scholarly journals Methylthioadenosine is Not Dramatically Elevated in MTAP-Homozygous Deleted Primary Glioblastomas

2019 ◽  
Author(s):  
Yasaman Barekatain ◽  
Victoria C. Yan ◽  
Jeffrey J. Ackroyd ◽  
Anton H. Poral ◽  
Theresa Tran ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Stromal Cells ◽  
Homozygous Deletion ◽  
Human Tumors ◽  
Normal Brain ◽  
List Type ◽  
Human Glioblastoma ◽  
Frequent Event

In BriefThe co-deletion of MTAP in the CDKN2A locus is a frequent event in diverse cancers including glioblastoma. Recent publications report that significant accumulations of the MTAP substrate, methylthioadenosine (MTA), can sensitize MTAP-deleted cancer cells to novel inhibitors of PRMT5 and MAT2A for targeted therapy against tumors with this particular genetic alteration. In this work, using comprehensive metabolomic profiling, we show that MTA is primarily secreted, resulting in exceedingly high levels of extracellular MTA in vitro. We further show that primary human glioblastoma tumors minimally accumulate MTA in vivo, which is likely explained by the metabolism of MTA by MTAP-competent stromal cells. Together, these data challenge whether the metabolic conditions required for therapies to exploit vulnerabilities associated MTAP deletions are present in primary human tumors, questioning their translational efficacy in the clinic.HighlightsMethylthioadenosine (MTA) is elevated in MTAP-deleted cancer cells in vitro, which provides a selective vulnerability to PRMT5 and MAT2A inhibitorsAccumulation of MTA in MTAP-deleted cancer cells is predominately extracellular, suggesting active secretion of MTA.MTAP-deleted primary human glioblastoma tumors show minimal intratumoral elevations of MTA, which is likely explained by secretion and metabolism by MTAP-competent stromal cells.SUMMARYHomozygous deletion of the CDK2NA locus frequently results in co-deletion of methylthioadenosine phosphorylase (MTAP) in many fatal cancers such as glioblastoma multiforme (GBM), resulting in elevations of the substrate metabolite, methylthioadenosine (MTA). To capitalize on such accumulations, therapeutic targeting of protein arginine methyltransferase 5 (PRMT5) and methionine adenosyl transferase (MAT2A) are ongoing. While extensively corroborated in vitro, the clinical efficacy of these strategies ultimately relies on equally significant accumulations of MTA in human tumors. Here, we show that in vitro accumulation of MTA is a predominately extracellular phenomenon, indicating secretion of MTA from MTAP-deleted cells. In primary human GBMs, we find that MTA levels are not significantly higher in MTAP-deleted compared to MTAP-intact tumors or normal brain tissue. Together, these findings highlight the metabolic discrepancies between in vitro models and primary human tumors and should thus be carefully considered in the development of the precision therapies targeting MTAP-homozygous deleted GBM.

scholarly journals Homozygous MTAP deletion in primary human glioblastoma is not associated with elevation of methylthioadenosine

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yasaman Barekatain ◽  
Jeffrey J. Ackroyd ◽  
Victoria C. Yan ◽  
Sunada Khadka ◽  
Lin Wang ◽  
...  
Keyword(s):  
Clinical Relevance ◽  
Homozygous Deletion ◽  
In Vitro Models ◽  
Arginine Methyltransferase ◽  
Human Glioblastoma ◽  
Primary Glioblastoma ◽  
Methylthioadenosine Phosphorylase ◽  
Metabolomic Profiling

AbstractHomozygous deletion of methylthioadenosine phosphorylase (MTAP) in cancers such as glioblastoma represents a potentially targetable vulnerability. Homozygous MTAP-deleted cell lines in culture show elevation of MTAP’s substrate metabolite, methylthioadenosine (MTA). High levels of MTA inhibit protein arginine methyltransferase 5 (PRMT5), which sensitizes MTAP-deleted cells to PRMT5 and methionine adenosyltransferase 2A (MAT2A) inhibition. While this concept has been extensively corroborated in vitro, the clinical relevance relies on exhibiting significant MTA accumulation in human glioblastoma. In this work, using comprehensive metabolomic profiling, we show that MTA secreted by MTAP-deleted cells in vitro results in high levels of extracellular MTA. We further demonstrate that homozygous MTAP-deleted primary glioblastoma tumors do not significantly accumulate MTA in vivo due to metabolism of MTA by MTAP-expressing stroma. These findings highlight metabolic discrepancies between in vitro models and primary human tumors that must be considered when developing strategies for precision therapies targeting glioblastoma with homozygous MTAP deletion.

