Sarah R. Klein

The Iron Chelator Dp44mT Causes DNA Damage and Selective Inhibition of Topoisomerase IIα in Breast Cancer Cells

Di-2-pyridylketone-4,4,-dimethyl-3-thiosemicarbazone (Dp44mT) is being developed as an iron chelator with selective anticancer activity. We investigated the mechanism whereby Dp44mT kills breast cancer cells, both as a single agent and in combination with doxorubicin. Dp44mT alone induced selective cell killing in the breast cancer cell line MDA-MB-231 when compared with healthy mammary epithelial cells (MCF-12A). It induces G(1) cell cycle arrest and reduces cancer cell clonogenic growth at nanomolar concentrations. Dp44mT, but not the iron chelator desferal, induces DNA double-strand breaks quantified as S139 phosphorylated histone foci (gamma-H2AX) and Comet tails induced in MDA-MB-231 cells. Doxorubicin-induced cytotoxicity and DNA damage were both enhanced significantly in the presence of low concentrations of Dp44mT. The chelator caused selective poisoning of DNA topoisomerase IIalpha (top2alpha) as measured by an in vitro DNA cleavage assay and cellular topoisomerase-DNA complex formation. Heterozygous Nalm-6 top2alpha knockout cells (top2alpha(+/-)) were partially resistant to Dp44mT-induced cytotoxicity compared with isogenic top2alpha(+/+) or top2beta(-/-) cells. Specificity for top2alpha was confirmed using top2alpha and top2beta small interfering RNA knockdown in HeLa cells. The results show that Dp44mT is cytotoxic to breast cancer cells, at least in part, due to selective inhibition of top2alpha. Thus, Dp44mT may serve as a mechanistically unique treatment for cancer due to its dual ability to chelate iron and inhibit top2alpha activity.

Glioblastoma Eradication Following Immune Checkpoint Blockade in an Orthotopic, Immunocompetent Model

Glioblastoma has been especially challenging to treat. In a systematic analysis of combinations of checkpoint therapies in a murine model, some single and dual immunotherapies increased intratumoral effectors, reduced suppressors, and eliminated the tumors. Inhibition of immune checkpoints, including cytotoxic T-lymphocyte antigen-4 (CTLA-4), programmed death-1 (PD-1), and its ligand PD-L1, has demonstrated exciting and durable remissions across a spectrum of malignancies. Combinatorial regimens blocking complementary immune checkpoints further enhance the therapeutic benefit. The activity of these agents for patients with glioblastoma, a generally lethal primary brain tumor associated with significant systemic and microenvironmental immunosuppression, is not known. We therefore systematically evaluated the antitumor efficacy of murine antibodies targeting a broad panel of immune checkpoint molecules, including CTLA-4, PD-1, PD-L1, and PD-L2 when administered as single-agent therapy and in combinatorial regimens against an orthotopic, immunocompetent murine glioblastoma model. In these experiments, we observed long-term tumor-free survival following single-agent anti–PD-1, anti–PD-L1, or anti–CTLA-4 therapy in 50%, 20%, and 15% of treated animals, respectively. Combination therapy of anti–CTLA-4 plus anti–PD-1 cured 75% of the animals, even against advanced, later-stage tumors. In long-term survivors, tumor growth was not seen upon intracranial tumor rechallenge, suggesting that tumor-specific immune memory responses were generated. Inhibitory immune checkpoint blockade quantitatively increased activated CD8+ and natural killer cells and decreased suppressive immune cells in the tumor microenvironment and draining cervical lymph nodes. Our results support prioritizing the clinical evaluation of PD-1, PD-L1, and CTLA-4 single-agent targeted therapy as well as combination therapy of CTLA-4 plus PD-1 blockade for patients with glioblastoma. Cancer Immunol Res; 4(2); 124–35. ©2015 AACR.

Critical Role of Autophagy in the Processing of Adenovirus Capsid-Incorporated Cancer-Specific Antigens

Adenoviruses are highly immunogenic and are being examined as potential vectors for immunotherapy. Infection by oncolytic adenovirus is followed by massive autophagy in cancer cells. Here, we hypothesize that autophagy regulates the processing of adenoviral proteins for antigen presentation. To test this hypothesis, we first examined the presentation of viral antigens by infected cells using an antibody cocktail of viral capsid proteins. We found that viral antigens were processed by JNK-mediated autophagy, and that autophagy was required for their presentation. Consistent with these results, splenocytes isolated from virus-immunized mice were activated by infected cells in an MHC II-dependent manner. We then hypothesize that this mechanism can be utilized to generate an efficient cancer vaccine. To this end, we constructed an oncolytic virus encompassing an EGFRvIII cancer-specific epitope in the adenoviral fiber. Infection of cancer cells with this fiber-modified adenovirus resulted in recognition of infected cancer cells by a specific anti-EGFRvIII antibody. However, inhibition of autophagy drastically decreased the capability of the specific antibody to detect the cancer-related epitope in infected cells. Our data suggest that combination of adenoviruses with autophagy inducers may enhance the processing and presentation of cancer-specific antigens incorporated into capsid proteins.

