Loss of BRD4 induces cell senescence in HSC/HPCs by deregulating histone H3 clipping.

Bromodomain-containing protein 4 (BRD4) is overexpressed and functionally implicated in various myeloid malignancies. However, the role of BRD4 in normal hematopoiesis remains largely unknown. Here, utilizing an inducible Brd4 knockout mouse model, we find that deletion of Brd4 (Brd4Δ/Δ ) in the hematopoietic system impairs hematopoietic stem cell (HSC) self-renewal and differentiation, which associates with cell cycle arrest and senescence. ATAC-seq analysis shows increased chromatin accessibility in Brd4Δ/Δ hematopoietic stem/progenitor cells (HSC/HPCs). Genome-wide mapping with cleavage under target and release using nuclease (CUT&RUN) assays demonstrate that increased global enrichment of H3K122ac and H3K4me3 in Brd4Δ/Δ HSC/HPCs is associated with the upregulation of senescence-specific genes. Interestingly, Brd4 deletion increases clipped H3 (cH3) which correlates with the upregulation of senescence-specific genes and results in a higher frequency of senescent HSC/HPCs. Re-expression of BRD4 reduces cH3 levels and rescues the senescence rate in Brd4Δ/Δ HSC/HPCs. This study unveils an important role of BRD4 in HSC/HPC function by preventing H3 clipping and suppressing senescence gene expression.

Kinome-Wide Virtual Screening by Multi-Task Deep Learning.

Deep learning is a machine learning technique to model high-level abstractions in data by utilizing a graph composed of multiple processing layers that experience various linear and non-linear transformations. This technique has been shown to perform well for applications in drug discovery, utilizing structural features of small molecules to predict activity. Here, we report a large-scale study to predict the activity of small molecules across the human kinome-a major family of drug targets, particularly in anti-cancer agents. While small-molecule kinase inhibitors exhibit impressive clinical efficacy in several different diseases, resistance often arises through adaptive kinome reprogramming or subpopulation diversity. Polypharmacology and combination therapies offer potential therapeutic strategies for patients with resistant diseases. Their development would benefit from a more comprehensive and dense knowledge of small-molecule inhibition across the human kinome. Leveraging over 650,000 bioactivity annotations for more than 300,000 small molecules, we evaluated multiple machine learning methods to predict the small-molecule inhibition of 342 kinases across the human kinome. Our results demonstrated that multi-task deep neural networks outperformed classical single-task methods, offering the potential for conducting large-scale virtual screening, predicting activity profiles, and bridging the gaps in the available data.

An overview of human single-cell RNA sequencing studies in neurobiological disease.

Neurobiological disorders are highly prevalent medical conditions that contribute to significant morbidity and mortality. Single-cell RNA sequencing (scRNA-seq) is a technique that measures gene expression in individual cells. In this review, we survey scRNA-seq studies of tissues from patients suffering from neurobiological disease. This includes postmortem human brains and organoids derived from peripheral cells. We highlight a range of conditions, including epilepsy, cognitive disorders, substance use disorders, and mood disorders. These findings provide new insights into neurobiological disease in multiple ways, including discovering novel cell types or subtypes involved in disease, proposing new pathophysiological mechanisms, uncovering novel drug targets, or identifying potential biomarkers. We discuss the quality of these findings and suggest potential future directions and areas open for additional research, including studies of non-cortical brain regions and additional conditions such as anxiety disorders, mood disorders, and sleeping disorders. We argue that additional scRNA-seq of tissues from patients suffering from neurobiological disease could advance our understanding and treatment of these conditions.

A dual aurora and lim kinase inhibitor reduces glioblastoma proliferation and invasion.

High rates of recurrence and treatment resistance in the most common malignant adult brain cancer, glioblastoma (GBM), suggest that monotherapies are not sufficiently effective. Combination therapies are increasingly pursued, but the possibility of adverse drug-drug interactions may preclude clinical implementation. Developing single molecules with multiple targets is a feasible alternative strategy to identify effective and tolerable pharmacotherapies for GBM. Here, we report the development of a novel, first-in-class, dual aurora and lim kinase inhibitor termed F114. Aurora kinases and lim kinases are involved in neoplastic cell division and cell motility, respectively. Due to the importance of these cellular functions, inhibitors of aurora kinases and lim kinases are being pursued separately as anti-cancer therapies. Using in vitro and ex vivo models of GBM, we found that F114 inhibits GBM proliferation and invasion. These results establish F114 as a promising new scaffold for dual aurora/lim kinase inhibitors that may be used in future drug development efforts for GBM, and potentially other cancers.

