Biology subjects

Day, B. W.

Publications and source records attributed to Day, B. W..

11 recordsLinked to original sources

bioRxiv2026

Pharmacologic inhibition of OLIG2 by CT-179 disrupts mitotic progression and enhances standard-of-care chemoradiotherapy in glioblastoma

Background: Glioblastoma (GBM) remains uniformly fatal. The basic helix-loop-helix transcription factor OLIG2 is universally expressed in diffuse glioma, sustains the tumor-propagating stem-like fraction, and opposes p53-mediated genotoxic responses, but has historically been considered undruggable. We characterized CT-179, a brain-penetrant small-molecule OLIG2 inhibitor, as a candidate therapeutic for GBM. Methods: Target engagement was assessed by cellular thermal shift assay, differential scanning fluorimetry, crosslinking, and electrophoretic mobility shift assay. Anti-tumor activity was evaluated across eleven patient-derived GBM cell lines, in zebrafish oligodendrocyte precursor cell (OPC)-derived tumors, and orthotopic and subcutaneous GBM models, including combinations with radiotherapy plus temozolomide and with selective Bcl-2 family inhibitors. Results: CT-179 engaged OLIG2 and altered the conformation of dimerized OLIG2 on chromatin without preventing dimerization or fully displacing OLIG2 from DNA. CT-179 reduced OLIG2-positive OPCs and OPC-derived tumors in zebrafish and inhibited proliferation across all eleven GBM lines (IC50 < 360 nM), depleting the stem-like fraction and inducing apoptosis through mitotic catastrophe, mitotic slippage, or a hybrid response. RNA-seq across three patient-derived glioma stem cell lines identified {beta}-tubulin gene isoforms as the most strongly CT-179-down-regulated transcripts at the early time point and showed correlated {beta}-tubulin and OLIG2 expression in TCGA glioblastoma specimens. Combination with the Mcl-1 inhibitor S63845 or the Bcl-2/Bcl-xL/Bcl-w inhibitor ABT-737 produced greater-than-additive cytotoxicity. Oral CT-179 crossed the blood-brain barrier and extended median survival as a single agent across two independent adult orthotopic patient-derived backgrounds (GBM8 and G06); intraperitoneal CT-179 was associated with reduced intratumoral OLIG2 and Ki-67 staining in GBM8 orthotopic xenografts. Combination of CT-179 with radiotherapy and temozolomide eliminated all GBM8 and 7 of 8 GBM4 subcutaneous xenografts without weight loss and significantly extended survival when combined with radiotherapy in MGMT-unmethylated GBM6 orthotopic xenografts (CT-179 administered intraperitoneally). Conclusions: CT-179 is a brain-penetrant inhibitor of an established GBM driver with single-agent and combination activity across several orthotopic models, supporting clinical development for GBM and translational evaluation in other OLIG2-dependent CNS malignancies.

cancer biology↗

bioRxiv2025

Shared molecular regulation of quiescence in neural and glioma stem cells reveals therapeutic vulnerabilities.

