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Glioblastomas (GBMs) are heterogeneous, treatment-resistant tumors driven by populations of cancer stem cells (CSCs). However, few molecular mechanisms critical for CSC population maintenance have been exploited for therapeutic development. We developed a spatially resolved loss-of-function screen in GBM patient-derived organoids to identify essential epigenetic regulators in the SOX2-enriched, therapy-resistant niche and identified WDR5 as indispensable for this population. WDR5 is a component of the WRAD complex, which promotes SET1 family-mediated Lys4 methylation of histone H3 (H3K4me), associated with positive regulation of transcription. In GBM CSCs, WDR5 inhibitors blocked WRAD complex assembly and reduced H3K4 trimethylation and expression of genes involved in CSC-relevant oncogenic pathways. H3K4me3 peaks lost with WDR5 inhibitor treatment occurred disproportionally on POU transcription factor motifs, including the POU5F1(OCT4)::SOX2 motif. Use of a SOX2/OCT4 reporter demonstrated that WDR5 inhibitor treatment diminished cells with high reporter activity. Furthermore, WDR5 inhibitor treatment and WDR5 knockdown altered the stem cell state, disrupting CSC in vitro growth and self-renewal, as well as in vivo tumor growth. These findings highlight the role of WDR5 and the WRAD complex in maintaining the CSC state and provide a rationale for therapeutic development of WDR5 inhibitors for GBM and other advanced cancers.

Synthetic lethality is a genetic interaction that results in cell death when two genetic deficiencies co-occur but not when either deficiency occurs alone, which can be co-opted for cancer therapeutics. Pairs of paralog genes are among the most straightforward potential synthetic–lethal interactions by virtue of their redundant functions. Here, we demonstrate a paralog-based synthetic lethality by targeting vaccinia-related kinase 1 (VRK1) in glioblastoma (GBM) deficient of VRK2, which is silenced by promoter methylation in approximately two thirds of GBM. Genetic knockdown of VRK1 in VRK2-null or VRK2-methylated cells resulted in decreased activity of the downstream substrate barrier to autointegration factor (BAF), a regulator of post-mitotic nuclear envelope formation. Reduced BAF activity following VRK1 knockdown caused nuclear lobulation, blebbing, and micronucleation, which subsequently resulted in G2–M arrest and DNA damage. The VRK1–VRK2 synthetic–lethal interaction was dependent on VRK1 kinase activity and was rescued by ectopic expression of VRK2. In VRK2-methylated GBM cell line–derived xenograft and patient-derived xenograft models, knockdown of VRK1 led to robust tumor growth inhibition. These results indicate that inhibiting VRK1 kinase activity could be a viable therapeutic strategy in VRK2-methylated GBM. Significance: A paralog synthetic–lethal interaction between VRK1 and VRK2 sensitizes VRK2-methylated glioblastoma to perturbation of VRK1 kinase activity, supporting VRK1 as a drug discovery target in this disease.

PDF file - 340K, Effects of BUB1B knockdown on in human astrocytes, RasV12 transformed astrocytes, Hela cells and RPE-1 cells

PDF file - 133K, BTIC and NSC growth phenotypes resulting from knockdown of PLK1 and pharmacological inhibition of Aurora B kinase

PDF file - 134K, Ras-Mapk signaling is inappropriately high BTICs and NHA-Ras cells

PDF file - 117K

PDF file - 39K, BUB1B knockdown prevents longer-term expansion BTICs and SSEA1+ BTIC subpopulations

PDF file - 120K, Supplemental data for Figure 5

XLS file - 42K, Candidate genes differentially required for expansion of G166-BTICs

PDF file - 27K, Measurement of mitotic transit times BTIC-G166 cells using live microscopy for mBUB1B allelic complementation experiments (from Figure 5)

PDF file - 74K, BUB1B and other mitotic checkpoint genes are up-regulated in BTIC isolates and Ras- transformed astrocytes

PDF file - 1.3MB, Examination of the effects of BUB1B knockdown on mitotic checkpoint activity

PDF file - 1MB, Additional information showing cancer cells have added requirement on BUB1B for chromosome alignment and KT-MT attachment

Metabolic dysregulation drives tumor initiation in a subset of glioblastomas harboring isocitrate dehydrogenase (IDH) mutations, but metabolic alterations in glioblastomas with wild-type IDH are poorly understood. MYC promotes metabolic reprogramming in cancer, but targeting MYC has proven notoriously challenging. Here, we link metabolic dysregulation in patient-derived brain tumor–initiating cells (BTIC) to a nexus between MYC and mevalonate signaling, which can be inhibited by statin or 6-fluoromevalonate treatment. BTICs preferentially express mevalonate pathway enzymes, which we find regulated by novel MYC-binding sites, validating an additional transcriptional activation role of MYC in cancer metabolism. Targeting mevalonate activity attenuated RAS-ERK–dependent BTIC growth and self-renewal. In turn, mevalonate created a positive feed-forward loop to activate MYC signaling via induction of miR-33b. Collectively, our results argue that MYC mediates its oncogenic effects in part by altering mevalonate metabolism in glioma cells, suggesting a therapeutic strategy in this setting. Cancer Res; 77(18); 4947–60. ©2017 AACR.

