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Glioblastoma (GBM) is a complex ecosystem that includes a heterogeneous tumor population and the tumor-immune microenvironment (TIME), prominently containing tumor-associated macrophages (TAM) and microglia. Here, we demonstrated that β2-microglobulin (B2M), a subunit of the class I major histocompatibility complex (MHC-I), promotes the maintenance of stem-like neoplastic populations and reprograms the TIME to an anti-inflammatory, tumor-promoting state. B2M activated PI3K/AKT/mTOR signaling by interacting with PIP5K1A in GBM stem cells (GSC) and promoting MYC-induced secretion of transforming growth factor-β1 (TGFβ1). Inhibition of B2M attenuated GSC survival, self-renewal, and tumor growth. B2M-induced TGFβ1 secretion activated paracrine SMAD and PI3K/AKT signaling in TAMs and promoted an M2-like macrophage phenotype. These findings reveal tumor-promoting functions of B2M and suggest that targeting B2M or its downstream axis may provide an effective approach for treating GBM. Significance: β2-microglobulin signaling in glioblastoma cells activates a PI3K/AKT/MYC/TGFβ1 axis that maintains stem cells and induces M2-like macrophage polarization, highlighting potential therapeutic strategies for targeting tumor cells and the immunosuppressive microenvironment in glioblastoma.

Background Accumulating evidence demonstrated that nuclear factor erythroid 2-related factor 2 (NRF2) expression plays a crucial role in the proliferation, invasion and metastasis of hepatocellular carcinoma (HCC). However, research on the effect of NRF2 genetic polymorphism on the development of chronic hepatitis B (CHB), HBV-related liver cirrhosis (LC) and HCC is still missing. Methods A total of 673 individuals were included in the study and classified into four groups: 110 CHB cases, 86 LC cases, 260 HCC cases, and 217 healthy controls. ​The polymerase chain reaction-restriction fragment length polymorphism and DNA sequencing method were used to detect rs6721961 and rs6726395 polymorphisms. Results Patients carrying the T allele in rs6721961 were at a higher risk of HCC than individuals with the G allele compared to CHB patients (OR = 1.561, 95%CI: 1.003–2.430, P = 0.048). The statistically significant differences were also found in the rs6721961 GT genotype (OR = 2.298, 95% CI: 1.282–4.119, P = 0.005) and dominant model (OR = 2.039, 95% CI: 1.184–0.510, P = 0.010). Subgroup analysis also detected a significant association between the rs6721961 T allele and the development of HCC in older subjects (≥ 50 years) (OR = 2.148, 95% CI: 1.208–3.818, P = 0.009). Statistical analysis results indicated that subjects carrying haplotype G-A had a lower risk of HCC (OR = 0.700, 95% CI: 0.508–0.965, P = 0.028). Conclusions For the first time, our findings provide evidence that the NRF2 gene rs6721961 variation is a potential genetic marker of susceptibility to HCC.

Background Glioblastomas (GBMs) display striking dysregulation of metabolism to promote tumor growth. Glioblastoma stem cells (GSCs) adapt to regions of heterogeneous nutrient availability, yet display dependency on de novo cholesterol biosynthesis. The transcription factor Sterol Regulatory Element-Binding Protein 2 (SREBP2) regulates cholesterol biosynthesis enzymes and uptake receptors. Here, we investigate adaptive behavior of GSCs under different cholesterol supplies. Methods In silico analysis of patient tumors demonstrated enrichment of cholesterol synthesis associated with decreased angiogenesis. Comparative gene expression of cholesterol biosynthesis enzymes in paired GBM specimens and GSCs were performed. In vitro and in vivo loss-of-function genetic and pharmacologic assays were conducted to evaluate the effect of SREBP2 on GBM cholesterol biosynthesis, proliferation, and self-renewal. Chromatin immunoprecipitation quantitative real-time PCR was leveraged to map the regulation of SREBP2 to cholesterol biosynthesis enzymes and uptake receptors in GSCs. Results Cholesterol biosynthetic enzymes were expressed at higher levels in GBM tumor cores than in invasive margins. SREBP2 promoted cholesterol biosynthesis in GSCs, especially under starvation, as well as proliferation, self-renewal, and tumor growth. SREBP2 governed the balance between cholesterol biosynthesis and uptake in different nutrient conditions. Conclusions SREBP2 displays context-specific regulation of cholesterol biology based on its availability in the microenvironment with induction of cholesterol biosynthesis in the tumor core and uptake in the margin, informing a novel treatment strategy for GBM.

Glioblastoma (GBM) is the most lethal primary brain cancer characterized by therapeutic resistance, which is promoted by GBM stem cells (GSC). Here, we interrogated gene expression and whole-genome CRISPR/Cas9 screening in a large panel of patient-derived GSCs, differentiated GBM cells (DGC), and neural stem cells (NSC) to identify master regulators of GSC stemness, revealing an essential transcription state with increased RNA polymerase II–mediated transcription. The YY1 and transcriptional CDK9 complex was essential for GSC survival and maintenance in vitro and in vivo. YY1 interacted with CDK9 to regulate transcription elongation in GSCs. Genetic or pharmacologic targeting of the YY1–CDK9 complex elicited RNA m6A modification–dependent interferon responses, reduced regulatory T-cell infiltration, and augmented efficacy of immune checkpoint therapy in GBM. Collectively, these results suggest that YY1–CDK9 transcription elongation complex defines a targetable cell state with active transcription, suppressed interferon responses, and immunotherapy resistance in GBM. Significance: Effective strategies to rewire immunosuppressive microenvironment and enhance immunotherapy response are still lacking in GBM. YY1-driven transcriptional elongation machinery represents a druggable target to activate interferon response and enhance anti–PD-1 response through regulating the m6A modification program, linking epigenetic regulation to immunomodulatory function in GBM. This article is highlighted in the In This Issue feature, p. 275

Background Cancer cells including cancer stem cells exhibit a higher rate of ribosome biogenesis than normal cells to support rapid cell proliferation in tumors. However, the molecular mechanisms governing the preferential ribosome biogenesis in glioma stem cells (GSCs) remain unclear. In this work, we show that the novel INHAT repressor (NIR) promotes ribosomal DNA (rDNA) transcription to support GSC proliferation and glioblastoma (GBM) growth, suggesting that NIR is a potential therapeutic target for GBM. Methods Immunoblotting, immunohistochemical and immunofluorescent analysis were used to determine NIR expression in GSCs and human GBMs. Using shRNA-mediated knockdown, we assessed the role and functional significance of NIR in GSCs and GSC-derived orthotopic GBM xenografts. We further performed mass spectrometry analysis, chromatin immunoprecipitation, and other biochemical assays to define the molecular mechanisms by which NIR promotes GBM progression. Results Our results show that high expression of NIR predicts poor survival in GBM patients. NIR is enriched in the nucleoli of GSCs in human GBMs. Disrupting NIR markedly suppresses GSC proliferation and tumor growth by inhibiting rDNA transcription and pre-ribosomal RNA synthesis. In mechanistic studies, we find that NIR activates rDNA transcription to promote GSC proliferation by cooperating with Nucleolin (NCL) and Nucleophosmin 1 (NPM1), 2 important nucleolar transcription factors. Conclusions Our study uncovers a critical role of NIR-mediated rDNA transcription in the malignant progression of GBM, indicating that targeting this axis may provide a novel therapeutic strategy for GBM.

