WDR5 represents a therapeutically exploitable target for cancer stem cells in glioblastoma.

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.

A TOX-ic axis of epigenetic stem cell maintenance and chemoresistance in colon cancer.

Cancer stem cells drive tumor growth and survival via self-renewal and therapeutic resistance, but the upstream mechanisms are not well defined. In this issue of PLOS Biology, a study in colon cancer reveals a new signalling network that links epigenetic regulation to these phenotypes.

Therapeutic targeting of ependymoma as informed by oncogenic enhancer profiling.

Genomic sequencing has driven precision-based oncology therapy; however, the genetic drivers of many malignancies remain unknown or non-targetable, so alternative approaches to the identification of therapeutic leads are necessary. Ependymomas are chemotherapy-resistant brain tumours, which, despite genomic sequencing, lack effective molecular targets. Intracranial ependymomas are segregated on the basis of anatomical location (supratentorial region or posterior fossa) and further divided into distinct molecular subgroups that reflect differences in the age of onset, gender predominance and response to therapy. The most common and aggressive subgroup, posterior fossa ependymoma group A (PF-EPN-A), occurs in young children and appears to lack recurrent somatic mutations. Conversely, posterior fossa ependymoma group B (PF-EPN-B) tumours display frequent large-scale copy number gains and losses but have favourable clinical outcomes. More than 70% of supratentorial ependymomas are defined by highly recurrent gene fusions in the NF-κB subunit gene RELA (ST-EPN-RELA), and a smaller number involve fusion of the gene encoding the transcriptional activator YAP1 (ST-EPN-YAP1). Subependymomas, a distinct histologic variant, can also be found within the supratetorial and posterior fossa compartments, and account for the majority of tumours in the molecular subgroups ST-EPN-SE and PF-EPN-SE. Here we describe mapping of active chromatin landscapes in 42 primary ependymomas in two non-overlapping primary ependymoma cohorts, with the goal of identifying essential super-enhancer-associated genes on which tumour cells depend. Enhancer regions revealed putative oncogenes, molecular targets and pathways; inhibition of these targets with small molecule inhibitors or short hairpin RNA diminished the proliferation of patient-derived neurospheres and increased survival in mouse models of ependymomas. Through profiling of transcriptional enhancers, our study provides a framework for target and drug discovery in other cancers that lack known genetic drivers and are therefore difficult to treat.

Transcription elongation factors represent in vivo cancer dependencies in glioblastoma.

Glioblastoma is a universally lethal cancer with a median survival time of approximately 15 months. Despite substantial efforts to define druggable targets, there are no therapeutic options that notably extend the lifespan of patients with glioblastoma. While previous work has largely focused on in vitro cellular models, here we demonstrate a more physiologically relevant approach to target discovery in glioblastoma. We adapted pooled RNA interference (RNAi) screening technology for use in orthotopic patient-derived xenograft models, creating a high-throughput negative-selection screening platform in a functional in vivo tumour microenvironment. Using this approach, we performed parallel in vivo and in vitro screens and discovered that the chromatin and transcriptional regulators needed for cell survival in vivo are non-overlapping with those required in vitro. We identified transcription pause-release and elongation factors as one set of in vivo-specific cancer dependencies, and determined that these factors are necessary for enhancer-mediated transcriptional adaptations that enable cells to survive the tumour microenvironment. Our lead hit, JMJD6, mediates the upregulation of in vivo stress and stimulus response pathways through enhancer-mediated transcriptional pause-release, promoting cell survival specifically in vivo. Targeting JMJD6 or other identified elongation factors extends survival in orthotopic xenograft mouse models, suggesting that targeting transcription elongation machinery may be an effective therapeutic strategy for glioblastoma. More broadly, this study demonstrates the power of in vivo phenotypic screening to identify new classes of 'cancer dependencies' not identified by previous in vitro approaches, and could supply new opportunities for therapeutic intervention.

Genome-wide RNAi screens in human brain tumor isolates reveal a novel viability requirement for PHF5A.