Organoid stem cell gradients are stable over time.

Supplementary Data from VRK1 Is a Synthetic–Lethal Target in VRK2-Deficient Glioblastoma

Supplementary Figure from VRK1 Is a Synthetic–Lethal Target in VRK2-Deficient Glioblastoma

Supplementary Figure 1. Induction of differentiation by BMP4 decreased expression of mevalonate synthesis enzymes (HMGCR, PMVK, MVK, MVD, IDI1 and FDPS). Supplementary Figure 2. Gene expression data from TCGA GBM patients revealed a high degree of co-expression of HMGCR, PMVK, MVK, MVD, IDI1 and FDPS. Supplementary Figure 3. Ivy glioblastomama atlas project (Ivy GAP) database analysis showed mevalonate pathway genes are enriched in invasive gliomas Supplementary Figure 4. Simvastatin induces cell cycle arrest in BTICs. Supplementary Figure 5. Simvastatin does not affect DGC proliferation. Supplementary Figure 6. Autophagy analysis showed that Simvastatin does not induce autophagy in BTICs. Supplementary Figure 7. Senescence analysis showed that Simvastatin does not cause senescence. Supplementary Figure 8. Apoptosis/ Necrosis analysis showed Simvastatin induces apoptosis in BTICs. Supplementary Figure 9. Farnesyltransferase inhibitor (FTI-277, 20 μM) and Geranylgeranyltransferase I inhibitor (GGTI-298, 10 μM) treatment decreased BTIC growth and self-renewal. Supplementary Figure 10. Farnesyl pyrophosphate (FPP, 20 μm/ml) can partially rescue the growth defect of BTICs induced by targeting MYC expression. Supplementary Figure 11. MYC and miR-33b expression were decreased when xenografted T3691 BTICs were treated with simvastatin (50mg/kg). Supplementary Figure 12. MiR-33b inhibitor treatment partially rescues the MYC protein levels and cellular growth defect caused by simvastatin.

Many cancers feature cellular hierarchies that are driven by tumor-initiating cancer stem cells (CSC) and rely on complex interactions with the tumor microenvironment. Standard cell culture conditions fail to recapitulate the original tumor architecture or microenvironmental gradients and are not designed to retain the cellular heterogeneity of parental tumors. Here, we describe a three-dimensional culture system that supports the long-term growth and expansion of tumor organoids derived directly from glioblastoma specimens, including patient-derived primary cultures, xenografts, genetically engineered glioma models, or patient samples. Organoids derived from multiple regions of patient tumors retain selective tumorigenic potential. Furthermore, organoids could be established directly from brain metastases not typically amenable to in vitro culture. Once formed, tumor organoids grew for months and displayed regional heterogeneity with a rapidly dividing outer region of SOX2+, OLIG2+, and TLX+ cells surrounding a hypoxic core of primarily non-stem senescent cells and diffuse, quiescent CSCs. Notably, non-stem cells within organoids were sensitive to radiotherapy, whereas adjacent CSCs were radioresistant. Orthotopic transplantation of patient-derived organoids resulted in tumors displaying histologic features, including single-cell invasiveness, that were more representative of the parental tumor compared with those formed from patient-derived sphere cultures. In conclusion, we present a new ex vivo model in which phenotypically diverse stem and non-stem glioblastoma cell populations can be simultaneously cultured to explore new facets of microenvironmental influences and CSC biology. Cancer Res; 76(8); 2465–77. ©2016 AACR.

Extended Organoid Culture Methods

Synthetic lethality is a genetic interaction that results in cell death when two genetic deficiencies co-occur but not when either deficiency occurs alone, which can be co-opted for cancer therapeutics. Pairs of paralog genes are among the most straightforward potential synthetic–lethal interactions by virtue of their redundant functions. Here, we demonstrate a paralog-based synthetic lethality by targeting vaccinia-related kinase 1 (VRK1) in glioblastoma (GBM) deficient of VRK2, which is silenced by promoter methylation in approximately two thirds of GBM. Genetic knockdown of VRK1 in VRK2-null or VRK2-methylated cells resulted in decreased activity of the downstream substrate barrier to autointegration factor (BAF), a regulator of post-mitotic nuclear envelope formation. Reduced BAF activity following VRK1 knockdown caused nuclear lobulation, blebbing, and micronucleation, which subsequently resulted in G2–M arrest and DNA damage. The VRK1–VRK2 synthetic–lethal interaction was dependent on VRK1 kinase activity and was rescued by ectopic expression of VRK2. In VRK2-methylated GBM cell line–derived xenograft and patient-derived xenograft models, knockdown of VRK1 led to robust tumor growth inhibition. These results indicate that inhibiting VRK1 kinase activity could be a viable therapeutic strategy in VRK2-methylated GBM.Significance:A paralog synthetic–lethal interaction between VRK1 and VRK2 sensitizes VRK2-methylated glioblastoma to perturbation of VRK1 kinase activity, supporting VRK1 as a drug discovery target in this disease.

