Cancer cells upregulate anabolic processes to maintain high rates of cellular turnover. Limiting the supply of macromolecular precursors by targeting enzymes involved in biosynthesis is a promising ...strategy in cancer therapy. Several tumors excessively metabolize glutamine to generate precursors for nonessential amino acids, nucleotides, and lipids, in a process called glutaminolysis. Here we show that pharmacological inhibition of glutaminase (GLS) eradicates glioblastoma stem-like cells (GSCs), a small cell subpopulation in glioblastoma (GBM) responsible for therapy resistance and tumor recurrence. Treatment with small molecule inhibitors compound 968 and CB839 effectively diminished cell growth and in vitro clonogenicity of GSC neurosphere cultures. However, our pharmaco-metabolic studies revealed that only CB839 inhibited GLS enzymatic activity thereby limiting the influx of glutamine derivates into the TCA cycle. Nevertheless, the effects of both inhibitors were highly GLS specific, since treatment sensitivity markedly correlated with GLS protein expression. Strikingly, we found GLS overexpressed in in vitro GSC models as compared with neural stem cells (NSC). Moreover, our study demonstrates the usefulness of in vitro pharmaco-metabolomics to score target specificity of compounds thereby refining drug development and risk assessment.
Human induced pluripotent stem cells (hiPSCs) have emerged as a powerful tool for in vitro modelling of diseases with broad application in drug development or toxicology testing. These assays usually ...require large quantities of hiPSC, which can entail long-term storage via cryopreservation of the same cell charges. However, it is essential that cryopreservation does not oppose durable changes on the cells. In this project, we characterize one parameter of functionality of one that is well established in the field, in a different research context, an applied hiPSC line (iPS11), namely their resistance to a medium size library of chemo interventions (>160 drugs). We demonstrate that cells, before and after cryopreservation, do not change their relative overall drug response phenotypes, as defined by identification of the top 20 interventions causing dose-dependent reduction of cell growth. Importantly, also frozen cells that are exogenously enforced for stable overexpression of oncogenes myelocytomatosis (cMYC) or tumor protein 53 mutation (TP53R175H), respectively, are not changed in their relative top 20 drugs response compared to their non-frozen counterparts. Taken together, our results support iPSCs as a reliable in vitro platform for in vitro pharmacology, further raising hopes that this technology supports biomarker-associated drug development. Given the general debate on ethical and economic problems associated with the reproducibly crisis in biomedicine, our results may be of interest to a wider audience beyond stem cell research.
Glioblastoma is the most aggressive type of glioma. The Wingless (Wnt) signaling pathway has been shown to promote stem cell properties and resistance to radio- and chemotherapy in glioblastoma. ...Here, we demonstrate that pharmacological Wnt pathway inhibition using the porcupine inhibitor LGK974 acts synergistically with temozolomide (TMZ), the chemotherapeutic drug currently used as standard treatment for glioblastoma, to suppress
growth of glioma cells. Synergistic growth inhibition was independent of the
-alkylguanine DNA alkyltransferase (
) promoter methylation status. Transcriptomic analysis revealed that expression of aldehyde dehydrogenase 3A1 (
) was significantly down-regulated when cells were treated with LGK974 and TMZ. Suppressing ALDH3A1 expression increased the efficacy of TMZ and reduced clonogenic potential accompanied by decreased expression of stem cell markers CD133, Nestin and Sox2. Taken together, our study suggests that previous observations concerning Wnt signaling blockade to reduce chemoresistance in glioblastoma is at least in part mediated by inhibition of ALDH3A1.
Summary
Phytophthora infestans is a devastating pathogen in agricultural systems. Recently, an RNA silencing suppressor (PSR2, ‘Phytophthora suppressor of RNA silencing 2’) has been described in P. ...infestans. PSR2 has been shown to increase the virulence of Phytophthora pathogens on their hosts. This gene is one of the few effectors present in many economically important Phytophthora species. In this study, we investigated: (i) the evolutionary history of PSR2 within and between species of Phytophthora; and (ii) the interaction between sequence variation, gene expression and virulence. In P. infestans, the highest PiPSR2 expression was correlated with decreased symptom expression. The highest gene expression was observed in the biotrophic phase of the pathogen, suggesting that PSR2 is important during early infection. Protein sequence conservation was negatively correlated with host range, suggesting host range as a driver of PSR2 evolution. Within species, we detected elevated amino acid variation, as observed for other effectors; however, the frequency spectrum of the mutations was inconsistent with strong balancing selection. This evolutionary pattern may be related to the conservation of the host target(s) of PSR2 and the absence of known corresponding R genes. In summary, our study indicates that PSR2 is a conserved effector that acts as a master switch to modify plant gene regulation early during infection for the pathogen's benefit. The conservation of PSR2 and its important role in virulence make it a promising target for pathogen management.