scholarly journals BIOM-55. DGKζ-TARGETED REGULATION OF MIR-34A IN THE PROLIFERATION AND TUMORIGENICITY OF HUMAN GLIOBLASTOMA

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii13-ii13
Author(s):  
Wangxian Gu ◽  
Guoqing Wan ◽  
Yanjun Zheng ◽  
Xintong Yang ◽  
Peng Zhang ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Precancerous Lesions ◽  
Lipid Kinase ◽  
Human Glioblastoma ◽  
Kinase Substrate ◽  
Downstream Target ◽  
Protein Levels ◽  
Type Iv

Abstract Diacylglycerol kinase (DGK) is a lipid kinase that catalyzes the phosphorylation of diacylglycerol (DAG) to produce phosphatidic acid (PA), which uses ATP as a phosphate donor. Diacylglycerol kinases ζ(DGKζ) is characterized as specific type IV due to its myristoylated alanine-rich C-kinase substrate (MARCKS), ankyrin, and PDZ binding domain. Similar to other DGKs, DGKζ is also reported to be abnormally expressed in human colorectal cancer cells, and it is indispensable for the proliferation of cancer cells. However, its implications in human glioblastoma (GBM) is largely unknown. Both the mRNA and protein levels of DGKζ were significantly higher in GBM tissues than in precancerous lesions. Knockdown of DGKζ inhibited GBM cell proliferation, cell cycle and promoted apoptosis of GBM cells. Moreover, down-regulation of DGKζ markedly reduced in vitro colony formation and in vivo tumorigenic capability. Furthermore, we confirmed that DGKζ was the downstream target of miR-34a. The expression level of DGKζ was negatively correlated with miR-34a in GBM tissues. Overexpression of DGKζ reversed the tumor suppressive roles of miR-34a in GBM cells. Taken together, DGKζ can act as a potential prognostic biomarker for GBM patients and promote the growth of GBM cells was regulated by miR-34a, and it may represent a promising therapeutic target for patients with GBM.

scholarly journals OTME-9. Comprehensive Metabolic Profiling Of high MYC Medulloblastoma Reveals Key Differences Between In Vitro And In Vivo Glucose And Glutamine Usage

2021 ◽  
Vol 3 (Supplement_2) ◽  
pp. ii15-ii15
Author(s):  
Khoa Pham ◽  
Brad Poore ◽  
Allison Hanaford ◽  
Micah J Maxwell ◽  
Heather Sweeney ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Tca Cycle ◽  
Genetic Mutations ◽  
Metabolic Profiles ◽  
Normal Brain ◽  
Genomic Amplification ◽  
Cells In Culture ◽  
Orthotopic Xenograft

Abstract Reprograming of cellular metabolism is a hallmark of cancer. The metabolic alterations in cancer cells is not only defined by series of genetic mutations, but also reflecting the crosstalk between cancer cells and other factors in the microenvironment. Altering metabolism allows cancer cells to overcome unfavorable conditions, to proliferate and invade. Medulloblastoma is the most common malignant brain tumor of children. Genomic amplification of MYC is a hallmark of a subset of poor-prognosis medulloblastoma. However, the metabolism of high MYC amplified medulloblastoma subgroup remains underexplored. We performed comprehensive metabolic studies of human MYC-amplified medulloblastoma by comparing the metabolic profiles of tumor cells in different environments – in vitro, in flank xenografts and in orthotopic xenografts. Principal component analysis showed that the metabolic profiles of brain and flank high-MYC medulloblastoma tumors clustered closely together and separated away from normal brain and the high-MYC medulloblastoma cells in culture. Compared to normal brain, MYC-amplified medulloblastoma orthotopic xenograft tumors showed upregulation of nucleotide, hexosamine biosynthetic pathway (HBP), TCA cycle, and amino acid and glutathione pathways. There was significantly higher glucose up taking and usage in orthotopic xenograft tumor compared to flank xenograft and cells in culture. The data demonstrated that glucose was the main carbon source for the glutamate, glutamine and glutathione synthesis through the TCA cycle. The glutaminase ii pathway was the main pathway utilizing glutamine in MYC-amplified medulloblastoma in vivo. Glutathione was found as the most abundant upregulated metabolite. Glutamine derived glutathione was mainly synthesized through glutamine transaminase K (GTK) enzyme in vivo. In conclusion, we demonstrated that high MYC medulloblastoma adapt to different environments by altering its metabolic pathways despite carrying the same genetic mutations. Glutamine antagonists may have therapeutic applications in human patients.