Supplementary Figure Legends for Supplemental Figures 1-5

Comparison of the glioblastoma organoid growth format with tumorsphere culture and with mouse intracranial xenograft models.

Glioblastoma (GBM) is the most commonly occurring primary malignant brain cancer with an extremely poor prognosis. Intra-tumoral cellular and molecular diversity, as well as complex interactions between tumor microenvironments, can make finding effective treatments a challenge. Traditional adherent or sphere culture methods can mask such complexities, whereas three-dimensional organoid culture can recapitulate regional microenvironmental gradients. Organoids are a method of three-dimensional GBM culture that better mimics patient tumor architecture, contains phenotypically diverse cell populations, and can be used for medium-throughput experiments. Although three-dimensional organoid culture is more laborious and time-consuming compared to traditional culture, it offers unique benefits and can serve to bridge the gap between current in vitro and in vivo systems. Organoids have established themselves as invaluable tools in the arsenal of cancer biologists to better understand tumor behavior and mechanisms of resistance, and their applications only continue to grow. Here, details are provided about methods for generating and maintaining GBM organoids. Instructions of how to perform organoid sample embedding and sectioning using both frozen and paraffin-embedding techniques, as well as recommendations for immunohistochemistry and immunofluorescence protocols on organoid sections, and measurement of total organoid cell viability, are all also described.

International audience; Newborn screening for severe combined immunodeficiency (SCID) is now routinely performed in many countries across Europe and around the world. The number of T-cell receptor excision circles (TRECs) reflects T cell levels. TREC quantification is possible using dried blood spot (DBS) samples already collected from newborns to screen for other conditions. This method is very sensitive and highly specific. Data in the literature show that the survival rate for children with SCID is much higher when the disease is detected through early screening, as opposed to a later diagnosis. Newborns diagnosed with SCID may receive the appropriate care quickly, before the onset of serious infectious complications, which raises survival rates, improves quality of life, and limits side effects and treatment costs. At the request of the French Ministry of Health, France's National Authority for Health (Haute Autorité de Santé) is expected to issue recommendations on this topic soon. The nationwide DEPISTREC study, involving 48 maternity units across France, showed that routine SCID screening is feasible and effective. Such screening offers the additional benefit of also diagnosing non-SCID lymphopenia within the infant population.

BackgroundGlioblastoma (GBM) tumor cells modulate expression of iron-associated genes to enhance iron uptake from the surrounding microenvironment, driving proliferation and tumor growth. The homeostatic iron regulator (HFE) gene, encoding the iron sensing HFE protein, is upregulated in GBM and correlates with poor survival outcomes. However, the molecular mechanisms underlying these observations remain unclear. Identification of pathways for targeting iron dependence in GBM tumors is therefore a critical area of investigation.MethodsWe interrogated the impact of cell-intrinsic Hfe expression on proliferation and tumor growth through genetic loss and gain of function approaches in syngeneic mouse glioma models. We determined the expression of iron-associated genes and their relationship with survival in GBM using public datasets and identified differentially expressed pathways in Hfe knockdown cells through Nanostring transcriptional profiling.ResultsLoss of Hfe induced apoptotic cell death in vitro and inhibited tumor growth in vivo while overexpression of Hfe accelerated both proliferation and tumor growth. Analysis of iron gene signatures in Hfe knockdown cells revealed alterations in the expression of several iron-associated genes, suggesting global disruption of intracellular iron homeostasis. Analyzing differentially expressed pathways further identified oxidative stress as the top pathway upregulated with Hfe loss. Enhanced 55Fe uptake and generation of reactive oxygen species (ROS) were found with Hfe knockdown, implicating toxic iron overload resulting in apoptotic cell death.ConclusionsCollectively, these findings identify a novel role for HFE in regulating iron homeostasis in GBM tumors and provide a potential avenue for future therapeutic development.Key PointsHFE is an iron sensor that is upregulated in GBM and negatively impacts survival.HFE overexpression drives proliferation and tumor growth in vivo.Loss of HFE increases production of reactive oxygen species and induces apoptosis, extending survival in vivo.Importance of StudyDysregulation of iron metabolism is an important feature of GBM contributing to tumor growth and negatively impacting survival. The identification of key iron regulators controlling this process is therefore important for therapeutic targeting. We identify HFE as an important regulator of iron homeostasis in GBM and suggest a role for sexual dimorphism in HFE-mediated tumor iron regulation that ultimately results in differential survival outcomes. Our findings demonstrate that HFE drives tumor cell proliferation and survival in GBM and may be a viable target for modulating tumor iron flux and inducing apoptosis in tumor cells.

