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Background The tumor suppressor TP53 (p53) is frequently mutated, and its downstream effectors inactivated in many cancers, including glioblastoma (GBM). In tumors with wild-type status, p53 function is frequently attenuated by alternate mechanisms including amplification and overexpression of its key negative regulator, MDM2. We investigated the efficacy of the MDM2 inhibitor, BI-907828, in GBM patient-derived brain tumor stem cells (BTSCs) with different amplification statuses of MDM2, in vitro and in orthotopic xenograft models. Methods In vitro growth inhibition and on-target efficacy of BI-907828 were assessed by cell viability, co-immunoprecipitation assays, and western blotting. In vivo efficacy of BI-907828 treatments was assessed with qPCR, immunohistochemistry, and in intracranial xenograft models. Results BI-907828 decreases viability and induces cell death at picomolar concentrations in both MDM2 amplified and normal copy number TP53 wild-type BTSC lines. Restoration of p53 activity, including robust p21 expression and apoptosis induction, was observed in TP53 wild-type but not in TP53 mutant BTSCs. shRNA-mediated knock-down of TP53 in wild-type BTSCs abrogated the effect of BI-907828, confirming the specificity of the inhibitor. Pharmacokinetic-pharmacodynamic studies in orthotopic tumor-bearing severe combined immunodeficiency (SCID) mice demonstrated that a single 50 mg/kg p.o. dose of BI-907828 resulted in strong activation of p53 target genes p21 and MIC1. Long-term weekly or bi-weekly treatment with BI-907828 in orthotopic BTSC xenograft models was well-tolerated and improved survival both as a single-agent and in combination with temozolomide, with dose-dependent efficacy observed in the MDM2 amplified model. Conclusions BI-907828 provides a promising new therapeutic option for patients with TP53 wild-type primary brain tumors.

Cancer cells can metabolize glutamine to replenish TCA cycle intermediates, leading to a dependence on glutaminolysis for cell survival. However, a mechanistic understanding of the role that glutamine metabolism has on the survival of glioblastoma (GBM) brain tumor stem cells (BTSC) has not yet been elucidated. Here, we report that across a panel of 19 GBM BTSC lines, inhibition of glutaminase (GLS) showed a variable response from complete blockade of cell growth to absolute resistance. Surprisingly, BTSC sensitivity to GLS inhibition was a result of reduced intracellular glutamate triggering the amino acid deprivation response (AADR) and not due to the contribution of glutaminolysis to the TCA cycle. Moreover, BTSC sensitivity to GLS inhibition negatively correlated with expression of the astrocytic glutamate transporters EAAT1 and EAAT2. Blocking glutamate transport in BTSCs with high EAAT1/EAAT2 expression rendered cells susceptible to GLS inhibition, triggering the AADR and limiting cell growth. These findings uncover a unique metabolic vulnerability in BTSCs and support the therapeutic targeting of upstream activators and downstream effectors of the AADR pathway in GBM. Moreover, they demonstrate that gene expression patterns reflecting the cellular hierarchy of the tissue of origin can alter the metabolic requirements of the cancer stem cell population.Significance:Glioblastoma brain tumor stem cells with low astrocytic glutamate transporter expression are dependent on GLS to maintain intracellular glutamate to prevent the amino acid deprivation response and cell death.

Oral administration of AZD is compatible with TMZ chemotherapy in vivo.

AZD and TMZ administration do not significantly influence body weights of treated animals.

Purpose: The current standard of care for glioblastoma (GBM) involves a combination of surgery, radiotherapy, and temozolomide chemotherapy, but this regimen fails to achieve long-term tumor control. Resistance to temozolomide is largely mediated by expression of the DNA repair enzyme MGMT; however, emerging evidence suggests that inactivation of MSH6 and other mismatch repair proteins plays an important role in temozolomide resistance. Here, we investigate endogenous MSH6 mutations in GBM, anaplastic oligodendroglial tumor tissue, and corresponding brain tumor–initiating cell lines (BTIC).Experimental Design:MSH6 sequence and MGMT promoter methylation were determined in human tumor samples and BTICs. Sensitivity to temozolomide was evaluated in vitro using BTICs in the absence and presence of O6-benzylguanine to deplete MGMT. The influence of MGMT and MSH6 status on in vivo sensitivity to temozolomide was evaluated using intracranial BTIC xenografts.Results: We identified 11 previously unreported mutations in MSH6 in nine different glioma samples and six paired BTIC lines from adult patients. In addition, MSH6 mutations were documented in three oligodendrogliomas and two treatment-naïve gliomas, both previously unreported findings. These mutations were found to influence the sensitivity of BTICs to temozolomide both in vitro and in vivo, independent of MGMT promoter methylation status.Conclusions: These data demonstrate that endogenous MSH6 mutations may be present before alkylator therapy and occur in at least two histologic subtypes of adult glial neoplasms, with this report serving as the first to note these mutations in oligodendroglioma. These findings broaden our understanding of the clinical response to temozolomide in gliomas. Clin Cancer Res; 20(18); 4894–903. ©2014 AACR.

Molecular and genetic analysis of BTICs identifies subgroups with endogenous mutations in EGFR and PTEN.

Rapamycin partially inhibits growth of most BTICs irrespective of EGFR or PTEN mutational status.

Supplementary Figures S1-S6 with Legends

Pharmacological inhibition of EGFR and PI3K/mTORC1/2 signaling in BTICs.