Impaired Anaplerosis Is a Major Contributor to Glycolysis Inhibitor Toxicity in Glioma

2020 ◽  
Author(s):  
Sunada Khadka ◽  
Kenisha Arthur ◽  
Mykia Washington ◽  
Yasaman Barekatain ◽  
Jeff Ackroyd ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Single Agent ◽  
Tca Cycle ◽  
Redox Status ◽  
Homozygous Deletion ◽  
Metabolomic Profiling ◽  
Selective Sensitivity ◽  
Homozygous Deletions

Abstract Reprogramming of metabolic pathways is crucial to satisfy the bioenergetic and biosynthetic demands and maintain the redox status of rapidly proliferating cancer cells. In tumors, the tricarboxylic acid (TCA) cycle generates biosynthetic intermediates by oxidation of anaplerotic substrates, such as glucose-derived pyruvate and glutamine20 derived glutamate. We have previously documented that a subset of tumors with 1p36 homozygous deletion exhibit co-deletion of ENO1, in turn becoming extremely dependent on its redundant isoform ENO2 and sensitive to an overall enzymatic deficiency of enolase. Metabolomic profiling of ENO1-deleted glioma cells treated with an enolase inhibitor revealed a profound decrease in TCA cycle metabolites, which correlated with cell-line specific sensitivity to enolase inhibition, highlighting the importance of glycolysis derived pyruvate for anaplerosis. Correspondingly, the toxicity of the enolase inhibitor was significantly attenuated by exogenous supplementation of supraphysiological levels of anaplerotic substrates including pyruvate. These findings led us to hypothesize that cancer cells with ENO1 homozygous deletions treated with an enolase inhibitor might show exceptional sensitivity to inhibition of glutaminolysis because of reduced anaplerotic flow from glycolysis. We found that ENO1-deleted cells indeed exhibited selective sensitivity to the glutaminase inhibitor CB-839, and this sensitivity was also attenuated by exogenous supplementation of anaplerotic substrates including pyruvate. Despite these promising in vitro results, the antineoplastic effects of CB-839 as a single agent in ENO1-deleted xenograft tumors in vivo were modest in both intracranial orthotopic tumors, where the limited efficacy could be attributed to the blood brain barrier (BBB), and subcutaneous xenografts, where BBB penetration is not an issue. This contrasts with the enolase inhibitor HEX, which, despite its negative charge, achieved antineoplastic effects in both intracranial and subcutaneous tumors. Together, these data suggest that at least for 1p36-deleted gliomas, tumors in vivo—unlike cells in culture—show limited dependence on glutaminolysis and instead primarily depend on glycolysis for anaplerosis. Our findings reinforce the previously reported metabolic idiosyncrasies of the in vitro and in vivo environments as the potential reasons for the differential efficacy of metabolism targeted therapies in in vitro and in vivo systems.

scholarly journals CD146+CD107a+ Mesenchymal Stem/Stromal Cells with Signature Attributes Correlate to Therapeutic Potency as “First Responders” to Injury and Inflammation

2019 ◽  
Author(s):  
Annie C. Bowles ◽  
Dimitrios Kouroupis ◽  
Melissa A. Willman ◽  
Carlotta Perucca Orfei ◽  
Ashutosh Agarwal ◽  
...  
Keyword(s):  
Stromal Cells ◽  
Immune Cell ◽  
First Responders ◽  
Infrapatellar Fat Pad ◽  
M2 Macrophage ◽  
List Type ◽  
Fat Pad ◽  
Secretory Capacity