Quiescence, a reversible state of cell-cycle arrest, is an adaptive feature of many adult tissue stem cells, including those in the adult brain. In gliomas, brain tumour stem cells that reside in a quiescent state preferentially survive chemotherapy and radiotherapy, highlighting their critical role in therapy resistance and disease progression. To date, it remains unclear whether the molecular programs governing these states are functionally conserved between neural stem cells and brain tumour stem cells. Here, we establish novel in vitro models to study quiescence and find that glioma stem cells are markedly more resistant to entering quiescence than neural stem cells, suggesting that glioma stem cell quiescence more closely resembles a slow-cycling phenotype or shallow quiescence. Nonetheless, direct comparison of quiescent neural stem cells and quiescent/slow-cycling glioma stem cells, as they transition towards proliferation, reveals conserved gene expression trajectories, indicating shared molecular mechanisms. Furthermore, we find that pathways influencing quiescence in neural stem cells exert similar effects in glioma stem cells, underscoring the functional parallels between these populations. Finally, we identify that inhibition of TGF-{beta} signalling might provide an avenue to improve current standard-of-care treatments by targeting quiescent glioma stem cells. Note on version 2This version corrects two figure errors and expands the Methods. The conclusions of are unchanged. ImagesIn version 1, debris adjacent to the organoid had been digitally removed from the drug-treated image in Figure 6D. The unmodified original is restored here. Images within Figures 3A and 6D have each been processed identically to one another. Scattered cellular debris was present to a similar extent in control and drug-treated samples. O_FIG O_LINKSMALLFIG WIDTH=153 HEIGHT=200 SRC="FIGDIR/small/634421v2_fig6.gif" ALT="Figure 6"> View larger version (51K): org.highwire.dtl.DTLVardef@19aef83org.highwire.dtl.DTLVardef@e2c1e1org.highwire.dtl.DTLVardef@126bf22org.highwire.dtl.DTLVardef@ce7acd_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 6:C_FLOATNO TGF--R1 inhibition drives GSCs out of a quiescent state. (A) QBC395 cells were cultured in BMP4 quiescence media plus inhibitors for three days. Graphs show percentage of proliferating GSCs (SOX2+Ki67+) over total GSCs (SOX2+). Data were normalised to mean of quiescence media alone. (B) QBC395 cells were cultured in palbociclib quiescence media plus inhibitors for three days. Graphs show proportion of proliferating GSCs (SOX2+Ki67+) over total GSCs (SOX2+). Data were first normalised to vehicle control for each independent experiment and then quiescence media alone. (C) Schematic of glioblastoma organoid generation. Experiments began within 1-2 weeks of derivation to maximise cellular heterogeneity. Protocol adapted from Jacob and colleagues (60). (D) Image of glioblastoma organoid treated with 30 {micro}M LY-364397 or DMSO vehicle (control) for three days, stained for SOX2 (cyan) and Ki67 (magenta). Dashed lines demarcate organoid boundary. Scale bar: 100 {micro}m. Scattered cellular debris was observed in both control and drug-treated samples to a similar extent. (E) Proportion of proliferating GSCs (SOX2+Ki67+) over total GSCs (SOX2+) after glioblastoma organoids were cultured in the presence of inhibitors. Data were normalised to mean of respective vehicle control. Graphs in (A, B, E) show mean {+/-} SEM. Experiments in (A, B) represent independent experiments on separate passages (n=3-4). Dots in (E) represent individual organoids. Statistics: one-way repeated measures ANOVA with Holm-Sidaks multiple comparisons test in (A, B). Two-way ANOVA in (E) with multiple comparisons t-test. *P < 0.05, **P < 0.01, ***P < 0.001. Panel C created with BioRender (Agreement number: FY289ELRZ5). C_FIG O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=52 SRC="FIGDIR/small/634421v2_fig3.gif" ALT="Figure 3"> View larger version (21K): org.highwire.dtl.DTLVardef@1e04d0corg.highwire.dtl.DTLVardef@e25e26org.highwire.dtl.DTLVardef@1934ab0org.highwire.dtl.DTLVardef@10c77fb_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 3:C_FLOATNO BMP4 and palbociclib treatment induces patient-derived GSCs into shallow versus deep states of quiescence, respectively. (A) Treatment with 16 ng/mL BMP4 markedly reduces the proportion of proliferating NSCs (SOX2+Ki67+) in primary adult mouse SVZ cultures. (B) Treatment with 16 ng/mL BMP4 modestly reduces the proportion of proliferating GSCs (SOX2+Ki67+) in primary patient-derived cultures (QBC395 cell line). (C) Concentration-response curve of mouse NSCs and patient-derived GSCs (QBC395 cell line) treated with BMP4, measuring proportion of proliferating stem cells (SOX2+Ki67+) relative to control media. Data were normalised to mean of control. (D) Palbociclib treatment markedly reduces the proportion of proliferating GSCs (SOX2+Ki67+) relative to control media. Data were normalised to mean of control. GSC graphs in (C, D) show mean {+/-} SEM of independent experiments conducted on separate passages (n=3-4). NSC graph in (C) shows mean {+/-} SEM of individual cell lines derived from SVZ of different mice (n=4). Scale bar: 15 {micro}m in (A, B). Cross in (D) represents concentration-response at 200 nM. Statistics in (C): two-way ANOVA reporting main effect of cell-line. C_FIG DataThe glioblastoma organoid data in Figure 6E have been updated following re-imaging and blinded re-analysis. Reported effect sizes have changed slightly; the conclusions are unchanged. Figure S2 has likewise been updated following re-imaging and re-analysis. MethodsThe Methods have been expanded to describe the analysis more fully, including data exclusion criteria, normalisation, and blinding. Minor changes to figure legends and the main text are not itemised.

