local immune activation. This delivery translated to anti-neoplastic effects, which improved survival in a murine glioblastoma model. Our results indicate that virus-specific TRM are a significant part of the glioblastoma immune microenvironment and can be leveraged to promote anti-tumoral immunity.

INTRODUCTION Natural Killer (NK) cells play pivotal roles in cancer immune surveillance and harbor potent anti-tumor activities. We have engineered an induced pluripotent stem cells (iPSCs) derived, NK cell (termed FT538) that express 1) a high-affinity non-cleavable CD16 Fc receptor to facilitate antibody engagement, 2) a membrane-bound IL-15/IL-15 receptor fusion to enhance NK activity and persistence and 3) a knock-out of CD38 to enhance NK metabolic fitness. FT538 is produced in a GMP-compliant manner as an off-the-shelf immunotherapy. Clinical experiences with FT538 have shown evidence of durable anti-tumor immunity after single administration in select lymphoma patients. METHODS We performed pre-clinical studies to characterize the tumoricidal activity of FT573 against glioblastomas. RESULTS Without specific antibody to engage CD16, FT538 exhibited potent in vitro activity against ~80% of a panel of isocitrate dehydrogenase wild-type patient-derived xenograft (PDX) glioblastoma lines, including MGG8. When directly injected into the murine brain, 50% of the FT538 persisted for > 2 weeks without addition of exogenous cytokine. Moreover, the mice exhibited no evidence of neurologic compromise, and brains harvested after injection show no signs of FT538 activation. In vivo intratumoral injection of FT538 into an orthotopically established MGG8 glioblastoma caused a ~20 fold increase in granzyme B release by FT538, with a comparable increase of cleaved-caspase 3 in the tumor. In independent experiments, > 90% of mice orthotopically implanted MGG8 survived beyond 100 days after intra-tumoral FT538 while all mice treated with mock injection died by 60 days. For glioblastoma PDX refractory to FT538, such as MGG4, tumoricidal activity of FT583 can be induced in vitro and in vivo by the addition of a tri-specific engager targeting B7H3, a surface protein highly expressed in glioblastomas. CONCLUSION Our results demonstrate great promise of FT538 as a glioblastoma therapy, with plans for translation into a first-in-human clinical trial.

Precision medicine in oncology leverages clinical observations of exceptional response. Toward an understanding of the molecular features that define this response, we applied an integrated, multiplatform analysis of RNA profiles derived from clinically annotated glioblastoma samples. This analysis suggested that specimens from exceptional responders are characterized by decreased accumulation of microglia/macrophages in the glioblastoma microenvironment. Glioblastoma-associated microglia/macrophages secreted interleukin 11 (IL11) to activate STAT3-MYC signaling in glioblastoma cells. This signaling induced stem cell states that confer enhanced tumorigenicity and resistance to the standard-of-care chemotherapy, temozolomide (TMZ). Targeting a myeloid cell restricted an isoform of phosphoinositide-3-kinase, phosphoinositide-3-kinase gamma isoform (PI3Kγ), by pharmacologic inhibition or genetic inactivation disrupted this signaling axis by reducing microglia/macrophage-associated IL11 secretion in the tumor microenvironment. Mirroring the clinical outcomes of exceptional responders, PI3Kγ inhibition synergistically enhanced the anti-neoplastic effects of TMZ in orthotopic murine glioblastoma models. Moreover, inhibition or genetic inactivation of PI3Kγ in murine glioblastoma models recapitulated expression profiles observed in clinical specimens isolated from exceptional responders. Our results suggest key contributions from tumor-associated microglia/macrophages in exceptional responses and highlight the translational potential for PI3Kγ inhibition as a glioblastoma therapy.

Cancer-associated fibroblasts (CAFs) are activated fibroblasts constituting the major stromal components in many types of cancer. CAFs contribute to hallmarks of cancer such as proliferation, invasion and immunosuppressive tumor microenvironment, and are associated with poor prognosis of patients with cancer. However, in glioblastoma (GBM), the most common and aggressive primary malignant brain tumor, our knowledge about CAFs or CAF-like stromal cells is limited. Here, using commonly accepted CAF markers, we characterized CAF-like cell populations in clinical glioma specimens and datasets along with mouse models of GBM. We found that tumor-associated pericytes marked by co-expression of fibroblast activation protein α (FAP) and PDGFRβ represent major stromal cell subsets in both human GBM and mouse GBM models, while a fraction of mesenchymal neoplastic cells also express FAP in patient tumors. Since oncolytic viruses can kill cancer cells and simultaneously modulate the tumor microenvironment by impacting non-neoplastic populations such as immune cells and tumor vasculature, we further investigated the ability of oncolytic viruses to target GBM-associated stromal cells. An oncolytic adenovirus, ICOVIR15, carrying ∆24-E1A and an RGD-fiber, infects and depletes FAP+ pericytes as well as GBM cells in murine GBM. Our study thus identifies FAP+/PDGFRβ+ pericytes as a major CAF-like stromal cell population in GBM, and highlights the unique property of this oncolytic adenovirus to target both GBM cells and GBM-associated stromal FAP+ cells.

