Development of a Cx46 Targeting Strategy for Cancer Stem Cells

Gap-junction-mediated cell-cell communication enables tumor cells to synchronize complex processes. We previously found that glioblastoma cancer stem cells (CSCs) express higher levels of the gap junction protein Cx46 compared to non-stem tumor cells (non-CSCs) and that this was necessary and sufficient for CSC maintenance. To understand the mechanism underlying this requirement, we use point mutants to disrupt specific functions of Cx46 and find that Cx46-mediated gap-junction coupling is critical for CSCs. To develop a Cx46 targeting strategy, we screen a clinically relevant small molecule library and identify clofazimine as an inhibitor of Cx46-specific cell-cell communication. Clofazimine attenuates proliferation, self-renewal, and tumor growth and synergizes with temozolomide to induce apoptosis. Although clofazimine does not cross the blood-brain barrier, the combination of clofazimine derivatives optimized for brain penetrance with standard-of-care therapies may target glioblastoma CSCs. Furthermore, these results demonstrate the importance of targeting cell-cell communication as an anti-cancer therapy

ADAMDEC1 maintains a growth factor signaling loop in cancer stem cells

Glioblastomas (GBM) are lethal brain tumors where poor outcome is attributed to cellular heterogeneity, therapeutic resistance, and a highly infiltrative nature. These characteristics are preferentially linked to GBM cancer stem cells (GSCs), but how GSCs maintain their stemness is incompletely understood and the subject of intense investigation. Here, we identify a novel signaling loop that induces and maintains GSCs consisting of an atypical metalloproteinase, a disintegrin and metalloproteinase domain-like protein decysin 1 (ADAMDEC1), secreted by GSCs. ADAMDEC1 rapidly solubilizes fibroblast growth factor-2 (FGF2) to stimulate FGF receptor 1 (FGFR1) expressed on GSCs. FGFR1 signaling induces upregulation of Zinc-finger E-box-binding homeobox 1 (ZEB1) via ERK1/2 that regulates ADAMDEC1 expression through miR-203, creating a positive feedback loop. Genetic or pharmacological targeting of components of this axis attenuates self-renewal and tumor growth. These findings reveal a new signaling axis for GSC maintenance and highlight ADAMDEC1 and FGFR1 as potential therapeutic targets in GB

SerpinB3 Drives Cancer Stem Cell Survival in Glioblastoma

Despite therapeutic interventions for glioblastoma (GBM), cancer stem cells (CSCs) drive recurrence. The precise mechanisms underlying CSC resistance, namely inhibition of cell death, are unclear. We built on previous observations that the high cell surface expression of junctional adhesion molecule-A drives CSC maintenance and identified downstream signaling networks, including the cysteine protease inhibitor SerpinB3. Using genetic depletion approaches, we found that SerpinB3 is necessary for CSC maintenance, survival, and tumor growth, as well as CSC pathway activation. Knockdown of SerpinB3 also increased apoptosis and susceptibility to radiation therapy. SerpinB3 was essential to buffer cathepsin L-mediated cell death, which was enhanced with radiation. Finally, we found that SerpinB3 knockdown increased the efficacy of radiation in pre-clinical models. Taken together, our findings identify a GBM CSC-specific survival mechanism involving a cysteine protease inhibitor, SerpinB3, and provide a potential target to improve the efficacy of GBM therapies against therapeutically resistant CSCs

Factors Influencing the Differentiation of Human Monocytic Myeloid-Derived Suppressor Cells Into Inflammatory Macrophages

Monocytic myeloid-derived suppressor cells (mMDSC) accumulate within tumors where they create an immunosuppressive milieu that inhibits the activity of cytotoxic T and NK cells thereby allowing cancers to evade immune elimination. The toll-like receptors 7/8 agonist R848 induces human mMDSC to mature into inflammatory macrophage (MACinflam). This work demonstrates that TNFα, IL-6, and IL-10 produced by maturing mMDSC are critical to the generation of MACinflam. Neutralizing any one of these cytokines significantly inhibits R848-dependent mMDSC differentiation. mMDSC cultured in pro-inflammatory cytokine IFNγ or the combination of TNFα plus IL-6 differentiate into MACinflam more efficiently than those treated with R848. These mMDSC-derived macrophages exert anti-tumor activity by killing cancer cells. RNA-Seq analysis of the genes expressed when mMDSC differentiate into MACinflam indicates that TNFα and the transcription factors NF-κB and STAT4 are major hubs regulating this process. These findings support the clinical evaluation of R848, IFNγ, and/or TNFα plus IL-6 for intratumoral therapy of established cancers