Limitations in genetic stability and recapitulating accurate physiological disease properties challenge the utility of patient‐derived (PD) cancer models for reproducible and translational research. ...A portfolio of isogenic human induced pluripotent stem cells (hiPSCs) with different pan‐cancer relevant oncoprotein signatures followed by differentiation into lineage‐committed progenitor cells was genetically engineered. Characterization on molecular and biological level validated successful stable genetic alterations in pluripotency state as well as upon differentiation to prove the functionality of our approach. Meanwhile proposing core molecular networks possibly involved in early dysregulation of stem cell homeostasis, the application of our cell systems in comparative substance testing indicates the potential for cancer research such as identification of augmented therapy resistance of stem cells in response to activation of distinct oncogenic signatures.
Graphical and Lay Summary
Traditional cancer research relies on cell lines or primary cell cultures derived from tumor specimens. Based on the scientific concept that cancer arises from cells with stem cell properties, this work presents the development of an alternative human cancer cell platform using genetically customized human induced pluripotent stem cells from a healthy donor featuring stable oncogene activation. Furthermore, the suitability of this platform to generate oncogene activated tissue‐specific progenitor cells to support the development of targeted chemotherapies was shown.
Cancer cells upregulate anabolic processes to maintain high rates of cellular turnover. Limiting the supply of macromolecular precursors by targeting enzymes involved in biosynthesis is a promising ...strategy in cancer therapy. Several tumors excessively metabolize glutamine to generate precursors for nonessential amino acids, nucleotides, and lipids, in a process called glutaminolysis. Here we show that pharmacological inhibition of glutaminase (GLS) eradicates glioblastoma stem-like cells (GSCs), a small cell subpopulation in glioblastoma (GBM) responsible for therapy resistance and tumor recurrence. Treatment with small molecule inhibitors compound 968 and CB839 effectively diminished cell growth and in vitro clonogenicity of GSC neurosphere cultures. However, our pharmaco-metabolic studies revealed that only CB839 inhibited GLS enzymatic activity thereby limiting the influx of glutamine derivates into the TCA cycle. Nevertheless, the effects of both inhibitors were highly GLS specific, since treatment sensitivity markedly correlated with GLS protein expression. Strikingly, we found GLS overexpressed in in vitro GSC models as compared with neural stem cells (NSC). Moreover, our study demonstrates the usefulness of in vitro pharmaco-metabolomics to score target specificity of compounds thereby refining drug development and risk assessment.
To assure efficient MHC class I (MHC‐I) peptide loading, the peptide loading complex (PLC) recruits the peptide‐receptive form of MHC‐I, and in this process, tapasin (tpn) connects MHC‐I with the ...peptide transporter TAP and forms a stable disulfide bond with ERp57. Here, we describe an alternatively spliced tpn transcript lacking exon 3, observed in cells infected with human cytomegalovirus. Recognition of exon 3 was regulated via G‐runs, suggesting that members of the hnRNP (heterogeneous nuclear ribonucleoprotein)‐family regulate expression of the ΔExon3 variant of tpn. Exon 3 includes Cys‐95, which is responsible for the disulfide bond formation with ERp57 and, consequently, interaction of the ΔExon3 variant with ERp57 was strongly impaired. Although the ΔExon3 variant specifically stabilized TAP expression but not MHC‐I in tpn‐deficient cells, in tpn‐proficient cells, the ΔExon3 tpn reduced cell surface expression of the tpn‐dependent HLA‐B*44:02 allele; the stability of the tpn‐independent HLA‐B*44:05 was not affected. Most importantly, detailed analysis of the PLC revealed a simultaneous binding of the ΔExon3 variant and tpn to TAP, suggesting modification of PLC functions. Indeed, an altered MHC‐I ligandome was observed in HeLa cells overexpressing the ΔExon3 variant, highlighting the potential of the alternatively spliced tpn variant to impact CD8+ T‐cell responses.
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