Glioblastoma is a universally lethal cancer driven by glioblastoma stem cells (GSC). Here, we interrogated N6-methyladenosine (m6A) mRNA modifications in GSCs by methyl RNA immunoprecipitation followed by sequencing and transcriptome analysis, finding transcripts marked by m6A often upregulated compared with normal neural stem cells (NSC). Interrogating m6A regulators, GSCs displayed preferential expression, as well as in vitro and in vivo dependency, of the m6A reader YTHDF2, in contrast to NSCs. Although YTHDF2 has been reported to destabilize mRNAs, YTHDF2 stabilized MYC and VEGFA transcripts in GSCs in an m6A-dependent manner. We identified IGFBP3 as a downstream effector of the YTHDF2–MYC axis in GSCs. The IGF1/IGF1R inhibitor linsitinib preferentially targeted YTHDF2-expressing cells, inhibiting GSC viability without affecting NSCs and impairing in vivo glioblastoma growth. Thus, YTHDF2 links RNA epitranscriptomic modifications and GSC growth, laying the foundation for the YTHDF2–MYC–IGFBP3 axis as a specific and novel therapeutic target in glioblastoma. Significance: Epitranscriptomics promotes cellular heterogeneity in cancer. RNA m6A landscapes of cancer and NSCs identified cell type–specific dependencies and therapeutic vulnerabilities. The m6A reader YTHDF2 stabilized MYC mRNA specifically in cancer stem cells. Given the challenge of targeting MYC, YTHDF2 presents a therapeutic target to perturb MYC signaling in glioblastoma. This article is highlighted in the In This Issue feature, p. 211

Background Glioblastoma (GBM) is a highly lethal malignancy for which neoangiogenesis serves as a defining hallmark. The anti-VEGF antibody, bevacizumab, has been approved for the treatment of recurrent GBM, but resistance is universal. Methods We analyzed expression data of GBM patients treated with bevacizumab to discover potential resistance mechanisms. Patient-derived xenografts (PDXs) and cultures were interrogated for effects of phosphofructokinase-1, muscle isoform (PFKM) loss on tumor cell motility, migration, and invasion through genetic and pharmacologic targeting. Results We identified PFKM as a driver of bevacizumab resistance. PFKM functions dichotomize based on subcellular location: cytosolic PFKM interacted with KIF11, a tubular motor protein, to promote tumor invasion, whereas nuclear PFKM safeguarded genomic stability of tumor cells through interaction with NBS1. Leveraging differential transcriptional profiling, bupivacaine phenocopied genetic targeting of PFKM, and enhanced efficacy of bevacizumab in preclinical GBM models in vivo. Conclusion PFKM drives novel molecular pathways in GBM, offering a translational path to a novel therapeutic paradigm.

Meningiomas are the most common primary intracranial tumor with current classification offering limited therapeutic guidance. Here, we interrogated meningioma enhancer landscapes from 33 tumors to stratify patients based upon prognosis and identify novel meningioma-specific dependencies. Enhancers robustly stratified meningiomas into three biologically distinct groups (adipogenesis/cholesterol, mesodermal, and neural crest) distinguished by distinct hormonal lineage transcriptional regulators. Meningioma landscapes clustered with intrinsic brain tumors and hormonally responsive systemic cancers with meningioma subgroups, reflecting progesterone or androgen hormonal signaling. Enhancer classification identified a subset of tumors with poor prognosis, irrespective of histologic grading. Superenhancer signatures predicted drug dependencies with superior in vitro efficacy to treatment based upon the NF2 genomic profile. Inhibition of DUSP1, a novel and druggable meningioma target, impaired tumor growth in vivo. Collectively, epigenetic landscapes empower meningioma classification and identification of novel therapies. Significance: Enhancer landscapes inform prognostic classification of aggressive meningiomas, identifying tumors at high risk of recurrence, and reveal previously unknown therapeutic targets. Druggable dependencies discovered through epigenetic profiling potentially guide treatment of intractable meningiomas. This article is highlighted in the In This Issue feature, p. 1611

Glioblastoma is the most prevalent primary malignant brain tumor in adults and is characterized by poor prognosis and universal tumor recurrence. Effective glioblastoma treatments are lacking, in part due to somatic mutations and epigenetic reprogramming that alter gene expression and confer drug resistance. To investigate recurrently dysregulated genes in glioblastoma, we interrogated allele-specific expression (ASE), the difference in expression between two alleles of a gene, in glioblastoma stem cells (GSC) derived from 43 patients. A total of 118 genes were found with recurrent ASE preferentially in GSCs compared with normal tissues. These genes were enriched for apoptotic regulators, including schlafen family member 11 (SLFN11). Loss of SLFN11 gene expression was associated with aberrant promoter methylation and conferred resistance to chemotherapy and PARP inhibition. Conversely, low SLFN11 expression rendered GSCs susceptible to the oncolytic flavivirus Zika. This discovery effort based upon ASE revealed novel points of vulnerability in GSCs, suggesting a potential alternative treatment strategy for chemotherapy-resistant glioblastoma. Significance: Assessing allele-specific expression reveals genes with recurrent cis-regulatory changes that are enriched in glioblastoma stem cells, including SLFN11, which modulates chemotherapy resistance and susceptibility to the oncolytic Zika virus.

Glioblastoma ranks among the most aggressive and lethal of all human cancers. Functionally defined glioma stem cells (GSC) contribute to this poor prognosis by driving therapeutic resistance and maintaining cellular heterogeneity. To understand the molecular processes essential for GSC maintenance and tumorigenicity, we interrogated the superenhancer landscapes of primary glioblastoma specimens and in vitro GSCs. GSCs epigenetically upregulated ELOVL2, a key polyunsaturated fatty-acid synthesis enzyme. Targeting ELOVL2 inhibited glioblastoma cell growth and tumor initiation. ELOVL2 depletion altered cellular membrane phospholipid composition, disrupted membrane structural properties, and diminished EGFR signaling through control of fatty-acid elongation. In support of the translational potential of these findings, dual targeting of polyunsaturated fatty-acid synthesis and EGFR signaling had a combinatorial cytotoxic effect on GSCs. Significance: Glioblastoma remains a devastating disease despite extensive characterization. We profiled epigenomic landscapes of glioblastoma to pinpoint cell state–specific dependencies and therapeutic vulnerabilities. GSCs utilize polyunsaturated fatty-acid synthesis to support membrane architecture, inhibition of which impairs EGFR signaling and GSC proliferation. Combinatorial targeting of these networks represents a promising therapeutic strategy. See related commentary by Affronti and Wellen, p. 1161. This article is highlighted in the In This Issue feature, p. 1143