Background Recurrence after radiation therapy is nearly universal for glioblastomas, the most common form of adult brain cancer. The study aims to define clinically pertinent mechanisms underlying this recurrence. Methods microRNA (miRNA) profiling was performed using matched pre- and post-radiation treatment glioblastoma specimens from the same patients. All specimens harbored unmethylated O6-methylguanine-DNA methyltransferase promoters (umMGMT) and wild-type isocitrate dehydrogenase (wtIDH). The most altered miRNA, miR-603, was characterized. Findings While nearly all miRNAs remained unchanged after treatment, decreased levels of few, select miRNAs in the post-treatment specimens were observed, the most notable of which involved miR-603. Unbiased profiling of miR-603 targets revealed insulin-like growth factor 1 (IGF1) and IGF1 receptor (IGF1R). Ionizing radiation (IR) induced cellular export of miR-603 through extracellular vesicle (EV) release, thereby de-repressing IGF1 and IGF1R. This de-repression, in turn, promoted cancer stem-cell (CSC) state and acquired radiation resistance in glioblastomas. Export of miR-603 additionally de-repressed MGMT, a DNA repair protein responsible for detoxifying DNA alkylating agents, to promote cross-resistance to these agents. Ectopic miR-603 expression overwhelmed cellular capacity for miR-603 export and synergized with the tumoricidal effects of IR and DNA alkylating agents. Interpretation Profiling of matched pre- and post-treatment glioblastoma specimens revealed altered homeostasis of select miRNAs in response to radiation. Radiation-induced EV export of miR-603 simultaneously promoted the CSC state and up-regulated DNA repair to promote acquired resistance. These effects were abolished by exogenous miR-603 expression, suggesting potential for clinical translation. Funding NIH 1R01NS097649-01, 9R44GM128223-02, 1R01CA240953-01, the Doris Duke Charitable Foundation Clinical Scientist Development Award, The Sontag Foundation Distinguished Scientist Award, the Kimmel Scholar Award, and BWF 1006774.01 (C.C.C).

Glioblastoma multiforme (GBM) is among the most aggressive, treatment resistant cancers, and despite standard of care surgery, radiation and chemotherapy, is invariably fatal. GBM is marked by local and systemic immunosuppression, contributing to resistance to existing immunotherapies that have had success in other tumor types. Memory T cells specific for previous infections reside in tissues throughout the host, including the brain, and are capable of rapid and potent immune activation. Here, we show that virus-specific memory CD8+ T cells expressing tissue resident markers populate the mouse and human glioblastoma microenvironment. Reactivating virus-specific memory T cells through intra-tumoral delivery of adjuvant-free virus-derived peptide triggered local immune activation. This delivery translated to anti-neoplastic effects, which improved survival in a murine glioblastoma model. Our results indicate that virus-specific memory T cells are a significant part of the glioblastoma immune microenvironment and may be leveraged to promote anti-tumoral immunity. Supported by UMN SPORE Program Project Planning grant (DM, CCC), NCI 1R01CA238439 (DM), Humor to Fight the Tumor Foundation (JN), NCI 5P30CA023108-42 (PR)

INTRODUCTION Neuro-cognitive decline is near universal in glioblastoma patients and negatively impacts the quality of life for afflicted patients. Yet, there is little information on longitudinal effects of brain tumor growth on cerebral cortical function and network connectivity. OBJECTIVE To address this knowledge gap, we examined in vivo Ca2+ imaging in a transgenic murine glioblastoma model. METHODS Mesoscopic Ca2+ imaging was performed after implant of GL261 glioblastoma cells into a transgenic mice strain (Thy1-GCaMP6f) that expresses the fast calcium indicator GCaMP6f in Layer II/III and Layer V pyramidal neurons. Independent component analysis (ICA), correlation matrix and graph theory approaches were used to assess changes in network connectivity. RESULTS ICA defined canonical cerebral network consisting of nodal convergence and connectivity between nodes. The overall network structure remained unaltered after tumor implant. A decrease in the strength of connectivity was observed immediately following tumor implant. This temporary suppression was followed by progressive, global increase in the strength of nodal connectivity (p < 0.0001). By two weeks post-tumor implant, 50% of the nodes exhibited increased connectivity compared to baseline. Progressive activation of select nodes was also observed in the weeks following tumor implant (p < 0.01). In aggregate, these results suggest that activation of select network nodes as well as enhanced connectivity as means to compensate for the deleterious effects of glioblastoma growth. CONCLUSIONS Our results indicate that focal brain tumor growth induces a reorganization of both local and remote cortical activity. The finding bears pertinence to the pathogenesis of neuro-cognitive decline and tumor-associated epilepsy.

INTRODUCTION Exceptional responders refer to the rare subset of patients who derive extraordinary benefits from a therapy compared to most treated patients. Understanding the basis for such responses represents a key pillar in the framework of precision medicine. OBJECTIVE Here, we examine the molecular basis underlying exceptional responses in glioblastoma patients who underwent concurrent radiation and temozolomide (TMZ) therapy. METHODS mRNA profiles derived from clinically annotated glioblastoma specimens were analyzed by Single sample gene set enrichment (ssGSEA) and Gene Cluster Expression Summary Score (GCESS). Key results were confirmed using cell-based assays and murine glioblastoma models. RESULTS Integrated, multi-platform analyses of RNA profiles derived from clinically annotated glioblastoma samples revealed decreased accumulation of microglia/macrophages in the tumor microenvironment of exceptional responders. Further analysis revealed that glioblastoma-associated microglia/macrophages secreted interleukin 11 (IL11) activate a STAT3-MYC signaling axis in glioblastoma cells. This signaling facilitated glioblastoma transition into stem cell states that confer enhanced tumorigenicity as well as resistance to the TMZ. Pharmacologic inhibition or genetic inactivation of a myeloid cell restricted isoform of PI3K, PI3K-gamma, disrupted this signaling axis by suppressing microglia/macrophage accumulation and IL11 secretion in the glioblastoma microenvironment. Mirroring the clinical outcome of exceptional responders, PI3K-gamma inhibition synergistically enhanced the anti-neoplastic effects of TMZ in orthotopic murine glioblastoma models. Moreover, inhibition or genetic inactivation of PI3K-gamma in murine glioblastoma models recapitulated expression profiles observed in clinical specimens isolated from exceptional responders. CONCLUSIONS Our results suggest key contributions from tumor-associated microglia/macrophages in exceptional responses and highlight the translational potential for PI3K-gamma inhibition as a glioblastoma therapy.

