Glioblastoma Stem Cells Respond to Differentiation Cues but Fail to Undergo Commitment and Terminal Cell-Cycle Arrest.

Glioblastoma (GBM) is an aggressive brain tumor whose growth is driven by stemcell-like cells. BMP signaling triggers cell-cycle exit and differentiation of GBM stemcells (GSCs) and, therefore, might have therapeutic value. However, the epigenetic mechanisms that accompany differentiation remain poorly defined. It is also unclear whether cell-cycle arrest is terminal. Herewe find only a subset ofGSCcultures exhibit astrocyte differentiation in response to BMP. Although overtly differentiated non-cycling astrocytes are generated, they remain vulnerable to cell-cycle re-entry and fail to appropriately reconfigure DNA methylation patterns. Chromatin accessibility mapping identified loci that failed to alter in response to BMP and these were enriched in SOX transcription factor-binding motifs. SOX transcription factors, therefore, may limit differentiation commitment. A similar propensity for cell-cycle re-entry and de-differentiation was observed in GSC-derived oligodendrocyte-like cells. These findings highlight significant obstacles to BMP-induced differentiation as therapy forGBM

Regulome analysis in B-acute lymphoblastic leukemia exposes Core Binding Factor addiction as a therapeutic vulnerability.

Funder: Blood Cancer UK/Grant Number 16001The ETV6-RUNX1 onco-fusion arises in utero, initiating a clinically silent pre-leukemic state associated with the development of pediatric B-acute lymphoblastic leukemia (B-ALL). We characterize the ETV6-RUNX1 regulome by integrating chromatin immunoprecipitation- and RNA-sequencing and show that ETV6-RUNX1 functions primarily through competition for RUNX1 binding sites and transcriptional repression. In pre-leukemia, this results in ETV6-RUNX1 antagonization of cell cycle regulation by RUNX1 as evidenced by mass cytometry analysis of B-lineage cells derived from ETV6-RUNX1 knock-in human pluripotent stem cells. In frank leukemia, knockdown of RUNX1 or its co-factor CBFβ results in cell death suggesting sustained requirement for RUNX1 activity which is recapitulated by chemical perturbation using an allosteric CBFβ-inhibitor. Strikingly, we show that RUNX1 addiction extends to other genetic subtypes of pediatric B-ALL and also adult disease. Importantly, inhibition of RUNX1 activity spares normal hematopoiesis. Our results suggest that chemical intervention in the RUNX1 program may provide a therapeutic opportunity in ALL

Live-cell imaging screen to determine responses of 160 kinase inhibitors against normal and glioblastoma-derived neural stem cells.

<p>(A) Glioblastoma subtype gene expression signatures established Verhaak et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077053#pone.0077053-Verhaak1" target="_blank">[7]</a> (left panel) were assessed in a set of GNS cell lines (right panel). (B) Correlations between subtype centroid values determined by Verhaak et al. and gene expression in GNS cells. G144 exhibits clear correspondence to the ‘proneural’ subtype, whereas G166 and G179 have greater similarities to the mesenchymal and neural/mesenchymal subtypes, respectively. (C) Summary of screening strategy based on these three GNS cell lines and a genetically normal NS cell (CB660). (D) Proliferation curves generated for each compound over a 3–6 day period identify J101 (red line) as an agent that can selectively block expansion of GNS cells. (E) Significant events were identified affecting GNS cells but not NS cells, and cytotostatic/cytotoxic compounds reducing confluence in all GNS cells >2.2 standard deviations from the average of DMSO controls are shown (<i>P</i> = 0.01). The full data for the screen are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077053#pone.0077053.s007" target="_blank">Table S1</a>. (F) Example phase contrast images acquired for G179 and CB660 prior to treatment with J101 (0 h) and 60 h.</p

GNS cells arrest at prometaphase due to loss of Plk1 activity and failure of spindle assembly.

<p>(A) 812 (550 pan- and 262 phospho-specific) proteins were assessed before and after drug treatment by antibody microarrays. Significant hits are shown and their suggested interactions presented following pathway analysis (Ingenuity). Several kinases (bold) were affected including downstream components of the PDGFR signalling pathway. However, we also noted a suppression of levels of active Plk1 (shown in bold). (B) Western immunoblot for PLK1 and phospho-T210, and PLK2 in a panel of NS and GNS treated with DMSO, J101 or the PLK1 inhibitor BI2536. (C) Immunostaining for spindle protein (α-tubulin) and pHH3 confirms that arrest at prometaphase is associated with aberrant spindle formation. Failure of viable bipolar spindle formation was observed for G7 cells treated with J101 or BI 2536. Photomicrographs show aberrant representative spindles of different morphologies (blue, DAPI; green, (α-tubulin; red, PHH3). (D) Quantification of data from (C) indicates that cells treated with J101 or BI2536 fail to progress to metaphase.</p

JNJ-10198402 induces mitotic arrest in GNS cells but not in normal NS cells.