Glioblastoma (GBM) contains self-renewing GBM stem cells (GSC) potentially amenable to immunologic targeting, but chimeric antigen receptor (CAR) T-cell therapy has demonstrated limited clinical responses in GBM. Here, we interrogated molecular determinants of CAR-mediated GBM killing through whole-genome CRISPR screens in both CAR T cells and patient-derived GSCs. Screening of CAR T cells identified dependencies for effector functions, including TLE4 and IKZF2. Targeted knockout of these genes enhanced CAR antitumor efficacy. Bulk and single-cell RNA sequencing of edited CAR T cells revealed transcriptional profiles of superior effector function and inhibited exhaustion responses. Reciprocal screening of GSCs identified genes essential for susceptibility to CAR-mediated killing, including RELA and NPLOC4, the knockout of which altered tumor–immune signaling and increased responsiveness of CAR therapy. Overall, CRISPR screening of CAR T cells and GSCs discovered avenues for enhancing CAR therapeutic efficacy against GBM, with the potential to be extended to other solid tumors. Significance: Reciprocal CRISPR screening identified genes in both CAR T cells and tumor cells regulating the potency of CAR T-cell cytotoxicity, informing molecular targeting strategies to potentiate CAR T-cell antitumor efficacy and elucidate genetic modifications of tumor cells in combination with CAR T cells to advance immuno-oncotherapy. This article is highlighted in the In This Issue feature, p. 995

Purpose/Objective(s) To develop a tutoring program to help physician and physics residents to learn pancreas stereotactic body radiation therapy (SBRT) treatment planning via carefully collected cases and a series of specially designed knowledge models as teaching aid. Such programs are especially important during the Covid pandemic when most traditional hands-on medical teaching had to be moved online and asynchronous. Materials/Methods The pilot tutoring program was composed of 5 teaching cases (1 benchmark case and 4 teaching cases), an interactive knowledge module (IKM) and a performance grading system. The tutoring program started with the benchmark case completed by each trainee independently to benchmark the baseline skill level. This same case was re-tested to evaluate the performance and clinical planning readiness after the trainee completes the tutoring program. 4 teaching cases were included: simple (1), intermediate (2) and complex (1). All 5 cases have simultaneous integrated boost prescription of 25Gy to the elective volume and 33Gy to the gross tumor volume. The IKM included a dose-volume prediction knowledge model for pancreas SBRT and is integrated via a graphic user interface in the treatment planning system. The trainee can seek reference and guidance from the IKM during learning – mimicking the hands-on tutoring of human expert. The grading system summarize the plan quality by weighing key dosimetric endpoints and their relative importance. Grade point average (GPA) was introduced to qualitatively appraise the plan quality into A, B and C (within 3%, 3-10% and > 10% difference of clinical plan score, corresponding to 4, 3, 2 point respectively). 5 trainees with minimum planning experience completed the teaching course. Results Trainees achieved an average of 65.1% of total points (3.6 GPA) with 84 minutes planning time for the benchmark case pre-teaching, and improved to an average of 75.7% (4.2 GPA) using 48 minutes post-teaching. The clinical plan scored 72.7% of total points. All trainees improved their teaching plans’ scores after taking the virtual tutoring program. Post-teaching, all trainees received the GPA of A (clinical quality level) on the benchmark case planning. The total teaching time for each trainee ranged between 5 and 7 hours. Conclusion The tutoring program with knowledge support modules provides encouraging learning outcomes in pancreas SBRT planning for inexperienced planners. This AI-enabled virtual teaching tool could provide valuable addition to the traditional human resource heavy in-person teaching of IMRT and SBRT treatment planning.

Purpose/Objective(s) Applications involving AI and Machine Learning are rapidly improving our ability to plan treatments efficiently and effectively by aiding human planners and physicians during the iterative process of planning. These algorithms involve iterating and training over a large number of cases or situations and can become computationally intractable due to bottlenecks in the dose and fluence calculations. We investigate the effect of kernel truncation and sparsity on the dose and fluence calculation and present a lightweight portable algorithm for implementation into AI/ML applications. Materials/Methods We designed our algorithm around the idea of beamlet and voxel discrimination allowing our dose kernel to truncated and the resulting deposition matrix to be highly sparse. We validated our dose calculation against calculations of varying field-sizes in water and patient anatomy against the AAA algorithm. The fluence optimization was tested on the TG-119 prostate phantom with common dosimetric end-points. Results In a cubic water phantom, the calculation algorithm had errors of less than 1% for all field sizes except for field sizes larger than 30 cm. The errors for field sizes greater than 30 cm were only slightly larger than 1%. In a patient anatomy the dose calculation had a dose difference of less than 0.10 cGy with a dose of 1 cGy at the iso-center. The fluence optimizer produced a plan with the following dose-volume differences from the objectives: PTV .1%, Rectum 2.9%, and Bladder 2.85%. The truncation and sparsity introduced an up to 17x speed up over a full dense calculation. Conclusion The introduction of sparsity and truncation into the dose and fluence calculation produced a sizable speed-up while not diminishing accuracy allowing for the algorithm to be introduced into AI/ML applications which involve many calls to the dose/fluence calculation.

PURPOSE/OBJECTIVE(S) Artificial intelligence (AI) driven tools have been maturing in automating the radiation treatment planning process. To prepare for clinical deployment of these tools, it is essential to understand their robustness in clinical scenarios. This study works to fill the existing gap between research initiated and clinically ready AI tools by investigating a clinical assessment approach based on phantom design and planning complexity simulation. The hypothesis is that AI tool assessment program provides more clinically relevant and comprehensive evaluations beyond typical model validation studies. MATERIALS/METHODS A cylindrical digital phantom was designed in the treatment planning system with an axial diameter of 30 cm and length of 18 cm. The phantom contains key structures involved in pancreas SBRT including the PTV25Gy, PTV33Gy, C-loop, stomach, bowel and liver with their base shape and volume representing the average of 100 clinical SBRT patients. Phantom cases were synthesized to mimic real-life anatomical variations and overlaps through displacement, expansion, and rotation of PTVs and OARs. This study involved a total of 32 simulated cases to test a broad range of planning scenarios. A previously developed deep learning based automatic planning tool was assessed in this study. This AI tool is composed of two deep neural networks (NNs) which predict beam dose and fluence maps sequentially. The goal of treatment planning was to deliver 25 Gy to PTV25 and 33 Gy to PTV33 in 5 fractions via simultaneous integral boost (SIB) while limiting luminal OAR max dose to below 29 Gy. The AI-plan's quality was analyzed against the clinical evaluation criteria, which include PTV V100%, luminal OAR max dose using Dmax and D0.03cc. The passing rate of key clinical criteria were collected to quantify overall robustness. RESULTS For all scenarios, the mean PTV25 V25Gy of the AI plans was 96.7% while mean PTV33 V33 Gy was 82.2%. Large variation (16.3%) in PTV33 V33 Gy was observed due to anatomical variations, i.e., proximity of luminal structures to PTV33. Mean max dose was 28.55, 27.68, and 24.63 Gy for the C-loop, bowel and stomach respectively. Using D0.03cc as a surrogate for max dose, the value was 28.03, 27.12, and 23.84 Gy for the same respective structures. A max dose constraint of 29 Gy was achieved for 81.3% cases for the C-loop and stomach, and 78.1% for the bowel. Using D0.03cc as a surrogate for max dose, the passing rate was 90.6% for the C-loop and 81.3% for both the bowel and stomach. CONCLUSION The results showed promising robustness of AI planning tool for pancreas SBRT, providing important evidence of its readiness for clinical implementation. The established approach could guide the robustness testing and other clinical assessments of AI based treatment planning tools in general, which is an important component for safe clinical implementation.