Background Gliomas represent the most common brain tumors within the central nervous system, with glioblastoma being the most aggressive type.1 Conventional treatment combines several approaches including surgery, chemotherapy, and radiation.2 However, the prognosis for glioblastoma remains unfavorable, with only 5% of patients surviving more than 5 years post-diagnosis.3 Thus, new treatment approaches are urgently needed. Natural killer (NK) cells directly lyse malignantly transformed or virally infected cells and secrete inflammatory cytokines that polarize cytotoxic immunity. Allogeneic NK cell adoptive transfer has shown clinical benefit in patients with advanced cancer.4–7 However, limitations of this approach include relatively low numbers of donor NK cells that can be isolated during an apheresis and variability in the quality of NK cells between donors. To overcome these limitations, we have developed a GMP manufacturing strategy to mass produce NK cells from induced pluripotent stem cells (iPSCs) as an approach to off-the-shelf cancer immunotherapy. We refer to these cells as ‘iNK’ (iPSC-derived NK) cells. Here, we provide preclinical data demonstrating the efficacy of iNK cells for immunotherapy against glioblastoma. Methods We generated iNK cells using previously published methods.8–10 iNK cells were used as effectors against an array of patient-derived glioblastoma lines in 2-dimensional live imaging IncuCyte assays where iNK cell-mediated killing was observed over the course of 48 hours. To investigate iNK cell infiltration and cytotoxicity in a more physiological context that accounts for the 3-dimensional architecture of the tumor, we also performed live imaging IncuCyte assays using iNK cells as effectors against glioblastoma spheroids. To test the anti-tumor function of iNK cells in vivo, we implanted patient-derived glioblastoma cells into mice via intracranial injection. Seven days later, 5 mice received intratumoral injections of iNK cells, and 5 mice received vehicle alone (as a control; figure 1A). All mice were monitored for weight and survival over 100 days. Results iNK cells exhibited strong and sustained cytotoxicity against 6 primary patient-derived mesenchymal glioblastoma lines in 2-dimensional IncuCyte assays and complete infiltration and destruction of glioblastoma spheroids in 3-dimensional IncuCyte assays. In xenogeneic adoptive transfer experiments, all mice receiving intratumoral injections of iNK cells survived out to day 100, while all mice in the vehicle group became moribund and had to be sacrificed by day 60 (figure 1B). Conclusions iNK cells are highly cytotoxic against glioblastoma cells, and our preclinical in vivo data provides proof-of-concept for future clinical trials. Ethics Approval This project has been approved by the University of Minnesota IACUC. Approval ID: 1812-36595A References Louis D N, Perry A, Reifenberger G, von Deimling A, Figarella-Branger D, Cavenee W K, Ohgaki H, Wiestler O D, Kleihues P, Ellison D W. The 2016 world health organization classification of tumors of the central nervous system: a summary. Acta Neuropathol 2016;131:803–820. Stupp R, Mason W P, van den Bent M J, Weller M, Fisher B, Taphoorn M J B, Belanger K, Brandes A A, Marosi C, Bogdahn U, Curschmann J, Janzer R C, Ludwin S K, Gorlia T, Allgeier A, Lacombe D, Cairncross J G, Eisenhauer E, Mirimanoff R O, European Organization for Research and Treatment of Cancer Brain Tumor and Radiotherapy Groups; National Cancer Institute of Canada Clinical Trials Group. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 2005;352:987–996. Thakkar JP, Dolecek TA, Horbinski C, Ostrom QT, Lightner DD, Barnholz-Sloan JS, Villano JL. Epidemiologic and molecular prognostic review of glioblastoma. Cancer Epidemiol Biomarkers Prev 2017;23:1985–1996. Miller J S, Soignier Y, Panoskaltsis-Mortari A, McNearney S A, Yun G H, Fautsch S K, McKenna D, Le C, Defor T E, Burns L J, Orchard P J, Blazar B R, Wagner J E, Slungaard A, Weisdorf D J, Okazaki J, McGlave P B. Successful adoptive transfer and in vivo expansion of human haploidentical NK cells in patients with cancer. Blood 2005;105:3051–3057. Bachanova V, Cooley S, Defor T E, Verneris M R, Zhang B, McKenna D H, Curtsinger J, Panoskaltsis-Mortari A, Lewis D, Hippen K, McGlave P, Weisdorf D J, Blazar B R, Miller J S. Clearance of acute myeloid leukemia by haploidentical natural killer cells is improved using IL-2 diphtheria toxin fusion protein. Blood 2014;123:3855. Ciurea S O, Schafer J R, Bassett R, Denman C J, Cao K, Willis D, Rondon G, Chen J, Soebbing D, Kaur I, Gulbis A, Ahmed S, Rezvani K, Scpall E J, Lee D A, Champlin R E. Phase 1 clinical trial using mbIL21 ex vivo-expanded donor-derived NK cells after haploidentical transplant. Blood 2017;130:1857–1868. Romee R, Rosario M, Berrien-Elliott M M, Wagner J A, Jewell B A, Schappe T, Leong J W, Abdel-Latif S, Schneider S E, Willey S, Neal C C, Yu L, Oh T, Lee S, Mulder A, Cooper M A, Fehniger T A. Cytokine-induced memory-like natural killer cells exhibit enhanced responses against myeloid leukiemia. Sci Transl Med2016:8;375ra123. Valamehr B, Abujarour R, Robinson M, Le T, Robbins D, Shoemaker D, Flynn P. A novel platform to enable the high-throughput derivation and characterization of feeder-free human iPSCs. Sci Rep2012:2;213. Valamehr B, Robinson M, Abujarour R, Rezner B, Vranceanu F, Le T, Medcalf A, Lee T T, Fitch M, Robbins D, Flynn P. Platform for induction and maintenance of transgene-free hiPSCs resembling ground state pluripotent stem cells. Stem Cell Reports 2014;2:366–381. Zhu H, Blum R H, Bjordahl R, Gaidarova S, Rogers P, Lee T T, Abujarour R, Bonello G B, Wu J, Tsai P-F, Miller J S, Walcheck B, Valamehr B, Kaufman D S. Pluripotent stem cell-derived NK cells with high-affinity noncleavable CD16a mediate improved antitumor immunity. Blood 2020;135:399–410.

