Threonine-120 phosphorylation regulated by phosphoinositide-3-kinase/Akt and mammalian target of rapamycin pathway signaling limits the antitumor activity of mammalian sterile 20-like kinase 1.

Mst1/Stk4, a hippo-like serine-threonine kinase, is implicated in many cancers, including prostate cancer. However, the mechanisms regulating Mst1 remain obscure. Here, we characterized the effects of phospho-Thr-120 on Mst1 in prostate cancer cells. We demonstrated that phospho-Thr-120 did not alter the nuclear localization or cleavage of Mst1 in a LNCaP or castration-resistant C4-2 prostate tumor cell model, as revealed by a mutagenesis approach. Phospho-Thr-120 appeared to be specific to cancer cells and predominantly localized in the nucleus. In contrast, phospho-Thr-183, a critical regulator of Mst1 cell death, was exclusively found in the cytoplasm. As assessed by immunohistochemistry, a similar distribution of phospho-Mst1-Thr-120/Thr-183 was also observed in a prostate cancer specimen. In addition, the blockade of PI3K signaling by a small molecule inhibitor, LY294002, increased cytoplasmic phospho-Mst1-Thr-183 without having a significant effect on nuclear phospho-Mst1-Thr-120. However, the attenuation of mammalian target of rapamycin (mTOR) activity by a selective pharmacologic inhibitor, Ku0063794 or CCI-779, caused the up-regulation of nuclear phospho-Mst1-Thr-120 without affecting cytoplasmic phospho-Mst1-Thr-183. This suggests that PI3K and mTOR pathway signaling differentially regulate phospho-Mst1-Thr-120/Thr-183. Moreover, mutagenesis and RNAi data revealed that phospho-Thr-120 resulted in C4-2 cell resistance to mTOR inhibition and reduced the Mst1 suppression of cell growth and androgen receptor-driven gene expression. Collectively, these findings indicate that phospho-Thr-120 leads to the loss of Mst1 functions, supporting cancer cell growth and survival.

Gene therapy-mediated delivery of targeted cytotoxins for glioma therapeutics.

Restricting the cytotoxicity of anticancer agents by targeting receptors exclusively expressed on tumor cells is critical when treating infiltrative brain tumors such as glioblastoma multiforme (GBM). GBMs express an IL-13 receptor (IL13Rα2) that differs from the physiological IL4R/IL13R receptor. We developed a regulatable adenoviral vector (Ad.mhIL-4.TRE.mhIL-13-PE) encoding a mutated human IL-13 fused to Pseudomonas exotoxin (mhIL-13-PE) that specifically binds to IL13Rα2 to provide sustained expression, effective anti-GBM cytotoxicity, and minimal neurotoxicity. The therapeutic Ad also encodes mutated human IL-4 that binds to the physiological IL4R/IL13R without interacting with IL13Rα2, thus inhibiting potential binding of mhIL-13-PE to normal brain cells. Using intracranial GBM xenografts and syngeneic mouse models, we tested the Ad.mhIL-4.TRE.mhIL-13-PE and two protein formulations, hIL-13-PE used in clinical trials (Cintredekin Besudotox) and a second-generation mhIL-13-PE. Cintredekin Besudotox doubled median survival without eliciting long-term survival and caused severe neurotoxicity; mhIL-13-PE led to ∼40% long-term survival, eliciting severe neurological toxicity at the high dose tested. In contrast, Ad-mediated delivery of mhIL-13-PE led to tumor regression and long-term survival in over 70% of the animals, without causing apparent neurotoxicity. Although Cintredekin Besudotox was originally developed to target GBM, when tested in a phase III trial it failed to achieve clinical endpoints and revealed neurotoxicity. Limitations of Cintredekin Besudotox include its short half-life, which demanded frequent or continued administration, and binding to IL4R/IL13R, present in normal brain cells. These shortcomings were overcome by our therapeutic Ad, thus representing a significant advance in the development of targeted therapeutics for GBM.

A novel bicistronic high-capacity gutless adenovirus vector that drives constitutive expression of herpes simplex virus type 1 thymidine kinase and tet-inducible expression of Flt3L for glioma therapeutics.

