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Glioblastoma (GBM) is an immunologically cold tumor, but several immunotherapy-based strategies show promise, including the administration of ex vivo expanded and activated cytotoxic gamma delta T cells. Cytotoxicity is partially mediated through interactions with natural killer group 2D ligands (NKG2DL) on tumor cells. We sought to determine whether the addition of the blood-brain barrier penetrant PARP inhibitor niraparib to the standard of care DNA alkylator temozolomide (TMZ) could upregulate NKG2DL, thereby improving immune cell recognition. Changes in viability were consistent with prior publications as there was a growth inhibitory effect of the combination of TMZ and niraparib. However, decreases in viability did not always correlate with changes in NKG2DL mRNA. ULBP1/Mult-1 mRNA was increased with the combination therapy in comparison to either drug alone in two of the three cell types tested, even though viability was consistently decreased. mRNA expression correlated with protein levels and ULBP1/MULT-1 cell surface protein was significantly increased with TMZ and niraparib treatment in four of the five cell types tested. Gamma delta T cell-mediated cytotoxicity at a 10:1 effector-to-target ratio was significantly increased upon pretreatment of cells derived from a GBM PDX with TMZ and niraparib in comparison to the control or either drug alone. Together, these data demonstrate that the combination of PARP inhibition, DNA alkylation, and gamma delta T cell therapy has the potential for the treatment of GBM.

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Drug repurposing is promising because approving a drug for a new indication requires fewer resources than approving a new drug. Signature reversion detects drug perturbations most inversely related to the disease-associated gene signature to identify drugs that may reverse that signature. We assessed the performance and biological relevance of three approaches for constructing disease-associated gene signatures (i.e., limma, DESeq2, and MultiPLIER) and prioritized the resulting drug repurposing candidates for four low-survival human cancers. Our results were enriched for candidates that had been used in clinical trials or performed well in the PRISM drug screen. Additionally, we found that pamidronate and nimodipine, drugs predicted to be efficacious against the brain tumor glioblastoma (GBM), inhibited the growth of a GBM cell line and cells isolated from a patient-derived xenograft (PDX). Our results demonstrate that by applying multiple disease-associated gene signature methods, we prioritized several drug repurposing candidates for low-survival cancers.

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Sphingolipid metabolism is dysregulated in many cancers, allowing cells to evade apoptosis through increased sphingosine-1-phosphate (S1P) and decreased ceramides. Ceramidases hydrolyze ceramides to sphingosine, which is phosphorylated by sphingosine kinases to generate S1P. The S1P allows cells to evade apoptosis by shifting the equilibrium away from ceramides, which favor cell death. One tumor type that exhibits a shift in the sphingolipid balance towards S1P is glioblastoma (GBM), a highly aggressive brain tumor. GBMs almost always recur despite surgical resection, radiotherapy, and chemotherapy with temozolomide (TMZ). Understanding sphingolipid metabolism in GBM is still limited, and currently, there are no approved treatments to target dysregulation of sphingolipid metabolism in GBM. Carmofur, a derivative of 5-fluorouracil, inhibits acid ceramidase (ASAH1), a key enzyme in the production of S1P, and is in use outside the USA to treat colorectal cancer. We find that the mRNA for ASAH1, but not other ceramidases, is elevated in recurrent GBM. When TMZ-resistant GBM cells were treated with carmofur, decreased cell growth and increased apoptosis were observed along with cell cycle perturbations. RNA-sequencing identified decreases in cell cycle control pathways that were specific to TMZ-resistant cells. Furthermore, the transcription factor and G1 to S phase regulator, E2F8, was upregulated in TMZ-resistant versus parental GBM cells and inhibited by carmofur treatment in TMZ-resistant GBM cells, specifically. These data suggest a possible role for E2F8 as a mediator of carmofur effects in the context of TMZ resistance. These data suggest the potential utility of normalizing the sphingolipid balance in the context of recurrent GBM.