Oncolytic viruses (OVs) have been engineered to selectively replicate in cancer cells. While initially thought to exert its anti-cancer effects through direct cytolysis, it is increasingly appreciated that OVs interact with a multitude of cellular processes during its life cycle; FDA approved pharmacologic agents that modulate these cellular processes have been shown to augment the anti-neoplastic effects of OVs. Moreover, because of the release of tumor antigens as well as the innate immuno-stimulatory nature of viruses, OVs induce potent immune responses that augment the anti-tumor effects of FDA approved immunotherapies. There is mounting interest in OV as a platform for combinational anti-cancer therapy in this context.We will review pre-clinical and clinical data that demonstrate proof-of-principle and potential efficacy for OV-based combination therapies with FDA approved anti-cancer agents.While the cytolytic activity of OV remains a key driver for its anti-neoplastic effects, understanding the virus-host interactions may afford opportunities for potential synergism with FDA approved therapeutics that target these interactions. Most intriguingly, the immune stimulatory effects of OVs renders combination with FDA approved immunotherapies more potent. While there are growing clinical trials employing such combination therapy, meaningful advances in this paradigm will require improved understanding of virus-host interactions.

In response to a variety of cellular stresses, poly(ADP-ribose) polymerase 1 (PARP1) has vital roles in orchestrating DNA damage repair and preserving genomic integrity. Clinical activity of PARP inhibitors (PARPis) in BRCA1/2 mutant cancers validated the concept of synthetic lethality between PARP inhibition and deleterious BRCA1/2 mutations, leading to clinical approval of several PARPis. Preclinical and clinical studies aiming to broaden the therapeutic application of PARPis identified sensitivity biomarkers and rationale combination strategies that can target BRCA wild-type and homologous recombination (HR) DNA repair-proficient cancers, including central nervous system (CNS) malignancies. In this review, we summarize recent progress in PARPi therapy in brain tumors, and discuss current opportunities for, and challenges to, the use of PARPis in neuro-oncology.

Novel therapies to override chemo-radiation resistance in prostate cancer (PCa) are needed. Prostate cancer sphere-forming cells (PCSCs) (also termed prostate cancer stem-like cells) likely participate in tumor progression and recurrence, and they are important therapeutic targets. We established PCSC-enriched spheres by culturing human (DU145) and murine (TRAMP-C2) PCa cells in growth factor-defined serum-free medium, and we characterized stem-like properties of clonogenicity and tumorigenicity. The efficacy of two different oncolytic herpes simplex viruses (oHSVs) (G47Δ and MG18L) in PCSCs was tested alone and in combination with radiation; chemotherapy; and inhibitors of phosphoinositide 3-kinase (PI3K), Wnt, and NOTCH in vitro; and, G47Δ was tested with the PI3K inhibitor BKM120 in a PCSC-derived tumor model in vivo. PCSCs were more tumorigenic than serum-cultured parental cells. Human and murine PCSCs were sensitive to oHSV and BKM120 killing in vitro, while the combination was synergistic. oHSV combined with radiation, docetaxel, Wnt, or NOTCH inhibitors was not. In athymic mice bearing DU145 PCSC-derived tumors, the combination of intra-tumoral G47Δ and systemic BKM120 induced complete regression of tumors in 2 of 7 animals, and it exhibited superior anti-tumor activity compared to either monotherapy alone, with no detectable toxicity. oHSV synergizes with BKM120 in killing PCSCs in vitro, and the combination markedly inhibits tumor growth, even inducing regression in vivo. Keywords: prostate cancer, oncolytic virus, herpes virus, G47Δ, MG18L, prostate cancer stem-like cells, PI3K inhibitor, BKM120, TrampC2, DU145

