Hikmat Assi

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.

Gene Therapy for Brain Tumors: Basic Developments and Clinical Implementation

Glioblastoma multiforme (GBM) is the most common and deadliest of adult primary brain tumors. Due to its invasive nature and sensitive location, complete resection remains virtually impossible. The resistance of GBM against chemotherapy and radiotherapy necessitate the development of novel therapies. Gene therapy is proposed for the treatment of brain tumors and has demonstrated pre-clinical efficacy in animal models. Here we review the various experimental therapies that have been developed for GBM including both cytotoxic and immune stimulatory approaches. We also review the combined conditional cytotoxic immune stimulatory therapy that our lab has developed which is dependent on the adenovirus mediated expression of the conditional cytotoxic gene, Herpes Simplex Type 1 Thymidine Kinase (TK) and the powerful DC growth factor Fms-like tyrosine kinase 3 ligand (Flt3L). Combined delivery of these vectors elicits tumor cell death and an anti-tumor adaptive immune response that requires TLR2 activation. The implications of our studies indicate that the combined cytotoxic and immunotherapeutic strategies are effective strategies to combat deadly brain tumors and warrant their implementation in human Phase I clinical trials for GBM.

Preclinical Characterization of Signal Transducer and Activator of Transcription 3 Small Molecule Inhibitors for Primary and Metastatic Brain Cancer Therapy

Signal transducer and activator of transcription 3 (STAT3) has been implicated as a hub for multiple oncogenic pathways. The constitutive activation of STAT3 is present in several cancers, including gliomas (GBMs), and is associated with poor therapeutic responses. Phosphorylation of STAT3 triggers its dimerization and nuclear transport, where it promotes the transcription of genes that stimulate tumor growth. In light of this role, inhibitors of the STAT3 pathway are attractive therapeutic targets for cancer. To this end, we evaluated the STAT3-inhibitory activities of three compounds (CPA-7 [trichloronitritodiammineplatinum(IV)], WP1066 [(S,E)-3-(6-bromopyridin-2-yl)-2-cyano-N-(1-phenylethyl)acrylamide, C17H14BrN3O], and ML116 [4-benzyl-1-{thieno[2,3-d]pyrimidin-4-yl}piperidine, C18H19N3S]) in cultured rodent and human glioma cells, including GBM cancer stem cells. Our results demonstrate a potent induction of growth arrest in GBM cells after drug treatment with a concomitant induction of cell death. Although these compounds were effective at inhibiting STAT3 phosphorylation, they also displayed variable dose-dependent inhibition of STAT1, STAT5, and nuclear factor κ light-chain enhancer of activated B cells. The therapeutic efficacy of these compounds was further evaluated in peripheral and intracranial mouse tumor models. Whereas CPA-7 elicited regression of peripheral tumors, both melanoma and GBM, its efficacy was not evident when the tumors were implanted within the brain. Our data suggest poor permeability of this compound to tumors located within the central nervous system. WP1066 and ML116 exhibited poor in vivo efficacy. In summary, CPA-7 constitutes a powerful anticancer agent in models of peripheral solid cancers. Our data strongly support further development of CPA-7–derived compounds with increased permeability to enhance their efficacy in primary and metastatic brain tumors.

Assessing the Role of STAT3 in DC Differentiation and Autologous DC Immunotherapy in Mouse Models of GBM

