Systematic literature review reveals suboptimal use of chemical probes in cell-based biomedical research.

Chemical probes have reached a prominent role in biomedical research, but their impact is governed by experimental design. To gain insight into the use of chemical probes, we conducted a systematic review of 662 publications, understood here as primary research articles, employing eight different chemical probes in cell-based research. We summarised (i) concentration(s) at which chemical probes were used in cell-based assays, (ii) inclusion of structurally matched target-inactive control compounds and (iii) orthogonal chemical probes. Here, we show that only 4% of analysed eligible publications used chemical probes within the recommended concentration range and included inactive compounds as well as orthogonal chemical probes. These findings indicate that the best practice with chemical probes is yet to be implemented in biomedical research. To achieve this, we propose 'the rule of two': At least two chemical probes (either orthogonal target-engaging probes, and/or a pair of a chemical probe and matched target-inactive compound) to be employed at recommended concentrations in every study.

DYRK1A Negatively Regulates CDK5-SOX2 Pathway and Self-Renewal of Glioblastoma Stem Cells.

Glioblastoma display vast cellular heterogeneity, with glioblastoma stem cells (GSCs) at the apex. The critical role of GSCs in tumour growth and resistance to therapy highlights the need to delineate mechanisms that control stemness and differentiation potential of GSC. Dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) regulates neural progenitor cell differentiation, but its role in cancer stem cell differentiation is largely unknown. Herein, we demonstrate that DYRK1A kinase is crucial for the differentiation commitment of glioblastoma stem cells. DYRK1A inhibition insulates the self-renewing population of GSCs from potent differentiation-inducing signals. Mechanistically, we show that DYRK1A promotes differentiation and limits stemness acquisition via deactivation of CDK5, an unconventional kinase recently described as an oncogene. DYRK1A-dependent inactivation of CDK5 results in decreased expression of the stemness gene SOX2 and promotes the commitment of GSC to differentiate. Our investigations of the novel DYRK1A-CDK5-SOX2 pathway provide further insights into the mechanisms underlying glioblastoma stem cell maintenance.

Global phosphoproteomics reveals DYRK1A regulates CDK1 activity in glioblastoma cells.

Both tumour suppressive and oncogenic functions have been reported for dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A). Herein, we performed a detailed investigation to delineate the role of DYRK1A in glioblastoma. Our phosphoproteomic and mechanistic studies show that DYRK1A induces degradation of cyclin B by phosphorylating CDC23, which is necessary for the function of the anaphase-promoting complex, a ubiquitin ligase that degrades mitotic proteins. DYRK1A inhibition leads to the accumulation of cyclin B and activation of CDK1. Importantly, we established that the phenotypic response of glioblastoma cells to DYRK1A inhibition depends on both retinoblastoma (RB) expression and the degree of residual DYRK1A activity. Moderate DYRK1A inhibition leads to moderate cyclin B accumulation, CDK1 activation and increased proliferation in RB-deficient cells. In RB-proficient cells, cyclin B/CDK1 activation in response to DYRK1A inhibition is neutralized by the RB pathway, resulting in an unchanged proliferation rate. In contrast, complete DYRK1A inhibition with high doses of inhibitors results in massive cyclin B accumulation, saturation of CDK1 activity and cell cycle arrest, regardless of RB status. These findings provide new insights into the complexity of context-dependent DYRK1A signalling in cancer cells.

MerTK activity is not necessary for the proliferation of glioblastoma stem cells.