Purpose: Previous studies have shown that the PI3K/Akt/mTOR pathway is activated in up to 70% of breast cancer brain metastases, but there are no approved agents for affected patients. GDC-0084 is a brain penetrant, dual PI3K/mTOR inhibitor that has shown promising activity in a preclinical model of glioblastoma. The aim of this study was to analyze the efficacy of PI3K/mTOR blockade in breast cancer brain metastases models. Experimental Design: The efficacy of GDC-0084 was evaluated in PIK3CA-mutant and PIK3CA wild-type breast cancer cell lines and the isogenic pairs of PIK3CA wild-type and mutant (H1047R/+) MCF10A cells in vitro. In vitro studies included cell viability and apoptosis assays, cell-cycle analysis, and Western blots. In vivo, the effect of GDC-0084 was investigated in breast cancer brain metastasis xenograft mouse models and assessed by bioluminescent imaging and IHC. Results: In vitro, GDC-0084 considerably decreased cell viability, induced apoptosis, and inhibited phosphorylation of Akt and p70 S6 kinase in a dose-dependent manner in PIK3CA-mutant breast cancer brain metastatic cell lines. In contrast, GDC-0084 led only to growth inhibition in PIK3CA wild-type cell lines in vitro. In vivo, treatment with GDC-0084 markedly inhibited the growth of PIK3CA-mutant, with accompanying signaling changes, and not PIK3CA wild-type brain tumors. Conclusions: The results of this study suggest that the brain-penetrant PI3K/mTOR targeting GDC-0084 is a promising treatment option for breast cancer brain metastases with dysregulated PI3K/mTOR signaling pathway conferred by activating PIK3CA mutations. A national clinical trial is planned to further investigate the role of this compound in patients with brain metastases.

INTRODUCTION Neuro-cognitive decline is near universal in glioblastoma patients and negatively impacts the quality of life for afflicted patients. Yet, there is little information on longitudinal effects of brain tumor growth on cerebral cortical function and network connectivity. OBJECTIVE To address this knowledge gap, we examined in vivo Ca2+ flux imaging in a transgenic murine glioblastoma model. METHODS Mesoscopic Ca2+ imaging was performed after implant of GL261 glioblastoma cells into a transgenic mice strain (Thy1-GCaMP6f) that expresses the fast calcium indicator GCaMP6f in Layer II/III and Layer V pyramidal neurons. Independent component analysis (ICA), correlation matrix and graph theory approaches were used to assess changes in network connectivity. RESULTS ICA defined canonical cerebral network consisting of nodal convergence and connectivity between nodes. The overall network structure remained unaltered after tumor implant. A decrease in the strength of connectivity was observed immediately following tumor implant. This temporary suppression was followed by progressive, global increase in the strength of nodal connectivity (p < 0.0001). By two weeks post-tumor implant, 50% of the nodes exhibited increased connectivity compared to baseline. Progressive activation of select nodes was also observed in the weeks following tumor implant (p < 0.01). In aggregate, these results suggest that activation of select network nodes as well as enhanced connectivity as means to compensate for the deleterious effects of glioblastoma growth. CONCLUSIONS Our results indicate that focal brain tumor growth induces a reorganization of both local and remote cortical activity. The finding bear pertinence to the pathogenesis of neuro-cognitive decline and tumor associated epilepsy.

INTRODUCTION Release of exosomes and extracellular vesicles (EV) by glioblastomas plays key roles in modulating the tumor microenvironment and therapeutic response. Studies to date have largely focused on the impact of EV and exosomes on the recipient cell. Here, we demonstrate that EV-mediated export of a master-regulatory miRNA has fate-determining impacts on the cell releasing the EVs. METHODS microRNA (miRNA) profiling was performed using clinical glioblastoma specimens from the same patients derived pre- and post-standard of care treatment. Mechanism mediating altered miRNA homeostasis were assessed. RESULTS While the levels of nearly all miRNAs remained unchanged after standard-of-care treatment, decreased levels of few, select miRNAs were observed, including miR-603. In response to ionizing radiation (IR), but not temozolomide (TMZ), glioblastoma cell lines exhibited a time-dependent decrease in miR-603 levels. While miR-603 biogenesis and degradation remained unchanged after IR, IR induced an increase in EV-mediated export of miR-603. Profiling of miR-603 targets revealed that miR-603 repressed the insulin-like growth factor 1 (IGF1) and IGF1 receptor (IGF1R), genes required to maintain the cancer stem cell (CSC) state, as well as MGMT, the gene encoding a DNA repair protein that detoxifies temozolomide (TMZ) induced DNA damages. IR induced export of miR-603 de-repress IGF1/IGF1R to promote radiation resistance and de-repress MGMT to promote cross-resistance to TMZ and CCNU. Ectopic miR-603 expression overwhelmed cellular capacity for miR-603 export and synergized with the tumoricidal effects of IR and temozolomide (TMZ). CONCLUSIONS Radiation stimulated EV-mediated export of miR-603 to facilitate acquired resistance to IR and cross-resistance to DNA alkylating agents.

Overcoming the immunosuppressive tumor microenvironment remains a major impediment to successful cancer immunotherapy. Virus-specific memory T cells are positioned throughout the entire body to sense reinfection or recrudescence. Mouse models have demonstrated upon reencountering cognate antigen, these tissue resident memory T cells (TRM) induce a local immunostimulatory environment that activates and recruits innate and adaptive arms of the immune system, and we extend these functions to include recruitment of circulating antibody. Like healthy tissue, we observe that mouse and human tumors are commonly surveyed by virus-specific memory CD8+ T cells. This was seen in a range of tumor types including traditionally ‘immune-privilege’ tissues such as glioblastoma. Given the described immunostimualtory functions of antiviral TRM in healthy tissue, we tested if we could leverage antiviral CD8+ T cells in tumors as an immunotherapy. Local delivery of adjuvant-free peptide derived from previously encountered viruses successfully reactivated antiviral T cells within melanoma and glioblastoma tumors. This arrested growth of checkpoint blockade-resistant and poorly immunogenic tumors in mice. Antiviral T cell reactivation triggered antigen presentation and cytotoxic pathways within the tumor, activating T cells, dendritic cells and natural killer cells. Viral peptide treatment of ex vivo human tumors demonstrated upregulation of immune activation gene expression profiles similar to those observed in mice. Lastly, viral peptide therapy renders resistant mouse tumors susceptible to PD-L1 blockade. Thus, re-stimulating known antiviral immunity may provide a novel therapeutic approach for a broad range of tumors.

