Jinkyu Jung

Hes3 regulates cell number in cultures from glioblastoma multiforme with stem cell characteristics.

Tumors exhibit complex organization and contain a variety of cell populations. The realization that the regenerative properties of a tumor may be largely confined to a cell subpopulation (cancer stem cell) is driving a new era of anti-cancer research. Cancer stem cells from Glioblastoma Multiforme tumors express markers that are also expressed in non-cancerous neural stem cells, including nestin and Sox2. We previously showed that the transcription factor Hes3 is a marker of neural stem cells, and that its expression is inhibited by JAK activity. Here we show that Hes3 is also expressed in cultures from glioblastoma multiforme which express neural stem cell markers, can differentiate into neurons and glia, and can recapitulate the tumor of origin when transplanted into immunocompromised mice. Similar to observations in neural stem cells, JAK inhibits Hes3 expression. Hes3 RNA interference reduces the number of cultured glioblastoma cells suggesting a novel therapeutic strategy.

Cardiac glycosides suppress the maintenance of stemness and malignancy via inhibiting HIF-1α in human glioma stem cells

Tissue hypoxia contributes to solid tumor pathogenesis by activating a series of adaptive programs. We previously showed that hypoxia promotes the preferential expansion and maintenance of CD133 positive human glioma stem cells (GSC) in a hypoxia inducible factor 1 alpha (HIF-1α)-dependent mechanism. Here, we examined the activity of digitoxin (DT), a cardiac glycoside and a putative inhibitor of HIF-1α, on human GSC in vitro and in vivo. During hypoxic conditions (1% O2), we observed the effect of DT on the intracellular level of HIF-1α and the extracellular level of vascular endothelial growth factor (VEGF) in human GSC. We found that DT at clinically achievable concentrations, suppressed HIF-1α accumulation during hypoxic conditions in human GSC and established glioma cell lines. DT treatment also significantly attenuated hypoxia-induced expression of VEGF, a downstream target of HIF-1α. Exposure to DT also reduced hypoxia-induced activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway. Furthermore, DT potently inhibited neurosphere formation, and decreased CD133 expression even at concentrations that were not overtly cytotoxic. Lastly, treatment with DT reduced GSC engraftment in an in vivo xenograft model of glioblastoma. Intraperitoneal injections of DT significantly inhibited the growth of established glioblastoma xenografts, and suppressed expression of HIF-1α and carbonic anhydrase (CA9), a surrogate marker of hypoxia. Taken together, these results suggest that DT at clinically achievable concentration functions as an inhibitor of HIF-1α, worthy of further investigations in the therapy of glioblastoma.

Protein phosphatase 2A inhibition enhances radiation sensitivity and reduces tumor growth in chordoma

Background Standard therapy for chordoma consists of surgical resection followed by high-dose irradiation. Protein phosphatase 2A (PP2A) is a ubiquitously expressed serine/threonine phosphatase involved in signal transduction, cell cycle progression, cell differentiation, and DNA repair. LB100 is a small-molecule inhibitor of PP2A designed to sensitize cancer cells to DNA damage from irradiation and chemotherapy. A recently completed phase I trial of LB100 in solid tumors demonstrated its safety. Here, we show the therapeutic potential of LB100 in chordoma. Methods Three patient-derived chordoma cell lines were used: U-CH1, JHC7, and UM-Chor1. Cell proliferation was determined with LB100 alone and in combination with irradiation. Cell cycle progression was assessed by flow cytometry. Quantitative γ-H2AX immunofluorescence and immunoblot evaluated the effect of LB100 on radiation-induced DNA damage. Ultrastructural evidence for nuclear damage was investigated using Raman imaging and transmission electron microscopy. A xenograft model was established to determine potential clinical utility of adding LB100 to irradiation. Results PP2A inhibition in concert with irradiation demonstrated in vitro growth inhibition. The combination of LB100 and radiation also induced accumulation at the G2/M phase of the cell cycle, the stage most sensitive to radiation-induced damage. LB100 enhanced radiation-induced DNA double-strand breaks. Animals implanted with chordoma cells and treated with the combination of LB100 and radiation demonstrated tumor growth delay. Conclusions Combining LB100 and radiation enhanced DNA damage-induced cell death and delayed tumor growth in an animal model of chordoma. PP2A inhibition by LB100 treatment may improve the effectiveness of radiation therapy for chordoma.

Dexamethasone-induced immunosuppression: mechanisms and implications for immunotherapy