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Glioblastoma (GBM) tumor-associated macrophages (TAMs) provide a major immune cell population contributing to growth and immunosuppression via the production of proinflammatory factors, including IL-1. In this issue of the JCI, Chen, Giotti, and colleagues investigated loss of ll1b in the immune tumor microenvironment (TME) in GBM models driven by PDGFB expression and Nf1 knockdown. Survival was only improved in PDGFB-driven GBM models, suggesting that tumor cell genotype influenced the immune TME. IL-1ß in the TME increased PDGFB-driven GBM growth by increasing tumor-derived NF-κB, expression of monocyte chemoattractants, and increased infiltration of bone marrow-derived myeloid cells (BMDMs). In contrast, no requirement for IL-1ß was evident in Nf1-silenced tumors due to high basal levels of NF-κB and monocyte chemoattractants and increased infiltration of BMDM and TAMs. Notably, treatment of mice bearing PDGFB-driven GBM with anti-IL-1ß or an IL1R1 antagonist extended survival. These findings suggest that effective clinical immunotherapy may require differential targeting strategies.

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Treatment for the deadly brain tumor glioblastoma (GBM) has been improved through the non-invasive addition of alternating electric fields, called tumor treating fields (TTFields). Improving both progression-free and overall survival, TTFields are currently approved for treatment of recurrent GBMs as a monotherapy and in the adjuvant setting alongside TMZ for newly diagnosed GBMs. These TTFields are known to inhibit mitosis, but the full molecular impact of TTFields remains undetermined. Therefore, we sought to understand the ability of TTFields to disrupt the growth patterns of and induce kinomic landscape shifts in TMZ-sensitive and -resistant GBM cells. We determined that TTFields significantly decreased the growth of TMZ-sensitive and -resistant cells. Kinomic profiling predicted kinases that were induced or repressed by TTFields, suggesting possible therapy-specific vulnerabilities. Serving as a potential pro-survival mechanism for TTFields, kinomics predicted the increased activity of platelet-derived growth-factor receptor alpha (PDGFRα). We demonstrated that the addition of the PDGFR inhibitor, crenolanib, to TTFields further reduced cell growth in comparison to either treatment alone. Collectively, our data suggest the efficacy of TTFields in vitro and identify common signaling responses to TTFields in TMZ-sensitive and -resistant populations, which may support more personalized medicine approaches.

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The transfer of intact mitochondria between heterogeneous cell types has been confirmed in various settings, including cancer. However, the functional implications of mitochondria transfer on tumor biology are poorly understood. Here we show that mitochondria transfer is a prevalent phenomenon in glioblastoma (GBM), the most frequent and malignant primary brain tumor. We identified horizontal mitochondria transfer from astrocytes as a mechanism that enhances tumorigenesis in GBM. This transfer is dependent on network-forming intercellular connections between GBM cells and astrocytes, which are facilitated by growth-associated protein 43 (GAP43), a protein involved in neuron axon regeneration and astrocyte reactivity. The acquisition of astrocyte mitochondria drives an increase in mitochondrial respiration and upregulation of metabolic pathways linked to proliferation and tumorigenicity. Functionally, uptake of astrocyte mitochondria promotes cell cycle progression to proliferative G2/M phases and enhances self-renewal and tumorigenicity of GBM. Collectively, our findings reveal a host-tumor interaction that drives proliferation and self-renewal of cancer cells, providing opportunities for therapeutic development.

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Pancreatic neuroendocrine tumors (pNETs) are extremely diverse and highly vascularized neoplasms that arise from endocrine cells in the pancreas. The pNETs harbor a subpopulation of stem cell-like malignant cells, known as cancer stem cells (CSCs), which contribute to intratumoral heterogeneity and promote tumor maintenance and recurrence. In this study, we demonstrate that CSCs in human pNETs co-express protein kinase PKD1 and CD44. We further identify PKD1 signaling as a critical pathway in the control of CSC maintenance in pNET cells. PKD1 signaling regulates the expression of a CSC- and EMT-related gene signature and promotes CSC self-renewal, likely leading to the preservation of a subpopulation of CSCs at an intermediate EMT state. This suggests that the PKD1 signaling pathway may be required for the development of a unique CSC phenotype with plasticity and partial EMT. Given that the signaling networks connected with CSC maintenance and EMT are complex, and extend through multiple levels of regulation, this study provides insight into signaling regulation of CSC plasticity and partial EMT in determining the fate of CSCs. Inhibition of the PKD1 pathway may facilitate the elimination of specific CSC subsets, thereby curbing tumor progression and metastasis.