Epigenetic pathways and plasticity in brain tumors.

Clinical studies have shown that treating many primary brain tumors is challenging due in part to the lack of safe and effective compounds that cross the blood brain barrier (BBB) (Tan et al., 2018). However, if we were to imagine that we have ideal BBB penetrant compounds that target brain tumor cells selectively, recent studies suggest that those compounds may still not be effective due to the heterogenous nature of the tumors. In other words, there are many subsets of cells within a brain tumor, and compounds that target all those different populations are needed. This is a considerable challenge. Targeting of the cell-of-origin of these brain tumors is equally important. And yet another impediment we face is that brain tumor cells-of-origin may be protean and are able to differentiate into other cell types to drive recurrence. Therefore, an ideal BBB-penetrant compound targeting a cell-of-origin in a brain tumor may be ineffective due to the cell's ability to differentiate into another resistant cell type. One possible means of combating the plastic nature of these cells is targeting epigenetic pathways used by the cells to differentiate into other cell types along with standard treatment regimens. We summarize here some of the epigenetic pathways that have been shown to be active in three different primary brain tumors, glioblastoma (GBM), medulloblastoma (MB), and diffuse intrinsic pontine glioma (DIPG). We also compare recent single-cell RNA sequencing analyses of these tumors in order to identify common epigenetic pathways to treat the respective cells-of-origin for these tumors. Lastly, we discuss possible combination therapies that may be generalizable for treating these and other brain tumors using multi-omics approaches. While our focus on these three tumor types is not exhaustive and certainly other brain tumors can have similar mechanisms, there has been significant recent evidence linking epigenetics, plasticity, and intratumor heterogeneity in these tumors.

Prenatal arsenic exposure alters the placental expression of multiple epigenetic regulators in a sex-dependent manner.

BACKGROUND: Prenatal exposure to arsenic has been linked to a range of adverse health conditions in later life. Such fetal origins of disease are frequently the result of environmental effects on the epigenome, leading to long-term alterations in gene expression. Several studies have demonstrated effects of prenatal arsenic exposure on DNA methylation; however the impact of arsenic on the generation and decoding of post-translational histone modifications (PTHMs) is less well characterized, and has not been studied in the context of prenatal human exposures. METHODS: In the current study, we examined the effect of exposure to low-to-moderate levels of arsenic in a US birth cohort, on the expression of 138 genes encoding key epigenetic regulators in the fetal portion of the placenta. Our candidate genes included readers, writers and erasers of PTHMs, and chromatin remodelers. RESULTS: Arsenic exposure was associated with the expression of 27 of the 138 epigenetic genes analyzed. When the cohort was stratified by fetal sex, arsenic exposure was associated with the expression of 40 genes in male fetal placenta, and only 3 non-overlapping genes in female fetal placenta. In particular, we identified an inverse relationship between arsenic exposure and expression of the gene encoding the histone methyltransferase, PRDM6 (p < 0.001). Mutation of PRDM6 has been linked to the congenital heart defect, patent ductus arteriosus. CONCLUSIONS: Our findings suggest that prenatal arsenic exposure may have sex-specific effects on the fetal epigenome, which could plausibly contribute to its subsequent health impacts.

