Male Glioblastoma Research Articles

EPCO-47. SEX-BIASED BRD4 ACTIVITY DRIVES TUMORIGENESIS IN GLIOBLASTOMA

Abstract Consistent sex differences in incidence and outcome have been reported in cancers including brain tumors. Glioblastoma (GBM), the most common and aggressive primary brain tumor, occurs with higher incidence and shorter survival in males compared to females. Brd4 is essential for regulating transcriptome-wide gene expression and specifying cell identity. Using an isogenic murine GBM model, we have reported correlated transcriptome-wide sex differences in gene expression, Brd4-bound enhancer usage, and Brd4 localization to oncogenic and tumor suppressor transcription factors binding sites in male and female GBM cells, respectively. Sex-biased gene expression patterns were also evident in human glioblastoma. Inhibiting Brd4 function abrogates sex differences in these transcriptional states and concomitant differences in tumorigenic phenotype. Thus, sex-biased Brd4 activity drives sex differences in GBM rendering males and females differentially sensitive to BET inhibitors. These data indicate that Brd4 can exhibit dual functions as an oncogene or tumor suppressor, dependent upon cellular sex. To better understand how to globally target Brd4, we interrogated the role played by master transcriptional Brd4 regulators in coordinating this dual phenotype in GBM. Motif-based analysis revealed that Brd4 co-localized with oncogenes such as Bach1, and Klf5 at male-biased enhancers whereas female-biased enhancers were co-occupied with tumor suppressors, such as Smad4 and p53. Knockdown of Bach1 decreased clonogenic frequency in males and females. Conversely, knockdown of Smad4 increased proliferation and clonogenicity in males, further inducing the male tumorigenic phenotype, while no change was observed in females. These results indicate that Smad4 might be necessary but not sufficient to provide females with resistance to transformation. Ongoing epitranscriptomics investigating Brd4 re-localization and enhancer usage in knockdown cells will increase our understanding of Brd4 mechanism in GBM. Targeting sex-biased Brd4 function will identify novel combinatorial sex-specific therapeutics for brain tumors and could have immediate impact on the clinical treatment of GBM patients.

EPCO-49. SEX-SPECIFIC CHROMATIN REMODELING DRIVES TUMORIGENESIS IN GLIOBLASTOMA

Abstract Glioblastoma (GBM) is the most prevalent primary malignant brain cancer and has higher incidence and lower survival in males compared to females. The SWI/SNF chromatin remodeling complexes, including canonical BAF (cBAF), polybromo-associated BAF (PBAF), and non-canonical BAF (ncBAF), play a critical role in multiple cancers including gliomas. However, their role in gliomagenesis and progression remains largely unknown. We analyzed 18 human glioma specimens for BAF complex mRNA expression by CNS WHO grade and used these data to guide CRISPRi knockdown of BAF components in syngeneic murine male and female GBM astrocytes. BAF complex knockdown revealed marked sex-specific differences in proliferation. Specifically, Smarce1 or Smarcb1 knockdown (cBAF & PBAF) resulted in increased growth in male astrocytes but decreased growth in females. Brd7 (PBAF) knockdown enhanced growth in both sexes, while Brd9 (ncBAF) had minimal impact. We utilized extreme limiting dilution analysis (ELDA) to assess the clonogenic potential of GBM cells with depleted BAF components. Notably, cells with BRD7 knockdown displayed enhanced sphere-forming capacity in both male and female cells. Male cells with Smarce1or Smarcb1 knockdown showed increased clonogenic frequency, whereas females with Smarce1 knockdown had a marked reduction in sphere formation. Brd9 knockdown did not affect the sphere-forming capacity in male or female cells, in part due to compensatory plasticity of an alternate BAF complex. Epitranscriptomic analysis is ongoing and demonstrates sex-specific differences in chromatin accessibility and downstream gene expression programs based on BAF complex knockdown. Immunoprecipitation coupled with mass spectrometry was performed to elucidate the structure and assembly of the BAF complex in GBM cells. Understanding sex-specific mechanisms of BAF chromatin remodeling complex in glioma pathogenesis may yield critical insights for the development of precision therapeutics that have the potential to improve patient outcomes.

Integrative multi-omics characterization reveals sex differences in glioblastoma.