Background Clinical success of poly(ADP-ribose) polymerase inhibitors (PARP i ) has been limited to repair-deficient cancers and by resistance. Oncolytic herpes simplex viruses (oHSVs) selectively kill cancer cells, irrespective of mutation, and manipulate DNA damage responses (DDR). Here, we explore potential synthetic lethal-like interactions between oHSV and PARP i . Methods The efficacy of combining PARP i , oHSV MG18L, and G47Δ in killing patient-derived glioblastoma stem cells (GSCs) was assessed using cell viability assays and Chou-Talalay synergy analysis. Effects on DDR pathways, apoptosis, and cell cycle after manipulation with pharmacological inhibitors and lentivirus-mediated knockdown or overexpression were examined by immunoblotting and FACS. In vivo efficacy was evaluated in two GSC-derived orthotopic xenograft models (n = 7-8 per group). All statistical tests were two-sided. Results GSCs are differentially sensitive to PARP i despite uniform inhibition of PARP activity. oHSV sensitized GSCs to PARP i , irrespective of their PARP i sensitivity through selective proteasomal degradation of key DDR proteins; Rad51, mediating the combination effects; and Chk1. Rad51 degradation required HSV DNA replication. This synthetic lethal-like interaction increased DNA damage, apoptosis, and cell death in vitro and in vivo. Combined treatment of mice bearing PARP i -sensitive or -resistant GSC-derived brain tumors greatly extended median survival compared to either agent alone (vs olaparib: P ≤.001; vs MG18L: P = .005; median survival for sensitive of 83 [95% CI = 77 to 86], 94 [95% CI = 75 to 107], 102 [95% CI = 85 to 110], and 131 [95% CI = 108 to 170] days and for resistant of 54 [95% CI = 52 to 58], 56 [95% CI = 52 to 61], 62 [95% CI = 56 to 72], and 75 [95% CI = 64 to 90] days for mock, PARPi, oHSV, and combination, respectively). Conclusions The unique oHSV property to target multiple components of DDR generates cancer selective sensitivity to PARP i . This combination of oHSV with PARP i is a new anticancer strategy that overcomes the clinical barriers of PARP i resistance and DNA repair proficiency and is applicable not only to glioblastoma, an invariably lethal tumor, but also to other tumor types.

Penaeidin-2 (Pen-2) is an important antimicrobial peptide derived from the Pacific white shrimp, Penaeus vannamei, and possesses both antibacterial and antifungal activities. Recent studies suggest that recombinant penaeidins show similar activities to the native Pen-2 protein. Previous researches have shown that some antimicrobial peptides (AMPs) exhibit cytotoxic activity against cancer cells. To date, there have been no studies on the antitumor effects of Pen-2. This study evaluated the potential of recombinant pen-2 (rPen-2) in the selective killing of kidney cancer cell lines ACHN and A498, and its action mechanism. MTT assays found the maximal growth inhibition of HK-2, ACHN and A498 cells treated with 100 μg/mL rPen-2 at 48 h was 13.2%, 62.4%, and 70.4%, respectively. DNA-specific fluorescent dye staining showed a high percentage of apoptosis on cancer cells. Flow cytometry revealed that the apoptosis rate of HK-2, ACHN and A498 cells was 15.2%, 55.2%, and 61.5% at 48 h respectively, suggesting that rPen-2 induced higher apoptosis rate in cancer cells than in HK-2 cells. Laser confocal scanning microscopy demonstrated that the plasma membrane was the key site where rPen-2 interacted with and destroyed tumor cells. Scanning electron microscopy showed the morphologic changes of the cell membranes of kidney cancer cells treated with rPen-2. These results suggest that rPen-2 is a novel potential therapeutic agent that may be useful in treating kidney cancers.

Glioblastoma (World Health Organization grade IV) is an aggressive adult brain tumor that is inevitably fatal despite surgery, radiation, and chemotherapy. Treatment failures are attributed to combinations of cellular heterogeneity, including a subpopulation of often-resistant cancer stem cells, aberrant vasculature, and noteworthy immune suppression. Current preclinical models and treatment strategies do not incorporate or address all these features satisfactorily. Herein, we describe a murine glioblastoma stem cell (GSC) model that recapitulates tumor heterogeneity, invasiveness, vascularity, and immunosuppressive microenvironment in syngeneic immunocompetent mice and should prove useful for a range of therapeutic studies. Using this model, we tested a genetically engineered oncolytic herpes simplex virus that is armed with an immunomodulatory cytokine, interleukin 12 (G47∆-mIL12). G47Δ-mIL12 infects and replicates similarly to its unarmed oncolytic herpes simplex virus counterpart in mouse 005 GSCs in vitro, whereas in vivo, it significantly enhances survival in syngeneic mice bearing intracerebral 005 tumors. Mechanistically, G47∆-mIL12 targets not only GSCs but also increases IFN-γ release, inhibits angiogenesis, and reduces the number of regulatory T cells in the tumor. The increased efficacy is dependent upon T cells, but not natural killer cells. Taken together, our findings demonstrate that G47Δ-mIL12 provides a multifaceted approach to targeting GSCs, tumor microenvironment, and the immune system, with resultant therapeutic benefit in a stringent glioblastoma model.