Glioblastoma multiforme (GBM) is a deadly primary brain tumor. Conditional cytotoxic/immune-stimulatory gene therapy (Ad-TK and Ad-Flt3L) elicits tumor regression and immunological memory in rodent GBM models. Since the majority of patients enrolled in clinical trials would exhibit adenovirus immunity, which could curtail transgene expression and therapeutic efficacy, we used high-capacity adenovirus vectors (HC-Ads) as a gene delivery platform. Herein, we describe for the first time a novel bicistronic HC-Ad driving constitutive expression of herpes simplex virus type 1 thymidine kinase (HSV1-TK) and inducible Tet-mediated expression of Flt3L within a single-vector platform. We achieved anti-GBM therapeutic efficacy with no overt toxicities using this bicistronic HC-Ad even in the presence of systemic Ad immunity. The bicistronic HC-Ad-TK/TetOn-Flt3L was delivered into intracranial gliomas in rats. Survival, vector biodistribution, neuropathology, systemic toxicity, and neurobehavioral deficits were assessed for up to 1 year posttreatment. Therapeutic efficacy was also assessed in animals preimmunized against Ads. We demonstrate therapeutic efficacy, with vector genomes being restricted to the brain injection site and an absence of overt toxicities. Importantly, antiadenoviral immunity did not inhibit therapeutic efficacy. These data represent the first report of a bicistronic vector platform driving the expression of two therapeutic transgenes, i.e., constitutive HSV1-TK and inducible Flt3L genes. Further, our data demonstrate no promoter interference and optimum gene delivery and expression from within this single-vector platform. Analysis of the efficacy, safety, and toxicity of this bicistronic HC-Ad vector in an animal model of GBM strongly supports further preclinical testing and downstream process development of HC-Ad-TK/TetOn-Flt3L for a future phase I clinical trial for GBM.

Protective role of osteopontin in endodontic infection.

Endodontic infections are polymicrobial infections resulting in bone destruction and tooth loss. The host response to these infections is complex, including both innate and adaptive mechanisms. Osteopontin (OPN), a secreted, integrin-binding protein, functions in the regulation of immune responses and enhancement of leucocyte migration. We have assessed the role of OPN in the host response to endodontic infection using a well-characterized mouse model. Periapical bone loss associated with endodontic infection was significantly more severe in OPN-deficient mice compared with wild-type 3 weeks after infection, and was associated with increased areas of inflammation. Expression of cytokines associated with bone loss, interleukin-1alpha (IL-1alpha) and RANKL, was increased 3 days after infection. There was little effect of OPN deficiency on the adaptive immune response to these infections, as there was no effect of genotype on the ratio of bacteria-specific immunoglobulin G1 and G2a in the serum of infected mice. Furthermore, there was no difference in the expression of cytokines associated with T helper type 1/type2 balance: IL-12, IL-10 and interferon-gamma. In infected tissues, neutrophil infiltration into the lesion area was slightly increased in OPN-deficient animals 3 days after infection: this was confirmed by a significant increase in expression of neutrophil elastase in OPN-deficient samples at this time-point. We conclude that OPN has a protective effect on polymicrobial infection, at least partially because of alterations in phagocyte recruitment and/or persistence at the sites of infection, and that this molecule has a potential therapeutic role in polymicrobial infections.

Both cell-surface and secreted CSF-1 expressed by tumor cells metastatic to bone can contribute to osteoclast activation.

Tumors metastatic to the bone produce factors that cause massive bone resorption mediated by osteoclasts in the bone microenvironment. Colony stimulating factor (CSF-1) is strictly required for the formation and survival of active osteoclasts, and is frequently produced by tumor cells. Here we hypothesize that the CSF-1 made by tumor cells contributes to bone destruction in osteolytic bone metastases. We show that high level CSF-1 protected osteoclasts from suppressive effects of transforming growth factor beta (TGF-beta). r3T cells, a mouse mammary tumor cell line that forms osteolytic bone metastases, express abundant CSF-1 in vitro as both a secreted and a membrane-spanning cell-surface glycoprotein, and we show that both the secreted and the cell-surface form of CSF-1 made by r3T cells can support osteoclast formation in co-culture experiments in the presence of RankL. Mice with r3T bone metastases have elevated levels of both circulating and bone-associated CSF-1, and the majority of CSF-1 found in bone metastases is associated with the tumor cells. These results support the idea that tumor-cell produced CSF-1 contributes to osteoclast development and survival in bone metastasis.