To identify key regulators of human brain tumor maintenance and initiation, we performed multiple genome-wide RNAi screens in patient-derived glioblastoma multiforme (GBM) stem cells (GSCs). These screens identified the plant homeodomain (PHD)-finger domain protein PHF5A as differentially required for GSC expansion, as compared with untransformed neural stem cells (NSCs) and fibroblasts. Given PHF5A's known involvement in facilitating interactions between the U2 snRNP complex and ATP-dependent helicases, we examined cancer-specific roles in RNA splicing. We found that in GSCs, but not untransformed controls, PHF5A facilitates recognition of exons with unusual C-rich 3' splice sites in thousands of essential genes. PHF5A knockdown in GSCs, but not untransformed NSCs, astrocytes, or fibroblasts, inhibited splicing of these genes, leading to cell cycle arrest and loss of viability. Notably, pharmacologic inhibition of U2 snRNP activity phenocopied PHF5A knockdown in GSCs and also in NSCs or fibroblasts overexpressing MYC. Furthermore, PHF5A inhibition compromised GSC tumor formation in vivo and inhibited growth of established GBM patient-derived xenograft tumors. Our results demonstrate a novel viability requirement for PHF5A to maintain proper exon recognition in brain tumor-initiating cells and may provide new inroads for novel anti-GBM therapeutic strategies.

Luteolin inhibits Musashi1 binding to RNA and disrupts cancer phenotypes in glioblastoma cells.

RNA binding proteins have emerged as critical oncogenic factors and potential targets in cancer therapy. In this study, we evaluated Musashi1 (Msi1) targeting as a strategy to treat glioblastoma (GBM); the most aggressive brain tumor type. Msi1 expression levels are often high in GBMs and other tumor types and correlate with poor clinical outcome. Moreover, Msi1 has been implicated in chemo- and radio-resistance. Msi1 modulates a range of cancer relevant processes and pathways and regulates the expression of stem cell markers and oncogenic factors via mRNA translation/stability. To identify Msi1 inhibitors capable of blocking its RNA binding function, we performed a ~ 25,000 compound fluorescence polarization screen. NMR and LSPR were used to confirm direct interaction between Msi1 and luteolin, the leading compound. Luteolin displayed strong interaction with Msi1 RNA binding domain 1 (RBD1). As a likely consequence of this interaction, we observed via western and luciferase assays that luteolin treatment diminished Msi1 positive impact on the expression of pro-oncogenic target genes. We tested the effect of luteolin treatment on GBM cells and showed that it reduced proliferation, cell viability, colony formation, migration and invasion of U251 and U343 GBM cells. Luteolin also decreased the proliferation of patient-derived glioma initiating cells (GICs) and tumor-organoids but did not affect normal astrocytes. Finally, we demonstrated the value of combined treatments with luteolin and olaparib (PARP inhibitor) or ionizing radiation (IR). Our results show that luteolin functions as an inhibitor of Msi1 and demonstrates its potential use in GBM therapy.

RNA processing as an alternative route to attack glioblastoma.

Genomic analyses have become an important tool to identify new avenues for therapy. This is especially true for cancer types with extremely poor outcomes, since our lack of effective therapies offers no tangible clinical starting point to build upon. The highly malignant brain tumor glioblastoma (GBM) exemplifies such a refractory cancer, with only 15 month average patient survival. Analyses of several hundred GBM samples compiled by the TCGA (The Cancer Genome Atlas) have produced an extensive transcriptomic map, identified prevalent chromosomal alterations, and defined important driver mutations. Unfortunately, clinical trials based on these results have not yet delivered an improvement on outcome. It is, therefore, necessary to characterize other regulatory routes known for playing a role in tumor relapse and response to treatment. Alternative splicing affects more than 90% of the human coding genes and it is an important source for transcript variation and gene regulation. Mutations and alterations in splicing factors are highly prevalent in multiple cancers, demonstrating the potential for splicing to act as a tumor driver. As a result, numerous genes are expressed as cancer-specific splicing isoforms that are functionally distinct from the canonical isoforms found in normal tissue. These include genes that regulate cancer-critical pathways such as apoptosis, DNA repair, cell proliferation, and migration. Splicing defects can even induce genomic instability, a common characteristic of cancer, and a driver of tumor evolution. Importantly, components of the splicing machinery are targetable; multiple drugs can inhibit splicing factors or promote changes in splicing which could be exploited to begin improving clinical outcomes. Here, we review the current literature and present a case for exploring RNA processing as therapeutic route for the treatment of GBM.

EGFR amplification and EGFRvIII predict and participate in TAT-Cx43266-283 antitumor response in preclinical glioblastoma models.