White spot syndrome virus is a highly pathogenic virus that infects crayfish and other crustaceans. VP28 is one of its major envelope proteins, and plays a crucial role in viral infection. Cell-penetrating peptides are short peptides that facilitate cellular uptake of various molecular cargoes, and one well known example is TAT peptide from HIV-1 TAT protein. In this study, recombinant plasmids were constructed and transformed into Escherichia coli strain BL21 (DE3) to express TAT-VP28, VP28, TAT-VP28-EGFP and VP28-EGFP fusion proteins. Enzyme-linked immunosorbent assay (ELISA) and flow cytometry methods were used to confirm that TAT fusion proteins can translocate from the intestine to the hemolymph of the crayfish Cambarus clarkii. After immunization, activities of phenoloxidase and superoxide dismutase were analyzed, and it was found that rTAT-VP28 produced the most pronounced increase in both C. clarkii were vaccinated by oral administration of rTAT-VP28 and rVP28 for 7 and 14 days, and rTAT-VP28 resulted in the highest relative percent survival (RPS) (63.3% at 7 days, and 67.8% at 14 days), compared with rVP28 (44.4% at 7 days, and 53.6% at 14 days) following challenge with WSSV after the last day of feeding. This study reports the use of TAT-derived peptide as an oral delivery method of a subunit vaccine against WSSV in C. clarkii.

Breast cancer is the second most common primary tumor leading to brain metastases in adult cancer patients. Previous studies have shown that the PI3K/AKT/mTOR pathway in breast cancer brain metastasis is activated in up to 70% of analyzed tumors. However, there are no approved agents targeting this pathway in breast cancer brain metastases at present. GDC-0084 is a dual brain penetrant PI3K/mTOR-inhibitor that has already demonstrated promising response rates in a preclinical glioblastoma model. The aim of this study was to analyze the efficacy of this compound in a breast cancer brain metastases model in vitro and in vivo using PIK3CA-mutant and PIK3CA-wildtype cell lines. In vitro methods included cell viability, apoptosis, growth inhibition assays, cell cycle analysis, Western blots and immunohistochemistry. In vivo, the effect of GDC-0084 was evaluated in an orthotopic intracranial mouse model with bioluminescent imaging. GDC-0084 induced apoptosis in PIK3CA-mutant breast cancer brain metastases cell lines and growth inhibition in PIK3CA-wildtype cell lines in vitro and markedly inhibited tumor growth of PIK3CA-mutant cell lines in vivo. The results of this study highlight the importance of brain-penetrant agents targeting the PI3K/AKT/mTOR-pathway and suggest that GDC-0084 might be a promising treatment option for breast cancer brain metastases patients in the future.

Brain tumors, solid neoplasms within the brain or the central spinal canal, can often be debilitating and difficult to treat. Glioblastoma (GBM) is the most aggressive and deadliest primary brain tumor in adults, not responding to current therapies and with a very low percentage of 5-year survivors. Therefore, the development of new treatments is critical. Gene therapy is a treatment strategy that relies on the delivery of genetic material into cells for therapeutic purposes, and has demonstrated increasing success in preclinical models and clinical trials. Within this strategy, we include selectively replication-competent oncolytic viruses. While the oncolytic viruses act directly as antitumor agents due to virus replication, the effects are due to the transfer of viral genetic material to the cells. GBM is a good target for gene therapy because tumors remain locally within the brain and only rarely metastasize to other tissues; the majority of cells in the brain are postmitotic, which allows for specific targeting of dividing tumor cells; and tumors can often be accessed neurosurgically for administration of therapy. This chapter will focus on GBM and describe current gene therapy strategies used for brain tumors, including cytotoxic, immune-stimulatory, tumor microenvironment disrupting gene therapy, oncolytic viruses, small RNA interference, and delivery systems encompassing viruses, stem cells, and nanotechnology, as well as current challenges and future directions.

PARP inhibitors (PARPi) have been used alone or in combination with other agents for the treatment of tumors with homologous repair (HR) deficiencies. However, challenges remain for the treatment of tumors that are PARPi-resistant or HR-proficient. Glioblastoma (GBM) is an invariably lethal tumor that is not associated with HR deficiencies. GBM stem cells (GSCs), thought to be critical for tumor growth and resistance to therapy, can be isolated from GBM specimens and are representative of the patient's tumors. GSCs exhibit variable PARPi sensitivity, with at least half being resistant. In order to enhance the antitumor efficacy of PARPi for GBM, we examined the combination of PARPi with inhibitors of oncogenic or DNA damage pathways, including inhibitors of PTEN, PI3K, ATM, ATR, and temozolomide, some of which have been reported to sensitize cancer cells to PARPi. Only inhibitors of ATR (VE821, VE822, AZ20) synergized with PARPi in killing GSCs in vitro. VE822 inhibited ATR activity both in vitro and in vivo, and modestly, but significantly, extended survival in mice bearing GSC-derived tumors. However, ATR inhibitor (ATRi) in combination with PARPi further prolonged survival, compared to each drug alone, even in mice bearing PARPi-resistant GSC-derived tumors. This is the first report that ATRi alone or in combination with PARPi is effective in treating GBM and provides a rationale for clinical trials for GBM. Citation Format: Jianfang Ning, Hiroaki Wakimoto, Robert L. Martuza, Samuel D. Rabkin. ATR inhibitors synergize with PARP inhibitors in killing glioblastoma stem cells and treating glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1122. doi:10.1158/1538-7445.AM2017-1122