Preclinical investigation of combined gene-mediated cytotoxic immunotherapy and immune checkpoint blockade in glioblastoma

Background Combined immunotherapy approaches are promising cancer treatments. We evaluated anti-programmed cell death protein 1 (PD-1) treatment combined with gene-mediated cytotoxic immunotherapy (GMCI) performed by intratumoral injection of a prodrug metabolizing nonreplicating adenovirus (AdV-tk), providing in situ chemotherapy and immune stimulation. Methods The effects of GMCI on PD ligand 1 (PD-L1) expression in glioblastoma were investigated in vitro and in vivo. The efficacy of the combination was investigated in 2 syngeneic mouse glioblastoma models (GL261 and CT-2A). Immune infiltrates were analyzed by flow cytometry. Results GMCI upregulated PD-L1 expression in vitro and in vivo. Both GMCI and anti-PD-1 increased intratumoral T-cell infiltration. A higher percentage of long-term survivors was observed in mice treated with combined GMCI/anti-PD-1 relative to single treatments. Long-term survivors were protected from tumor rechallenge, demonstrating durable memory antitumor immunity. GMCI led to elevated interferon gamma positive T cells and a lower proportion of exhausted double positive PD1+TIM+CD8+ T cells. GMCI also increased PD-L1 levels on tumor cells and infiltrating macrophages/microglia. Our data suggest that anti-PD-1 treatment improves the effectiveness of GMCI by overcoming interferon-induced PD-L1-mediated inhibitory signals, and GMCI improves anti-PD-1 efficacy by increasing tumor-infiltrating T-cell activation. Conclusions Our data show that the GMCI/anti-PD-1 combination is well tolerated and effective in glioblastoma mouse models. These results support evaluation of this combination in glioblastoma patients.

Abstract 572: Inhibition of IDO1 with epacadostat enhances anti-tumor efficacy of PD-1 blockade in a syngeneic glioblastoma (GBM) model

Purpose: To determine if epacadostat, an oral indoleamine 2,3-dioxygenase (IDO1) inhibitor has therapeutic benefit against GBM when administered as single agent and with PD-1 blocking antibody. Methods: An initial survival experiment was performed to assess efficacy and was followed by an identical repeat experiment for validation. 1X10 5 luciferized GL261 cells, a murine GBM tumor line derived from intracerebral methylcholanthrene implantation, were stereotactically implanted intracranially in albino syngeneic C57BL/6 mice. Mice with increasing bioluminescence on days 3 and 6 were randomized (n=8/group) to receive treatment beginning on day 6: anti-PD-1 (332.8H3, mouse IgG1; 500 μg intraperitoneal (IP) on day 6, 250 μg q 3 days X 7); epacadostat (Incyte Corporation, orally dosed at 300 mg/kg/day for 5 days on/2days off for 3 weeks); anti-PD-1 + epacadostat; and control therapy (isotype IgG antibody IP and 0.5% methocel in water). Tumor response assessments were performed by quantifying bioluminescence and survival. A re-challenge experiment was performed in long-term survivors to assess for tumor immune responses capable of preventing relapse. All long-term surviving mice (defined as ≥ 100 days) from the efficacy experiment were injected with 1X10 5 GL261 cells in the contralateral hemisphere and followed for survival. Results: In both preclinical efficacy experiments, median survival in the epacadostat monotherapy group did not differ from controls (approximately 30 days). Four of 8 mice (50%) treated with anti-PD-1 were long-term survivors in both efficacy experiments. In the epacadostat plus anti-PD-1 combination group, 81% of the mice were long-term survivors (7 of 8 in experiment 1 and 6 of 8 in experiment 2). Of note, none of the long-term surviving mice developed evidence of tumor; thus the median survival among the anti-PD-1 and epacadostat plus anti-PD-1 combination groups were both > 100 days. In the re-challenge study, all of the mice who underwent GL261 re-inoculation survived > 100 more days with no evidence of tumor recurrence. Conclusions: IDO1 inhibition with epacadostat increased the eradication rate of anti-PD-1 therapy in an orthotopic syngeneic GBM model and long term survivors rejected tumor following orthotopic re-challenge. Further combinatorial studies incorporating IDO inhibitor therapy for GBM, including mechanistic studies, are warranted. Citation Format: David A. Reardon, Prafulla C. Gokhale, Sarah R. Klein, Kristen L. Jones, Paul T. Kirschmeier, Maria Speranza, Holly Koblish, Peggy Scherle, Lance Leopold, Robert Newton, Gordon J. Freeman. Inhibition of IDO1 with epacadostat enhances anti-tumor efficacy of PD-1 blockade in a syngeneic glioblastoma (GBM) model [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 572. doi:10.1158/1538-7445.AM2017-572

