Purpose: Glioblastoma is one of the most lethal cancers in humans, and with existing therapy, survival remains at 14.6 months. Current barriers to successful treatment include their infiltrative behavior, extensive tumor heterogeneity, and the presence of a stem-like population of cells, termed brain tumor-initiating cells (BTIC) that confer resistance to conventional therapies.Experimental Design: To develop therapeutic strategies that target BTICs, we focused on a repurposing approach that explored already-marketed (clinically approved) drugs for therapeutic potential against patient-derived BTICs that encompass the genetic and phenotypic heterogeneity of glioblastoma observed clinically.Results: Using a high-throughput in vitro drug screen, we found that montelukast, clioquinol, and disulfiram (DSF) were cytotoxic against a large panel of patient-derived BTICs. Of these compounds, disulfiram, an off-patent drug previously used to treat alcoholism, in the presence of a copper supplement, showed low nanomolar efficacy in BTICs including those resistant to temozolomide and the highly infiltrative quiescent stem-like population. Low dose DSF-Cu significantly augmented temozolomide activity in vitro, and importantly, prolonged in vivo survival in patient-derived BTIC models established from both newly diagnosed and recurrent tumors. Moreover, we found that in addition to acting as a potent proteasome inhibitor, DSF-Cu functionally impairs DNA repair pathways and enhances the effects of DNA alkylating agents and radiation. These observations suggest that DSF-Cu inhibits proteasome activity and augments the therapeutic effects of DNA-damaging agents (temozolomide and radiation).Conclusions: DSF-Cu should be considered as an adjuvant therapy for the treatment of patients with glioblastoma in both newly diagnosed and recurrent settings.

We have previously reported that the CD8+ T cell response elicited by recombinant adenovirus vaccination displayed a delayed contraction in the spleen. In our current study, we demonstrate that this unusual kinetic is a general phenomenon observed in multiple tissues. Phenotypic analysis of transgene-specific CD8+ T cells present 30 days postimmunization with recombinant adenovirus revealed a population with evidence of partial exhaustion, suggesting that the cells had been chronically exposed to Ag. Although Ag expression could no longer be detected 3 wk after immunization, examination of Ag presentation within the draining lymph nodes demonstrated that APCs were loaded with Ag peptide for at least 40 days postimmunization, suggesting that Ag remains available to the system for a prolonged period, although the exact source of this Ag remains to be determined. At 60 days postimmunization, the CD8+ T cell population continued to exhibit a phenotype consistent with partially exhausted effector memory cells. Nonetheless, these CD8+ T cells conferred sterilizing immunity against virus challenge 7–12 wk postimmunization, suggesting that robust protective immunity can be provided by CD8+ T cells with an exhausted phenotype. These data demonstrate that prolonged exposure to Ag may not necessarily impair protective immunity and prompt a re-evaluation of the impact of persistent exposure to Ag on T cell function.

Despite a deeper molecular understanding, human glioblastoma remains one of the most treatment refractory and fatal cancers. It is known that the presence of macrophages and microglia impact glioblastoma tumorigenesis and prevent durable response. Herein we identify the dual function cytokine IL-33 as an orchestrator of the glioblastoma microenvironment that contributes to tumorigenesis. We find that IL-33 expression in a large subset of human glioma specimens and murine models correlates with increased tumor-associated macrophages/monocytes/microglia. In addition, nuclear and secreted functions of IL-33 regulate chemokines that collectively recruit and activate circulating and resident innate immune cells creating a pro-tumorigenic environment. Conversely, loss of nuclear IL-33 cripples recruitment, dramatically suppresses glioma growth, and increases survival. Our data supports the paradigm that recruitment and activation of immune cells, when instructed appropriately, offer a therapeutic strategy that switches the focus from the cancer cell alone to one that includes the normal host environment.

Excellular hemoglobin is an extremely active oxidant of low-density lipoproteins (LDL), a phenomenon explained so far by different mechanisms. In this study, we analyzed the mechanism of met-hemoglobin oxidability by comparing its mode of operation with other hemoproteins, met-myoglobin and horseradish peroxidase (HRP) or with free hemin. The kinetics of met-hemoglobin activity toward LDL lipids and protein differed from that of met-myoglobin and HRP, both quantitatively and qualitatively. Those differences were further clarified by analyzing heme transfer from the above-mentioned hemoproteins to LDL. It appeared that met-hemoglobin transferred most of its hemin to LDL, and the presence of H(2)O(2) accelerated the process. In contrast, met-myoglobin partially released hemin, but only in the presence of H(2)O(2), while HRP could not transfer heme at all. The minor amount of hemin transferred from met-myoglobin to LDL sufficed to trigger ApoB oxidation, forming covalent aggregates via inter-bityrosines. This indicated that heme bound to high affinity site(s) is responsible for oxidation. LDL components providing the sites were analyzed by binding heme-CO monomers to LDL. Soret spectra revealed that the high affinity site of monomeric hemin is located on the LDL protein, ApoB. The complex heme-CO-ApoB underwent instantaneous oxidation to hemin-ApoB, and the bound hemin then slowly disintegrated in conjunction with LDL oxidation. Hemopexin prevented LDL oxidation by trapping hemoprotein transferable heme. We concluded that met-hemoglobin exerts its oxidative activity on LDL via transfer of heme, which serves as a vehicle for iron insertion into the LDL protein, leading to formation of atherogenic LDL aggregates.