Oncolytic viruses (OV) are a class of antitumor agents that selectively kill tumor cells while sparing normal cells. Oncolytic herpes simplex virus (oHSV) has been investigated in clinical trials for patients with the malignant brain tumor glioblastoma for more than a decade. These clinical studies have shown the safety of oHSV administration to the human brain, however, therapeutic efficacy of oHSV as a single treatment remains unsatisfactory. Factors that could hamper the anti-glioblastoma efficacy of oHSV include: attenuated potency of oHSV due to deletion or mutation of viral genes involved in virulence, restricting viral replication and spread within the tumor; suboptimal oHSV delivery associated with intratumoral injection; virus infection-induced inflammatory and cellular immune responses which could inhibit oHSV replication and promote its clearance; lack of effective incorporation of oHSV into standard-of-care, and poor knowledge about the ability of oHSV to target glioblastoma stem cells (GSCs). In an attempt to address these issues, recent research efforts have been directed at: (1) design of new engineered viruses to enhance potency, (2) better understanding of the role of the cellular immunity elicited by oHSV infection of tumors, (3) combinatorial strategies with different antitumor agents with a mechanistic rationale, (4) “armed” viruses expressing therapeutic transgenes, (5) use of GSC-derived models in oHSV evaluation, and (6) combinations of these. In this review, we will describe the current status of oHSV clinical trials for glioblastoma, and discuss recent research advances and future directions toward successful oHSV-based therapy of glioblastoma.

We have recently described a new murine model of glioblastoma, generated by the implantation of syngeneic glioblastoma stem cells into immunocompetent mice, that recapitulates the salient histopathological and immunological features of the human disease. We employed this model to demonstrate the multifaceted activity of an oncolytic herpes simplex virus genetically modified to express interleukin-12, G47∆-IL12.

Protective immune responses in shrimp induced by DNA vaccines against white spot syndrome virus (WSSV) with intramuscular injection have been reported in recent reports. In this study, we investigated the utilities of attenuated Salmonella enterica serovar Typhimurium (Salmonella typhimurium) as a bactofection vehicle for the oral delivery of a DNA vaccine plasmid to crayfish (Cambarus clarkii). The DNA vaccine plasmid pcDNA3.1-VP28, encoding viral envelope protein VP28, was transformed to an attenuated S. typhimurium strain SV4089 and the resulting recombinant bacteria named SV/pcDNA3.1-VP28 were used to orally immunize crayfish with coated feed. Successful delivery of the DNA vaccine plasmid was shown by the isolation of recombinant bacteria SV/pcDNA3.1-VP28 from the vaccinated crayfish. The distribution analysis of plasmid pcDNA3.1-VP28 in different tissues revealed the effective release of DNA vaccine plasmid into crayfish. RT-PCR and immunoflurescence results confirmed the expression of protein VP28 in the vaccinated crayfish. Challenge experiments with WSSV at 7, 15, 25 days post-vaccination demonstrated significant protection in immunized crayfish with relative survival rate 83.3%, 66.7% and 56.7%, respectively. Studies on stability and safety of SV/pcDNA3.1-VP28 showed the recombinant bacteria could exist in crayfish at least 7 days but not more than 10 days and without any observable harm to the host. Our study here demonstrates, for the first time, the ability of attenuated Salmonella as a live vector to orally deliver a DNA vaccine against WSSV into the arthropod crayfish and provides a new way to design more practical strategies for the control of WSSV and other invertebrate pathogens.

Glioblastoma (GBM) is an invariably fatal brain tumor in adults and remains a great challenge for preclinical and clinical research. PARP inhibitors (PARPi) have been used alone or in combination with genotoxic agents for the treatment of cancers with homologous recombination (HR) repair deficiencies. Oncolytic herpes simplex viruses (oHSVs) are genetically-engineered to selectively replicate in cancer cells, and previous studies have shown that oHSV infection of glioblastoma stem cells (GSC) induces DNA damage and compromises HR DNA repair. Therefore, we hypothesized that PARPi and oHSV would synergistically kill GSCs through inducing lethal DNA damage. Here, we show that PARPi consistently inhibited PARylation in a panel of patient-derived GSC lines. Surprisingly, half of GSCs (4 of 8) were sensitive to PARPi killing, with the other half being resistant. When combined with 2 different oHSVs, MG18L and G47Δ, PARPi olaparib and oHSV synergistically killed olaparib-sensitive GSCs in vitro. Moreover, olaparib sensitized PARPi-resistant GSCs to the cytotoxic effects of MG18L or G47Δ, leading to increased DNA damage and cell death. While monotherapy with systemic olaparib or intratumoral injection of MG18L increased survival in an orthotopic xenograft model generated with olaparib-sensitive GSCs, combination treatment greatly extended survival over either monotherapy alone, with concomitant induction of robust DNA damage and apoptosis in the tumors. Furthermore, in an orthotopic model with olaparib-resistant GSCs, in which olaparib monotherapy was not effective, combinatorial therapy of olaparib and MG18L was significantly more efficacious than MG18L alone in prolonging lifespan. Our studies demonstrate that PARPi have anti-GSC activity in about half of GBMs tested, and combining PARPi with oHSV targets DNA damage responses and induces a synthetic lethal effect in vitro and in vivo against both PARPi-sensitive and -resistant GSCs. This report describes a new therapeutic strategy for GBM and supports investigations of this combination in the clinic. Citation Format: Jianfang Ning, Hiroaki Wakimoto, Robert Martuza, Samuel Rabkin. PARP inhibitors sensitize glioblastoma stem cells to oncolytic herpes simplex virus therapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4227. doi:10.1158/1538-7445.AM2015-4227