BACKGROUND: Glioblastoma (GBM) commonly displays epidermal growth factor receptor (EGFR) alterations (mainly amplification and EGFRvIII) and TAT-Cx43266-283 is a Src-inhibitory peptide with antitumor properties in preclinical GBM models. Given the link between EGFR and Src, the aim of this study was to explore the role of EGFR in the antitumor effects of TAT-Cx43266-283. METHODS: The effect of TAT-Cx43266-283, temozolomide (TMZ), and erlotinib (EGFR inhibitor) was studied in patient-derived GBM stem cells (GSCs) and murine neural stem cells (NSCs) with and without EGFR alterations, in vitro and in vivo. EGFR alterations were analyzed by western blot and fluorescence in situ hybridization in these cells, and compared with Src activity and survival in GBM samples from The Cancer Genome Atlas. RESULTS: The effect of TAT-Cx43266-283 correlated with EGFR alterations in a set of patient-derived GSCs and was stronger than that exerted by TMZ and erlotinib. In fact, TAT-Cx43266-283 only affected NSCs with EGFR alterations, but not healthy NSCs. EGFR alterations correlated with Src activity and poor survival in GBM patients. Finally, tumors generated from NSCs with EGFR alterations showed a decrease in growth, invasiveness, and vascularization after treatment with TAT-Cx43266-283, which enhanced the survival of immunocompetent mice. CONCLUSIONS: Clinically relevant EGFR alterations are predictors of TAT-Cx43266-283 response and part of its mechanism of action, even in TMZ- and erlotinib-resistant GSCs. TAT-Cx43266-283 targets NSCs with GBM-driver mutations, including EGFR alterations, in an immunocompetent GBM model in vivo, suggesting a promising effect on GBM recurrence. Together, this study represents an important step toward the clinical application of TAT-Cx43266-283.

RNA splicing analysis deciphers developmental hierarchies and reveals therapeutic targets in adult glioma.

Widespread alterations in RNA alternative splicing (AS) have been identified in adult gliomas. However, their regulatory mechanism, biological significance, and therapeutic potential remain largely elusive. Here, using a computational approach with both bulk and single-cell RNA-Seq, we uncover a prognostic AS signature linked with neural developmental hierarchies. Using advanced iPSC glioma models driven by glioma driver mutations, we show that this AS signature could be enhanced by EGFRvIII and inhibited by in situ IDH1 mutation. Functional validations of 2 isoform switching events in CERS5 and MPZL1 show regulations of sphingolipid metabolism and SHP2 signaling, respectively. Analysis of upstream RNA binding proteins reveals PTBP1 as a key regulator of the AS signature where targeting of PTBP1 suppresses tumor growth and promotes the expression of a neuron marker TUJ1 in glioma stem-like cells. Overall, our data highlights the role of AS in affecting glioma malignancy and heterogeneity and its potential as a therapeutic vulnerability for treating adult gliomas.

Homeostatic iron regulatory protein drives glioblastoma growth via tumor cell-intrinsic and sex-specific responses.

Background: Glioblastoma (GBM) displays alterations in iron that drive proliferation and tumor growth. Iron regulation is complex and involves many regulatory mechanisms, including the homeostatic iron regulator (HFE) gene, which encodes the homeostatic iron regulatory protein. While HFE is upregulated in GBM and correlates with poor survival outcomes, the function of HFE in GBM remains unclear. Methods: We interrogated the impact of cell-intrinsic Hfe expression on proliferation and survival of intracranially implanted animals through genetic gain- and loss-of-function approaches in syngeneic mouse glioma models, along with in vivo immune assessments. We also determined the expression of iron-associated genes and their relationship to survival in GBM using public data sets and used transcriptional profiling to identify differentially expressed pathways in control compared to Hfe-knockdown cells. Results: Overexpression of Hfe accelerated GBM proliferation and reduced animal survival, whereas suppression of Hfe induced apoptotic cell death and extended survival, which was more pronounced in females and associated with attenuation of natural killer cells and CD8+ T cell activity. 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. Further analysis of differentially expressed pathways revealed oxidative stress as the top pathway upregulated following Hfe loss. Hfe knockdown indeed resulted in enhanced 55Fe uptake and generation of reactive oxygen species. Conclusions: These findings reveal an essential function for HFE in GBM cell growth and survival, as well as a sex-specific interaction with the immune response.

Tissue factor is a critical regulator of radiation therapy-induced glioblastoma remodeling.