Purpose: The EGFR and PI3K/mTORC1/2 pathways are frequently altered in glioblastoma (GBM), but pharmacologic targeting of EGFR and PI3K signaling has failed to demonstrate efficacy in clinical trials. Lack of relevant models has rendered it difficult to assess whether targeting these pathways might be effective in molecularly defined subgroups of GBMs. Here, human brain tumor–initiating cell (BTIC) lines with different combinations of endogenous EGFR wild-type, EGFRvIII, and PTEN mutations were used to investigate response to the EGFR inhibitor gefitinib, mTORC1 inhibitor rapamycin, and dual mTORC1/2 inhibitor AZD8055 alone and in combination with temozolomide (TMZ)Experimental Design:In vitro growth inhibition and cell death induced by gefitinib, rapamycin, AZD8055, and TMZ or combinations in human BTICs were assessed by alamarBlue, neurosphere, and Western blotting assays. The in vivo efficacy of AZD8055 was assessed in subcutaneous and intracranial BTIC xenografts. Kaplan–Meier survival studies were performed with AZD8055 and in combination with TMZ.Results: We confirm that gefitinib and rapamycin have modest effects in most BTIC lines, but AZD8055 was highly effective at inhibiting Akt/mTORC2 activity and dramatically reduced the viability of BTICs regardless of their EGFR and PTEN mutational status. Systemic administration of AZD8055 effectively inhibited tumor growth in subcutaneous BTIC xenografts and mTORC1/2 signaling in orthotopic BTIC xenografts. AZD8055 was synergistic with the alkylating agent TMZ and significantly prolonged animal survival.Conclusion: These data suggest that dual inhibition of mTORC1/2 may be of benefit in GBM, including the subset of TMZ-resistant GBMs. Clin Cancer Res; 20(22); 5756–67. ©2014 AACR.

Supplementary Figure 1. Detection of MSH6 somatic mutations by cDNA and genomic DNA sequencing. A-D, cDNA sequencing (A) revealed a heterozygous T1219I mutation that was also confirmed by sequencing the genomic DNA of cell line 119 (B) and the fresh frozen tumor from patient 119 (C). The T1219I mutation was not present in genomic DNA from a blood sample of patient 119 (D) demonstrating it to be of somatic rather than germline origin.

Supplementary tables S1-S4. Supplementary Table S1. Primer sets for qPCR. Supplementary Table S2. Astrocyte gene signature gene list. Supplementary Table S3. OPC gene signature gene list. Supplementary Table S4. Early RGC gene signature gene list.

Gefitinib inhibits growth of EGFRvIII and PTENwt BTICs.

Supplementary Figure Legends from Dual mTORC1/2 Blockade Inhibits Glioblastoma Brain Tumor Initiating Cells In Vitro and In Vivo and Synergizes with Temozolomide to Increase Orthotopic Xenograft Survival

Effect of 2-HG on cell proliferation of IDH1 wild-type cells for the U87 (A) and NHA (B) models.

Comparison of PDH activity in the various models used in this study.

Brain tumour stem cell population in glioblastoma (GBM) display key cancer stem cell characteristics of high self-renewal and drug resistance that are maintained by the coordinated functions of epigenetic and molecular regulators. Yet, specific epigenetic mechanisms that, in collaboration with relevant molecular pathways, help maintain a stem-like state in BTSCs remain poorly understood. Here, we identify HDAC2 as a foremost epigenetic regulator in BTSCs that specifically utilizes the transforming growth factor-β (TGF-β) pathway related proteins, SMAD3-SKI, for remodelling BTSC chromatin accessibility and transcriptional programs to facilitate their stemness and tumorigenic potentials. Our initial drug screening revealed that selective inhibition of HDAC1 and 2 with romidepsin was effective in targeting BTSC viability, cell proliferation and self-renewal in vitro. Using CRISPR-cas9 knockout and shRNA knockdown strategies, we further demonstrated that loss of HDAC2 disrupts an epigenetic and molecular coordination between HDAC2 and SMAD-SKI proteins, which negatively impacts BTSC survival, cell proliferation and self-renewal in vitro and improves median survival in orthotopic xenograft mouse models. Loss of HDAC2 showed reduction in the protein abundance of transcriptional regulator, SMAD3 and negative regulator protein, SKI. However, overexpression of SMAD3 in HDAC2 deficient BTSCs could partially rescues their cell functional deficits. These findings suggest that context-specific epigenetic regulations by HDAC2 and its interaction with the critical transcriptional regulators, SMAD3-SKI, maintains the stemness and growth characteristics of BTSCs. Further HDAC2 overexpression increases cell proliferation and self-renewal abilities in normal neural stem cells (NSCs). These findings thus support the role of HDAC2 as a key epigenetic determinant of stemness in normal NSCs and of cancer stem cell characteristics and tumorigenic potential in BTSCs. Collectively, our data raises the potential that disruption of the coordinated mechanisms regulated by HDAC2-SMAD3-SKI axis may be an effective therapeutic approach for targeting GBM BTSCs.

Glioblastoma (GBM) is a deadly cancer in which cancer stem cells (CSCs) sustain tumor growth and contribute to therapeutic resistance. Protein arginine methyltransferase 5 (PRMT5) has recently emerged as a promising target in GBM. Using two orthogonal-acting inhibitors of PRMT5 (GSK591 or LLY-283), we show that pharmacological inhibition of PRMT5 suppresses the growth of a cohort of 46 patient-derived GBM stem cell cultures, with the proneural subtype showing greater sensitivity. We show that PRMT5 inhibition causes widespread disruption of splicing across the transcriptome, particularly affecting cell cycle gene products. We identify a GBM splicing signature that correlates with the degree of response to PRMT5 inhibition. Importantly, we demonstrate that LLY-283 is brain-penetrant and significantly prolongs the survival of mice with orthotopic patient-derived xenografts. Collectively, our findings provide a rationale for the clinical development of brain penetrant PRMT5 inhibitors as treatment for GBM., The arginine methyltransferase PRMT5 is over-expressed in cancer and has a role in the maintenance of stem cells. Here, the authors show that PRMT5 inhibitors can block the growth of patient derived glioblastoma stem cell cultures in vitro and in vivo, suggesting that PRMT5 inhibition may be a useful therapeutic strategy