Cellular microenvironments, particularly those found in tumors, elicit a tolerogenic DC phenotype which can attenuate immune responses. Central to this process is the STAT3-mediated signaling cascade. As a transcription factor and oncogene, STAT3 promotes the expression of genes which allow tumor cells to proliferate, migrate and evade apoptosis. More importantly, activation of STAT3 in tumor infiltrating immune cells has been shown to be responsible, in part, for their immune-suppressed phenotype. The ability of STAT3 to orchestrate a diverse set of immunosuppressive instructions has made it an attractive target for cancer vaccines. Using a conditional hematopoietic knockout mouse model of STAT3, we evaluated the impact of STAT3 gene ablation on the differentiation of dendritic cells from bone marrow precursors. We also assessed the impact of STAT3 deletion on phagocytosis, maturation, cytokine secretion and antigen presentation by GM-CSF derived DCs in vitro. In addition to in vitro studies, we compared the therapeutic efficacy of DC vaccination using STAT3 deficient DCs to wild type counterparts in an intracranial mouse model of GBM. Our results indicated the following pleiotropic functions of STAT3: hematopoietic cells which lacked STAT3 were unresponsive to Flt3L and failed to differentiate as DCs. In contrast, STAT3 was not required for GM-CSF induced DC differentiation as both wild type and STAT3 null bone marrow cells gave rise to similar number of DCs. STAT3 also appeared to regulate the response of GM-CSF derived DCs to CpG. STAT3 null DCs expressed high levels of MHC-II, secreted more IL-12p70, IL-10, and TNFα were better antigen presenters in vitro. Although STAT3 deficient DCs displayed an enhanced activated phenotype in culture, they elicited comparable therapeutic efficacy in vivo compared to their wild type counterparts when utilized in vaccination paradigms in mice bearing intracranial glioma tumors.

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, GBMs 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.

Lentiviral-Induced High-Grade Gliomas in Rats: The Effects of PDGFB, HRAS-G12V, AKT, and IDH1-R132H

In human gliomas, the RTK/RAS/PI(3)K signaling pathway is nearly always altered. We present a model of experimental gliomagenesis that elucidates the contributions of genes involved in this pathway (PDGF-B ligand, HRAS-G12V, and AKT). We also examine the effect on gliomagenesis by the potential modifier gene, IDH1-R132H. Injections of lentiviral-encoded oncogenes induce de novo gliomas of varying penetrance, tumor progression, and histological grade depending on the specific oncogenes used. Our model mimics hallmark histological structures of high-grade glioma, such as pseudopalisades, glomeruloid microvascular proliferation, and diffuse tumor invasion. We use our model of gliomagenesis to test the efficacy of an experimental brain tumor gene therapy. Our model allowed us to test the contributions of oncogenes in the RTK/RAS/PI(3)K pathway, and their potential modification by over-expression of mutated IDH1, in glioma development and progression in rats. Our model constitutes a clinically relevant system to study gliomagenesis, the effects of modifier genes, and the efficacy of experimental therapeutics.

Abstract 3195: STAT3 inhibition using shRNA inhibits GBM proliferation, cell migration, anchorage-independent growth of mouse, rat, and human stem-like cells in vitro; and it induces long term survival and anti-GBM immunity in vivo

The purpose of this study was to elucidate the role of Signal Transducers and Activators of Transcription 3 (STAT3) signaling on the tumor microenvironment in the most commonly occurring and aggressive primary brain tumor, Glioblastoma Multiforme (GBM), including preclinical testing of inhibition of STAT3 expression in vivo in syngeneic and patient derived xenograft (PDX) models of GBM. GBM is genetically heterogeneous, but always overexpresses genes that are vital to cell cycle regulation, cell growth and proliferation, cell invasion, and angiogenesis. STAT proteins are transcription factors associated with gene regulation and expression signatures that are implicated in several survival pathways that enable human GBMs to grow in the brain parenchyma. In addition, STAT3 has been identified as a central mechanism in tumor-induced immunosuppression in GBM and other cancers. We studied the effects of STAT3 inhibition via shRNA down-regulation in vivo and in vitro using patient derived primary glioma cells (HF2303 & MGG8) in conjunction with other glioma lines from humans, mice, and rats (U251, GL26, and CNS-1, respectively). In this study, we found that GBM cells harboring down-regulated STAT3 signaling exhibit delayed proliferation, increased apoptosis, and anchorage independence. In vivo, STAT3 inhibition resulted in increased survival rates when tumor cells were treated with the STAT3 shRNA both pre- and post-GBM implantation (syngeneic GBM model). Our data demonstrate that STAT3 has a profound influence on the GBM microenvironment, which prevents the host from clearing the tumor. Down-regulating this signaling pathway using gene therapeutic strategies allows for infiltration of immune cells, decreased invasion, and a decrease in GBM cells’ proliferation that leads to the tumor elimination in ∼83% of the animals, this was associated with the development of an effective anti-tumor immunity that prevents tumor recurrence. Given the phenotype of STAT3 inhibition in in vitro assays and its success in in vivo GBM models, inhibition of STAT3 expression using shRNA and gene therapy technologies constitutes an attractive strategy for preclinical development as a potent therapeutic target for GBM. Citation Format: Nathan T. VanderVeen, Nicholas Raja, Elizabeth Yi, James Curtin, Peter Chockley, Hikmat Assi, Jonathan Savakus, Tom Mikkelsen, Samuel Rabkin, Pedro R. Lowenstein, Maria G. Castro. STAT3 inhibition using shRNA inhibits GBM proliferation, cell migration, anchorage-independent growth of mouse, rat, and human stem-like cells in vitro; and it induces long term survival and anti-GBM immunity in vivo. [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 3195. doi:10.1158/1538-7445.AM2015-3195