Radiation therapy (RT) provides therapeutic benefits for patients with glioblastoma (GBM), but inevitably induces poorly understood global changes in GBM and its microenvironment (TME) that promote radio-resistance and recurrence. Through a cell surface marker screen, we identified that CD142 (tissue factor or F3) is robustly induced in the senescence-associated ß-galactosidase (SA-ßGal)-positive GBM cells after irradiation. F3 promotes clonal expansion of irradiated SA-ßGal+ GBM cells and orchestrates oncogenic TME remodeling by activating both tumor-autonomous signaling and extrinsic coagulation pathways. Intratumoral F3 signaling induces a mesenchymal-like cell state transition and elevated chemokine secretion. Simultaneously, F3-mediated focal hypercoagulation states lead to activation of tumor-associated macrophages (TAMs) and extracellular matrix (ECM) remodeling. A newly developed F3-targeting agent potently inhibits the aforementioned oncogenic events and impedes tumor relapse in vivo. These findings support F3 as a critical regulator for therapeutic resistance and oncogenic senescence in GBM, opening potential therapeutic avenues.

Patient-derived glioblastoma organoids reflect tumor heterogeneity and treatment sensitivity.

Background: Treatment resistance and tumor relapse are the primary causes of mortality in glioblastoma (GBM), with intratumoral heterogeneity playing a significant role. Patient-derived cancer organoids have emerged as a promising model capable of recapitulating tumor heterogeneity. Our objective was to develop patient-derived GBM organoids (PGO) to investigate treatment response and resistance. Methods: GBM samples were used to generate PGOs and analyzed using whole-exome sequencing (WES) and single-cell karyotype sequencing. PGOs were subjected to temozolomide (TMZ) to assess viability. Bulk RNA sequencing was performed before and after TMZ. Results: WES analysis on individual PGOs cultured for 3 time points (1-3 months) showed a high inter-organoid correlation and retention of genetic variants (range 92.3%-97.7%). Most variants were retained in the PGO compared to the tumor (range 58%-90%) and exhibited similar copy number variations. Single-cell karyotype sequencing demonstrated preservation of genetic heterogeneity. Single-cell multiplex immunofluorescence showed maintenance of cellular states. TMZ treatment of PGOs showed a differential response, which largely corresponded with MGMT promoter methylation. Differentially expressed genes before and after TMZ revealed an upregulation of the JNK kinase pathway. Notably, the combination treatment of a JNK kinase inhibitor and TMZ demonstrated a synergistic effect. Conclusions: Overall, these findings demonstrate the robustness of PGOs in retaining the genetic and phenotypic heterogeneity in culture and the application of measuring clinically relevant drug responses. These data show that PGOs have the potential to be further developed into avatars for personalized adaptive treatment selection and actionable drug target discovery and as a platform to study GBM biology.

Patient-derived explants as tumor models.

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.

Maintaining Human Glioblastoma Cellular Diversity Ex vivo using Three-Dimensional Organoid Culture.

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.

Three-dimensional organoid culture unveils resistance to clinical therapies in adult and pediatric glioblastoma.

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.

Surgical resection of glioblastomas induces pleiotrophin-mediated self-renewal of glioblastoma stem cells in recurrent tumors.

BACKGROUND: Glioblastomas are highly resistant to therapy, and virtually all patients experience tumor recurrence after standard-of-care treatment. Surgical tumor resection is a cornerstone in glioblastoma therapy, but its impact on cellular phenotypes in the local postsurgical microenvironment has yet to be fully elucidated. METHODS: We developed a preclinical orthotopic xenograft tumor resection model in rats with integrated 18F-FET PET/CT imaging. Primary and recurrent tumors were subject to bulk and single-cell RNA sequencing. Differentially expressed genes and pathways were investigated and validated using tissue specimens from the xenograft model, 23 patients with matched primary/recurrent tumors, and a cohort including 190 glioblastoma patients. Functional investigations were performed in vitro with multiple patient-derived cell cultures. RESULTS: Tumor resection induced microglia/macrophage infiltration, angiogenesis as well as proliferation and upregulation of several stem cell-related genes in recurrent tumor cells. Expression changes of selected genes SOX2, POU3F2, OLIG2, and NOTCH1 were validated at the protein level in xenografts and early recurrent patient tumors. Single-cell transcriptomics revealed the presence of distinct phenotypic cell clusters in recurrent tumors which deviated from clusters found in primary tumors. Recurrent tumors expressed elevated levels of pleiotrophin (PTN), secreted by both tumor cells and tumor-associated microglia/macrophages. Mechanistically, PTN could induce tumor cell proliferation, self-renewal, and the stem cell program. In glioblastoma patients, high PTN expression was associated with poor overall survival and identified as an independent prognostic factor. CONCLUSION: Surgical tumor resection is an iatrogenic driver of PTN-mediated self-renewal in glioblastoma tumor cells that promotes therapeutic resistance and tumor recurrence.