cancer biology↗

bioRxiv2025

Histone methyltransferase PRDM9 promotes survival of drug-tolerant persister cells in glioblastoma

Chemotherapy often kills a large fraction of cancer cells but leaves behind a small population of drug- tolerant persister cells. These persister cells survive drug treatments through reversible, non-genetic mechanisms and cause tumour recurrence upon cessation of therapy. Here, we report a drug tolerance mechanism regulated by the germ-cell-specific H3K4 methyltransferase PRDM9. Through histone proteomic, transcriptomic, lipidomic, and ChIP-sequencing studies combined with CRISPR knockout and phenotypic drug screen, we identified that chemotherapy-induced PRDM9 upregulation promotes metabolic rewiring in glioblastoma stem cells, leading to chemotherapy tolerance. Mechanistically, PRDM9-dependent H3K4me3 at cholesterol biosynthesis genes enhances cholesterol biosynthesis, which persister cells rely on to maintain homeostasis under chemotherapy- induced oxidative stress and lipid peroxidation. PRDM9 inhibition, combined with chemotherapy, resulted in strong anti-cancer efficacy in preclinical glioblastoma models, significantly enhancing the magnitude and duration of the antitumor response by eliminating persisters. These findings demonstrate a previously unknown role of PRDM9 in promoting metabolic reprogramming that enables the survival of drug-tolerant persister cells.

cancer biology↗

bioRxiv2024

Spatial Transcriptomic Sequencing of a DIPG-infiltrated Brainstem reveals Key Invasion Markers and Novel Ligand-Receptor Pairs contributing to Tumour to TME Crosstalk

Emerging spatially-resolved sequencing technologies offer unprecedented possibilities to study cellular functionality and organisation, transforming our understanding of health and disease. The necessity to understand healthy and diseased tissues in its entirety becomes even more evident for the human brain, the most complex organ in the body. The brains cellular architecture and corresponding functions are tightly regulated. However, when intercellular communications are altered by pathologies, such as brain cancer, these microenvironmental interactions are disrupted. DIPG is a brainstem high-grade glioma arising in young children and is universally fatal. Major disease obstacles include intratumoural genetic and cellular heterogeneity as well as a highly invasive phenotype. Recent breakthrough studies have highlighted the vital oncogenic capacity of brain cancer cells to functionally interact with the central nervous system (CNS). This CNS-crosstalk crucially contributes to tumour cell invasion and disease progression. Ongoing worldwide efforts seek to better understand these cancer-promoting CNS interactions to develop more effective DIPG anti-cancer therapies. In this study, we performed spatial transcriptomic analysis of a complete tumour-infiltrated brainstem from a single DIPG patient. Gene signatures from ten sequential tumour regions were analysed to assess disease progression and to study DIPG cell interactions with the tumour microenvironment (TME). We leveraged this unique DIPG dataset to evaluate genes significantly correlated with invasive tumour distal regions versus the proximal tumour initiation site. Furthermore, we assessed novel ligand-receptor pairs that actively promote DIPG tumour progression via crosstalk with endothelial, neuronal and immune cell communities, which can be utilised to support future research efforts in this area of high unmet need.

cancer biology↗

bioRxiv2023

ATR inhibition using gartisertib enhances cell death and synergises with temozolomide and radiation in patient-derived glioblastoma cell lines

Glioblastoma cells can restrict the DNA-damaging effects of temozolomide (TMZ) and radiation therapy (RT) using the DNA damage response (DDR) mechanism which activates cell cycle arrest and DNA repair pathways. Ataxia-telangiectasia and Rad3-Related protein (ATR) plays a pivotal role in the recognition of DNA damage induced by chemotherapy and radiation causing downstream DDR activation. Here, we investigated the activity of gartisertib, a potent ATR inhibitor, alone and in combination with TMZ and/or RT in 12 patient-derived glioblastoma cell lines. We showed that gartisertib alone potently reduced the cell viability of glioblastoma cell lines, where sensitivity was associated with the frequency of DDR mutations and higher expression of the G2 cell cycle pathway. ATR inhibition significantly enhanced cell death in combination with TMZ and RT and was shown to have higher synergy than TMZ+RT treatment. MGMT promoter unmethylated and TMZ+RT resistant glioblastoma cells were also more sensitive to gartisertib. Analysis of gene expression from gartisertib treated glioblastoma cells identified the upregulation of innate immune-related pathways. Overall, this study identifies ATR inhibition as a strategy to enhance the DNA-damaging ability of glioblastoma standard treatment, while providing preliminary evidence that ATR inhibition induces an innate immune gene signature that warrants further investigation.