ABSTRACTCD146+ bone marrow–derived Mesenchymal Stem/Stromal Cells (BM-MSC) play key roles in the perivascular niche, skeletogenesis and hematopoietic support, however elucidation of therapeutic potency has yet to be determined. Here, inflammatory challenge to crude BM-MSC captured a baseline of signatures including enriched expression of CD146+ with CD107a+, CXCR4+, and LepR+, transcriptional profile, enhanced secretory capacity, robust secretome and immunomodulatory function with stimulated target immune cells. These responses were significantly more pronounced in CD146+ (POS)-selected subpopulation than in the CD146- (NEG). Mechanistically, POS uniquely mediated robust immunosuppression while inducing significant frequencies of Naïve and Regulatory T cells in vitro. Moreover, POS promoted a pivotal M1-to-M2 macrophage shift in vivo, ameliorating inflammation/fibrosis of joint synovium and fat pad of the knee, failed by NEG. This study provides high-content evidence of CD146+CD107a+ BM-MSC, herein deemed ‘first responders’ to inflammation, as the underrepresented subpopulation within crude BM-MSC with innately higher secretory capacity and therapeutic potency.HIGHLIGHTSSignature phenotypic, transcriptional, and secretome profiles were identified and enriched in human CD146+ (POS)-selected subpopulation in response to inflammationInflammatory challenge consistently altered stemness (LIF) and differentiation master regulators (SOX9, RUNX2, PPARγ) in crude, POS, and NEG BM-MSC, and deduced unique expressions in POS compared to NEGPOS BM-MSC mediated the strongest immunomodulation, e.g. target immune cell suppression, Treg induction, diminished T cell differentiationPOS BM-MSC promoted the largest M1-to-M2 shift in vivo alleviating induced synovitis and infrapatellar fat pad fibrosis of the knee

scholarly journals Gli1+ mesenchymal stromal cells modulate epithelial metaplasia in lung fibrosis

2019 ◽  
Author(s):  
Monica Cassandras ◽  
Chaoqun Wang ◽  
Jaymin Kathiriya ◽  
Tatsuya Tsukui ◽  
Peri Matatia ◽  
...  
Keyword(s):  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Hedgehog Signaling ◽  
List Type ◽  
Basal Cells ◽  
Human Lungs ◽  
Metaplastic Epithelium ◽  
Organ Fibrosis

AbstractOrgan fibrosis is often accompanied by aberrant epithelial reprogramming, culminating in a transformed barrier composed of scar and metaplastic epithelium. Understanding how the scar promotes an abnormal epithelial response could better inform strategies to reverse the fibrotic damage. Here we show that Gli1+ mesenchymal stromal cells (MSCs), previously shown to contribute to myofibroblasts in the scar, promote metaplastic differentiation of airway progenitors into KRT5+ basal cells in vitro and in vivo. During fibrotic repair, Gli1+ MSCs integrate hedgehog activation to promote metaplastic KRT5 differentiation by upregulating BMP antagonism in the progenitor niche. Restoring the balance towards BMP activation attenuated metaplastic KRT5+ differentiation while promoting adaptive alveolar differentiation. Finally, fibrotic human lungs demonstrate altered BMP activation in the metaplastic epithelium. These findings show that Gli1+ MSCs integrate hedgehog signaling as a rheostat to control BMP activation in the progenitor niche to determine regenerative outcome in fibrosis.HighlightsGli1+ MSCs are required for metaplastic airway progenitor differentiation into KRT5+ basal cells.Hedgehog activation of MSCs promotes KRT5 differentiation of airway progenitors by suppressing BMP activation.Restoring BMP activation attenuates metaplastic KRT5 differentiationMetaplastic KRT5+ basal cells in human fibrotic lungs demonstrate altered BMP activation.

scholarly journals A 3D Heterotypic Multicellular Tumor Spheroid Assay Platform to Discriminate Drug Effects on Stroma versus Cancer Cells

2019 ◽  
Vol 25 (3) ◽  
pp. 265-276
Author(s):  
Zoe Weydert ◽  
Madhu Lal-Nag ◽  
Lesley Mathews-Greiner ◽  
Christoph Thiel ◽  
Henrik Cordes ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Stromal Cells ◽  
High Throughput Screening ◽  
Drug Testing ◽  
Therapeutic Window ◽  
Multicellular Tumor Spheroid ◽  
Cytotoxic Effects ◽  
Primary Tumors