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One of the least-investigated areas of brain pathology research is glycosylation, which is a critical regulator of cell surface protein structure and function. ß-Galactoside α2,6-sialyltransferase (ST6GAL1) is the primary enzyme that α2,6 sialylates N-glycosylated proteins destined for the plasma membrane or secretion, thereby modulating cell signaling and behavior. We demonstrate a potentially novel, protumorigenic role for α2,6 sialylation and ST6GAL1 in the deadly brain tumor glioblastoma (GBM). GBM cells with high α2,6 sialylation exhibited increased in vitro growth and self-renewal capacity and decreased mouse survival when orthotopically injected. α2,6 Sialylation was regulated by ST6GAL1 in GBM, and ST6GAL1 was elevated in brain tumor-initiating cells (BTICs). Knockdown of ST6GAL1 in BTICs decreased in vitro growth, self-renewal capacity, and tumorigenic potential. ST6GAL1 regulates levels of the known BTIC regulators PDGF Receptor ß (PDGFRB), Activated Leukocyte Cell Adhesion Molecule, and Neuropilin, which were confirmed to bind to a lectin-recognizing α2,6 sialic acid. Loss of ST6GAL1 was confirmed to decrease PDGFRB α2,6 sialylation, total protein levels, and the induction of phosphorylation by PDGF-BB. Thus, ST6GAL1-mediated α2,6 sialylation of a select subset of cell surface receptors, including PDGFRB, increases GBM growth.

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Patients presenting with metastatic hepatoblastoma have limited treatment options and survival rates as low as 25%. We previously demonstrated that Proviral Integration site in Maloney murine leukemia virus 3 (PIM3) kinase promotes tumorigenesis and cancer cell stemness in hepatoblastoma. In this study, we assessed the role of PIM3 kinase in promoting hepatoblastoma metastasis. We utilized a tail vein injection model of metastasis to evaluate the effect of CRISPR/Cas9-mediated PIM3 knockout, stable overexpression of PIM3, and pharmacologic PIM inhibition on the formation of lung metastasis. In vivo studies revealed PIM3 knockout impaired the formation of lung metastasis: 5 out of 6 mice injected with wild type hepatoblastoma cells developed lung metastasis while none of the 7 mice injected with PIM3 knockout hepatoblastoma cells developed lung metastasis. PIM3 overexpression in hepatoblastoma increased the pulmonary metastatic burden in mice and mechanistically, upregulated the phosphorylation and cell surface expression of CXCR4, a key receptor in the progression of cancer cell metastasis. CXCR4 blockade with AMD3100 decreased the metastatic phenotype of PIM3 overexpressing cells, indicating that CXCR4 contributed to PIM3's promotion of hepatoblastoma metastasis. Clinically, PIM3 expression correlated positively with CXCR4 expression in primary hepatoblastoma tissues. In conclusion, we have shown PIM3 kinase promotes the metastatic phenotype of hepatoblastoma cells through upregulation of CXCR4 cell surface expression and these findings suggest that targeting PIM3 kinase may provide a novel therapeutic strategy for metastatic hepatoblastoma.

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The Golgi-sialyltransferase ST6Gal1 (ßgalactosidase α2,6 sialyltransferase 1), adds the negatively charged sugar, sialic acid, to the terminal galactose of N-glycosylated proteins. Upregulation of ST6Gal1 is observed in many malignancies, and a large body of research has determined that ST6Gal1-mediated α2,6 sialylation impacts cancer hallmarks. ST6Gal1 affects oncogenic behaviors including sustained proliferation, enhanced self-renewal, epithelial-to-mesenchymal transition, invasion, and chemoresistance. However, there are relatively few ST6GaL1 related signaling pathways that are well-established to mediate these biologies: greater delineation of specific targets and signaling mechanisms that are orchestrated by ST6Gal1 is needed. The aim of this review is to provide a summary of our current understanding of select oncogenic signaling pathways and targets affected by ST6Gal1.