Abstract B84: Preclinical analysis of combinatorial glioblastoma therapy with the prodrug-mediated gene therapy vector AdV-TK and immune checkpoint inhibition

Early clinical trial data show that blockade of PD-1 signaling leads to significant anticancer responses in a subset of patients in certain cancer types. While the brain has traditionally been considered to be an immune-privileged site, evidence supporting the use of immunotherapeutics in brain tumors has been rapidly accumulating. Given that virus-based cancer therapies can be immunostimulatory and immune checkpoint inhibitors block the body9s natural checkpoint response, the combination of these two approaches offers a potentially advantageous interaction. One of the molecular underpinnings of T-cell exhaustion is the expression of Programmed Death-1 (PD-1) on T-cells that recognizes its ligand PD-L1. AdV-TK is an immunostimulatory virus-based approach, known as Gene-Mediated Cytotoxic Immunotherapy (GMCI), that involves the intra-tumoral delivery of a non-replicating adenoviral vector carrying the Herpes virus thymidine kinase gene (TK) followed by administration of an anti-herpetic prodrug (ganciclovir GCV) and recently showed encouraging results in a Phase II trial in glioblastoma (Wheeler et al., 2016). The immunological component results from the delivery vehicle being a virus, the mode of cell death, through both necrosis and apoptosis, and the pro-immunogenic properties of the TK protein. We confirm that this approach induces glioblastoma cell death and a consistent anti-tumor immune stimulation. Not surprisingly, however, this immune stimulation also leads to increase in cell surface of immune checkpoint inhibitory ligands on tumor cells, including PD-L1, detected by flow cytometry and immunohistochemistry. We show that GMCI induces a type-I interferon response, and using IFN decoy we demonstrated that the release of IFNβ in vitro is at least partially responsible for autocrine/paracrine PD-L1 up-regulation both in human and mouse glioblastoma cell lines. In vivo studies using an intracranial GL261 model showed high numbers of long term survivors in the GMCI/PD-1 combination (11/14), compared with GMCI (6/16), anti-PD-1 (5/12) and untreated (0/11). In addition, long term survival mice were no longer able to form tumors after rechallenge indicating the establishment of anti-tumor immunity. Finally, tumor infiltrating lymphocytes after GMCI showed an increase in CD8+, CD8+/GranzymeB+, and IFNγ+ cells suggestive of cytotoxic T-cell activation. However, there was also a significant increase in CD4+, CD4+/FoxP3+, and IL-10 indicating a significant infiltration by Tregs, releasing immunosuppressive cytokines. Additionally, there was a significant increase in PD-1+ /TIM3+ T-cells, indicative of an immunosuppressive microenvironment. Overall, our data show that GMCI/anti-PD-1 combinatorial therapy is effective in a syngeneic tumor model, and strongly support clinical trials of GMCI/checkpoint inhibitor combinations in glioblastoma patients. Citation Format: Maria Carmela Speranza, Franz Ricklefs, Carmela Passaro, Sarah R. Klein, Kazue Kasai, Johanna Kaufmann, Hiroshi Nakashima, Bronisz Agnieszka, Estuardo Aguilar-Cordova, Brian W. Guzik, Gordon J. Freeman, David A. Reardon, Patrick Wen, E. Antonio Chiocca, Sean E. Lawler. Preclinical analysis of combinatorial glioblastoma therapy with the prodrug-mediated gene therapy vector AdV-TK and immune checkpoint inhibition. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B84.