Serum long noncoding RNA HOTAIR as a novel diagnostic and prognostic biomarker in glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most common and aggressive malignant adult primary brain tumor. Despite surgical resection followed by radiotherapy and chemotherapy, the median survival rate is approximately 14 months. Although experimental therapies are in clinical trials for GBM, there is an urgent need for a peripheral GBM biomarker for measuring treatment response. As we have previously demonstrated that the long noncoding RNA HOX Transcript Antisense Intergenic RNA, or HOTAIR, is dysregulated in GBM and required for GBM cell proliferation, we hypothesized that HOTAIR expression may be utilized as a peripheral biomarker for GBM. HOTAIR expression was measured in serum from 43 GBM and 40 controls using quantitative real-time PCR (qRT-PCR). The PCR products were subsequently subcloned into pCR™4-TOPO®TA vectors for DNA sequencing. A ROC curve was also generated to examine HOTAIR's prognostic value. The amount of HOTAIR in serum exosomes and exosome-depleted supernatant was calculated by qRT-PCR. The relative HOTAIR expression was also investigated in 15 pairs of GBM serum and tumors. We detected HOTAIR in serum from GBM patients. HOTAIR levels in serum samples from GBM patients was significantly higher than in the corresponding controls (P < 0.0001). The area under the ROC curve distinguishing GBM patients from controls was 0.913 (95% CI: 0.845-0.982, P < 0.0001), with 86.1% sensitivity and 87.5% specificity at the cut-off value of 10.8. HOTAIR expression was significantly correlated with high grade brain tumors. In addition, Pearson correlation analysis indicated a medium correlation of serum HOTAIR levels and the corresponding tumor HOTAIR levels (r = 0.734, P < 0.01). We confirmed via sequencing that the amplified HOTAIR from serum contained the HOTAIR sequence and maps to the known HOTAIR locus at 12q13. The serum-derived exosomes contain HOTAIR and the purified exosomes were validated by western blot and nanoparticle tracking analysis. Importantly, our results demonstrate that serum HOTAIR can be used as a novel prognostic and diagnostic biomarker for GBM.

The Bromodomain protein BRD4 controls HOTAIR, a long noncoding RNA essential for glioblastoma proliferation.

Bromodomain and extraterminal (BET) domain proteins have emerged as promising therapeutic targets in glioblastoma and many other cancers. Small molecule inhibitors of BET bromodomain proteins reduce expression of several oncogenes required for Glioblastoma Multiforme (GBM) progression. However, the mechanism through which BET protein inhibition reduces GBM growth is not completely understood. Long noncoding RNAs (lncRNAs) are important epigenetic regulators with critical roles in cancer initiation and malignant progression, but mechanistic insight into their expression and regulation by BET bromodomain inhibitors remains elusive. In this study, we used Helicos single molecule sequencing to comprehensively profile lncRNAs differentially expressed in GBM, and we identified a subset of GBM-specific lncRNAs whose expression is regulated by BET proteins. Treatment of GBM cells with the BET bromdomain inhibitor I-BET151 reduced levels of the tumor-promoting lncRNA HOX transcript antisense RNA (HOTAIR) and restored the expression of several other GBM down-regulated lncRNAs. Conversely, overexpression of HOTAIR in conjunction with I-BET151 treatment abrogates the antiproliferative activity of the BET bromodomain inhibitor. Moreover, chromatin immunoprecipitation analysis demonstrated binding of Bromodomain Containing 4 (BRD4) to the HOTAIR promoter, suggesting that BET proteins can directly regulate lncRNA expression. Our data unravel a previously unappreciated mechanism through which BET proteins control tumor growth of glioblastoma cells and suggest that modulation of lncRNA networks may, in part, mediate the antiproliferative effects of many epigenetic inhibitors currently in clinical trials for cancer and other diseases.

The BET bromodomain inhibitor I-BET151 acts downstream of smoothened protein to abrogate the growth of hedgehog protein-driven cancers.

Epigenetic enzymes modulate signal transduction pathways in different biological contexts. We reasoned that epigenetic regulators might modulate the Hedgehog (HH) signaling pathway, a main driver of cell proliferation in various cancers including medulloblastoma. To test this hypothesis, we performed an unbiased small-molecule screen utilizing an HH-dependent reporter cell line (Light2 cells). We incubated Light2 cells with small molecules targeting different epigenetic modulators and identified four histone deacetylase inhibitors and a bromodomain and extra terminal domain (BET) protein inhibitor (I-BET151) that attenuate HH activity. I-BET151 was also able to inhibit the expression of HH target genes in Sufu(-/-) mouse embryonic fibroblasts, in which constitutive Gli activity is activated in a Smoothened (Smo)-independent fashion, consistent with it acting downstream of Smo. Knockdown of Brd4 (which encodes one of the BET proteins) phenocopies I-BET151 treatment, suggesting that Brd4 is a regulator of the HH signaling pathway. Consistent with this suggestion, Brd4 associates with the proximal promoter region of the Gli1 locus, and does so in a manner that can be reversed by I-BET151. Importantly, I-BET151 also suppressed the HH activity-dependent growth of medulloblastoma cells, in vitro and in vivo. These studies suggest that BET protein modulation may be an attractive therapeutic strategy for attenuating the growth of HH-dependent cancers, such as medulloblastoma.