MerTK has been identified as a promising target for therapeutic intervention in glioblastoma. Genetic studies documented a range of oncogenic processes that MerTK targeting could influence, however robust pharmacological validation has been missing. The aim of this study was to assess therapeutic potential of MerTK inhibitors in glioblastoma therapy. Unlike previous studies, our work provides several lines of evidence that MerTK activity is dispensable for glioblastoma growth. We observed heterogeneous responses to MerTK inhibitors that could not be correlated to MerTK inhibition or MerTK expression in cells. The more selective MerTK inhibitors UNC2250 and UNC2580A lack the anti-proliferative potency of less-selective inhibitors exemplified by UNC2025. Functional assays in MerTK-high and MerTK-deficient cells further demonstrate that the anti-cancer efficacy of UNC2025 is MerTK-independent. However, despite its efficacy in vitro, UNC2025 failed to attenuate glioblastoma growth in vivo. Gene expression analysis from cohorts of glioblastoma patients identified that MerTK expression correlates negatively with proliferation and positively with quiescence genes, suggesting that MerTK regulates dormancy rather than proliferation in glioblastoma. In summary, this study demonstrates the importance of orthogonal inhibitors and disease-relevant models in target validation studies and raises a possibility that MerTK inhibitors could be used to target dormant glioblastoma cells.

Histone lysine demethylases and their functions in cancer.

Histone lysine demethylases (KDMs) are enzymes that remove the methylation marks on lysines in nucleosomes' histone tails. These changes in methylation marks regulate gene transcription during both development and malignant transformation. Depending on which lysine residue is targeted, the effect of a given KDM on gene transcription can be either activating or repressing, and KDMs can regulate the expression of both oncogenes and tumour suppressors. Thus, the functions of KDMs can be regarded as both oncogenic and tumour suppressive, contingent on cell context and the enzyme isoform. Finally, KDMs also demethylate nonhistone proteins and have a variety of demethylase-independent functions. These epigenetic and other mechanisms that KDMs control make them important regulators of malignant tumours. Here, we present an overview of eight KDM subfamilies, their most-studied lysine targets and selected recent data on their roles in cancer stem cells, tumour aggressiveness and drug tolerance.

MK2 Inhibition Induces p53-Dependent Senescence in Glioblastoma Cells.

MAPK-activated protein kinase 2 (MK2) has diverse roles in cancer. In response to chemotherapy, MK2 inhibition is synthetically lethal to p53-deficiency. While TP53 deletion is rare in glioblastomas, these tumors often carry TP53 mutations. Here, we show that MK2 inhibition strongly attenuated glioblastoma cell proliferation through p53wt stabilization and senescence. The senescence-inducing efficacy of MK2 inhibition was particularly strong when cells were co-treated with the standard-of-care temozolomide. However, MK2 inhibition also increased the stability of p53 mutants and enhanced the proliferation of p53-mutant stem cells. These observations reveal that in response to DNA damaging chemotherapy, targeting MK2 in p53-mutated cells produces a phenotype that is distinct from the p53-deficient phenotype. Thus, MK2 represents a novel drug target in 70% glioblastomas harboring intact TP53 gene. However, targeting MK2 in tumors with TP53 mutations may accelerate disease progression. These findings are highly relevant since TP53 mutations occur in over 50% of all cancers.

Targeting Cancer Cell Dormancy.

Cancer cell dormancy is a process whereby cells enter reversible cell cycle arrest, termed quiescence. Quiescence is essential for cancer cells to acquire additional mutations, to survive in a new environment and initiate metastasis, to become resistant to cancer therapy, and to evade immune destruction. Thus, dormant cancer cells are considered to be responsible for cancer progression. As we start to understand the mechanisms that enable quiescence, we can begin to develop pharmacological strategies to target dormant cancer cells. Herein, we summarize the major molecular mechanisms underlying the dormancy of disseminated tumor cells and drug-tolerant persister cells. We then analyze the current pharmacological strategies aimed (i) to keep cancer cells in the harmless dormant state, (ii) to reactivate dormant cells to increase their susceptibility to anti-proliferative drugs, and (iii) to eradicate dormant cancer cells.

Lower Tubulin Expression in Glioblastoma Stem Cells Attenuates Efficacy of Microtubule-Targeting Agents.