HMGB1 mediates endogenous TLR2 activation and brain tumor regression.

BACKGROUND: Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor that carries a 5-y survival rate of 5%. Attempts at eliciting a clinically relevant anti-GBM immune response in brain tumor patients have met with limited success, which is due to brain immune privilege, tumor immune evasion, and a paucity of dendritic cells (DCs) within the central nervous system. Herein we uncovered a novel pathway for the activation of an effective anti-GBM immune response mediated by high-mobility-group box 1 (HMGB1), an alarmin protein released from dying tumor cells, which acts as an endogenous ligand for Toll-like receptor 2 (TLR2) signaling on bone marrow-derived GBM-infiltrating DCs. METHODS AND FINDINGS: Using a combined immunotherapy/conditional cytotoxic approach that utilizes adenoviral vectors (Ad) expressing Fms-like tyrosine kinase 3 ligand (Flt3L) and thymidine kinase (TK) delivered into the tumor mass, we demonstrated that CD4(+) and CD8(+) T cells were required for tumor regression and immunological memory. Increased numbers of bone marrow-derived, tumor-infiltrating myeloid DCs (mDCs) were observed in response to the therapy. Infiltration of mDCs into the GBM, clonal expansion of antitumor T cells, and induction of an effective anti-GBM immune response were TLR2 dependent. We then proceeded to identify the endogenous ligand responsible for TLR2 signaling on tumor-infiltrating mDCs. We demonstrated that HMGB1 was released from dying tumor cells, in response to Ad-TK (+ gancyclovir [GCV]) treatment. Increased levels of HMGB1 were also detected in the serum of tumor-bearing Ad-Flt3L/Ad-TK (+GCV)-treated mice. Specific activation of TLR2 signaling was induced by supernatants from Ad-TK (+GCV)-treated GBM cells; this activation was blocked by glycyrrhizin (a specific HMGB1 inhibitor) or with antibodies to HMGB1. HMGB1 was also released from melanoma, small cell lung carcinoma, and glioma cells treated with radiation or temozolomide. Administration of either glycyrrhizin or anti-HMGB1 immunoglobulins to tumor-bearing Ad-Flt3L and Ad-TK treated mice, abolished therapeutic efficacy, highlighting the critical role played by HMGB1-mediated TLR2 signaling to elicit tumor regression. Therapeutic efficacy of Ad-Flt3L and Ad-TK (+GCV) treatment was demonstrated in a second glioma model and in an intracranial melanoma model with concomitant increases in the levels of circulating HMGB1. CONCLUSIONS: Our data provide evidence for the molecular and cellular mechanisms that support the rationale for the clinical implementation of antibrain cancer immunotherapies in combination with tumor killing approaches in order to elicit effective antitumor immune responses, and thus, will impact clinical neuro-oncology practice.

PD-L1 checkpoint blockade delivered by retroviral replicating vector confers anti-tumor efficacy in murine tumor models.

Immune checkpoint inhibitors (CPIs) are associated with a number of immune-related adverse events and low response rates. We provide preclinical evidence for use of a retroviral replicating vector (RRV) selective to cancer cells, to deliver CPI agents that may circumvent such issues and increase efficacy. An RRV, RRV-scFv-PDL1, encoding a secreted single chain variable fragment targeting PD-L1 can effectively compete with PD-1 for PD-L1 occupancy. Cell binding assays showed trans-binding activity on 100% of cells in culture when infection was limited to 5% RRV-scFv-PDL1 infected tumor cells. Further, the ability of scFv PD-L1 to rescue PD-1/PD-L1 mediated immune suppression was demonstrated in a co-culture system consisting of human-derived immune cells and further demonstrated in several syngeneic mouse models including an intracranial tumor model. These tumor models showed that tumors infected with RRV-scFv-PD-L1 conferred robust and durable immune-mediated anti-tumor activity comparable or superior to systemically administered anti-PD-1 or anti PD-L1 monoclonal antibodies. Importantly, the nominal level of scFv-PD-L1 detected in serum is ∼50-150 fold less than reported for systemically administered therapeutic antibodies targeting immune checkpoints. These results support the concept that RRV-scFv-PDL1 CPI strategy may provide an improved safety and efficacy profile compared to systemic monoclonal antibodies of currently approved therapies.