Abstract 457: Depletion of glioma infiltrating myeloid derived suppressor cells promotes anti-tumor T cell responses

MDSCs represent a population of immature myeloid cells at various stages of differentiation that have the potential to inhibit anti-tumor T cell immunity. We demonstrate the accumulation of MDSCs in GL26 and M7-induced glioma (GBM) bearing mice. Absolute numbers of Ly-6G+ (Gr-1high) MDSCs showed a 200 fold increase within the tumor microenvironment (TME) 28 days post-tumor implantation. The numbers of Ly-6C+ (Gr-1low) MDSCs also showed a similar trend within the TME. While this massive influx of MDSCs was noted within intracranial tumors, MDSC levels did not increase in the dLNs, spleen or bone marrow (BM) of intracranial tumor bearing mice. MDSC numbers were significantly elevated in the blood of GL26 and M7 intracranial tumor bearing mice at 28 days. While both Gr-1high and Gr-1low MDSCs isolated from the TME of GL26 intracranial tumor bearing mice inhibited antigen-specific T cell proliferation, Gr-1low MDSC were found to be more efficient. Gr-1high or Gr-1low MDSCs from the bone marrow of intracranial tumor bearing mice failed to suppress antigen-specific T cell proliferation suggesting that TME derived factors may activate MDSCs to exert their immune-suppressive properties. In vivo, depletion of Gr-1+ cells enhanced the median survival of GBM bearing mice. Furthermore, when combined with Ad-TK + Ad-FLT3L immune-gene therapy, Gr-1+ depletion significantly enhanced the frequency of tumor-specific T cells within the TME and spleen and increased IFN-γ production by splenic T cells. Our data therefore indicates that inhibiting the accumulation of MDSCs with in the GBM TME promotes the generation of robust anti-tumor immunity. Preliminary experiments to determine the mechanism of MDSC trafficking to the TME point towards the receptor CXCR2 and its ligand CXCL1. Microarray analysis of glioma cell lines showed elevated levels of CXCL1 mRNA. Additionally a culture of primary mixed glial cells also produced CXCL1 when stimulated with GBM cell lysates. SB225002, a CXCR2 inhibitor suppressed the migration of MDSCs towards GBM cells in an in vitro migration assay. Overall, our data suggests that strategies that inhibit MDSC recruitment to the GBM TME and/or block their activity could enhance the T cell mediated tumor clearance and provide survival benefit. Work supported by grants from NIH-NINDS Citation Format: Neha Kamran, Youping Li, Mariela Moreno-Ayala, Hikmat Assi, Marianela Candolfi, Marta Dzaman, Pedro Lowenstein, Maria Castro. Depletion of glioma infiltrating myeloid derived suppressor cells promotes anti-tumor T cell responses. [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 457. doi:10.1158/1538-7445.AM2015-457