Glioblastomas harbor diverse cell populations, including rare glioblastoma stem cells (GSCs) that drive tumorigenesis. To characterize functional diversity within this population, we performed single-cell RNA sequencing on >69,000 GSCs cultured from the tumors of 26 patients. We observed a high degree of inter- and intra-GSC transcriptional heterogeneity that could not be fully explained by DNA somatic alterations. Instead, we found that GSCs mapped along a transcriptional gradient spanning two cellular states reminiscent of normal neural development and inflammatory wound response. Genome-wide CRISPR–Cas9 dropout screens independently recapitulated this observation, with each state characterized by unique essential genes. Further single-cell RNA sequencing of >56,000 malignant cells from primary tumors found that the majority organize along an orthogonal astrocyte maturation gradient yet retain expression of founder GSC transcriptional programs. We propose that glioblastomas grow out of a fundamental GSC-based neural wound response transcriptional program, which is a promising target for new therapy development. Pugh and colleagues use single-cell RNA sequencing, CRISPR screens and functional assays to define a gradient of developmental and wound-response cell states in glioblastoma stem cells, revealing insights into glioblastoma origins and potential therapeutic targets.

Telomere maintenance is a universal hallmark of cancer. Most tumors including low-grade oligodendrogliomas use telomerase reverse transcriptase (TERT) expression for telomere maintenance while astrocytomas use the alternative lengthening of telomeres (ALT) pathway. Although TERT and ALT are hallmarks of tumor proliferation and attractive therapeutic targets, translational methods of imaging TERT and ALT are lacking. Here we show that TERT and ALT are associated with unique 1H-magnetic resonance spectroscopy (MRS)-detectable metabolic signatures in genetically-engineered and patient-derived glioma models and patient biopsies. Importantly, we have leveraged this information to mechanistically validate hyperpolarized [1-13C]-alanine flux to pyruvate as an imaging biomarker of ALT status and hyperpolarized [1-13C]-alanine flux to lactate as an imaging biomarker of TERT status in low-grade gliomas. Collectively, we have identified metabolic biomarkers of TERT and ALT status that provide a way of integrating critical oncogenic information into non-invasive imaging modalities that can improve tumor diagnosis and treatment response monitoring., Telomerase expression and the alternative lengthening of telomeres pathway are hallmarks of cancer. Here, the authors show that, in primary brain tumors, these features are correlated with metabolic signatures detectable by metabolic imaging, suggesting that they can be used to non-invasively monitor telomere maintenance in brain tumours.

Cancer cells can metabolize glutamine to replenish TCA cycle intermediates, leading to a dependence on glutaminolysis for cell survival. However, a mechanistic understanding of the role that glutamine metabolism has on the survival of glioblastoma (GBM) brain tumor stem cells (BTSC) has not yet been elucidated. Here, we report that across a panel of 19 GBM BTSC lines, inhibition of glutaminase (GLS) showed a variable response from complete blockade of cell growth to absolute resistance. Surprisingly, BTSC sensitivity to GLS inhibition was a result of reduced intracellular glutamate triggering the amino acid deprivation response (AADR) and not due to the contribution of glutaminolysis to the TCA cycle. Moreover, BTSC sensitivity to GLS inhibition negatively correlated with expression of the astrocytic glutamate transporters EAAT1 and EAAT2. Blocking glutamate transport in BTSCs with high EAAT1/EAAT2 expression rendered cells susceptible to GLS inhibition, triggering the AADR and limiting cell growth. These findings uncover a unique metabolic vulnerability in BTSCs and support the therapeutic targeting of upstream activators and downstream effectors of the AADR pathway in GBM. Moreover, they demonstrate that gene expression patterns reflecting the cellular hierarchy of the tissue of origin can alter the metabolic requirements of the cancer stem cell population. Significance: Glioblastoma brain tumor stem cells with low astrocytic glutamate transporter expression are dependent on GLS to maintain intracellular glutamate to prevent the amino acid deprivation response and cell death.

The PI3K/AKT/mTOR pathway activation plays a central role in glioblastoma multiforme (GBM) development and progression, and in resistance to anti-cancer therapies. Inhibition of the PI3K pathway has been shown to sensitize cultured glioma cells and tumor xenografts to the effects of temozolomide (TMZ) and radiation. Vistusertib is an oral inhibitor of mTORC1/2 complexes. The primary objective of this Canadian Cancer Trials Group phase I study was to determine the recommended phase II dose (RP2D) of vistusertib in patients with GBM receiving TMZ at first progression following primary treatment. Vistusertib was administered at a starting dose of 100 mg bid 2 days on/5 days off weekly with TMZ 150 mg/m2 daily for 5 days/28-days cycle. Dose escalation was according to a 3 + 3 design. Secondary objectives included assessment of vistusertib safety and toxicity profile, and preliminary efficacy. 15 patients were enrolled in the study (median age 66 (range 51–77), females 8). Vistusertib 125 mg BID in combination with TMZ 150 mg/m2 daily for 5 days was well tolerated. Vistusertib treatment-related adverse events were generally grade 1–2, with the most frequently reported being fatigue, gastrointestinal symptoms, and rash. Of 13 response evaluable patients, 1 patient (8%) had a partial response ongoing at 7.6 months of follow-up, and 5 patients had stable disease (38%) as best response (median duration 9.6 months, range 3.7-not yet reached). Six-month progression-free survival (PFS) rate was 26.6%. Combination of vistusertib with TMZ in GBM patients at first recurrence demonstrated a favorable safety profile at the tested dose levels.