Cell size increased in tissues from transgenic mice overexpressing a cell surface growth-related and cancer-specific hydroquinone oxidase, tNOX, with protein disulfide-thiol interchange activity.

tNOX (ENOX2), a cancer-specific and growth-related cell surface protein with protein disulfide-thiol interchange and hydroquinone (NADH) oxidase activities was overexpressed in a transgenic mouse model. Female transgenic mice grew faster than wild type as did embryonic fibroblast cells prepared from the transgenic mice. The tissue expression of tNOX mRNA was greatest in heart, lung and liver. When these tissues were analyzed for cell size, the cells from the tissues of transgenic animals were, on average, 20% larger in surface area than cells from corresponding wild-type tissues. Also analyzed were cells of intestine, spleen and kidney in which tNOX overexpression was observed but to a lesser extent. Cell size was increased as well with intestine and kidney but less so with spleen. At the end of the study, carcass weights of the transgenic animals were greater than those of wild type. This increase in carcass weight was reflected in an increase in femur weight and thickness in both male and female transgenic mice but not in femur length. Other carcass parameters such as skin weight and body fat or body fluids were unchanged or changes were insufficient to account for the increased carcass weight. The findings are consistent with the property of tNOX observed in studies with cultured cells as contributing to the enlargement phase of cell growth.

Efficient Therapeutic Protein Expression Using Retroviral Replicating Vector with 2A Peptide in Cancer Models.

Toca 511, a retroviral replicating vector (RRV), uses an internal ribosomal entry site (IRES) to express an optimized yeast cytosine deaminase (yCD2), which converts 5-fluorocytosine to 5-fluorouracil. This configuration is genetically stable in both preclinical mouse models and human clinical trials. However, the use of IRES (∼600 bp) restricts choices of therapeutic transgenes due to limits in RRV genome size. This study replaced IRES with 2A peptides derived from picornaviruses with or without a GSG linker. The data show that GSG-linked 2A (g2A) peptide resulted in higher polyprotein separation efficiency than non-GSG linked 2A peptide. The study also shows that RRV can tolerate insertion of two separate 2A peptides to allow expression of two transgenes without compromising the assembly and function of the virus in addition to insertion of a single 2A peptide to confirm genetic stability with yCD2, green fluorescent protein, and HSV-1 thymidine kinase. In a parallel comparison of the RRV-IRES-yCD2 and RRV-g2A-yCD2 configurations, the study shows the yCD2 protein expressed from RRV-g2A-yCD2 has higher activity, resulting in a higher survival benefit in an intracranial tumor mouse model. These data enable a wider range of potential product candidates that could be developed using the RRV platform.

A Retroviral Replicating Vector Encoding Cytosine Deaminase and 5-FC Induces Immune Memory in Metastatic Colorectal Cancer Models.

Treatment of tumors with Toca 511, a gamma retroviral replicating vector encoding cytosine deaminase, followed by 5-fluorocytosine (5-FC) kills tumors by local production of 5-fluorouracil (5-FU). In brain tumor models, this treatment induces systemic anti-tumor immune responses and long-term immune-mediated survival. Phase 1 Toca 511 and Toca FC (extended-release 5-FC) clinical trials in patients with recurrent high-grade glioma show durable complete responses and promising survival data compared to historic controls. The work described herein served to expand on our earlier findings in two models of metastatic colorectal carcinoma (mCRC). Intravenous (i.v.) delivery of Toca 511 resulted in substantial tumor-selective uptake of vector into metastatic lesions. Subsequent treatment with 5-FC resulted in tumor shrinkage, improved survival, and immune memory against future rechallenge with the same CT26 CRC cell line. Similar results were seen in a brain metastasis model of mCRC. Of note, 5-FC treatment resulted in a significant decrease in myeloid-derived suppressor cells (MDSCs) in mCRC tumors in both the liver and brain. These results support the development of Toca 511 and Toca FC as a novel immunotherapeutic approach for patients with mCRC. A phase 1 study of i.v. Toca 511 and Toca FC in solid tumors, including mCRC, is currently underway (NCT02576665).