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Key molecular regulators of acquired radiation resistance in recurrent glioblastoma (GBM) are largely unknown, with a dearth of accurate preclinical models. To address this, we generated 8 GBM patient-derived xenograft (PDX) models of acquired radiation therapy-selected (RTS) resistance compared with same-patient, treatment-naive (radiation-sensitive, unselected; RTU) PDXs. These likely unique models mimic the longitudinal evolution of patient recurrent tumors following serial radiation therapy. Indeed, while whole-exome sequencing showed retention of major genomic alterations in the RTS lines, we did detect a chromosome 12q14 amplification that was associated with clinical GBM recurrence in 2 RTS models. A potentially novel bioinformatics pipeline was applied to analyze phenotypic, transcriptomic, and kinomic alterations, which identified long noncoding RNAs (lncRNAs) and targetable, PDX-specific kinases. We observed differential transcriptional enrichment of DNA damage repair pathways in our RTS models, which correlated with several lncRNAs. Global kinomic profiling separated RTU and RTS models, but pairwise analyses indicated that there are multiple molecular routes to acquired radiation resistance. RTS model-specific kinases were identified and targeted with clinically relevant small molecule inhibitors. This cohort of in vivo RTS patient-derived models will enable future preclinical therapeutic testing to help overcome the treatment resistance seen in patients with GBM.

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Glioblastoma (GBM) remains one of the most aggressive cancers, partially due to its ability to migrate into the surrounding brain. The sphingolipid balance, or the balance between ceramides and sphingosine-1-phosphate, contributes to the ability of GBM cells to migrate or invade. Of the ceramidases which hydrolyze ceramides, acid ceramidase (ASAH1) is highly expressed in GBM samples compared to non-tumor brain. ASAH1 expression also correlates with genes associated with migration and focal adhesion. To understand the role of ASAH1 in GBM migration, we utilized shRNA knockdown and observed decreased migration that did not depend upon changes in growth. Next, we inhibited ASAH1 using carmofur, a clinically utilized small molecule inhibitor. Inhibition of ASAH1 by carmofur blocks in vitro migration of U251 (GBM cell line) and GBM cells derived from patient-derived xenografts (PDXs). RNA-sequencing suggested roles for carmofur in MAPK and AKT signaling. We found that carmofur treatment decreases phosphorylation of AKT, but not of MAPK. The decrease in AKT phosphorylation was confirmed by shRNA knockdown of ASAH1. Our findings substantiate ASAH1 inhibition using carmofur as a potential clinically relevant treatment to advance GBM therapeutics, particularly due to its impact on migration.

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Under cellular distress, multiple facets of normal homeostatic signaling are altered or disrupted. In the context of the immune landscape, external and internal stressors normally promote the expression of natural killer group 2 member D (NKG2D) ligands that allow for the targeted recognition and killing of cells by NKG2D receptor-bearing effector populations. The presence or absence of NKG2D ligands can heavily influence disease progression and impact the accessibility of immunotherapy options. In cancer, tumor cells are known to have distinct regulatory mechanisms for NKG2D ligands that are directly associated with tumor progression and maintenance. Therefore, understanding the regulation of NKG2D ligands in cancer will allow for targeted therapeutic endeavors aimed at exploiting the stress response pathway. In this review, we summarize the current understanding of regulatory mechanisms controlling the induction and repression of NKG2D ligands in cancer. Additionally, we highlight current therapeutic endeavors targeting NKG2D ligand expression and offer our perspective on considerations to further enhance the field of NKG2D ligand biology.