Description, PD-1 on glioblastoma cells promotes brain tumor growth, and therapeutic antibodies cannot suppress its proliferative effects., Brain tumor–initiating cells (BTICs) drive glioblastoma growth through not fully understood mechanisms. Here, we found that about 8% of cells within the human glioblastoma microenvironment coexpress programmed cell death 1 (PD-1) and BTIC marker. Gain- or loss-of-function studies revealed that tumor-intrinsic PD-1 promoted proliferation and self-renewal of BTICs. Phosphorylation of tyrosines within the cytoplasmic tail of PD-1 recruited Src homology 2–containing phosphatase 2 and activated the nuclear factor kB in BTICs. Notably, the tumor-intrinsic promoting effects of PD-1 did not require programmed cell death ligand 1(PD-L1) ligation; thus, the therapeutic antibodies inhibiting PD-1/PD-L1 interaction could not overcome the growth advantage of PD-1 in BTICs. Last, BTIC-intrinsic PD-1 accelerated intracranial tumor growth, and this occurred in mice lacking T and B cells. These findings point to a critical role for PD-1 in BTICs and uncover a nonimmune resistance mechanism of patients with glioblastoma to PD-1– or PD-L1–blocking therapies.

Glioblastoma (GBM) is the most aggressive adult brain tumor, with a median survival of 15 months despite current treatments. We recently established that the epigenetic regulator Disruptor of Telomeric Silencing-1-Like (DOT1L) was essential for the growth of brain tumor stem cells (BTSCs), which are thought to underlie GBM tumor initiation and treatment resistance. Given the previously recognized importance of DOT1L histone methylation for the regulation of the similarly rare and aggressive childhood cancer, Mixed Lineage Leukemia (MLL), we interrogated for common mechanisms in both BTSCs and MLL cells, that overlap due to the epigenetic role of DOT1L. To gain a more detailed perspective of the importance of the DOT1L epigenetic mark in BTSCs, we performed a chemogenomic screen using the DOT1L inhibitor, EPZ-5676. Results from this screen revealed genes from transcriptional and epigenetic complexes required for a therapeutic response to DOT1L inhibition in BTSCs. Gene targeting approaches and growth assays further identified the Polycomb Repressive Complex 2 (PRC2) as a common determining factor for the growth response of both BTSCs and MLL cells following DOT1L inhibition. Furthermore, analysis of the chromatin accessibility changes regulated by DOT1L and PRC2 histone methylation identified both shared and unique epigenetic characteristics of GBM and MLL. The extent to which these shared mechanisms underpin the pathogenic process in these distinct diseases is being further investigated by assessing the divergence in transcriptional responses that emerge from this common epigenetic phenomenon. The findings from this study will provide insight into the importance of shared epigenetic mechanisms that underlie the tumorigenesis of unique cancers affecting the brain and blood.

Glioblastoma (GBM) is a devastating brain cancer with a median overall survival of a mere 12-15 months. Patients receive the standard of care treatment, comprised of maximum surgical resection, ionizing radiation, and temozolomide chemotherapy, however the tumor inevitably recurs. Recurrent GBM is more invasive, difficult to resect, and treatment resistant. GBM brain tumor stem cells (BTSCs), a sub-population of stem-like tumor cells with the ability to self-renew and differentiate into a heterogeneous tumor, are thought to be at the root of recurrent disease. BTSCs isolated from primary and recurrent tumors from the same patient are rare due to fewer resections of recurrent tumors and reduced quality of recurrent tumor samples used to initiate cell lines. As a result, the process of tumor recurrence is vastly understudied in BTSCs. To further understand the process of recurrence and elucidate potential mechanisms of invasion and treatment resistance, we explored global transcriptomics in 40 primary versus 17 recurrent BTSC cultures. Additionally, we profiled changes at the chromatin level in a subset of primary versus recurrent BTSCs using ATAC sequencing. Several pathways were found to be upregulated in recurrent GBM BTSCs, including innate immune signaling at the mRNA and chromatin level, which may mediate the aggressive nature of recurrent BTSCs. Moreover, these signaling changes may be unique to the stem cell population within the tumor, as they are not observed when profiling primary versus recurrent bulk tumors. We are currently targeting these pathways genetically and pharmacologically in primary and recurrent GBM BTSCs established from the same patient and have uncovered mechanisms for treatment resistance, invasion, and recurrence. Our work investigating global signaling changes in primary versus recurrent BTSCs holds promise for uncovering new therapeutic targets or biomarkers for treatment of recurrent GBM.

Background Telomerase reverse transcriptase (TERT) is essential for tumor proliferation, including in low-grade oligodendrogliomas (LGOGs). Since TERT is silenced in normal cells, it is also a therapeutic target. Therefore, noninvasive methods of imaging TERT are needed. Here, we examined the link between TERT expression and metabolism in LGOGs, with the goal of leveraging this information for noninvasive magnetic resonance spectroscopy (MRS)-based metabolic imaging of LGOGs. Methods Immortalized normal human astrocytes with doxycycline-inducible TERT silencing, patient-derived LGOG cells, orthotopic tumors, and LGOG patient biopsies were studied to determine the mechanistic link between TERT expression and glucose metabolism. The ability of hyperpolarized [U-13C, U-2H]-glucose to noninvasively assess TERT expression was tested in live cells and orthotopic tumors. Results TERT expression was associated with elevated glucose flux through the pentose phosphate pathway (PPP), elevated NADPH, which is a major product of the PPP, and elevated glutathione, which is maintained in a reduced state by NADPH. Importantly, hyperpolarized [U-13C, U-2H]-glucose metabolism via the PPP noninvasively reported on TERT expression and response to TERT inhibition in patient-derived LGOG cells and orthotopic tumors. Mechanistically, TERT acted via the sirtuin SIRT2 to upregulate the glucose transporter GLUT1 and the rate-limiting PPP enzyme glucose-6-phosphate dehydrogenase. Conclusions We have, for the first time, leveraged a mechanistic understanding of TERT-associated metabolic reprogramming for noninvasive imaging of LGOGs using hyperpolarized [U-13C, U-2H]-glucose. Our findings provide a novel way of imaging a hallmark of tumor immortality and have the potential to improve diagnosis and treatment response assessment for LGOG patients.