Casein kinase 1δ-dependent Wee1 protein degradation.

Eukaryotic mitotic entry is controlled by Cdk1, which is activated by the Cdc25 phosphatase and inhibited by Wee1 tyrosine kinase, a target of the ubiquitin proteasome pathway. Here we use a reporter of Wee1 degradation, K328M-Wee1-luciferase, to screen a kinase-directed chemical library. Hit profiling identified CK1δ-dependent Wee1 degradation. Small-molecule CK1δ inhibitors specifically disrupted Wee1 destruction and arrested HeLa cell proliferation. Pharmacological inhibition, siRNA knockdown, or conditional deletion of CK1δ also reduced Wee1 turnover. Thus, these studies define a previously unappreciated role for CK1δ in controlling the cell cycle.

Epigenetic pathways and glioblastoma treatment: insights from signaling cascades.

There is an urgent need to identify novel therapies for glioblastoma (GBM) as most therapies are ineffective. A first step in this process is to identify and validate targets for therapeutic intervention. Epigenetic modulators have emerged as attractive drug targets in several cancers including GBM. These epigenetic regulators affect gene expression without changing the DNA sequence. Recent studies suggest that epigenetic regulators interact with drivers of GBM cell and stem-like cell proliferation. These drivers include components of the Notch, Hedgehog, and Wingless (WNT) pathways. We highlight recent studies connecting epigenetic and signaling pathways in GBM. We also review systems and big data approaches for identifying patient specific therapies in GBM. Collectively, these studies will identify drug combinations that may be effective in GBM and other cancers.

RAPID resistance to BET inhibitors is mediated by FGFR1 in glioblastoma.

Bromodomain and extra-terminal domain (BET) proteins are therapeutic targets in several cancers including the most common malignant adult brain tumor glioblastoma (GBM). Multiple small molecule inhibitors of BET proteins have been utilized in preclinical and clinical studies. Unfortunately, BET inhibitors have not shown efficacy in clinical trials enrolling GBM patients. One possible reason for this may stem from resistance mechanisms that arise after prolonged treatment within a clinical setting. However, the mechanisms and timeframe of resistance to BET inhibitors in GBM is not known. To identify the temporal order of resistance mechanisms in GBM we performed quantitative proteomics using multiplex-inhibitor bead mass spectrometry and demonstrated that intrinsic resistance to BET inhibitors in GBM treatment occurs rapidly within hours and involves the fibroblast growth factor receptor 1 (FGFR1) protein. Additionally, small molecule inhibition of BET proteins and FGFR1 simultaneously induces synergy in reducing GBM tumor growth in vitro and in vivo. Further, FGFR1 knockdown synergizes with BET inhibitor mediated reduction of GBM cell proliferation. Collectively, our studies suggest that co-targeting BET and FGFR1 may dampen resistance mechanisms to yield a clinical response in GBM.

Triptolide and its pro-drug Minnelide target high-risk MYC amplified medulloblastoma in preclinical models.

Most children with medulloblastoma (MB) achieve remission, but some face very aggressive metastatic tumors. Their dismal outcome highlights the critical need to advance therapeutic approaches that benefit such high-risk patients. Minnelide, a clinically relevant analog of the natural product triptolide, has oncostatic activity in both preclinical and early clinical settings. Despite its efficacy and tolerable toxicity, this compound has not been evaluated in MB. Utilizing a bioinformatic dataset that integrates cellular drug response data with gene expression, we predicted that Group 3 (G3) MB, which has a poor five-year survival, would be sensitive to triptolide/Minnelide. We subsequently showed that both triptolide and Minnelide attenuate the viability of G3 MB cells ex vivo. Transcriptomic analyses identified MYC signaling, a pathologically relevant driver of G3 MB, as a downstream target of this class of drugs. We validated this MYC dependency in G3 MB cells and showed that triptolide exerts its efficacy by reducing both MYC transcription and MYC protein stability. Importantly, Minnelide acted on MYC to reduce tumor growth and leptomeningeal spread, which resulted in improved survival of G3 MB animal models. Moreover, Minnelide improved the efficacy of adjuvant chemotherapy, further highlighting its potential for the treatment of MYC-driven G3 MB patients.

BET protein inhibition promotes non-myeloid cell mediated neuroprotection after rodent spinal cord contusion.