ECTO-NOX target for the anticancer isoflavene phenoxodiol.

Phenoxodiol, a synthetic isoflavene with clinical efficacy in the management of ovarian and other forms of human cancer, blocked the activity of a cancer-specific and growth-related cell surface ECTO-NOX protein with both oxidative (hydroquinone) and protein disulfide-thiol interchange activity designated tNOX. Purified recombinant tNOX bound phenoxodiol with high affinity (Kd of 50 nM). The tNOX protein appeared to be both necessary and sufficient for the cancer-specific cytotoxicity of phenoxodiol. Growth inhibition of fibroblasts from embryos of mice expressing a tNOX transgene, but not from wild-type mice, was inhibited by phenoxodiol followed by apoptosis. Both the oxidative and protein disulfide-thiol interchange activities that alternate to generate the complex set of oscillations with a period length of 22 min (24 min for the constitutive counterpart CNOX) that characterize ECTO-NOX proteins respond to phenoxodiol. Oxidation of NADH or reduced coenzyme Q10 was rapidly blocked by phenoxodiol. In contrast, the protein disulfidethiol interchange activity measured either by the restoration of activity to scrambled and inactive RNase or from the cleavage of dithiodipyridine (EC50 of 50 nM) was inhibited progressively over an interval of 60 min that spanned three cycles of activity. Inhibition of the latter paralleled the inhibition of cell enlargement and the consequent inability of inhibited cells to initiate traverse of the cell cycle. Activities of constitutive ECTO-NOX (CNOX) forms of either cancer or noncancer cells were unaffected by phenoxodiol to help explain how the cytotoxic effects of phenoxodiol may be restricted to cancer cells.

Mapping the STK4/Hippo signaling network in prostate cancer cell.

Dysregulation of MST1/STK4, a key kinase component of the Hippo-YAP pathway, is linked to the etiology of many cancers with poor prognosis. However, how STK4 restricts the emergence of aggressive cancer remains elusive. Here, we investigated the effects of STK4, primarily localized in the cytoplasm, lipid raft, and nucleus, on cell growth and gene expression in aggressive prostate cancer. We demonstrated that lipid raft and nuclear STK4 had superior suppressive effects on cell growth in vitro and in vivo compared with cytoplasmic STK4. Using RNA sequencing and bioinformatics analysis, we identified several differentially expressed (DE) genes that responded to ectopic STK4 in all three subcellular compartments. We noted that the number of DE genes observed in lipid raft and nuclear STK4 cells were much greater than cytoplasmic STK4. Our functional annotation clustering showed that these DE genes were commonly associated with oncogenic pathways such as AR, PI3K/AKT, BMP/SMAD, GPCR, WNT, and RAS as well as unique pathways such as JAK/STAT, which emerged only in nuclear STK4 cells. These findings indicate that MST1/STK4/Hippo signaling restricts aggressive tumor cell growth by intersecting with multiple molecular pathways, suggesting that targeting of the STK4/Hippo pathway may have important therapeutic implications for cancer.

STAT3 and STAT5A are potential therapeutic targets in castration-resistant prostate cancer.

Mechanisms of castration-resistant prostate cancer (CRPC) are not well understood, thus hindering rational-based drug design. Activation of STAT3/5A, key components of the JAK/STAT pathway, is implicated in aggressive PC, yet their clinical relevance in CRPC remains elusive. Here, we evaluated the possible role of STAT3/5A in CRPC using immunological, quantitative mRNA expression profiling, and pharmacological methods. We observed a strong nuclear immunoreactivity for STAT3 and STAT5A in 93% (n=14/15) and 80% (n=12/15) of CRPC cases, respectively, compared with benign prostatic hyperplasia (BPH). We demonstrated that PC cells express varying levels of STAT3 and STAT5A transcripts. In addition, we demonstrate that pimozide, a psychotropic drug and an indirect inhibitor of STAT5, attenuated PC cells growth, and induced apoptosis in a dose-dependent manner. Furthermore, our analysis of the PC public data revealed that the STAT3/5A genes were frequently amplified in metastatic CRPC. These findings suggest that STAT3/5A potentially serves as a predictive biomarker to evaluate the therapeutic efficacy of a cancer drug targeting the JAK/STAT pathway. Since the JAK/STAT and AR pathways are suggested to be functionally synergistic, inhibition of the JAK/STAT signaling alone or together with AR may lead to a novel treatment modality for patients with advanced PC.