Purpose/objective(s) With many deep learning (DL) models being developed for clinical applications, it is important to understand their behavior and clinical consequence. This study aims to collect insights of the relationship between fluence map prediction error and its dosimetric impacts in a DL-based AI agent for H&N IMRT planning. Materials/methods An AI agent has been implemented to generate IMRT plans via fluence map prediction, bypassing inverse optimization. While the prostate IMRT plans generated by the agent were comparable to clinical plans in quality, the application into H&N patients exhibited large variations in the plan quality due to higher anatomy complexity. As the DL model's output is fluence maps of an IMRT plan, standard error analyses were focused on the differences between the predicted and ground truth fluence maps, i.e., prediction error. However, the ultimate plan evaluation is based on clinical criteria such as DVHs and dose distributions. Therefore, the AI agent's performance in clinics is subjected to complex and non-intuitive relationships between fluence map prediction error and corresponding dose distribution changes, and warrants thorough investigation. In this study, a series of tests were designed to collect insights of the impact of DL model performance on plan's dosimetric quality. The fluence map prediction error was analyzed for its dosimetric effects using five error decomposition modes:1) ground truth fluence intensity bands in 5 threshold levels, 2) predicted fluence intensity bands in 5 threshold levels, 3) ground truth fluence gradient bands (high and low), 4) Fourier space bands (frequency bands) in 8 threshold levels, and 5) Fourier space circles (below certain frequency) in 8 threshold levels. The DL model was trained with 216 cases and tested with 15 additional cases. PTV and OAR dosimetric metrics were analyzed by Spearman's rank tests (P = 0.05). Results Most PTV-related metrics were significantly correlated with the error components. Among the different decomposition modes, the Fourier space circle radii have large Spearman's coefficients with PTV metrics, suggesting that they were best able to extract error components that reveal plan quality impacts. The low-frequency error within a Fourier space circle of radius = 32 pixels (20% of Fourier space) had the most significant impact on overall plan quality and PTV heterogeneity. Conclusion The fluence map prediction error analysis is critical to evaluate the AI agent performance. Such insight will help with fine-tuning the DL models in architecture design and loss function selection.

Glioblastomas are highly lethal cancers, containing self-renewing glioblastoma stem cells (GSC). Here, we show that GSCs, differentiated glioblastoma cells (DGC), and nonmalignant brain cultures all displayed robust circadian rhythms, yet GSCs alone displayed exquisite dependence on core clock transcription factors, BMAL1 and CLOCK, for optimal cell growth. Downregulation of BMAL1 or CLOCK in GSCs induced cell-cycle arrest and apoptosis. Chromatin immunoprecipitation revealed that BMAL1 preferentially bound metabolic genes and was associated with active chromatin regions in GSCs compared with neural stem cells. Targeting BMAL1 or CLOCK attenuated mitochondrial metabolic function and reduced expression of tricarboxylic acid cycle enzymes. Small-molecule agonists of two independent BMAL1–CLOCK negative regulators, the cryptochromes and REV-ERBs, downregulated stem cell factors and reduced GSC growth. Combination of cryptochrome and REV-ERB agonists induced synergistic antitumor efficacy. Collectively, these findings show that GSCs co-opt circadian regulators beyond canonical circadian circuitry to promote stemness maintenance and metabolism, offering novel therapeutic paradigms. Significance: Cancer stem cells are highly malignant tumor-cell populations. We demonstrate that GSCs selectively depend on circadian regulators, with increased binding of the regulators in active chromatin regions promoting tumor metabolism. Supporting clinical relevance, pharmacologic targeting of circadian networks specifically disrupted cancer stem cell growth and self-renewal. This article is highlighted in the In This Issue feature, p. 1469

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.

// Qi Xie 1 , Qiulian Wu 1 , Stephen C. Mack 1 , Kailin Yang 1, 2 , Leo Kim 1 , Christopher G. Hubert 1 , William A. Flavahan 1, 3, 4, 5 , Chengwei Chu 1, 6 , Shideng Bao 1, 2 , Jeremy N. Rich 1, 2 1 Department of Stem Cell Biology and Regenerative Medicine, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, USA 2 Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, OH 44195, USA 3 Department of Pathology and Center for Cancer Research, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, USA 4 Broad Institute of Harvard and Massachusetts Institute of Technology (MIT), Cambridge, MA 02142, USA 5 Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA 6 Division of Neurosurgery, Department of Surgery, Kaohsiung Medical University Hospital, Kaohsiung 80756, Taiwan Correspondence to: Jeremy N. Rich, e-mail: drjeremyrich@gmail.com Keywords: cancer stem cell, glioblastoma, glioma, tumor initiating cell, CDC20 Received: March 05, 2015 Accepted: April 16, 2015 Published: April 28, 2015 ABSTRACT Glioblastoma is the most prevalent and lethal primary intrinsic brain tumor. Glioblastoma displays hierarchical arrangement with a population of self-renewing and tumorigenic glioma tumor initiating cells (TICs), or cancer stem cells. While non-neoplastic neural stem cells are generally quiescent, glioblastoma TICs are often proliferative with mitotic control offering a potential point of fragility. Here, we interrogate the role of cell-division cycle protein 20 (CDC20), an essential activator of anaphase-promoting complex (APC) E3 ubiquitination ligase, in the maintenance of TICs. By chromatin analysis and immunoblotting, CDC20 was preferentially expressed in TICs relative to matched non-TICs. Targeting CDC20 expression by RNA interference attenuated TIC proliferation, self-renewal and in vivo tumor growth. CDC20 disruption mediated its effects through induction of apoptosis and inhibition of cell cycle progression. CDC20 maintains TICs through degradation of p21 CIP1/WAF1 , a critical negative regulator of TICs. Inhibiting CDC20 stabilized p21 CIP1/WAF1 , resulting in repression of several genes critical to tumor growth and survival, including CDC25C, c-Myc and Survivin. Transcriptional control of CDC20 is mediated by FOXM1, a central transcription factor in TICs. These results suggest CDC20 is a critical regulator of TIC proliferation and survival, linking two key TIC nodes – FOXM1 and p21 CIP1/WAF1 — elucidating a potential point for therapeutic intervention.

Glioma stem-like cells (GSC) are a subpopulation of cells in tumors that are believed to mediate self-renewal and relapse in glioblastoma (GBM), the most deadly form of primary brain cancer. In radiation oncology, hyperthermia is known to radiosensitize cells, and it is reemerging as a treatment option for patients with GBM. In this study, we investigated the mechanisms of hyperthermic radiosensitization in GSCs by a phospho-kinase array that revealed the survival kinase AKT as a critical sensitization determinant. GSCs treated with radiation alone exhibited increased AKT activation, but the addition of hyperthermia before radiotherapy reduced AKT activation and impaired GSC proliferation. Introduction of constitutively active AKT in GSCs compromised hyperthermic radiosensitization. Pharmacologic inhibition of PI3K further enhanced the radiosensitizing effects of hyperthermia. In a preclinical orthotopic transplant model of human GBM, thermoradiotherapy reduced pS6 levels, delayed tumor growth, and extended animal survival. Together, our results offer a preclinical proof-of-concept for further evaluation of combined hyperthermia and radiation for GBM treatment. Cancer Res; 75(8); 1760–9. ©2015 AACR.