Chromatin accessibility discriminates stem from mature cell populations, enabling the identification of primitive stem-like cells in primary tumors, such as glioblastoma (GBM) where self-renewing cells driving cancer progression and recurrence are prime targets for therapeutic intervention. We show, using single-cell chromatin accessibility, that primary human GBMs harbor a heterogeneous self-renewing population whose diversity is captured in patient-derived glioblastoma stem cells (GSCs). In-depth characterization of chromatin accessibility in GSCs identifies three GSC states: Reactive, Constructive, and Invasive, each governed by uniquely essential transcription factors and present within GBMs in varying proportions. Orthotopic xenografts reveal that GSC states associate with survival, and identify an invasive GSC signature predictive of low patient survival, in line with the higher invasive properties of Invasive state GSCs compared to Reactive and Constructive GSCs as shown by in vitro and in vivo assays. Our chromatin-driven characterization of GSC states improves prognostic precision and identifies dependencies to guide combination therapies.

BackgroundThe EGFR pathway is frequently mutated in glioblastoma (GBM). However, to date, EGFR therapies have not demonstrated efficacy in clinical trials. Poor brain penetration of conventional inhibitors, lack of patient stratification for EGFR status, and mechanisms of resistance are likely responsible for the failure of EGFR-targeted therapy. We aimed to address these elements in a large panel of molecularly diverse patient-derived GBM brain tumor stem cells (BTSCs).MethodsIn vitro growth inhibition and on-target efficacy of afatinib, pacritinib, or a combination were assessed by cell viability, neurosphere formation, cytotoxicity, limiting dilution assays, and western blotting. In vivo efficacy was assessed with mass spectrometry, immunohistochemistry, magnetic resonance imaging, and intracranial xenograft models.ResultsWe show that afatinib and pacritinib decreased BTSC growth and sphere-forming capacity in vitro. Combinations of the 2 drugs were synergistic and abrogated the activation of STAT3 signaling observed upon EGFR inhibition in vitro and in vivo. We further demonstrate that the brain-penetrant EGFR inhibitor, afatinib, improved survival in EGFRvIII mt orthotopic xenograft models. However, upregulation of the oncogenic STAT3 signaling pathway was observed following afatinib treatment. Combined inhibition with 2 clinically relevant drugs, afatinib and pacritinib, synergistically decreased BTSC viability and abrogated this compensatory mechanism of resistance to EGFR inhibition. A significant decrease in tumor burden in vivo was observed with the combinatorial treatment.ConclusionsThese data demonstrate that brain-penetrant combinatorial therapies targeting the EGFR and STAT3 signaling pathways hold therapeutic promise for GBM.

In glioblastoma (GBM), brain tumor stem cells (BTSCs) encompass heterogenous populations of multipotent, self-renewing, and tumorigenic cells, which have been proposed to be at the root of therapeutic resistance and recurrence. While the functional significance of BTSC heterogeneity remains to be fully determined, we previously distinguished relatively quiescent stem-like precursor state from the more aggressive progenitor-like precursor state. In the present study, we hypothesized that progenitor-like BTSCs arise from stem-like precursors through a mesenchymal transition and drive post-treatment recurrence. We first demonstrate that progenitor-like BTSCs display a more mesenchymal transcriptomic profile. Moreover, we show that both mesenchymal GBMs and progenitor-like BTSCs are characterized by over-activated STAT3/EMT pathways and that SLUG is the primary epithelial to mesenchymal transition (EMT) transcription factor directly regulated by STAT3 in BTSCs. SLUG overexpression in BTSCs enhances invasiveness, promotes inflammation, and shortens survival. Importantly, SLUG overexpression in a quiescent stem-like BTSC line enhances tumorigenesis. Finally, we report that recurrence is associated with SLUG-induced transcriptional changes in both BTSCs and GBM patient samples. Collectively, our findings show that a STAT3-driven precursor state transition, mediated by SLUG, may prime BTSCs to initiate more aggressive mesenchymal recurrence. Targeting the STAT3/SLUG pathway may maintain BTSCs in a quiescent stem-like precursor state, delaying recurrence and improving survival in GBM.

Glioblastoma multiforme (GBM) is the most deadly brain tumor, and currently lacks effective treatment options. Brain tumor-initiating cells (BTICs) and orthotopic xenografts are widely used in investigating GBM biology and new therapies for this aggressive disease. However, the genomic characteristics and molecular resemblance of these models to GBM tumors remain undetermined. We used massively parallel sequencing technology to decode the genomes and transcriptomes of BTICs and xenografts and their matched tumors in order to delineate the potential impacts of the distinct growth environments. Using data generated from whole-genome sequencing of 201 samples and RNA sequencing of 118 samples, we show that BTICs and xenografts resemble their parental tumor at the genomic level but differ at the mRNA expression and epigenomic levels, likely due to the different growth environment for each sample type. These findings suggest that a comprehensive genomic understanding of in vitro and in vivo GBM model systems is crucial for interpreting data from drug screens, and can help control for biases introduced by cell-culture conditions and the microenvironment in mouse models. We also found that lack of MGMT expression in pretreated GBM is linked to hypermutation, which in turn contributes to increased genomic heterogeneity and requires new strategies for GBM treatment.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive, incurable cancer with a 20% 1 year survival rate. While standard-of-care therapy can prolong life in a small fraction of cases, PDAC is inherently resistant to current treatments, and novel therapies are urgently required. Histone deacetylase (HDAC) inhibitors are effective in killing pancreatic cancer cells in in vitro PDAC studies, and although there are a few clinical studies investigating combination therapy including HDAC inhibitors, no HDAC drug or combination therapy with an HDAC drug has been approved for the treatment of PDAC. We developed an inhibitor of HDACs, AES-135, that exhibits nanomolar inhibitory activity against HDAC3, HDAC6, and HDAC11 in biochemical assays. In a three-dimensional coculture model, AES-135 kills low-passage patient-derived tumor spheroids selectively over surrounding cancer-associated fibroblasts and has excellent pharmacokinetic properties in vivo. In an orthotopic murine model of pancreatic cancer, AES-135 prolongs survival significantly, therefore representing a candidate for further preclinical testing.