Transgenic mouse line overexpressing the cancer-specific tNOX protein has an enhanced growth and acquired drug-response phenotype.

tNOX, a novel cell surface protein related to unregulated growth and drug response of cancer cells, has been proposed as the cellular target for the anticancer action of various quinone site inhibitors with anticancer activity including the polyphenol (-)-epigallocatechin-3-gallate (EGCg). A transgenic mouse line overexpressing tNOX was generated to determine its overall growth phenotype and susceptibility to EGCg. Cultured noncancer cells lack tNOX and are unresponsive to EGCg. Overexpression of tNOX in cultured noncancer cells through transfection resulted in both enhanced growth and an acquired inhibitory response to EGCg. The tNOX transgenic mouse line was developed using a phCMV2 vector with the hemagglutinin (HA) tag. Transgenic mice exhibited both an enhanced growth rate and a response to EGCg not observed with wild-type mice. Female transgenic mice grew twice as fast as wild type, and growth was reflected in an overall increased carcass weight. Administration of EGCg in the drinking water [500 mg/kg body weight (BW)] reduced the growth rate of the transgenic mice to that of wild-type mice. The findings provide in situ validation of the hypothesis that tNOX represents a necessary and sufficient molecular target as the basis for the protective and potential cancer therapeutic benefits of EGCg.

Toca 511 plus 5-fluorocytosine in combination with lomustine shows chemotoxic and immunotherapeutic activity with no additive toxicity in rodent glioblastoma models.

BACKGROUND: Toca 511, a gamma retroviral replicating vector encoding cytosine deaminase, used in combination with 5-fluorocytosine (5-FC) kills tumor by local production of 5-fluorouracil (5-FU), inducing local and systemic immunotherapeutic response resulting in long-term survival after cessation of 5-FC. Toca 511 and Toca FC (oral extended-release 5-FC) are under investigation in patients with recurrent high-grade glioma. Lomustine is a treatment option for patients with high-grade glioma. METHODS: We investigated the effects of lomustine combined with Toca 511 + 5-FC in syngeneic orthotopic glioma models. Safety and survival were evaluated in immune-competent rat F98 and mouse Tu-2449 models comparing Toca 511 + 5-FC to lomustine + 5-FC or the combination of Toca 511 + 5-FC + lomustine. After intracranial implantation of tumor, Toca 511 was delivered transcranially followed by cycles of intraperitoneal 5-FC with or without lomustine at the first or fourth cycle. RESULTS: Coadministration of 5-FC with lomustine was well tolerated. In F98, combination Toca 511 + 5-FC and lomustine increased median survival, but "cures" were not achieved. In Tu-2449, combination Toca 511 + 5-FC and lomustine increased median survival and resulted in high numbers of cure. Rejection of tumor rechallenge occurred after treatment with Toca 511 + 5-FC or combined with lomustine, but not with lomustine + 5-FC. Mixed lymphocyte-tumor cell reactions using splenocytes from cured animals showed robust killing of target cells in an effector:target ratio-dependent manner with Toca 511 + 5-FC and Toca 511 + 5-FC + lomustine day 10. CONCLUSION: The combination of Toca 511 + 5-FC and lomustine shows promising efficacy with no additive toxicity in murine glioma models. Immunotherapeutic responses resulting in long-term survival were preserved despite lomustine-related myelosuppression.

Temozolomide does not impair gene therapy-mediated antitumor immunity in syngeneic brain tumor models.