Spinal cord injuries (SCI) often lead to multiple neurological deficits as a result from the initial trauma and also the secondary damage that follows. Despite abundant preclinical data proposing anti-inflammatory therapies to minimize secondary injury and improve functional recovery, the field still lacks an effective neuroprotective treatment. Epigenetic proteins, such as bromodomain and extraterminal domain (BET) proteins, are emerging as new targets to regulate inflammation. More importantly, pharmacological inhibition of BET proteins suppresses pro-inflammatory gene transcription after SCI. In this study, we tested the therapeutic potential of inhibiting BET proteins after SCI with clinically relevant compounds, and investigated the role of the BET protein BRD4 in macrophages during progression of SCI pathology. Systemic inhibition of BET proteins with I-BET762 significantly reduced lesion size 8 weeks after a contusion injury in rats. However, we observed no histological or locomotor improvements after SCI when we deleted Brd4 in macrophages through the use of myeloid-specific Brd4 knockout mice or after macrophage-targeted pharmacological BET inhibition. Taken together, our data indicate that systemic I-BET762 treatment is neuroprotective, and the histopathological improvement observed is likely to be a result of effects on non-macrophage targets. Expanding our understanding on the role of BET proteins after SCI is necessary to identify novel therapeutic targets that can effectively promote repair after SCI.

Multiprotein GLI Transcriptional Complexes as Therapeutic Targets in Cancer.

The Hedgehog signaling pathway functions in both embryonic development and adult tissue homeostasis. Importantly, its aberrant activation is also implicated in the progression of multiple types of cancer, including basal cell carcinoma and medulloblastoma. GLI transcription factors function as the ultimate effectors of the Hedgehog signaling pathway. Their activity is regulated by this signaling cascade via their mRNA expression, protein stability, subcellular localization, and ultimately their transcriptional activity. Further, GLI proteins are also regulated by a variety of non-canonical mechanisms in addition to the canonical Hedgehog pathway. Recently, with an increased understanding of epigenetic gene regulation, novel transcriptional regulators have been identified that interact with GLI proteins in multi-protein complexes to regulate GLI transcriptional activity. Such complexes have added another layer of complexity to the regulation of GLI proteins. Here, we summarize recent work on the regulation of GLI transcriptional activity by these novel protein complexes and describe their relevance to cancer, as such GLI regulators represent alternative and innovative druggable targets in GLI-dependent cancers.

A Druggable UHRF1/DNMT1/GLI Complex Regulates Sonic Hedgehog-Dependent Tumor Growth.

Dysregulation of Sonic hedgehog (SHH) signaling drives the growth of distinct cancer subtypes, including medulloblastoma (MB). Such cancers have been treated in the clinic with a number of clinically relevant SHH inhibitors, the majority of which target the upstream SHH regulator, Smoothened (SMO). Despite considerable efficacy, many of these patients develop resistance to these drugs, primarily due to mutations in SMO. Therefore, it is essential to identify druggable, signaling components downstream of SMO to target in SMO inhibitor resistant cancers. We utilized an integrated functional genomics approach to identify epigenetic regulators of SHH signaling and identified a novel complex of Ubiquitin-like with PHD and RING finger domains 1 (UHRF1), DNA methyltransferase 1 (DNMT1), and GLI proteins. We show that this complex is distinct from previously described UHRF1/DNMT1 complexes, suggesting that it works in concert to regulate GLI activity in SHH driven tumors. Importantly, we show that UHRF1/DNMT1/GLI complex stability is targeted by a repurposed FDA-approved therapy, with a subsequent reduction in the growth of SHH-dependent MB ex vivo and in vivo. IMPLICATIONS: This work describes a novel, druggable UHRF1/DNMT1/GLI complex that regulates SHH-dependent tumor growth, and highlights an FDA-approved drug capable of disrupting this complex to attenuate tumor growth.

A multiparametric pharmacogenomic strategy for drug repositioning predicts therapeutic efficacy for glioblastoma cell lines.