Immunotherapies for Brain Cancer: From Preclinical Models to Human Trials

Glioblastoma Multiforme (GBM) is the most common and aggressive primary brain tumor. Every year ~22,000 patients are diagnosed with GBM in the US, and less than 5% survive 5 years post-diagnosis. Thus, novel therapeutic approaches are urgently needed to improve the outcome in these patients. Immunotherapy has the potential of stimulating the immune system to eliminate GBM cells that might have spread throughout the brain. Here we will discuss the latest advances in preclinical immunotherapy for glioma, which involve the local delivery of pro-inflammatory cytokines, such as Flt3L, Type I IFNs, IL-2, IL-4, and IL-12 using gene therapy vectors and neural stem cells, or the blockade of immune-suppressive mediators such as TGF-beta, FasL and phosphorylated STAT3. Novel immunotherapeutic approaches have also been assessed in clinical trials implemented in GBM patients. These involve the systemic or local adoptive transfer of autologous immune cells activated ex vivo back into the patient, and the administration of dendritic cell vaccines loaded with tumor peptides or cells, which induce active immunity against GBM. Preclinical and clinical findings so far indicate that immunotherapy improves anti-tumor immunity in preclinical GBM models and patients, which makes it a valuable adjuvant in the treatment of GBM.

Abstract 187: Absence of S100A9 confers survival advantage in an aggressive de novo mouse model of glioblastoma multiforme

Glioblastoma multiforme (GBM) is the most common (60%) primary brain tumor in adults and constitutes a major challenge for both patients and clinicians, with a median survival of only 15-21 months when treated with surgery, radiation therapy and temozolomide. To improve patient outcome, a promising and safe avenue of adjuvant treatment is immune therapy with dendritic cell vaccines or adoptive T cell transfer. These treatments are however hindered by the immune suppressive environment induced by GBMs. Understanding the mechanisms by which GBM suppress the anti-tumor immune response is paramount in order to develop successful immune therapies. The Sleeping Beauty transposase system was used to modify the genetic makeup of stem cells in the sub-ventricular zone of neonatal mice and thus induce de novo glioblastomas with several combinations of genes. The most aggressive tumors where induced when combining NRAS and SV-40 Large T antigen (NLgT) generating invasive tumors which show the histological hallmarks human GBM (WHO grade IV) with pseudo-pallisading necrosis, neovascularization and hemorrhages, rendering the mice moribund with a median survival of 30 days. Myeloid derived suppressive cells, a heterogeneous population of immature bone marrow derived cells, are induced by cancers and other consumptive diseases and strongly inhibit adaptive and innate immune responses. We show that MDSCs isolated from de novo GBMs, inhibit antigen-specific and antigen non-specific T cell proliferation. Tumor infiltrating MDSCs highly express the pro-inflammatory secreted calcium binding protein S100A9 and its cognate receptors RAGE and TLR4. In bone marrow cultures, conditioned media from primary cell lines derived from NLgT tumors induce a marked expansion (60-70%) of myeloid derived suppressor cells (MDSCs) and enhance the expression of S100A9. Mice deficient for S100A9 with de novo NLgT tumors show an increased survival compared to wild-type animals (median survival= 47days, a 56% improvement). Analysis of tumor-infiltrating mononuclear cells in moribund NLgT wild-type and S100A9KO mice shows no significant difference in percent infiltrating MDSCs, macrophages and dendritic cells (% of CD45+), however the percent of CD8+ cytotoxic T lymphocytes is increased in S100A9 KO (16% vs. 6% p=0.0228). In addition, expression of MHCII is increased in macrophages and dendritic cells from S100A9 KO mice when compared to wild type mice. Taken together these data suggest that the absence of S100A9 confers a survival advantage by allowing a stronger anti-tumor immune response with:(1) increased number of cytotoxic T lymphocytes and (2) increased maturation of antigen presenting cells. Experiments are currently underway to identify the detailed cellular and molecular events, which give rise to this phenotype. This study was supported by NIH/RO1 NS057711 and NS074387. Citation Format: Alexandra Calinescu, Hikmat Assi, Bradley Kolb, Carl Koschmann, Pedro R. Lowenstein, John Ohlfest, Maria G. Castro. Absence of S100A9 confers survival advantage in an aggressive de novo mouse model of glioblastoma multiforme. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 187. doi:10.1158/1538-7445.AM2014-187