PURPOSE: Glioblastoma multiforme is the most common primary brain cancer in adults. Chemotherapy with temozolomide (TMZ) significantly prolongs the survival of patients with glioblastoma multiforme. However, the three-year survival is still approximately 5%. Herein, we combined intratumoral administration of an adenoviral vector expressing Flt3L (Ad-Flt3L) with systemic temozolomide to assess its impact on therapeutic efficacy. EXPERIMENTAL DESIGN: Wild-type or immunodeficient mice bearing intracranial glioblastoma multiforme or metastatic melanoma were treated with an intratumoral injection of Ad-Flt3L alone or in combination with the conditionally cytotoxic enzyme thymidine kinase (Ad-TK), followed by systemic administration of ganciclovir and temozolomide. We monitored survival and measured the tumor-infiltrating immune cells. RESULTS: Although treatment with temozolomide alone led to a small improvement in median survival, when used in combination with gene therapy-mediated immunotherapy, it significantly increased the survival of tumor-bearing mice. The antitumor effect was further enhanced by concomitant intratumoral administration of Ad-TK, leading to 50% to 70% long-term survival in all tumor models. Although temozolomide reduced the content of T cells in the tumor, this did not affect the therapeutic efficacy. The antitumor effect of Ad-Flt3L+Ad-TK+TMZ required an intact immune system because the treatment failed when administered to knock out mice that lacked lymphocytes or dendritic cells. CONCLUSIONS: Our results challenge the notion that chemotherapy leads to a state of immune-suppression which impairs the ability of the immune system to mount an effective antitumor response. Our work indicates that temozolomide does not inhibit antitumor immunity and supports its clinical implementation in combination with immune-mediated therapies.

Plasmacytoid dendritic cells in the tumor microenvironment: immune targets for glioma therapeutics.

Adenovirus-mediated delivery of the immune-stimulatory cytokine Flt3L and the conditionally cytotoxic thymidine kinase (TK) induces tumor regression and long-term survival in preclinical glioma (glioblastoma multiforme [GBM]) models. Flt3L induces expansion and recruitment of plasmacytoid dendritic cells (pDCs) into the brain. Although pDCs can present antigen and produce powerful inflammatory cytokines, that is, interferon α (IFN-α), their role in tumor immunology remains debated. Thus, we studied the role of pDCs and IFN-α in Ad.TK/GCV+ Ad.Flt3L-mediated anti-GBM therapeutic efficacy. Our data indicate that the combined gene therapy induced recruitment of plasmacytoid DCs (pDCs) into the tumor mass; which were capable of in vivo phagocytosis, IFN-α release, and T-cell priming. Thus, we next used either pDCs or an Ad vector encoding IFN-α delivered within the tumor microenvironment. When rats were treated with Ad.TK/GCV in combination with pDCs or Ad-IFN-α, they exhibited 35% and 50% survival, respectively. However, whereas intracranial administration of Ad.TK/GCV + Ad.Flt3L exhibited a high safety profile, Ad-IFN-α led to severe local inflammation, with neurologic and systemic adverse effects. To elucidate whether the efficacy of the immunotherapy was dependent on IFN-α-secreting pDCs, we administered an Ad vector encoding B18R, an IFN-α antagonist, which abrogated the antitumoral effect of Ad.TK/GCV + Ad.Flt3L. Our data suggest that IFN-α release by activated pDCs plays a critical role in the antitumor effect mediated by Ad.TK/GCV + Ad.Flt3L. In summary, taken together, our results demonstrate that pDCs mediate anti-GBM therapeutic efficacy through the production of IFN-α, thus manipulation of pDCs constitutes an attractive new therapeutic target for the treatment of GBM.

Gene therapy-mediated reprogramming tumor infiltrating T cells using IL-2 and inhibiting NF-κB signaling improves the efficacy of immunotherapy in a brain cancer model.