Glioblastoma (GBM) is the most lethal brain cancer containing highly tumorigenic glioma stem cells (GSCs). As GSCs contribute to malignant behaviors including tumor angiogenesis, cancer invasion, therapeutic resistance and tumor recurrence, targeting GSCs may significantly improve GBM treatment. Here we demonstrate that the glycogen synthase kinase 3β (GSK3β) promotes the BMX-mediated STAT3 signaling to maintain GSCs and thus targeting GSK3β impairs GBM tumor growth. GSK3β is preferentially activated in GSCs relative to matched non-stem tumor cells (NSTCs) and neural progenitor cells (NPCs). Functional inhibition of GSK3β by its inhibitors or shRNA reduced activating phosphorylation of BMX kinase and STAT3 in GSCs but not in NPCs that express little BMX. Disrupting GSK3β inhibited GSC tumorsphere formation and induced cell death in GSCs but showed little effect on NPCs. Importantly, disruption of GSK3β potently suppressed GSC-driven tumor growth in vivo. Ectopic expression of a constitutively active STAT3 (STAT3-C) rescued the effects caused by GSK3β disruption, supporting that GSK3β mediates through STAT3 activation to maintain the GSC phenotype. Collectively, these data indicate that GSK3β is a potential therapeutic target of GSCs. Thus, pharmacological inhibition of GSK3β may potently disrupt GSCs to overcome therapeutic resistance and improve GBM treatment.

Glioblastoma is a universally lethal cancer with a median survival of approximately 15 months. Despite substantial efforts to define druggable targets, there are no therapeutic options that meaningfully extend glioblastoma patient lifespan. 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 (PDX) 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 necessary 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, supporting targeting the transcription elongation machinery as a 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, which could supply untapped opportunities for therapeutic intervention.

Glioma stem cells (GSCs) represent highly tumorigenic, self-renewing cells that contribute to tumor invasion, angiogenesis, and therapeutic resistance. Using comparative gene expression analysis from validated GSC models, we found two DNA helicases – DNA helicase B (HELB) and ATP-dependent helicase (ATRX) – were preferentially expressed in GSCs relative to matched differentiated glioma cells (DGCs). While ATRX mutation has been associated with diffuse astrocytomas, its role in the glioma cellular hierarchy has not been investigated to date, whereas HELB has not been studied in gliomas. We confirmed the differential expression of HELB and ATRX using RT-PCR and immunoblotting of matched GSC and DGC cultures, as well as neural stem cells, with concordance in active histone marks measured by histone 3 lysine 27 acetyl (H3K27ac). Targeting either HELB or ATRX in GSCs reduced cellular proliferation, induced apoptosis, and blocked self-renewal. In contradistinction, HELB overexpression in DGCs or neural stem cells increased cellular proliferation. Supporting the potential therapeutic value of these targets, the survival of mice bearing intracranial GSCs was significantly extended upon targeting of either HELB or ATRX. Clinical significance of HELB was noted with an association with the mesenchymal transcriptional subgroup of glioblastoma and reduced survival. Collectively, these results suggest that GSCs may preferentially express helicases due to differences in proliferation and endogenous DNA damage relative to the tumor bulk, potentially informing the development of novel therapeutic targeting of therapeutically resistant glioblastoma cells.

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.

Malignant gliomas are complex systems containing a number of factors that drive tumor initiation and progression, including genetic aberrations that lead to extensive cellular heterogeneity within the neoplastic compartment. Mouse models recapitulate these genetic aberrations, but readily observable heterogeneity remains challenging.To interrogate cellular heterogeneity in mouse glioma models, we utilized a replication-competent avian sarcoma-leukosis virus long terminal repeat with splice acceptor/tumor virus A (RCAS-tva) system to generate spontaneous mouse gliomas that contained a Sox2-enhanced green fluorescent protein (EGFP) reporter. Glial fibrillary acidic protein-tva mice were crossed with Sox2-EGFP mice, and tumors were initiated that contained a subpopulation of Sox2-EGFP-high cells enriched for tumor-initiating cell properties such as self-renewal, multilineage differentiation potential, and perivascular localization.Following implantation into recipient mice, Sox2-EGFP-high cells generated tumors containing Sox2-EGFP-high and Sox2-EGFP-low cells. Kinomic analysis of Sox2-EGFP-high cells revealed activation of known glioma signaling pathways that are strongly correlated with patient survival including platelet-derived growth factor receptor beta, phosphoinositide-3 kinase, and vascular endothelial growth factor. Our functional analysis identified active feline sarcoma (Fes) signaling in Sox2-EGFP-high cells. Fes negatively correlated with glioma patient survival and was coexpressed with Sox2-positive cells in glioma xenografts and primary patient-derived tissue.Our RCAS-tva/Sox2-EGFP model will empower closer examination of cellular heterogeneity and will be useful for identifying novel glioma pathways as well as testing preclinical treatment efficacy.

Glioblastoma stem cells (GSCs) reprogram glucose metabolism by hijacking high-affinity glucose uptake to survive in a nutritionally dynamic microenvironment. Here, we trace metabolic aberrations in GSCs to link core genetic mutations in glioblastoma to dependency on de novo pyrimidine synthesis. Targeting the pyrimidine synthetic rate-limiting step enzyme carbamoyl-phosphate synthetase 2, aspartate transcarbamyolase, dihydroorotase (CAD) or the critical downstream enzyme, dihydroorotate dehydrogenase (DHODH) inhibited GSC survival, self-renewal, and in vivo tumor initiation through the depletion of the pyrimidine nucleotide supply in rodent models. Mutations in EGFR or PTEN generated distinct CAD phosphorylation patterns to activate carbon influx through pyrimidine synthesis. Simultaneous abrogation of tumor-specific driver mutations and DHODH activity with clinically approved inhibitors demonstrated sustained inhibition of metabolic activity of pyrimidine synthesis and GSC tumorigenic capacity. Higher expression of pyrimidine synthesis genes portend poor prognosis of glioblastoma patients. Collectively, our results demonstrate a therapeutic approach of precision medicine through targeting the nexus between driver mutations and metabolic reprogramming in cancer stem cells.

// Akio Soeda 1 , Justin Lathia 2 , Brian J. Williams 3 , Qiulian Wu 2 , Joseph Gallagher 2 , Andreas Androutsellis-Theotokis 4 , Amber J. Giles 5 , Chunzhang Yang 5 , Zhengping Zhuang 5 , Mark R. Gilbert 5 , Jeremy N. Rich 2 and Deric M. Park 5 1 Department of Neurosurgery, Gifu University, Gifu, Japan 2 Department of Stem Cell Biology and Regenerative Medicine, Cleveland Clinic Foundation, Cleveland, OH, USA 3 Department of Neurosurgery, University of Louisville, Louisville, KY, USA 4 Division of Stem Cell Biology, Technische Universitat Dresden, Dresden, Germany 5 Neuro-Oncology Branch, National Cancer Institute, NIH, Bethesda, MD, USA Correspondence to: Deric M. Park, email: deric.park@nih.gov Keywords: glioma, cancer stem cell, p38 MAPK, EGFR, quiescence Received: July 10, 2016 Accepted: March 19, 2017 Published: March 31, 2017 ABSTRACT EGFR pathway is upregulated in malignant gliomas, and its downstream signaling is important for self-renewal of glioma cancer stem-like cells (GSC). p38 mitogen-activated protein kinase (MAPK) signaling, a stress-activated signaling cascade with suppressive and permissive effects on tumorigenesis, can promote internalization and ubiquitin ligase mediated degradation of EGFR. In this study, we investigated the role of p38 MAPK signaling on the self-renewal of GSCs with the hypothesis that inhibition may lead to enhanced self-renewal capacity by retention of EGFR. Inhibition of p38 MAPK pathway led to increase in EGFR expression but surprisingly, reduced proliferation. Additional functional evaluation revealed that p38 inhibition was associated with decrease in cell death and maintenance of undifferentiated state. Further probing the effect of p38 inhibition demonstrated attenuation of EGFR downstream signaling activity in spite of prolonged surface expression of the receptor. In vitro observations were confirmed in xenograft in vivo experiments. These data suggest that p38 MAPK control of EGFR signaling activity may alter GSC cell cycle state by regulating quiescence and passage into transit amplifying state.