VRK1 Is a Synthetic-Lethal Target in VRK2-Deficient Glioblastoma.

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.

High-resolution profiling of homing endonuclease binding and catalytic specificity using yeast surface display.

Experimental analysis and manipulation of protein-DNA interactions pose unique biophysical challenges arising from the structural and chemical homogeneity of DNA polymers. We report the use of yeast surface display for analytical and selection-based applications for the interaction between a LAGLIDADG homing endonuclease and its DNA target. Quantitative flow cytometry using oligonucleotide substrates facilitated a complete profiling of specificity, both for DNA-binding and catalysis, with single base pair resolution. These analyses revealed a comprehensive segregation of binding specificity and affinity to one half of the pseudo-dimeric interaction, while the entire interface contributed specificity at the level of catalysis. A single round of targeted mutagenesis with tandem affinity and catalytic selection steps provided mechanistic insights to the origins of binding and catalytic specificity. These methods represent a dynamic new approach for interrogating specificity in protein-DNA interactions.

Altered lipid metabolism marks glioblastoma stem and non-stem cells in separate tumor niches.

Glioblastoma (GBM) displays marked cellular and metabolic heterogeneity that varies among cellular microenvironments within a tumor. Metabolic targeting has long been advocated as a therapy against many tumors including GBM, but how lipid metabolism is altered to suit different microenvironmental conditions and whether cancer stem cells (CSCs) have altered lipid metabolism are outstanding questions in the field. We interrogated gene expression in separate microenvironments of GBM organoid models that mimic the transition between nutrient-rich and nutrient-poor pseudopalisading/perinecrotic tumor zones using spatial-capture RNA-sequencing. We revealed a striking difference in lipid processing gene expression and total lipid content between diverse cell populations from the same patient, with lipid enrichment in hypoxic organoid cores and also in perinecrotic and pseudopalisading regions of primary patient tumors. This was accompanied by regionally restricted upregulation of hypoxia-inducible lipid droplet-associated (HILPDA) gene expression in organoid cores and pseudopalisading regions of clinical GBM specimens, but not lower-grade brain tumors. CSCs have low lipid droplet accumulation compared to non-CSCs in organoid models and xenograft tumors, and prospectively sorted lipid-low GBM cells are functionally enriched for stem cell activity. Targeted lipidomic analysis of multiple patient-derived models revealed a significant shift in lipid metabolism between GBM CSCs and non-CSCs, suggesting that lipid levels may not be simply a product of the microenvironment but also may be a reflection of cellular state. CSCs had decreased levels of major classes of neutral lipids compared to non-CSCs, but had significantly increased polyunsaturated fatty acid production due to high fatty acid desaturase (FADS1/2) expression which was essential to maintain CSC viability and self-renewal. Our data demonstrate spatially and hierarchically distinct lipid metabolism phenotypes occur clinically in the majority of patients, can be recapitulated in laboratory models, and may represent therapeutic targets for GBM.

Glioblastoma Stem Cells: Driving Resilience through Chaos.

Glioblastoma is an aggressive and heterogeneous tumor in which glioblastoma stem cells (GSCs) are at the apex of an entropic hierarchy and impart devastating therapy resistance. The high entropy of GSCs is driven by a permissive epigenetic landscape and a mutational landscape that revokes crucial cellular checkpoints. The GSC population encompasses a complex array of diverse microstates that are defined and maintained by a wide variety of attractors including the complex tumor ecosystem and therapeutic intervention. Constant dynamic transcriptional fluctuations result in a highly adaptable and heterogeneous entity primed for therapy evasion and survival. Analyzing the transcriptional, epigenetic, and metabolic landscapes of GSC dynamics in the context of a stochastically fluctuating tumor network will provide novel strategies to target resistant populations of GSCs in glioblastoma.