Immune-mediated gene therapy using adenovirus expressing Flt3 ligand and thymidine kinase followed by ganciclovir administration (Flt3/TK) effectively elicits tumor regression in preclinical glioma models. Herein, we assessed new strategies to optimize Flt3L/TK therapeutic efficacy in a refractory RG2 orthotopic glioblastoma model. Specifically, we aimed to optimize the therapeutic efficacy of Flt3L/TK treatment in the RG2 model by overexpressing the following genes within the brain tumor microenvironment: 1) a TK mutant with enhanced cytotoxicity (SR39 mutant TK), 2) Flt3L-IgG fusion protein that has a longer half-life, 3) CD40L to stimulate DC maturation, 4) T helper cell type 1 polarizing dendritic cell cytokines interleukin-12 or C-X-C motif ligand 10 chemokine (CXCL)-10, 5) C-C motif ligand 2 chemokine (CCL2) or C-C motif ligand 3 chemokine (CCL3) to enhance dendritic cell recruitment into the tumor microenvironment, 6) T helper cell type 1 cytokines interferon-γ or interleukin-2 to enhance effector T-cell functions, and 7) IκBα or p65RHD (nuclear factor kappa-B [NF-κB] inhibitors) to suppress the function of Foxp3+ Tregs and enhanced effector T-cell functions. Anti-tumor immunity and tumor specific effector T-cell functions were assessed by cytotoxic T lymphocyte assay and intracellular IFN-γ staining. Our data showed that overexpression of interferon-γ or interleukin-2, or inhibition of the nuclear factor kappa-B within the tumor microenvironment, enhanced cytotoxic T lymphocyte-mediated immune responses and successfully extended the median survival of rats bearing intracranial RG2 when combined with Flt3L/TK. These findings indicate that enhancement of T-cell functions constitutes a critical therapeutic target to overcome immune evasion and enhance therapeutic efficacy for brain cancer. In addition, our study provides novel targets to be used in combination with immune-therapeutic strategies for glioblastoma, which are currently being tested in the clinic.

Targeted toxins for glioblastoma multiforme: pre-clinical studies and clinical implementation.

Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults. GBM is very aggressive due to its poor cellular differentiation and invasiveness, which makes complete surgical resection virtually impossible. Therefore, GBM's invasive nature as well as its intrinsic resistance to current treatment modalities makes it a unique therapeutic challenge. Extensive examination of human GBM specimens has uncovered that these tumors overexpress a variety of receptors that are virtually absent in the surrounding non-neoplastic brain. Human GBMs overexpress receptors for cytokines, growth factors, ephrins, urokinase-type plasminogen activator (uPA), and transferrin, which can be targeted with high specificity by linking their ligands with highly cytotoxic molecules, such as Diptheria toxin and Pseudomonas exotoxin A. We review the preclinical development and clinical translation of targeted toxins for GBM. In view of the clinical experience, we conclude that although these are very promising therapeutic modalities for GBM patients, efforts should be focused on improving the delivery systems utilized in order to achieve better distribution of the immuno-toxins in the tumor/resection cavity. Delivery of targeted toxins using viral vectors would also benefit enormously from improved strategies for local delivery.

Gene therapy and targeted toxins for glioma.

The most common primary brain tumor in adults is glioblastoma. These tumors are highly invasive and aggressive with a mean survival time of 15-18 months from diagnosis to death. Current treatment modalities are unable to significantly prolong survival in patients diagnosed with glioblastoma. As such, glioma is an attractive target for developing novel therapeutic approaches utilizing gene therapy. This review will examine the available preclinical models for glioma including xenographs, syngeneic and genetic models. Several promising therapeutic targets are currently being pursued in pre-clinical investigations. These targets will be reviewed by mechanism of action, i.e., conditional cytotoxic, targeted toxins, oncolytic viruses, tumor suppressors/oncogenes, and immune stimulatory approaches. Preclinical gene therapy paradigms aim to determine which strategies will provide rapid tumor regression and long-term protection from recurrence. While a wide range of potential targets are being investigated preclinically, only the most efficacious are further transitioned into clinical trial paradigms. Clinical trials reported to date are summarized including results from conditionally cytotoxic, targeted toxins, oncolytic viruses and oncogene targeting approaches. Clinical trial results have not been as robust as preclinical models predicted; this could be due to the limitations of the GBM models employed. Once this is addressed, and we develop effective gene therapies in models that better replicate the clinical scenario, gene therapy will provide a powerful approach to treat and manage brain tumors.