cancer biology↗

bioRxiv2023

Molecular Determinants of Calcitriol Signaling and Sensitivity in Glioma Stem-like Cells

Glioblastoma is the most common primary brain cancer in adults and represents one of the worst cancer diagnosis for the patients. Suffering from a poor prognosis and limited treatment options, tumor recurrences are virtually inevitable. Additionally, treatment resistance is very common for this disease and worsens the prognosis. These and others factors are hypothesized to be largely due to the fact that glioblastoma cells are known to be able to obtain stem-like traits and thereby driving these phenotypes. Recently, we could show that the in vitro and ex vivo treatment of glioblastoma stem-like cells with the hormonally active form of Vitamin D3, Calcitriol (1,25(OH)2-vitamin D3) can block stemness in a subset of cell lines and reduce tumor growth. Here, we expanded our cell panel to over 40 different cultures and can show that, while half of the tested cell lines are sensitive, a quarter can be classified as high-responders. Using genetic and proteomic analysis, we further determined that treatment success can be partially explained by specific polymorphism of the Vitamin D3 receptor and that high-responders display a proteome suggestive of blockade of stemness, as well as migratory potential.

cancer biology↗

bioRxiv2022

Targeting OLIG2 increases therapeutic responses in SHH medulloblastoma mouse models and patient-derived medulloblastoma organoids

Recurrence after therapy is the primary life-threatening complication of medulloblastoma. In Sonic Hedgehog (SHH)-subgroup medulloblastoma, OLIG2-expressing tumour stem cells are crucial to recurrence. We investigated the potential of the small-molecule OLIG2 inhibitor CT-179 to decrease recurrence in patient-derived organoids, mice genetically-engineered to develop SHH-driven MB, and mice with MB patient-derived xenograft (PDX) tumours. We found that OLIG2 mRNA significantly correlated with poor survival in patients with SHH-MB, but not other subgroups. CT-179 rapidly downregulated OLIG2 protein in vitro and displayed nanomolar IC50 values. CT-179 arrested MB cells at G2/M, with degradation of cyclin B1 and phospho-CDK1 inducing apoptosis. In vivo CT-179 induced similar cell cycle changes in MBs in Smo-mutant mice and significantly increased mouse survival. In both MB organoids and mouse models, CT-179 combined with radiotherapy showed greater efficacy than either treatment alone. These data highlight the potential for OLIG2-targeted therapy to improve MB outcomes by targeting recurrent disease.

cancer biology↗

bioRxiv2021

A short ERK5 isoform modulates nucleocytoplasmic shuttling of active ERK5 and associates with poor survival in breast cancer

BackgroundThe nucleocytoplasmic shuttling of ERK5 has gained recent attention as a regulator of its diverse roles in cancer progression but the exact mechanisms for this shuttling are still under investigation. MethodsUsing in vitro, in vivo and in silico studies, we investigated the roles of shorter ERK5 isoforms in regulating the nucleocytoplasmic shuttling of active phosphorylated-ERK5 (pERK5). Retrospective cohorts of primary and metastatic breast cancer cases were used to evaluate the association of the subcellular localization of pERK5 with clinicopathological features. ResultsExtranuclear localization of pERK5 was observed during cell migration in vitro and at the invasive fronts of metastatic tumors in vivo. The nuclear and extranuclear cell fractions contained different isoforms of pERK5, which are encoded by splice variants expressed in breast and other cancers in the TCGA data. One isoform, isoform-3, lacks the C-terminal transcriptional domain and the nuclear localization signal. The co-expression of isoform-3 and full-length ERK5 associated with high epithelial-to-mesenchymal transition (EMT) and poor patient survival. Experimentally, expressing isoform-3 with full-length ERK5 in breast cancer cells increased cell migration, drove EMT and led to tamoxifen resistance. In breast cancer patient samples, pERK5 showed variable subcellular localizations where its extranuclear localization associated with aggressive clinicopathological features, metastasis, and poor survival. ConclusionOur studies support a model of ERK5 nucleocytoplasmic shuttling driven by splice variants in an interplay between mesenchymal and epithelial states during metastasis. Using ERK5 as a biomarker and a therapeutic target should account for its splicing and context-dependent biological functions. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=97 SRC="FIGDIR/small/436061v1_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@1491ed5org.highwire.dtl.DTLVardef@1875fbeorg.highwire.dtl.DTLVardef@9edbbcorg.highwire.dtl.DTLVardef@16c34c8_HPS_FORMAT_FIGEXP M_FIG C_FIG ERK5 isoform-3 expression deploys active ERK5 (pERK5) outside the nucleus to facilitate EMT and cell migration. In cells dominantly expressing isoform-1, pERK5 shuttles to the nucleus to drive cell expansion.