The nearly universal recurrence of glioblastoma (GBM) is driven in part by a treatment-resistant subpopulation of GBM stem cells (GSC). To identify improved therapeutic possibilities, we combined the EGFR/HER2 inhibitor lapatinib with a novel small molecule, CBL0137, which inhibits FACT (facilitates chromatin transcription), a histone chaperone complex predominantly expressed in undifferentiated cells. Lapatinib and CBL0137 synergistically inhibited the proliferation of patient-derived GBM cells. Compared with non–stem tumor cells (NSTC) enriched from the same specimens, the GSCs were extremely sensitive to CBL0137 monotherapy or FACT knockdown. FACT expression was elevated in GSCs compared with matched NSTCs and decreased in GSCs upon differentiation. Acute exposure of GSCs to CBL0137 increased asymmetric cell division, decreased GSC marker expression, and decreased the capacity of GSCs to form tumor spheres in vitro and to initiate tumors in vivo. Oral administration of CBL0137 to mice bearing orthotopic GBM prolonged their survival. Knockdown of FACT reduced the expression of genes encoding several core stem cell transcription factors (SOX2, OCT4, NANOG, and OLIG2), and FACT occupied the promoters of these genes. FACT expression was elevated in GBM tumors compared with non-neoplastic brain tissues, portended a worse prognosis, and positively correlated with GSC markers and stem cell gene expression signatures. Preferential targeting of GSCs by CBL0137 and synergy with EGFR inhibitors support the development of clinical trials combining these two agents in GBM. Cancer Res; 76(8); 2432–42. ©2016 AACR.

Glioblastomas are lethal cancers characterized by florid angiogenesis promoted in part by glioma stem cells (GSCs). Because hypoxia regulates angiogenesis, we examined hypoxic responses in GSCs. We now demonstrate that hypoxia-inducible factor HIF2alpha and multiple HIF-regulated genes are preferentially expressed in GSCs in comparison to non-stem tumor cells and normal neural progenitors. In tumor specimens, HIF2alpha colocalizes with cancer stem cell markers. Targeting HIFs in GSCs inhibits self-renewal, proliferation, and survival in vitro, and attenuates tumor initiation potential of GSCs in vivo. Analysis of a molecular database reveals that HIF2A expression correlates with poor glioma patient survival. Our results demonstrate that GSCs differentially respond to hypoxia with distinct HIF induction patterns, and HIF2alpha might represent a promising target for antiglioblastoma therapies.

Glioblastoma (GBM) therapeutic resistance is driven in part by tumor-initiating, cancer stem cells (CSCs) which 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 tumor cellular heterogeneity. We demonstrate a 3D culture system that supports long-term growth and expansion of tumor organoids derived directly from GBM specimens.1 Tumor organoids grow for months and display 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, heterogeneous, and rare quiescent CSCs. Unlike patient-matched sphere cultures, orthotopic transplantation of patient-derived organoids resulted in tumors strikingly similar to the parental tumor. Non-stem cells within organoids are sensitive to radiation therapy whereas directly adjacent CSCs are radioresistant, an example of granular therapeutic response previously only demonstrable in whole tumors. We have further developed and leveraged a method of 3D region-specific labeling of live CSCs within organoids to perform high-throughput functional screens for genes essential to glioblastoma survival within distinct niches. These niche-essential genes better reflect glioblastoma-lethal gene targets in vivo.2 Our novel ex-vivo systems preserve phenotypically diverse stem and non-stem cell populations side-by-side amidst varying microenvironmental gradients similar to patient tumors, allowing exploration of new CSC biology. These methods and screening techniques are further applicable to a wide range of disease models that require a complex microenvironment to maintain physiologic relevance. 1Hubert et. al. Cancer Research 2016, 2Miller et. al. Nature 2017

Post-transcriptional regulation of gene expression is well-controlled across normal tissues. Dysregulation of these regulatory events contributes to tumorigenesis and progression of cancer. RNA binding proteins (RBPs), as trans-acting factors, play a pivotal role in post-transcriptional RNA modification. We interrogated the TCGA glioblastoma microarray dataset, comparing glioblastoma to non-tumor with a differential expression level of at least twofold for RBP genes to identify candidate RBP genes that could contribute to glioblastoma malignancy. 36 RBP genes were significantly upregulated and 12 RBP genes were downregulated in glioblastoma. Among these RBP candidates, the Zinc Finger Protein 36, C3H1 Type-Like 2 or ZFP36L2, significantly correlated with poor survival. However, the biological function of ZFP36L2 is poorly understood. Here, we found that ZFP36L2 was highly expressed in the stem-like glioma cells, glioma stem cells (GSCs), compared to normal brain tissues. Targeting ZFP36L2 by short hairpin RNAs inhibited GSC proliferation, decreased self-renewal capacity in vitro, and inhibited tumor formation in vivo. Depletion of ZFP36L2 caused S-phase and G2/M-phase cell cycle arrest. Interrogating the upstream regulation of ZFP36L2 expression, we found CEBP/β overexpressed in GSC as a transcription factor binding to the ZFP36L2 promoter and regulated the ZFP36L2 expression. Downstream, HuR, another RNA binding protein encoded by the ELAVL1 gene, regulated ZFP36L2 at the mRNA level. Collectively, we are elucidating a novel RNA binding protein network essential for GSC maintenance, informing gene-regulatory mechanisms offering new opportunities for developing mechanistic based target therapeutics in neuro-oncology.

Glioblastomas are lethal cancers defined by angiogenesis and pseudopalisading necrosis. Here, we demonstrate that these features are associated with distinct transcriptional programs, with vascular regions showing a proneural profile and hypoxic regions a mesenchymal pattern. As these regions harbor glioma stem cells (GSCs), we investigated the epigenetic regulation of these two niches. Proneural, perivascular GSCs activated EZH2, whereas mesenchymal GSCs in hypoxic regions expressed BMI1 protein, which promoted cellular survival under stress. Using both genetic and pharmacologic inhibition, we found that proneural GSCs are selectively sensitive to EZH2 disruption, whereas mesenchymal GSCs are sensitive to BMI1 inhibition. Given that glioblastomas contain both proneural and mesenchymal GSCs, combined EZH2 and BMI1 targeting proved more effective than either agent alone both in culture and in vivo, suggesting that strategies that simultaneously target multiple epigenetic regulators within glioblastomas may be necessary to overcome resistance to therapies caused by intratumoral heterogeneity.