Metabolic imaging of brain tumors using 13C Magnetic Resonance Spectroscopy (MRS) of hyperpolarized [1-13C] pyruvate is a promising neuroimaging strategy which, after a decade of preclinical success in glioblastoma (GBM) models, is now entering clinical trials in multiple centers. Typically, the presence of GBM has been associated with elevated hyperpolarized [1-13C] lactate produced from [1-13C] pyruvate, and response to therapy has been associated with a drop in hyperpolarized [1-13C] lactate. However, to date, lower grade gliomas had not been investigated using this approach. The most prevalent mutation in lower grade gliomas is the isocitrate dehydrogenase 1 (IDH1) mutation, which, in addition to initiating tumor development, also induces metabolic reprogramming. In particular, mutant IDH1 gliomas are associated with low levels of lactate dehydrogenase A (LDHA) and monocarboxylate transporters 1 and 4 (MCT1, MCT4), three proteins involved in pyruvate metabolism to lactate. We therefore investigated the potential of 13C MRS of hyperpolarized [1-13C] pyruvate for detection of mutant IDH1 gliomas and for monitoring of their therapeutic response. We studied patient-derived mutant IDH1 glioma cells that underexpress LDHA, MCT1 and MCT4, and wild-type IDH1 GBM cells that express high levels of these proteins. Mutant IDH1 cells and tumors produced significantly less hyperpolarized [1-13C] lactate compared to GBM, consistent with their metabolic reprogramming. Furthermore, hyperpolarized [1-13C] lactate production was not affected by chemotherapeutic treatment with temozolomide (TMZ) in mutant IDH1 tumors, in contrast to previous reports in GBM. Our results demonstrate the unusual metabolic imaging profile of mutant IDH1 gliomas, which, when combined with other clinically available imaging methods, could be used to detect the presence of the IDH1 mutation in vivo., Graphical abstract Image 1, Highlights • Metabolic imaging of mutant IDH1 gliomas using hyperpolarized 13C MRS is described. • In contrast to GBM, mutant IDH1 gliomas produce no hyperpolarized [1-13C] lactate. • Hyperpolarized [1-13C] lactate is not reduced by treatment in mutant IDH1 tumors. • Mutant IDH1 gliomas present an unusual metabolic imaging profile.

The brain tumor Glioblastoma (GBM) is a virtual death sentence for anyone who is diagnosed, with a median survival time of 12-15 months with standard treatments1 and a 5 year survival rate of under 10% 2. There is a strong body of evidence supporting the existence of stem like cells, termed glioma stem cells (GSCs), that can repopulate the tumor after removal and therapy application3-6. Thus, GSCs present a tantalizing target for potential therapies against GBM. However, studies of GSCs have shown heterogeneity at the level of the transcriptome and drug response5,7, suggesting that there is significant biological variation that translates into differential sensitivity to various drugs. A full characterization of biological heterogeneity may aid in the search for targeted therapies. Here, we profile the transcriptomes of 72 patient derived GSC cultures, and obtain scRNA-seq on 29 cultures from 26 patients (> 69,000 cells) plus 5 GBM tumors (> 14,000 cells). With this data, we find two anticorrelated transcriptional programs in the GSC cultures, one associated with immune or injury response related pathways and the other with neural developmental pathways. We then compare the GSC cultures to patient tumors in the scRNA-seq data, and find that a portion of GBM tumor cells are similar to GSC cultures. We find that a gradient between GSC and astrocyte programs separates cells with stemness properties from those that are not stem-like, and that within stem-like tumor cells and non stem-like tumor cells a gradient between the neural developmental and immune related programs exists as was seen for the cultured GSCs. In GSCs, we also find epigenetic variation in DNA methylation associated with the developmental and immune related programs. Overall these data suggest variation between two biological programs manifests at the level of gene expression and epigenetic regulation in GSCs in tumors. 1. Stupp, R. et al. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N. Engl. J. Med. 352, 987-996 (2005). 2. Brennan, C. W. et al. The somatic genomic landscape of glioblastoma. Cell 155, 462-477 (2013). 3. Singh, S. K. et al. Identification of human brain tumour initiating cells. Nature 432, 396-401 (2004). 4. Chen, J. et al. A restricted cell population propagates glioblastoma growth after chemotherapy. Nature 488, 522-526 (2012). 5. Lan, X. et al. Fate mapping of human glioblastoma reveals an invariant stem cell hierarchy. Nature 549, 227-232 (2017). 6. Patel, A. P. et al. Single-cell RNA-seq highlights intratumoral heterogeneity in primary glioblastoma. Science 344, 1396-1401 (2014). 7. Meyer, M. et al. Single cell-derived clonal analysis of human glioblastoma links functional and genomic heterogeneity. Proc. Natl. Acad. Sci. U. S. A. 112, 851-856 (2015). Citation Format: Owen K. Whitley, Laura M. Richards, Florence Cavalli, Paul Guilhamon, Fiona Coutinho, Michelle Kushida, H. Artee Luchman, Samuel Weiss, Mathieu Lupien, Peter Dirks, Trevor Pugh, Gary Bader. Revelation of shared transcriptional gradients in glioblastoma tumors and cultured glioma stem cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5724.