cancer biology↗

bioRxiv2020

Immunophenotyping of HER4/YAP axis in breast cancer brain metastasis

Brain metastasis (BrM) is a devastating diagnosis for patients with breast cancer. Identification of immunophenotypical features specific to BrM is crucial for developing effective new therapeutic interventions. Yes-associated protein (YAP) mediates downstream effects of the neuregulin receptor, HER4, on breast cancer cell proliferation in vitro. HER4 is frequently over-expressed in BrM, but the relationship with YAP has not been investigated. This study examined the HER4/YAP axis in patient samples (n=41) from matched primary breast and metastatic brain tumours. Immunohistochemistry analysis revealed HER4 was highly phosphorylated in BrM compared to matched primary tumours (p=0.0022), and this was strongly associated with expression and phosphorylation of dimerization partners HER2 and HER3 (p<0.0001). When compared to a general breast cancer cohort (n=373), we found HER4 activation to be brain metastasis specific (p<0.0001). However, YAP was frequently phosphorylated in both HER4-activated primary breast tumours and brain metastases, suggesting that pro-proliferative YAP signaling is not a major consequence of HER4 activation in these tumours. These results display the complexity of expression of the HER family receptors and the downstream pathways in BrM and suggest simultaneous targeting of multiple receptors might be more advantageous.

pathology↗

bioRxiv2019

Transcription factors NFIA and NFIB induce cellular differentiation high-grade astrocytoma

Astrocytomas are composed of heterogeneous cell populations. Compared to grade IV glioblastoma, low-grade astrocytomas have more differentiated cells and are associated with a better prognosis. Therefore, inducing cellular differentiation may serve as a therapeutic strategy. The nuclear factor one (NFI) transcription factors are essential for normal astrocytic differentiation and therefore could be effectors of cellular differentiation in glioblastoma. We analysed expression of family members NFIA and NFIB using high-grade astrocytoma mRNA expression datasets, and with immunofluorescence co-staining. Their expression is reduced in glioblastomas and is associated with differentiated and mature astrocyte-like cells at a cellular level. Furthermore, induction of NFI expression is sufficient to promote cellular differentiation in patient-derived glioblastoma xenografts. Our findings indicate that NFI proteins may have an endogenous pro-differentiative function in astrocytomas, similar to their role in normal development. Overall, our study establishes a basis for further investigation of targeting NFI-mediated differentiation as a potential differentiation therapy.

cancer biology↗

bioRxiv2019

Direct evidence for transport of RNA from the mouse brain to the germline and offspring

BackgroundThe traditional concept that heritability occurs exclusively from the transfer of germline-restricted genetics is being challenged by the increasing accumulation of evidence confirming the existence of experience-dependent transgenerational inheritance. However, questions remain unanswered as to how heritable information can be passed from somatic cells. Previous studies have implicated the critical involvement of RNA in heritable transgenerational effects and the high degree of mobility and genomic impact of RNAs in all organisms is an attractive model for the efficient transfer of genetic information.\n\nResultsWe hypothesized that RNA may be transported from a somatic tissue, in this case the brain, of an adult male mouse to the germline, and subsequently to offspring. To investigate this, we injected one hemisphere of the male mouse striatum with an AAV1/9 virus expressing human pre-MIR941 (MIR941). After 2, 8 and 16 weeks following injection, we used an LNA-based qPCR system to detect the presence of virus and human MIR941 in brain, peripheral tissues and offspring, from injected male mice mated with uninjected females. Virus was never detected outside of the brain. Verification of single bands of the correct size for MIR941 was performed using Sanger sequencing while quantitation demonstrated that a small percentage ([~] 1-8%) of MIR941 is transported to the germline and to offspring in about a third of the cases.\n\nConclusionsWe show that somatic RNA can be transported to the germline and passed on to offspring, thereby providing additional evidence of a role for RNA in somatic cell-derived transgenerational effects.

evolutionary biology↗