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BACKGROUND/PURPOSE: Metastatic hepatoblastoma continues to pose a significant treatment challenge, primarily because the precise mechanisms involved in metastasis are not fully understood, making cell lines and preclinical models that depict the progression of disease and metastasis-related biology paramount. We aimed to generate and characterize a metastatic hepatoblastoma cell line to create a model for investigation of the molecular mechanisms associated with metastasis. MATERIALS/METHODS: Using a murine model of serial tail vein injections of the human hepatoblastoma HuH6 cell line, non-invasive bioluminescence imaging, and dissociation of metastatic pulmonary lesions, we successfully established and characterized the metastatic human hepatoblastoma cell line, HLM_3. RESULTS: The HLM_3 cells exhibited enhanced tumorigenicity and invasiveness, both in vitro and in vivo compared to the parent HuH6 cell line. Moreover, HLM_3 metastatic hepatoblastoma cells exhibited a stem cell-like phenotype and were more resistant to the standard chemotherapeutic cisplatin. CONCLUSION: This newly described metastatic hepatoblastoma cell line offers a novel tool to study mechanisms of tumor metastasis and evaluate new therapeutic strategies for metastatic hepatoblastoma.

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Gene knockout of the master regulator of mitochondrial fission, Drp1, prevents neoplastic transformation. Also, mitochondrial fission and its opposing process of mitochondrial fusion are emerging as crucial regulators of stemness. Intriguingly, stem/progenitor cells maintaining repressed mitochondrial fission are primed for self-renewal and proliferation. Using our newly derived carcinogen transformed human cell model, we demonstrate that fine-tuned Drp1 repression primes a slow cycling 'stem/progenitor-like state', which is characterized by small networks of fused mitochondria and a gene-expression profile with elevated functional stem/progenitor markers (Krt15, Sox2 etc) and their regulators (Cyclin E). Fine tuning Drp1 protein by reducing its activating phosphorylation sustains the neoplastic stem/progenitor cell markers. Whereas, fine-tuned reduction of Drp1 protein maintains the characteristic mitochondrial shape and gene-expression of the primed 'stem/progenitor-like state' to accelerate neoplastic transformation, and more complete reduction of Drp1 protein prevents it. Therefore, our data highlights a 'goldilocks' level of Drp1 repression supporting stem/progenitor state dependent neoplastic transformation.

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Controlling reactive oxygen species (ROS) at sustainable levels can drive multiple facets of tumor biology, including within the cancer stem cell (CSC) population. Tight regulation of ROS is one key component in CSCs that drives disease recurrence, cell signaling, and therapeutic resistance. While ROS are well-appreciated to need oxygen and are a product of oxidative phosphorylation, there are also important roles for ROS under hypoxia. As hypoxia promotes and sustains major stemness pathways, further consideration of ROS impacts on CSCs in the tumor microenvironment is important. Furthermore, glycolytic shifts that occur in cancer and may be promoted by hypoxia are associated with multiple mechanisms to mitigate oxidative stress. This altered metabolism provides survival advantages that sustain malignant features, such as proliferation and self-renewal, while producing the necessary antioxidants that reduce damage from oxidative stress. Finally, disease recurrence is believed to be attributed to therapy resistant CSCs which can be quiescent and have changes in redox status. Effective DNA damage response pathways and/or a slow-cycling state can protect CSCs from the genomic catastrophe induced by irradiation and genotoxic agents. This review will explore the delicate, yet complex, relationship between ROS and its pleiotropic role in modulating the CSC.

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Treatment for the lethal primary adult brain tumor glioblastoma (GBM) includes the chemotherapy temozolomide (TMZ), but TMZ resistance is common and correlates with promoter methylation of the DNA repair enzyme O-6-methylguanine-DNA methyltransferase (MGMT). To improve treatment of GBMs, including those resistant to TMZ, we explored the potential of targeting dopamine receptor signaling. We found that dopamine receptor 3 (DRD3) is expressed in GBM and is also a previously unexplored target for therapy. We identified novel antagonists of DRD3 that decreased the growth of GBM xenograft-derived neurosphere cultures with minimal toxicity against human astrocytes and/or induced pluripotent stem cell-derived neurons. Among a set of DRD3 antagonists, we identified two compounds, SRI-21979 and SRI-30052, that were brain penetrant and displayed a favorable therapeutic window analysis of The Cancer Genome Atlas data demonstrated that higher levels of DRD3 (but not DRD2 or DRD4) were associated with worse prognosis in primary, MGMT unmethylated tumors. These data suggested that DRD3 antagonists may remain efficacious in TMZ-resistant GBMs. Indeed, SRI-21979, but not haloperidol, significantly reduced the growth of TMZ-resistant GBM cells. Together our data suggest that DRD3 antagonist-based therapies may provide a novel therapeutic option for the treatment of GBM.