Circadian rhythms are self-sustainable endogenous 24 hours oscillations, present in every living system and cell, driving daily cycles of biological process, including cell division and metabolism. Circadian rhythm disruption by shift work or core clock genes dysfunction is associated with high risk of tumor development, indicating a putative tumor-suppressive role of circadian rhythm. Using a subpopulation of stem-like cells, coming from human glioblastoma displaying cellular hierarchies, we characterized the role of core clock genes in these stem-like cells. We found that circadian rhythm transcriptional factors Clock and Bmal1 in glioblastoma stem-like cells (GSC) is required for the growth of GSC in vitro and in vivo. Disruption of canonical circadian pathway components produce anti-brain tumor effects, including decreased proliferation, impaired self-renew, increased cell apoptosis and depletion of GSC. Loss of Bmal1 and Clock led to mitochondrial respiration dysregulation and impaired the tricarboxylic acid cycle by regulating genes and enzymes expression in this process. Our finding established a novel role for the core circadian rhythm genes in glioblastoma stem-like cells.

The nearly universal tumor recurrence seen in glioblastoma is driven by a myriad of factors including the invasive nature of the disease and a cellular heterogeneity that includes a more treatment resistant population termed glioblastoma stem cells (GSCs). In an effort to identify novel therapeutic targets for GBM with potential efficacy against the GSC sub-population, we queried the mRNA levels of mitotic regulators between GSCs and the neoplastic cells without stem cell characteristic, or non-GSCs. One target, the mitotic kinesin, Eg5, was consistently elevated in GSCs on both the mRNA and protein levels with increased expression correlated with higher-grade glioma and poor prognosis. Highly specific inhibitors of Eg5 are available and several have moved to phase I and II clinical trials in patients with solid malignancies. We found that GSCs exhibited a greater sensitivity to the Eg5 inhibitor ispinesib compared to non-GSCs from the same tumor and that inhibition of Eg5 compromised the key stem cell characteristics of self-renewal and tumor initiation. Using an orthotopic xenograft model, we demonstrated the first in vivo evidence for efficacy of Eg5 inhibition on glioblastoma tumor progression. An unexpected impact on invasion was observed following Eg5 inhibition resulting in the identification of a hitherto unappreciated role for Eg5 outside of cellular proliferation. We therefore propose that Eg5 inhibitors hold promise in preventing treatment resistance and recurrence in GBM and may provide a novel therapeutic target for GSCs.

Glioblastomas are highly lethal brain tumors containing glioma stem cells (GSCs) that display stem cell-like properties. The molecular link between stemness and malignancy in GSCs is not fully understood. c-Myc appears to be the dominant driver linking self-renewal to neoplasia, but how c-Myc is regulated in GSCs is not defined. We previously demonstrated that the zinc finger and X-linked transcription factor (ZFX) controls c-Myc expression to maintain GSC self-renewal and tumorigenic potential. Down-regulation of ZFX by shRNA reduced c-Myc expression and potently inhibited GSC self-renewal and tumor growth in vivo. Ectopic expression of c-Myc to its endogenous level rescued the phenotype caused by ZFX disruption, demonstrating that ZFX controls c-Myc expression to maintain GSC properties. Here we show a post-translational regulation of c-Myc by deubiquitylation in GSCs. Deubiquitylation of c-Myc in GSCs stabilizes c-Myc protein to promote the maintenance of the GSC phenotype. Blocking c-Myc deubiquitylation disrupted GSC self-renewal and tumorigenic potential. These data indicated that c-Myc is regulated at both transcriptional and post-translational levels in GSCs and c-Myc is a dominant driver linking self-renewal to malignancy.

BACKGROUND: Glioblastoma (GBM) is the most lethal type of primary brain tumor with florid angiogenesis. GBMs display striking cellular hierarchies containing self-renewing glioma stem cells (GSCs) that are highly tumorigenic. GSCs not only produce elevated levels of VEGF to promote tumor angiogenesis but also contribute to cellular compartments of tumor vessels. METHODS: 1. In vivo cell lineage tracing with constitutive and Desmin promoter-driven GFP or RFP fluorescent reporters. 2. Selective elimination of GSC-derived pericytes with Desmin promoter-driven HsvTK. 3. GSC-derived GBM xenograft models. RESULTS: Cell lineage tracing with constitutive and lineage specific fluorescent reporters demonstrated that GSCs generate the majority of vascular pericytes in vivo. Selective elimination of GSC-derived pericytes disrupted tumor vessels and potently suppressed GSC tumor growth. Thus, GSCs can function as pericyte progenitors and contribute to vasculature formation and maintenance in GBMs. CONCLUSIONS: The ability of GSCs to generate the majority of pericytes allows active tumor angiogenesis to promote rapid tumor growth in GBMs. GSC-derived pericytes also play a critical role in the resistance to the anti-angiogenic therapy targeting endothelial cells only, suggesting that selective targeting of GSC-derived pericytes may significantly improve the efficacy and specificity of the anti-angiogenic treatment. SECONDARY CATEGORY: n/a.

Glioblastomas display cellular hierarchies with self-renewing tumor-initiating cells (TIC), also known as cancer stem cells, at the apex. Although the TIC hypothesis remains controversial and the functional assays to define the TIC phenotype are evolving, we and others have shown that TICs may contribute to therapeutic resistance, tumor spread, and angiogenesis. The identification of TICs has been informed by the use of markers characterized in normal stem cells, but this approach has an inherent limitation to selectively identify TICs. To develop reagents that enrich TICs but not matched non-TICs or tissue-specific stem cells, we adopted Cell-Systematic Evolution of Ligands by Exponential Enrichment (Cell-SELEX) to identify glioblastoma TIC-specific nucleic acid probes—aptamers—that specifically bind TICs. In this study, using Cell-SELEX with positive selection for TICs and negative selection for non-TICs and human neural progenitor cells, we identified TIC aptamers that specifically bind to TICs with excellent dissociation constants (Kd). These aptamers select and internalize into glioblastoma cells that self-renew, proliferate, and initiate tumors. As aptamers can be modified to deliver payloads, aptamers may represent novel agents that could selectively target or facilitate imaging of TICs. Cancer Res; 73(15); 4923–36. ©2013 AACR.

Introduction: Different cancer cell compartments often communicate through soluble factors to facilitate tumor growth. Glioma stem cells (GSCs) are a subset of tumor cells that resist standard therapy to contribute to disease progression. How GSCs employ a distinct secretory program to communicate with and nurture each other over the non-stem tumor cell (NSTC) population is not well defined. Methods/Materials: GSCs were enriched from patient specimens and functionally validated. We used a candidate approach to screen for secreted factors that were preferentially used by GSCs but not NSTCs. Expression was assessed by Western blot, and confirmed by immunohistochemistry and immunofluorescence in vitro and in human and mouse GBM tissues. Ligand-receptor interactions were confirmed by co-immunoprecipitation. Rac activity was assessed by Rac-GTP pulldown. Co-culture epistasis experiments were performed using knockdown and recombinant protein approaches. Glioma stem cell marker analysis (CD133, Sox2, Olig2), cell viability (ATP-dependent assays), apoptosis (TUNEL, PI/AnnexinV, cleaved caspase 3/7), self-renewal (tumorsphere formation and limiting dilution assay) and invasion (transwell migration assay) were performed. GSCs were separated in PlexinD1 hi and lo populations by FACS. Radiation (3 Gy) was delivered by Cs-137 irradiator. GSCs were used to establish orthotopic xenograft models of glioblastoma and tumor growth and animal survival were assessed. Results: We have found that GSCs preferentially secrete Sema3C and coordinately express PlexinA2/D1 receptors to activate Rac1/NF-κB signaling in an autocrine and paracrine loop to promote their own survival. Importantly, Sema3C is not expressed in neural progenitor cells (NPCs) or NSTCs. Disruption of Sema3C induced apoptosis of GSCs, but not NPCs or NSTCs, and suppressed tumor growth in orthotopic models of glioblastoma. Introduction of activated Rac1 rescued the Sema3C knockdown phenotype in vivo. Furthermore, GSCs that expressed high levels of Plexin receptors were more resistant to radiation, and Sema3C and Plexin receptors were upregulated after irradiation. Together, these data support that Sema3C signaling contributes to radiation resistance. Conclusions: Our study supports targeting Sema3C to break this GSC-specific autocrine/paracrine loop to improve glioblastoma treatment, potentially with a high therapeutic index. Citation Format: Jianghong Man, Jocelyn Shoemake, Qiulian Wu, Jennifer Yu. Sema3C promotes glioma stem cell survival and radioresistance. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2525.