BackgroundCorticosteroids are routinely utilized to alleviate edema in patients with intracranial lesions and are first-line agents to combat immune-related adverse events (irAEs) that arise with immune checkpoint blockade treatment. However, it is not known if or when corticosteroids can be administered without abrogating the efforts of immunotherapy. The purpose of this study was to evaluate the impact of dexamethasone on lymphocyte activation and proliferation during checkpoint blockade to provide guidance for corticosteroid use while immunotherapy is being implemented as a cancer treatment.MethodsLymphocyte proliferation, differentiation, and cytokine production were evaluated during dexamethasone exposure. Human T cells were stimulated through CD3 ligation and co-stimulated either directly by CD28 ligation or by providing CD80, a shared ligand for CD28 and CTLA-4. CTLA-4 signaling was inhibited by antibody blockade using ipilimumab which has been approved for the treatment of several solid tumors. The in vivo effects of dexamethasone during checkpoint blockade were evaluated using the GL261 syngeneic mouse intracranial model, and immune populations were profiled by flow cytometry.ResultsDexamethasone upregulated CTLA-4 mRNA and protein in CD4 and CD8 T cells and blocked CD28-mediated cell cycle entry and differentiation. Naïve T cells were most sensitive, leading to a decrease of the development of more differentiated subsets. Resistance to dexamethasone was conferred by blocking CTLA-4 or providing strong CD28 co-stimulation prior to dexamethasone exposure. CTLA-4 blockade increased IFNγ expression, but not IL-2, in stimulated human peripheral blood T cells exposed to dexamethasone. Finally, we found that CTLA-4 blockade partially rescued T cell numbers in mice bearing intracranial gliomas. CTLA-4 blockade was associated with increased IFNγ-producing tumor-infiltrating T cells and extended survival of dexamethasone-treated mice.ConclusionsDexamethasone-mediated T cell suppression diminishes naïve T cell proliferation and differentiation by attenuating the CD28 co-stimulatory pathway. However, CTLA-4, but not PD-1 blockade can partially prevent some of the inhibitory effects of dexamethasone on the immune response.

Abstract A11: Ipilimumab protects T cells from the antiproliferative effects of dexamethasone

Background: Checkpoint inhibitor blockade, designed to activate antitumor immune cells, is gaining enthusiasm as a potential treatment modality for patients with brain tumors. However, adjuvant therapies aimed at killing tumor (i.e., chemotherapy) or reducing tumor-related edema (i.e., corticosteroids) are often cytotoxic to lymphocytes. Yet, whether tumor-specific T cells are harmed during corticosteroid treatment is not known. Methods: The effects of dexamethasone on healthy donor T cells was tested in vitro for T cell proliferation, cell cycle analysis, glucose uptake, transcriptional changes, and protein expression. The effects of dexamethasone and CTLA-4 antibody blockade were tested in vivo using the GL261 murine glioblastoma model. Results: Here we show that dexamethasone blocks proliferation of naive human T cells but not memory T cells. We find that dexamethasone inhibits early stages of T cell proliferation by impairing CD28-mediated cell cycle entry. Dexamethasone induced a fourfold increase in surface CTLA-4 during T cell stimulation, and neutralizing CTLA-4 with ipilimumab overcame the dexamethasone-induced blockade of cell cycle entry in vitro. Further, CTLA-4 blockade in combination with dexamethasone provided a survival benefit in vivo to mice bearing orthotopic GL261 brain tumors. Intriguingly, early dexamethasone treatment afforded the greatest survival advantage Conclusions: These findings shed light on the T cell-specific effects of dexamethasone and suggest that antigen-experienced T cells are resistant to anti-proliferative effects of corticosteroids. These findings have important implications for patients receiving immune therapy who may benefit from the anti-inflammatory properties of dexamethasone. Citation Format: Amber J. Giles, Marsha-Kay N.M. Hutchinson, Heather Sonnemann, Caitlin M. Reid, Jinkyu Jung, Wei Zhang, Hua Song, Rolanda Bailey, Dionne Davis, Deric M. Park, Mario Roederer, Mark R. Gilbert. Ipilimumab protects T cells from the antiproliferative effects of dexamethasone [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A11.

Abstract 1533: Transcriptional inhibition of brachyury in chordoma is associated with adoption of quiescent phenotype

BACKGROUND: Brachyury is a mesoderm specification transcription factor involved in notochord development and overexpressed in a variety of cancers, including chordoma. High levels of brachyury protein expression in cancers is associated with a poor prognosis in part due to its role in mediating epithelial-mesenchymal transition (EMT). TG02 is a multikinase inhibitor that targets transcriptional regulation of cyclin-dependent kinases (CDKs). Because chordoma is characterized by a relative paucity of genomic mutations and largely driven by the super-enhancer activity of brachyury expression, we investigated the downstream effect of a transcriptional inhibitor, TG02, in chordomas. METHODS: Established chordoma cell lines, UCH-1 and UM-Chor1, were exposed to increasing concentrations of TG02 to determine effect on brachyury protein expression. qPCR was used to analyze brachyury mRNA expression as well as the mesenchymal proteins, vimentin and fibronectin, typically associated with brachyury expression in tumor cells. Cell migration was evaluated using wound healing assay. CellTiterGlo Luminescence Assay was used to quantify cell viability. RESULTS: TG02 down-regulated protein expression of brachyury in a dose-dependent manner. Attenuation of brachyury expression was seen within 4 hours and persisted for 5 days upon single exposure to a clinically relevant concentration of TG02. Brachyury downregulation did not affect cell count or viability, and was associated with a quiescent phenotype, including impaired migration. Expression of vimentin and fibronectin, both associated with EMT, was also suppressed within 4 hours of TG02 treatment and persisted for 24 hours. CONCLUSIONS: Inhibition of brachyury expression and its downstream signaling is associated with quiescent behavior of chordoma cells. Pharmacologically induced transcriptional targeting of brachyury using TG02, a potent CDK9 inhibitor, represents a potential therapeutic strategy. Citation Format: Ashlee N. Seldomridge, Jinkyu Jung, Heather M. Sonnemann, Amber Giles, Kristan Meetze, Mark R. Gilbert, Claudia M. Palena, Deric M. Park. Transcriptional inhibition of brachyury in chordoma is associated with adoption of quiescent phenotype [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 1533. doi:10.1158/1538-7445.AM2017-1533