BACKGROUND: Poor prognosis of glioblastoma patients and the extensive heterogeneity of glioblastoma at both the molecular and cellular level necessitates developing novel individualized treatment modalities via genomics-driven approaches. METHODS: This study leverages numerous pharmacogenomic and tissue databases to examine drug repositioning for glioblastoma. RNA-seq of glioblastoma tumor samples from The Cancer Genome Atlas (TCGA, n = 117) were compared to "normal" frontal lobe samples from Genotype-Tissue Expression Portal (GTEX, n = 120) to find differentially expressed genes (DEGs). Using compound gene expression data and drug activity data from the Library of Integrated Network-Based Cellular Signatures (LINCS, n = 66,512 compounds) CCLE (71 glioma cell lines), and Chemical European Molecular Biology Laboratory (ChEMBL) platforms, we employed a summarized reversal gene expression metric (sRGES) to "reverse" the resultant disease signature for GBM and its subtypes. A multiparametric strategy was employed to stratify compounds capable of blood-brain barrier penetrance with a favorable pharmacokinetic profile (CNS-MPO). RESULTS: Significant correlations were identified between sRGES and drug efficacy in GBM cell lines in both ChEMBL(r = 0.37, P < .001) and Cancer Therapeutic Response Portal (CTRP) databases (r = 0.35, P < 0.001). Our multiparametric algorithm identified two classes of drugs with highest sRGES and CNS-MPO: HDAC inhibitors (vorinostat and entinostat) and topoisomerase inhibitors suitable for drug repurposing. CONCLUSIONS: Our studies suggest that reversal of glioblastoma disease signature correlates with drug potency for various GBM subtypes. This multiparametric approach may set the foundation for an early-phase personalized -omics clinical trial for glioblastoma by effectively identifying drugs that are capable of reversing the disease signature and have favorable pharmacokinetic and safety profiles.

Activation domain-dependent degradation of somatic Wee1 kinase.

Cell cycle progression is dependent upon coordinate regulation of kinase and proteolytic pathways. Inhibitors of cell cycle transitions are degraded to allow progression into the subsequent cell cycle phase. For example, the tyrosine kinase and Cdk1 inhibitor Wee1 is degraded during G(2) and mitosis to allow mitotic progression. Previous studies suggested that the N terminus of Wee1 directs Wee1 destruction. Using a chemical mutagenesis strategy, we report that multiple regions of Wee1 control its destruction. Most notably, we find that the activation domain of the Wee1 kinase is also required for its degradation. Mutations in this domain inhibit Wee1 degradation in somatic cell extracts and in cells without affecting the overall Wee1 structure or kinase activity. More broadly, these findings suggest that kinase activation domains may be previously unappreciated sites of recognition by the ubiquitin proteasome pathway.

The tumour suppressor RASSF1A regulates mitosis by inhibiting the APC-Cdc20 complex.

The tumour suppressor gene RASSF1A is frequently silenced in lung cancer and other sporadic tumours as a result of hypermethylation of a CpG island in its promoter. However, the precise mechanism by which RASSF1A functions in cell cycle regulation and tumour suppression has remained unknown. Here we show that RASSF1A regulates the stability of mitotic cyclins and the timing of mitotic progression. RASSF1A localizes to microtubules during interphase and to centrosomes and the spindle during mitosis. The overexpression of RASSF1A induced stabilization of mitotic cyclins and mitotic arrest at prometaphase. RASSF1A interacts with Cdc20, an activator of the anaphase-promoting complex (APC), resulting in the inhibition of APC activity. Although RASSF1A does not contribute to either the Mad2-dependent spindle assembly checkpoint or the function of Emi1 (ref. 1), depletion of RASSF1A by RNA interference accelerated the mitotic cyclin degradation and mitotic progression as a result of premature APC activation. It also caused a cell division defect characterized by centrosome abnormalities and multipolar spindles. These findings implicate RASSF1A in the regulation of both APC-Cdc20 activity and mitotic progression.

Organoid Models of Glioblastoma and Their Role in Drug Discovery.

Glioblastoma (GBM) is a devastating adult brain cancer with high rates of recurrence and treatment resistance. Cellular heterogeneity and extensive invasion of surrounding brain tissues are characteristic features of GBM that contribute to its intractability. Current GBM model systems do not recapitulate some of the complex features of GBM and have not produced sufficiently-effective treatments. This has cast doubt on the effectiveness of current GBM models and drug discovery paradigms. In search of alternative pre-clinical GBM models, various 3D organoid-based GBM model systems have been developed using human cells. The scalability of these systems and potential to more accurately model characteristic features of GBM, provide promising new avenues for pre-clinical GBM research and drug discovery efforts. Here, we review the current suite of organoid-GBM models, their individual strengths and weaknesses, and discuss their future applications with an emphasis on compound screening.