Tumors contain heterogeneous neoplastic cells and diverse stromal elements that collectively function as dynamic ecosystems, and this complicates predictive modeling ex vivo. LeBlanc et al. utilize single-cell analysis to demonstrate that patient-derived explants replicate tumor cell diversity and transient stromal cell types in patient surgical specimens. This suggests that patient-derived explants can be valuable as tumor models.

Highlights • Glioblastoma organoid cultures preserve diversity of proliferative cell phenotypes. • Heterogeneous 3D cultures recapitulate resistance to clinical GBM therapeutics. • Patient specimens show different behavior depending on 2D vs 3D growth., Background Glioblastoma (GBM) is the most common primary brain tumor with a dismal prognosis. The inherent cellular diversity and interactions within tumor microenvironments represent significant challenges to effective treatment. Traditional culture methods such as adherent or sphere cultures may mask such complexities whereas three-dimensional (3D) organoid culture systems derived from patient cancer stem cells (CSCs) can preserve cellular complexity and microenvironments. The objective of this study was to determine if GBM organoids may offer a platform, complimentary to traditional sphere culture methods, to recapitulate patterns of clinical drug resistance arising from 3D growth. Methods Adult and pediatric surgical specimens were collected and established as organoids. We created organoid microarrays and visualized bulk and spatial differences in cell proliferation using immunohistochemistry (IHC) staining, and cell cycle analysis by flow cytometry paired with 3D regional labeling. We tested the response of CSCs grown in each culture method to temozolomide, ibrutinib, lomustine, ruxolitinib, and radiotherapy. Results GBM organoids showed diverse and spatially distinct proliferative cell niches and include heterogeneous populations of CSCs/non-CSCs (marked by SOX2) and cycling/senescent cells. Organoid cultures display a comparatively blunted response to current standard-of-care therapy (combination temozolomide and radiotherapy) that reflects what is seen in practice. Treatment of organoids with clinically relevant drugs showed general therapeutic resistance with drug- and patient-specific antiproliferative, apoptotic, and senescent effects, differing from those of matched sphere cultures. Conclusions Therapeutic resistance in organoids appears to be driven by altered biological mechanisms rather than physical limitations of therapeutic access. GBM organoids may therefore offer a key technological approach to discover and understand resistance mechanisms of human cancer cells.

Glioblastomas (GBMs) are heterogeneous, treatment-resistant tumors that are driven by populations of cancer stem cells (CSCs). Despite their importance for tumor growth, few molecular mechanisms critical for CSC population maintenance have been exploited for therapeutic development. We developed a spatially resolved loss-of-function screen in GBM patient-derived organoids to identify essential epigenetic regulators in the SOX2-enriched, therapy resistant niche and identified WDR5 as indispensable for this population. WDR5 is a component of the WRAD complex, which promotes SET1-family-mediated Lys4 methylation of histone H3, associated with positive regulation of transcription. In GBM CSC models, WDR5 inhibitors blocked WRAD complex assembly and reduced H3K4 trimethylation and expression of genes involved in CSC-relevant oncogenic pathways. H3K4me3 peaks lost with WDR5 inhibitor treatment occurred disproportionally on POU transcription factor motifs, including the POU5F1(OCT4)::SOX2 motif. We incorporated a SOX2/OCT4 motif driven GFP reporter system into our CSC cell models and found that WDR5 inhibitor treatment diminished reporter activity. Further, WDR5 inhibitor treatment altered the stem cell state, disrupting CSCin vitrogrowth and self-renewal as well asin vivotumor growth. These findings highlight the role of WDR5 and the WRAD complex in maintaining the CSC state and provide a rationale for therapeutic development of WDR5 inhibitors for GBM and other advanced cancers.SignificanceIn this study, we perform an epigenetic-focused functional genomics screen in glioblastoma organoids and identify WDR5 as an essential epigenetic regulator in the SOX2-enriched, therapy resistant cancer stem cell niche.Graphical Abstract

The complexity of glioblastoma is becoming increasingly recognized. Three recent studies used single-cell approaches that integrate cellular states, transcriptional trajectories, and metabolic alterations to uncover multiple dimensions of cellular and molecular heterogeneity and provide a framework for additional functional investigation and therapeutic development.