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

SummarySuccessful glioblastoma (GBM) therapies have remained elusive due to limitations in understanding mechanisms of growth and survival of the tumorigenic population. Using CRISPR-Cas9 approaches in patient-derived GBM stem cells to interrogate function of the coding genome, we identify diverse actionable pathways responsible for growth that reveal the gene-essential circuitry of GBM stemness. In particular, we describe the Sox developmental transcription factor family; H3K79 methylation by DOT1L; and ufmylation stress responsiveness programs as essential for GBM stemness. Additionally, we find mechanisms of temozolomide resistance and sensitivity that could lead to combination strategies with this standard of care treatment. By reaching beyond static genome analysis of bulk tumors, with a genome wide functional approach, we dive deep into a broad range of biological processes to provide new understanding of GBM growth and treatment resistance.SignificanceGlioblastoma (GBM) remains an incurable disease despite an increasingly thorough depth of knowledge of the genomic and epigenomic alterations of bulk tumors. Evidence from multiple approaches support that GBM reflects an aberrant developmental hierarchy, with GBM stem cells (GSCs), fueling tumor growth and invasion. The properties of this tumor subpopulation may also in part explain treatment resistance and disease recurrence. Unfortunately, we still have a limited knowledge of the molecular circuitry of these cells and progress has been slow as we have not been able, until recently, to interrogate function at the genome-wide scale. Here, using parallel genome-wide CRISPR-Cas9 screens, we identify the essential genes for GSC growth. Further, by screening in the presence of low and high dose temozolomide, we identify mechanisms of drug resistance and sensitivity. These functional screens in patient derived cells reveal new aspects of GBM biology and identify a diversity of actionable targets such as genes governing stem cell traits, epigenome regulation and the response to stress stimuli.

The median survival for patients diagnosed with Glioblastoma (GBM) is only 14 months, due to recurrence, despite current treatment options of surgery, radio- and chemo- therapies. Recurrence results from the invasion of GBM cancer cells away from the initial cancerous mass and may be due to brain tumor stem cells (BTSCs) that are resistant to therapy and capable of tumor initiation. Recently, epigenetic-based therapies have become of great interest given that, unlike genomic mutations, epigenetic alterations are potentially reversible. Disrupter of telomeric silencing-1-like (DOT1L) is the only known histone methyltransferase responsible for histone-3-lysine-79 methylation (H3K79me), an epigenetic mark associated with active gene transcription. Previous studies investigating the therapeutic implications of targeting DOT1L in cancer have shown that its inhibition in leukemia results in cancer cell death and, in solid cancers, decreases metastasis. We investigated the role of DOT1L in GBM BTSCs. Long term treatment with the DOT1L inhibitor EPZ-5676 decreased H3K79me2 levels in BTSCs and altered BTSC sphere morphology, sphere initiating frequency, proliferation, migration and invasion. To investigate the mechanism by which DOT1L may regulate GBM tumorigenesis and growth we performed qPCR, flow cytometry, RNA-sequencing and H3K79me2 ChIP-sequencing of BTSC lines, following treatment with EPZ-5676. We found that DOT1L inhibition decreases the expression of GBM stem and progenitor cell genes and increases the expression of genes associated with neuronal and astrocytic differentiation. Furthermore, H3K79me2 ChIP sequencing revealed that DOT1L histone methylation was reduced within the gene body of stem and progenitor cell genes. Flank xenograft studies, of mice treated with EPZ-5676, revealed that DOT1L inhibition promotes neuronal and astrocytic differentiation in tumors. Ongoing pre-clinical studies are assessing the effect of DOT1L inhibition on survival in an orthotopic xenograft model of GBM and investigating rational combinatorial targeting strategies, with a long-term aim of preventing GBM recurrence and improving overall patient survival.

Glioblastoma multiforme (GBM) is the most aggressive adult primary brain tumor. Despite a treatment regimen involving surgical resection, chemotherapy, and radiation, GBM patients have a median survival of approximately 15 months. This poor outcome highlights the need for improved therapeutic approaches for GBM patients. A subpopulation of cells that exhibit stem cell properties, termed brain tumor stem cells (BTSCs), are hypothesized to be the source of recurrence and resistance due to their inherent ability to resist treatment, invade into normal tissue, and re-populate a tumor. We have recently found that a subset of BTSC lines are uniquely dependent on glutamine metabolism for survival and maintenance of their stem cell properties. This dependence on glutamine differs from the more conventional view that cancer cells rely on increased glucose metabolism. Inhibiting glutamine metabolism in the sensitive subset of BTSC lines, by targeting the enzyme glutaminase (GLS) that deaminates glutamine to produce glutamate, results in decreased growth, viability, and self-renewal. We have found that inhibition of GLS decreases intracellular glutamate levels and leads to amino acid depletion. Replenishing the amino acid pool using bovine serum albumin rescues the decrease in BTSC growth following GLS inhibition. Intriguingly, the sensitive subset of BTSC lines have low expression of the glutamate transporter termed excitatory amino acid transporter 2 (EAAT2), limiting their glutamate uptake. We are exploring the relationship between reduced glutamate uptake and glutamine dependence; specifically, by asking whether GLS inhibition-resistant BTSC lines become glutamine-dependent after exposure to glutamate transport inhibitors. Taken together, our findings point to a unique dependence on glutamine metabolism for amino acid synthesis, in a subset of BTSC lines. Our long-term goal is to pursue this glutamine dependence as a novel therapeutic strategy to target the cells at the root of this aggressive and lethal disease.