IMMU-57. EFFECT OF THE ONCOMETABOLITE D2HG ON MYELOID-DERIVED SUPPRESSOR CELL POPULATIONS

Abstract Mutations in the isocitrate dehydrogenase 1/2 (IDH1/2) enzyme correlate with better prognosis of gliomas, partly due to enhanced infiltration of nonimmunosuppressive granulocytic cells compared to IDH wild-type (IDHwt) tumors. Altered cytokine/chemokine secretion from tumor cells and reprogramming of myeloid cell metabolism are among the factors that drive differences in immune profiles of IDHwt vs. IDHmut tumors. IDH mutant protein produces the oncometabolite D-2-hydroxyglutarate (D2HG) instead of the canonical alpha-ketoglutarate (αKG), a co-factor for histone and DNA demethylases. Since D2HG is known to be secreted into the tumor microenvironment (TME), we interrogated the effect of this metabolite on the epigenetic landscape and behavior of granulocytic and monocytic myeloid-derived suppressor cells (g/mMDSCs). Co-culturing bone marrow cells with either IDHmut or IDHwt glioma cells demonstrated that mMDSCs, which we previously showed to be the abundant tumor infiltrating MDSC subtype in IDHwt tumors, but not gMDSCs specifically experience higher levels of apoptosis when exposed to IDHmut cells. Treatment of bone marrow cells with physiologically relevant concentrations of either D2HG or αKG demonstrated that D2HG re-capitulates the pro-apoptotic effect on mMDSCs. Furthermore, co-cultures of IDHmut glioma cells and bone-marrow cells induced a pronounced increase in global methylation on mMDSCs specifically, and we observed that mMDSCs and gMDSCs differentially express histone demethylases via western blotting. Collectively, these results suggest that D2HG can differentially impact mMDSC vs gMDSC biology by epigenetic reprogramming. Future studies will elucidate the specific targets of D2HG in mMDSCs that renders these cells more susceptible to apoptosis with the goal of identifying the mechanisms that can be modulated to down-regulate mMDSC infiltration and/or survival within the TME

The missing link? LGMN connects hypoxia and immunosuppression in glioblastoma

In this issue, Pang and colleagues1 identify the protease legumain as a potential immunotherapy target in glioblastoma that drives tumor-associated macrophages in response to hypoxia

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<p>Monocytic myeloid-derived suppressor cells (mMDSC) accumulate within tumors where they create an immunosuppressive milieu that inhibits the activity of cytotoxic T and NK cells thereby allowing cancers to evade immune elimination. The toll-like receptors 7/8 agonist R848 induces human mMDSC to mature into inflammatory macrophage (MAC_inflam). This work demonstrates that TNFα, IL-6, and IL-10 produced by maturing mMDSC are critical to the generation of MAC_inflam. Neutralizing any one of these cytokines significantly inhibits R848-dependent mMDSC differentiation. mMDSC cultured in pro-inflammatory cytokine IFNγ or the combination of TNFα plus IL-6 differentiate into MAC_inflam more efficiently than those treated with R848. These mMDSC-derived macrophages exert anti-tumor activity by killing cancer cells. RNA-Seq analysis of the genes expressed when mMDSC differentiate into MAC_inflam indicates that TNFα and the transcription factors NF-κB and STAT4 are major hubs regulating this process. These findings support the clinical evaluation of R848, IFNγ, and/or TNFα plus IL-6 for intratumoral therapy of established cancers.</p

IMMU-35. G-AMINOBUTYRIC ACID DRIVES GLIOBLASTOMA IN FEMALES BY ACTIVATING THE COMPLEMENT SYSTEM

Abstract Interaction between tumor and neural cells drives the progression of glioblastoma (GBM), which is characterized by enhanced immunosuppressive myeloid cell accumulation. Our previous results revealed that myeloid-derived suppressor cell subsets (MDSCs) play a central role in tumorigenesis in a sex-specific manner, with monocytic MDSCs (mMDSCs) accumulating in male tumors and granulocytic MDSCs (gMDSCs) correlating with a worse outcome in females. However, host factors governing sex differences in MDSC subset activity remained unknown. Here, we report that g-aminobutyric acid (GABA) receptor expression by gMDSCs informs their sex-specific behavior. We observed that only female gMDSCs but not male gMDSCs or mMDSCs from either sex respond to GABA by upregulating the antiviral innate immune pathway. As part of this signature, GABA-stimulated female gMDSCs had higher complement 1qa (C1qa) expression. To test the potential role of the GABA pathway on GBM outcome, we tested the GABA analogue pregabalin in preclinical models. Female mice treated with pregabalin succumbed to disease earlier, consistent with our previous observation that gMDSCs have a central role in GBM progression in females. In contrast, pregabalin treatment had no effect in male mice with tumors or immunocompromised female mice. To further evaluate whether C1q is a relevant physiological target, we performed single-cell sequencing of tumor-infiltrating versus circulating MDSC subsets and detected high levels of C1q expression in gMDSCs from tumors. C1q KO transgenic female mice had significantly better outcomes compared to wild-type littermates upon implantation with syngeneic mouse GBM lines, while C1q expression status did not impact the outcome in males. Correspondingly, only in female patients C1q expression correlated with poor prognosis. Collectively, these results identify a neuron-immune-cancer communication axis and indicate that GABA and C1q alter anti-tumor immune responses in a sex-specific manner. These results support the future assessment of the GABA pathway and complement inhibitors as potential immunotherapy agents

'Slicing' glioblastoma drivers with the Swiss cheese model

The Swiss cheese model is used to assess risks and explain accidents in a variety of industries. This model can be applied to dissect the homeostatic mechanisms whose cumulative dysregulation contributes to disease states, including cancer. Using glioblastoma (GBM) as an exemplar, we discuss how specific protumorigenic mechanisms collectively drive disease by affecting genomic integrity, epigenetic regulation, metabolic homeostasis, and antitumor immunity. We further highlight how host factors, such as hormonal differences and aging, impact this process, and the interplay between these 'system failures' that enable tumor progression and foster therapeutic resistance. Finally, we examine therapies that consider the interactions between these elements, which may comprise more effective approaches given the multifaceted protumorigenic mechanisms that drive GBM