Abstract 2688: Multiplex IHC detection of immune checkpoint receptors in the tumor microenvironment

As immune checkpoint blockade has been shown to partially reverse the exhausted T cell phenotype and consequently lead to a decrease in tumor burden, there is a need for an understanding of this T cell type. Using recently developed, highly validated antibodies, we have developed a fluorescent multiplex, TSA-based assay in order to examine the interaction of PD-L1 with PD-1-expressing exhausted T cells in various tumor types. Here, we construct a seven-color multiplex panel in order to simultaneously visualize cytokeratin (the tumor mask), DAPI (nuclear counterstain), CD8 (cytotoxic T cell marker), the exhausted T cell markers PD-1, Tim-3 and Lag-3, and the ligand for PD-1, PD-L1. This technique not only enabled the concurrent detection of these markers, but also provided high-resolution visualization of interactions between PD-1-expressing CD8+ T cells and PD-L1-expressing CD68+ macrophages in the tumor microenvironment. The seven-plex panel was applied to FFPE tumor microarrays (TMAs) consisting of breast, lung and ovarian tumor tissue and each core was subsequently analyzed for the distribution, co-localization, frequency and proximity of these targets in relation to one another. While we often visualized co-expression of Lag-3 and PD-1 on T cells, Tim-3 was frequently observed on PD-L1+ macrophages in several tumor types. This data provides valuable insight into the co-expression profiles of these markers in multiple tumor types and has implications for the use of combination therapies that aim to target both the innate and adaptive immune systems. Citation Format: Jennifer E. Ziello, Sarah R. Klein, Emily Alonzo, Herbert Haack. Multiplex IHC detection of immune checkpoint receptors in the tumor microenvironment [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 2688. doi:10.1158/1538-7445.AM2017-2688

Abstract A075: Preclinical analysis of combinatorial glioblastoma therapy with the prodrug-mediated gene therapy vector AdV-TK and immune checkpoint inhibition in GBM therapy

While the brain has traditionally been considered to be an immune-privileged site, evidence supporting the use of immunotherapeutics has been rapidly accumulating. Given that virus-based cancer therapies can be immunostimulatory and immune-checkpoint inhibitors block tumor-induced T-cell exhaustion, the combination of these two approaches offers a potentially synergistic interaction. One of the molecular underpinnings of T-cell exhaustion is the expression of Programmed Death-1 (PD1) on T-cells that recognizes its ligand PD-L1. AdV-tk is an immunostimulatory virus-based approach, known as Gene-Mediated Cytotoxic Immunotherapy (GMCI), that involves the intra-tumoral delivery of a non-replicating adenoviral vector carrying the Herpes virus thymidine kinase gene(TK) followed by administration of an anti-herpetic prodrug(ganciclovir-GCV) and recently showed encouraging results in a Phase II trial in glioblastoma(Wheeler et al.,2016). To provide a rationale for this therapeutic combination we investigated PD-L1 expression during GMCI therapy in human and mouse glioma cells in vitro and found that there was a consistent increase in cell surface PD-L1 levels. Interestingly, this was not associated with an increase of mRNA or protein. We also show that GMCI induces a type-I interferon response, and that the release of IFNβ is at least partially responsible for autocrine/paracrine PD-L1 up-regulation. In vivo studies using an intracranial GL261 model showed high levels of long term survivors in the GMCI/PD1 combination (11/14), compared with GMCI (6/16), anti-PD1 (5/12) and controls (0/11). In addition, tumor infiltrating lymphocytes after GMCI showed an increase in CD8+, CD8+/GranzymeB+, and CD8+/IFNγ+/TNFα+cells suggestive of cytotoxic T-cell activation. However, there was also a significant increase in CD4+, CD4+/FoxP3+, and IL-10 indicating a significant infiltration by Tregs, releasing immunosuppressive cytokines. Additionally, there was a significant increase in PD1+/TIM3+ T-cells, indicative of an immunosuppressive microenvironment. Overall, our data show that GMCI/anti-PD1 combinatorial therapy is effective in a syngeneic tumor model, and strongly support clinical trials of GMCI/checkpoint inhibitor combinations in glioblastoma patients. Citation Format: Maria Carmela Speranza, Kazue Kasai, Franz Ricklefs, Sarah R. Klein, Carmela Passaro, Hiroshi Nakashima, Johanna Kaufmann, Agnieszka Bronisz, Estuardo Aguilar-Cordova, Brian W. Guzik, Gordon J. Freeman, David A. Reardon, Patrick Wen, E. Antonio Chiocca, Sean E. Lawler. Preclinical analysis of combinatorial glioblastoma therapy with the prodrug-mediated gene therapy vector AdV-TK and immune checkpoint inhibition in GBM therapy [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A075.