<p>(A) Segmentation of phase contrast images with high-content analysis software (CellProfiler & CellProfiler Analyst). Objects were assigned into different classes/bins. Interphase cells (blue) and mitoses (red; ‘i–iii’). Erroneously segmented debris or dead cells (green; ‘x’) are isolated and discarded from event counts. (B) Relative number of mitoses scored within 48 h from the start of the experiment for each line and all 160 inhibitors. (C) Kinetics of change in total cell number and mitosis for G179. J101 significantly increased the number of mitoses in G179, but not CB660, without parallel increases in cell number (top panel; blue dots), whereas cells with mitotic morphology increased dramatically during the first 1–2 days (bottom panel; red dots).</p

p53 null mouse NS cells display increased sensitivity to Plk1 inhibitors.

<p>(A) Wild-type and GFP positive p53^−/− mouse NS cells were co-cultured in equal numbers. Both J101 and BI2536 preferentially affect GFP-positive p53 mutant cells. (B) Quantification of flow cytometry data over a 6-day timecourse. (C) Acute genetic deletion of <i>tp53</i> sensitizes mouse NS cells to BI 2536 treatment in the absence of aneuploidy. Cells were sensitised to BI 2536 treatment following loss of p53, similarly to an independent p53^−/− NS cell line (used in panel A–B). * <i>P</i><0.05. *** <i>P</i><0.001 (<i>n</i> = 4; two biological and two technical replicates). (D) Immunoblotting before and after Cre delivery confirms efficient excision of p53 (P = passage number).</p

Alternative Plk1 inhibitors in clinical development compare favourably with BI 2536 in selectivity against GNS cells and blood-brain barrier permeability.

<p>(A) Co-culture of equal numbers of GFP-transfected G144 cell lines together with wild-type CB660 cells. In the presence of BI2536 GNS cells are selectively lost in the culture, while normal NS cells continue to proliferate. (B) DAPI staining of day 10 cultures of GNS or NS cells confirms that NS cells continue to undergo normal mitosis (anaphase events in red circles), without evidence of mitotic slippage and lagging chromosomes. (C) Two other Plk1 inhibitors in clinical development are selective against GNS cells (G7) compared to foetal NS cells (CB11130 and CB11171; chosen as they have a similar doubling time to G7). (D) Relative numbers of GNS cells arrested in mitosis after 48 h treatment with Plk1 inhibitors using an <i>in vitro</i> blood-brain barrier co-culture model. Inhibitors were added directly or via cell culture inserts containing a confluent layer of endothelial cells, and anti-mitotic responses were assessed in G7. J101 displayed poor blood-brain barrier permeability (<i>P</i><0.001). GSK461364 performed similarly to BI 2536. Values shown are percentages of pHH3-positive cells relative to no-insert value of GSK 461364 treated GNS cells (<i>n</i> = 3). (E) Dose-response curves and IC50 values for specific Plk1 inhibitors BI 2536 and GSK 461363. NS and GNS cells were treated with different concentrations of the Plk1 inhibitors BI 2536 and GSK461364. Five days after treatment the total number of viable cells was counted and normalised to DMSO control values. Calculation of IC50 values confirmed the differential effect of both inhibitors on NS and GNS cells, with G7 being most sensitive.</p

Immunocytochemistry confirms arrest at prometaphase in response to J101.

<p>(A) Phospho-histone H3 (pHH3) staining (green) for CB660, G179, G166, G144 and G7 after following treatment with DMSO or J101 (100 nM) for 24 h with nuclear counterstaining using DAPI (blue). (B) Mitotically arrested cells (G7), were immunostained for pHH3 (white), Lamin B (green) and counterstained with rhodamine-phalloidin to visualise actin (red). Arrows indicate the fragmented nuclear membrane, a feature of prometaphase, in mitotic cells as similar in DMSO controls and following inhibitor treatment. (C) Quantification of the ratio between pHH3 stained and total cell numbers (DAPI) from experiments in panel A. Greater sensitivity of GNS cells is observed across seven different cell lines.</p