Three-dimensional (3D) cell culture models are thought to mimic the physiological and pharmacological properties of tissues in vivo more accurately than two-dimensional cultures on plastic dishes. For the development of cancer therapies, 3D spheroid models are being created to reflect the complex histology and physiology of primary tumors with the hopes that drug responses will be more similar to and as predictive as those obtained in vivo. The effect of additional cell types in tumors, such as stromal cells, and the resulting heterotypic cell–cell crosstalk can be investigated in these heterotypic 3D cell cultures. Here, a high-throughput screening-compatible drug testing platform based on 3D multicellular spheroid models is described that enables the parallel assessment of toxicity on stromal cells and efficacy on cancer cells by drug candidates. These heterotypic microtissue tumor models incorporate NIH3T3 fibroblasts as stromal cells that are engineered with a reporter gene encoding secreted NanoLUC luciferase. By tracking the NanoLUC signal in the media over time, a time-related measurement of the cytotoxic effects of drugs on stromal cells over the cancer cells was possible, thus enabling the identification of a therapeutic window. An in vitro therapeutic index parameter is proposed to help distinguish and classify those compounds with broad cytotoxic effects versus those that are more selective at targeting cancer cells.

Biofabricated tumor microenvironments for studying colorectal cancer in vitro and in vivo.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. e14689-e14689
Author(s):  
Mahesh Devarasetty ◽  
Samuel Herberg ◽  
Anthony Dominijanni ◽  
Ethan Willey-Shelkey ◽  
Aleksander Skardal ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cancer Cells ◽  
Cancer Cell ◽  
Stromal Cells ◽  
Cell Phenotype ◽  
Micro Structure ◽  
Therapeutic Development

e14689 Background: Microenvironmental mechanics have a tremendous effect on the progression, phenotype, and therapeutic response of cancer cells positioning it as a high-potential target for novel therapeutic development. Laboratory modeling of the microenvironment and its multitude of effects is imperative for developing new avenues of anti-cancer therapy that can target non-traditional vectors such as the extracellular matrix (ECM) and stromal cells. Researchers have developed in vitro models of the tumor microenvironment (TME) to meet this need. While in vitro modeling is an important step in therapeutic development, there are few studies that validate in vitro generated results to gold-standard in vivo models, and further, to patient-derived data. Previously, we have developed a model of the colorectal tumor microenvironment and found a connection between collagen ultrastructure and cancer cell phenotype. Using this characterized organoid model, we implant bioengineered TMEs into mice to track long-term growth and progression of cancer and compare our results to clinical biopsies. Methods: Tumor organoids are produced by combining stromal cells and type I collagen. Cancer cell spheroids are embedded into the organoid for long term observation. Organoids are either observed in vitro or implanted subcutaneously into mice for in vivo tracking. Results: Organoids retain structure and viability during long term culture in vitro and in vivo, and embedded cancer cells respond significantly differently depending on the architecture of the surrounding TME. Cancer cells assume a mesenchymal, invasive, and proliferative phenotype in unorganized TMEs, and revert to an epithelial phenotype in an ordered TME. In addition, analysis of biopsied tissue, across tumor grade, demonstrates a correlation between cancer cell phenotype and microenvironmental architecture. Conclusions: In all this is the first study to establish a connection between TME micro-structure and cancer cell phenotype consistently across three distinct research modalities. These results have the potential to pave the way for utilizing bioengineered microenvironmental models as therapeutic development platforms and for targeting TME micro-structure to control colorectal cancer cell progression.

LACK OF EFFECT OF CORTICOSTEROIDS ON THE IN VIVO DISAPPEARANCE OF SULFHYDRYL-INHIBITED AND ANTIBODY-COATED ERYTHROCYTES

1964 ◽  
Vol 47 (3_Suppl) ◽  
pp. S28-S36
Author(s):  
Kailash N. Agarwal
Keyword(s):  
Red Cells ◽  
List Type ◽  
Red Cell

ABSTRACT Red cells were incubated in vitro with sulfhydryl inhibitors and Rhantibody with and without prior incubation with prednisolone-hemisuccinate. These erythrocytes were labelled with Cr51 and P32 and their disappearance in vivo after autotransfusion was measured. Prior incubation with prednisolone-hemisuccinate had no effect on the rate of red cell disappearance. The disappearance of the cells was shown to take place without appreciable intravascular destruction.

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