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The multifaceted roles of metabolism in invasion have been investigated across many cancers. The brain tumor glioblastoma (GBM) is a highly invasive and metabolically plastic tumor with an inevitable recurrence. The neuronal glucose transporter 3 (GLUT3) was previously reported to correlate with poor glioma patient survival and be upregulated in GBM cells to promote therapeutic resistance and survival under restricted glucose conditions. It has been suggested that the increased glucose uptake mediated by GLUT3 elevation promotes survival of circulating tumor cells to facilitate metastasis. Here we suggest a more direct role for GLUT3 in promoting invasion that is not dependent upon changes in cell survival or metabolism. Analysis of glioma datasets demonstrated that GLUT3, but not GLUT1, expression was elevated in invasive disease. In human xenograft derived GBM cells, GLUT3, but not GLUT1, elevation significantly increased invasion in transwell assays, but not growth or migration. Further, there were no changes in glycolytic metabolism that correlated with invasive phenotypes. We identified the GLUT3 C-terminus as mediating invasion: substituting the C-terminus of GLUT1 for that of GLUT3 reduced invasion. RNA-seq analysis indicated changes in extracellular matrix organization in GLUT3 overexpressing cells, including upregulation of osteopontin. Together, our data suggest a role for GLUT3 in increasing tumor cell invasion that is not recapitulated by GLUT1, is separate from its role in metabolism and survival as a glucose transporter, and is likely broadly applicable since GLUT3 expression correlates with metastasis in many solid tumors.

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Tumor microenvironments are the result of cellular alterations in cancer that support unrestricted growth and proliferation and result in further modifications in cell behavior, which are critical for tumor progression. Angiogenesis and therapeutic resistance are known to be modulated by hypoxia and other tumor microenvironments, such as acidic stress, both of which are core features of the glioblastoma microenvironment. Hypoxia has also been shown to promote a stem-like state in both non-neoplastic and tumor cells. In glial tumors, glioma stem cells (GSCs) are central in tumor growth, angiogenesis, and therapeutic resistance, and further investigation of the interplay between tumor microenvironments and GSCs is critical to the search for better treatment options for glioblastoma. Accordingly, we summarize the impact of hypoxia and acidic stress on GSC signaling and biologic phenotypes, and potential methods to inhibit these pathways.

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Glioblastoma (GBM) is an aggressive malignancy with limited effectiveness of standard of care therapies including surgery, radiation, and temozolomide chemotherapy necessitating novel therapeutics. Unfortunately, GBMs also harbor several signaling alterations that protect them from traditional therapies that rely on apoptotic programmed cell death. Because almost all GBM tumors have dysregulated phosphoinositide signaling as part of that process, we hypothesized that peptide mimetics derived from the phospholipid binding domain of Myristoylated alanine-rich C-kinase substrate (MARCKS) could serve as a novel GBM therapeutic. Using molecularly classified patient-derived xenograft (PDX) lines, cultured in stem-cell conditions, we demonstrate that cell permeable MARCKS effector domain (ED) peptides potently target all GBM molecular classes while sparing normal human astrocytes. Cell death mechanistic testing revealed that these peptides produce rapid cytotoxicity in GBM that overcomes caspase inhibition. Moreover, we identify a GBM-selective cytolytic death mechanism involving plasma membrane targeting and intracellular calcium accumulation. Despite limited relative partitioning to the brain, tail-vein peptide injection revealed tumor targeting in intracranially implanted GBM PDX. These results indicate that MARCKS ED peptide therapeutics may overcome traditional GBM resistance mechanisms, supporting further development of similar agents.

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