Sensitivity to microtubule-targeting agents (MTAs) varies among cancers and predicting the response of individual cancer patients to MTAs remains challenging. As microtubules possess vast molecular heterogeneity generated by tubulin isotypes and their post-translational modifications, we questioned whether this heterogeneity can impact MTA sensitivity. We investigated microtubule heterogeneity in 15 glioblastoma cell lines and measured sensitivity of orthogonal MTAs using a per-division growth rate inhibition method that corrects for the confounding effects of variable cell proliferation rates. We found that the tubulin profile is unique for each glioblastoma cell line and that the total α- and ß-tubulin levels impact on MTA sensitivity. The baseline levels of α- and ß-tubulin were up to 20% lower in cells that were not effectively killed by MTAs. We report that lower α/ß-tubulin expression is associated with lack of cell differentiation and increased expression of stemness markers. The dedifferentiated stem-like cells with low α/ß-tubulin levels survive MTAs treatment via reversible nonmutational dormancy. Our findings provide novel insights into the relationships between microtubules and MTAs and lay a foundation for better understanding of the sensitivity of cancer cells to MTAs.

Anti-proliferative and cytotoxic activities of the flavonoid isoliquiritigenin in the human neuroblastoma cell line SH-SY5Y.

Neuroblastoma is a common childhood cancer with high mortality. We evaluated the capacity of the flavonoid, isoliquiritigenin (4,2',4'-trihydroxychalcone; ISL) to inhibit cellular proliferation and migration in the human neuroblastoma cell line SH-SY5Y. Incubation of cultured SH-SY5Y cells with 20-100 µM ISL decreased cell confluency (15-70%) after 24 h incubation, while 10-100 µM ISL (24 h) depleted intracellular ATP stores (15-90% vs vehicle-treated control) after 24 h incubation. ISL-mediated cell toxicity did not involve intracellular caspase 3/7 activation, externalization of phosphatidylserine on the cell membrane or stimulation of TNF and IL-1ß release, all indicating that the flavonoid did not induce apoptosis. Pre-treatment of cells with necrostatin-1, a necroptosis inhibitor, significantly restored ATP levels (ATP levels increased 12-42%) in ISL-treated neuroblastoma cells indicative of enhanced viability. By contrast, RIP1 phosphorylation status remained unchanged in cells treated with ISL although the intracellular ratio of phosphorylated/total parental RIP1 increased after ISL treatment on SH-SY5Y cells indicating that ISL decreased levels of native RIP1. In addition, ISL treatment inhibited SH-SY5Y cell migration/proliferation in a scratch assay and arrested cell cycle transition by significantly decreasing the number of cells in G0/G1 phase and increasing populations by ~10% in S (primarily) and G2/M (lesser extent) phases. The intracellular ratio of phosphorylated/total ERK 1/2 and p38 remained unchanged after ISL treatment (up to 40 µM); ERK activation was only determined at ISL dose well above the experimental IC50 value as judged by ELISA analyses and this did not correlate with ISL cytotoxicity at lower dose <40 µM; Western blot assay confirmed the detection of phosphorylated (p-)ERK1/2 and (p-)p38 in ISL treated cells. Together the results suggest that ISL exerts anti-proliferative and cytotoxic activity on SHSY5Y cells through the loss of ATP, induction of cell cycle arrest, and cell death largely via a necroptotic mechanism in the absence of apoptotic activity.

Synthesis and in vitro evaluation of diverse heterocyclic diphenolic compounds as inhibitors of DYRK1A.

Dual-specificity tyrosine phosphorylation-related kinase 1A (DYRK1A) is a dual-specificity protein kinase that catalyses phosphorylation and autophosphorylation. Higher DYRK1A expression correlates with cancer, in particular glioblastoma present within the brain. We report here the synthesis and biological evaluation of new heterocyclic diphenolic derivatives designed as novel DYRK1A inhibitors. The generation of these heterocycles such as benzimidazole, imidazole, naphthyridine, pyrazole-pyridines, bipyridine, and triazolopyrazines was made based on the structural modification of the lead DANDY and tested for their ability to inhibit DYRK1A. None of these derivatives showed significant DYRK1A inhibition but provide valuable knowledge around the importance of the 7-azaindole moiety. These data will be of use for developing further structure-activity relationship studies to improve the selective inhibition of DYRK1A.