Glioblastoma (GBM), the most prevalent type of primary intrinsic brain cancer in adults, remains universally fatal despite maximal therapy, including radiotherapy and chemotherapy. Cytotoxic therapy generates double stranded DNA breaks (DSBs), most commonly repaired by homologous recombination (HR). We hypothesized that cancer cells coopt meiotic repair machinery as DSBs are generated during meiosis and repaired by molecular complexes distinct from genotoxic responses in somatic tissues. Indeed, we found that gliomas express meiotic repair genes and their expression informed poor prognosis. We interrogated the function of Disrupted Meiotic cDNA1 (DMC1), a homolog of RADiation sensitive51 (RAD51), the primary recombinase used in mitotic cells to search and recombine with the homologous DNA template. DMC1, whose only known function is as an HR recombinase, was expressed by GBM cells and induced by radiation. While targeting DMC1 in non-neoplastic cells minimally altered cell growth, DMC1 depletion in GBM cells decreased proliferation, induced activation of Checkpoint kinase 1 (CHK1) and expression of p21CIP1/WAF1, and increased replication protein A (RPA) foci, suggesting increased replication stress. Combining loss of DMC1 with ionizing radiation (IR) inhibited activation of DNA damage responses and increased radiosensitivity. Furthermore, loss of DMC1 reduced tumor growth and prolonged survival in vivo. Our results suggest that cancers coopt meiotic genes to augment survival under genotoxic stress, offering molecular targets with high therapeutic indices. Citation Format: Maricruz Rivera, Qiulian Wu, Petra Hamerlik, Anita Hjelmeland, Shideng Bao, Jeremy Rich. Acquisition of meiotic DNA repair regulators maintain genome stability in glioblastoma. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr B44.

SummaryGlioblastomas display cellular hierarchies containing tumor-propagating glioblastoma stem cells (GSCs). STAT3 is a critical signaling node in GSC maintenance but molecular mechanisms underlying STAT3 activation in GSCs are poorly defined. Here we demonstrate that the bone marrow X-linked (BMX) nonreceptor tyrosine kinase activates STAT3 signaling to maintain self-renewal and tumorigenic potential of GSCs. BMX is differentially expressed in GSCs relative to nonstem cancer cells and neural progenitors. BMX knockdown potently inhibited STAT3 activation, expression of GSC transcription factors, and growth of GSC-derived intracranial tumors. Constitutively active STAT3 rescued the effects of BMX downregulation, supporting that BMX signals through STAT3 in GSCs. These data demonstrate that BMX represents a GSC therapeutic target and reinforces the importance of STAT3 signaling in stem-like cancer phenotypes.

Malignant gliomas are lethal cancers that display striking cellular heterogeneity. A highly tumorigenic glioma tumor subpopulation, termed cancer stem cells or tumor-initiating cells, promotes therapeutic resistance and tumor angiogenesis. Therefore, targeting cancer stem cells may improve patient survival. We interrogated the role of a neuronal cell adhesion molecule, L1CAM, in glioma stem cells as L1CAM regulates brain development and is expressed in gliomas. L1CAM+ and CD133+ cells cosegregated in gliomas, and levels of L1CAM were higher in CD133+ glioma cells than normal neural progenitors. Targeting L1CAM using lentiviral-mediated short hairpin RNA (shRNA) interference in CD133+ glioma cells potently disrupted neurosphere formation, induced apoptosis, and inhibited growth specifically in glioma stem cells. We identified a novel mechanism for L1CAM regulation of cell survival as L1CAM knockdown decreased expression of the basic helix-loop-helix transcription factor Olig2 and up-regulated the p21WAF1/CIP1 tumor suppressor in CD133+ glioma cells. To determine if targeting L1CAM was sufficient to reduce glioma stem cell tumor growth in vivo, we targeted L1CAM in glioma cells before injection into immunocompromised mice or directly in established tumors. In each glioma xenograft model, shRNA targeting of L1CAM expression in vivo suppressed tumor growth and increased the survival of tumor-bearing animals. Together, these data show that L1CAM is required for maintaining the growth and survival of CD133+ glioma cells both in vitro and in vivo, and L1CAM may represent a cancer stem cell–specific therapeutic target for improving the treatment of malignant gliomas and other brain tumors. [Cancer Res 2008;68(15):6043–8]

Cellular and molecular regulation of the immune system is exquisitely controlled in the brain and disrupted in neoplasia. Glioblastoma (GBM) has an immunosuppressive phenotype, and despite the accumulation of immune cells in the tumor microenvironment, tumor growth persists. Myeloid derived suppressor cells (MDSCs) are a class of immature immune cells that mitigate cytotoxic T cell responses and promote immunosuppression in a variety of cancers. MDSCs are increased in the peripheral blood of GBM patients, but their role in the GBM microenvironment is undefined. We observed co-localization between arginase (Arg1) producing immunosuppressive MDSCs and cancer stem cells (CSCs) in GBM patient specimens and xenografts, leading to the hypothesis that CSCs stimulate MDSCs to promote immunosuppression. Intracranial injections of CSCs into mice led to GBM formation, expansion of the Arg1-producing MDSCs within the marrow of GBM-bearing mice, and increased levels of MDSCs within the GBM microenvironment. CSC conditioned media increased Arg1 production of murine marrow MDSCs which were then able to modulate cytotoxic T cell responses in co-culture models. Screening of conditioned media revealed cytokines secreted selectively by CSCs that activated MDSCs suppression. Removal of CSC-secreted factors decreased MDSCs in the GBM microenvironment, increased cytotoxic T cell infiltration, and had a pronounced survival benefit. Peripheral targeting of MDSCs through a sub-therapeutic dose of 5-fluorouracil (5-FU) in an immunocompetent syngeneic mouse model led to a similar phenotype to CSC-secreted factor removal in the GBM microenvironment, and an even greater survival benefit. Taken together, our results suggest a critical role of MDSC-mediated immunosuppression in GBM, a novel paradigm of immunomodulation by CSCs, and demonstrate that peripheral removal of MDSCs leads to increased T cell infiltration into GBM, and consequently suggest a novel chemotherapeutic strategy that is not generally toxic to bone marrow and that is not dependent on agents crossing the blood brain barrier.