Background: Glioblastoma (GBM) is a fatal cancer that has eluded major therapeutic advances. Failure to make progress may reflect the absence of a human GBM model that could be used to test compounds for anti-GBM activity. In this respect, the development of brain tumor-initiating cell (BTIC) cultures is a step forward because BTICs appear to capture the molecular diversity of GBM better than traditional glioma cell lines. Here, we perform a comparative genomic and genetic analysis of BTICs and their parent tumors as preliminary evaluation of the BTIC model. Methods: We assessed single nucleotide polymorphisms (SNPs), genome-wide copy number variations (CNVs), gene expression patterns, and molecular subtypes of 11 established BTIC lines and matched parent tumors. Results: Although CNV differences were noted, BTICs retained the major genomic alterations characteristic of GBM. SNP patterns were similar between BTICs and tumors. Importantly, recurring SNP or CNV alterations specific to BTICs were not seen. Comparative gene expression analysis and molecular subtyping revealed differences between BTICs and GBMs. These differences formed the basis of a 63-gene expression signature that distinguished cells from tumors; differentially expressed genes primarily involved metabolic processes. We also derived a set of 73 similarly expressed genes; these genes were not associated with specific biological functions. Conclusions: Although not identical, established BTIC lines preserve the core molecular alterations seen in their parent tumors, as well as the genomic hallmarks of GBM, without acquiring recurring BTIC-specific changes.

Mutant isocitrate dehydrogenase 1 (IDH1) catalyzes the production of 2-hydroxyglutarate but also elicits additional metabolic changes. Levels of both glutamate and pyruvate dehydrogenase (PDH) activity have been shown to be affected in U87 glioblastoma cells or normal human astrocyte (NHA) cells expressing mutant IDH1, as compared with cells expressing wild-type IDH1. In this study, we show how these phenomena are linked through the effects of IDH1 mutation, which also reprograms pyruvate metabolism. Reduced PDH activity in U87 glioblastoma and NHA IDH1 mutant cells was associated with relative increases in PDH inhibitory phosphorylation, expression of pyruvate dehydrogenase kinase-3, and levels of hypoxia inducible factor-1α. PDH activity was monitored in these cells by hyperpolarized 13C-magnetic resonance spectroscopy (13C-MRS), which revealed a reduction in metabolism of hyperpolarized 2-13C-pyruvate to 5-13C-glutamate, relative to cells expressing wild-type IDH1. 13C-MRS also revealed a reduction in glucose flux to glutamate in IDH1 mutant cells. Notably, pharmacological activation of PDH by cell exposure to dichloroacetate (DCA) increased production of hyperpolarized 5-13C-glutamate in IDH1 mutant cells. Furthermore, DCA treatment also abrogated the clonogenic advantage conferred by IDH1 mutation. Using patient-derived mutant IDH1 neurosphere models, we showed that PDH activity was essential for cell proliferation. Taken together, our results established that the IDH1 mutation induces an MRS-detectable reprogramming of pyruvate metabolism, which is essential for cell proliferation and clonogenicity, with immediate therapeutic implications. Cancer Res; 75(15); 2999–3009. ©2015 AACR.

Summary In glioblastoma multiforme (GBM), brain-tumor-initiating cells (BTICs) with cancer stem cell characteristics have been identified and proposed as primordial cells responsible for disease initiation, recurrence, and therapeutic resistance. However, the extent to which individual, patient-derived BTIC lines reflect the heterogeneity of GBM remains poorly understood. Here we applied a stem cell biology approach and compared self-renewal, marker expression, label retention, and asymmetric cell division in 20 BTIC lines. Through cluster analysis, we identified two subgroups of BTIC lines with distinct precursor states, stem- or progenitor-like, predictive of survival after xenograft. Moreover, stem and progenitor transcriptomic signatures were identified, which showed a strong association with the proneural and mesenchymal subtypes, respectively, in the TCGA cohort. This study proposes a different framework for the study and use of BTIC lines and provides precursor biology insights into GBM., Graphical Abstract, Highlights • BTICs are extensively characterized using a stem cell approach • Two groups of BTIC lines are identified: stem-like and progenitor-like • Progenitor-like BTICs lead to strikingly shorter survival in xenografted mice • Stem- and progenitor-like profiles associate with proneural and mesenchymal subtypes, In this article, Weiss and colleagues show that multivariate analysis of otherwise heterogeneous stem cell characteristics allow segregation of BTIC lines into two groups: stem- and progenitor-like. Remarkably, these groups are predictive of survival, with progenitor-like lines leading to strikingly shorter survival in xenografts. Interestingly, stem- and progenitor-like groups also associated with proneural and mesenchymal GBM subtypes, respectively.

Although various lines of evidence suggest that oxidative stress plays a role in human prostate cancer initiation and progression, there is a paucity of direct evidence for its role in tumor initiation. To begin to address this issue, we developed a novel tumorigenesis model by reducing the expression of multiple selenoproteins (SPs) in mouse prostatic epithelium. This was accomplished via the prostate-specific deletion of Trsp, a gene that encodes a transfer RNA (Sec tRNA) required for the insertion of selenocysteine residues into SPs during their translation. By 6 weeks of age, Trsp-deficient mice exhibited widespread prostatic intraepithelial neoplasia lesions in all prostatic lobes, which then progressed to high-grade dysplasia and microinvasive carcinoma by 24 weeks. In contrast to other murine prostate cancer models, Trsp-deficient mice required neither the deletion of a tumor suppressor nor the transgenic introduction of an oncogene for prostatic intraepithelial neoplasia lesion development. In keeping with the antioxidant functions of several SPs, we found increases in lipid peroxidation markers in Trsp-deficient epithelial cells. This novel model of prostate neoplasia provides evidence for the existence of a selenoprotein or selenoproteins capable of acting as a tumor suppressor in the murine prostate.