Glioblastoma (GBM) is the most common and lethal primary brain tumor in adults, with limited treatment modalities and poor prognosis. Recent studies have highlighted the importance of considering sex differences in cancer incidence, prognosis, molecular disparities, and treatment outcomes across various tumor types, including colorectal adenocarcinoma, lung adenocarcinoma, and GBM. We performed comprehensive analyses of large-scale multi-omics data (genomic, transcriptomic, and proteomic data) from TCGA, GLASS, and CPTAC to investigate the genetic and molecular determinants that contribute to the unique clinical properties of male and female GBM patients. Our results revealed several key differences, including enrichments of MGMT promoter methylation, which correlated with increased overall and post-recurrence survival and improved response to chemotherapy in female patients. Moreover, female GBM exhibited a higher degree of genomic instability, including aneuploidy and tumor mutational burden. Integrative proteomic and phosphor-proteomic characterization uncovered sex-specific protein abundance and phosphorylation activities, including EGFR activation in males and SPP1 hyperphosphorylation in female patients. Lastly, the identified sex-specific biomarkers demonstrated prognostic significance, suggesting their potential as therapeutic targets. Collectively, our study provides unprecedented insights into the fundamental modulators of tumor progression and clinical outcomes between male and female GBM patients and facilitates sex-specific treatment interventions. Highlights Female GBM patients were characterized by increased MGMT promoter methylation and favorable clinical outcomes compared to male patients. Female GBMs exhibited higher levels of genomic instability, including aneuploidy and TMB. Each sex-specific GBM is characterized by unique pathway dysregulations and molecular subtypes. EGFR activation is prevalent in male patients, while female patients are marked by SPP1 hyperphosphorylation.

Sex-Biased T-cell Exhaustion Drives Differential Immune Responses in Glioblastoma.

Immunotherapies in patients with GBM have been unsuccessful due to a variety of factors, including the highly immunosuppressive tumor microenvironment in GBM. This study demonstrates that sex-biased T-cell behaviors are predominantly intrinsically regulated, further suggesting sex-specific approaches can be leveraged to potentially improve the therapeutic efficacy of immunotherapy in GBM. See related commentary by Alspach, p. 1966. This article is featured in Selected Articles from This Issue, p. 1949.

EPCO-29. KLF5 IS REQUIRED FOR THE SEX-DIFFERENCES ACROSS MULTIPLE TUMORIGENIC PHENOTYPES IN PEDIATRIC GLIOBLASTOMA (GBM)

Abstract High-grade glioblastoma (GBM) is a highly heterogeneous, malignant cancer with no cure to date. A sex-biased incidence and therapeutic response is reported (1) for GBM with a male to female incidence ratio of 1.6:1 and female patients having a 4-6 month survival advantage (2) compared to male patients. In our current work, we sought to determine the molecular basis for the sex-bias in incidence and therapeutic response using an isogenic murine model of GBM with dual loss of neurofibromin 1 (NF1) and P53 (DNP53) in astrocytes. Our model yielded GBM from the murine astrocytes in a sex-biased manner, with male GBM cells exhibiting a highly significant proliferative, migratory, in-vivo tumorigenicity, stem-cell enrichment rate and therapeutic resistance compared to female GBM cells. Using transposon calling cards, we mapped Brd4 enhancer usage across the genome in male, compared to female GBM cells and identified key transcription factors that differentially enriched at male-biased, compared to female-biased Brd4 bound enhancers. Interestingly, female-biased Brd4-bound enhancers were enriched for transcription factors with tumor suppressor roles including P53 and SMAD4, whereas male-biased Brd4-bound enhancers were enriched for transcription factors with oncogenic roles, including Oct4, MYC and KLF5. Genetic or chemical silencing of KLF5 reduced the tumorigenic potential in male GBM with rates of cellular proliferation, survival, migration and stem-cell frequency significantly reduced to levels that resemble female GBM. Using transposon calling cards, we mapped KLF5 genomic localization in male and female GBM cells and identified significant peaks that affiliated with differentially expressed genes between the sexes. Interestingly, genes induced by KLF5 in the male cells were affiliated with poor survival in patients with various cancers, whereas those induced by KLF5 in the female cells were affiliated with favorable prognosis. This signifies the need to explore molecular mechanisms by which KLF5 drives these sex differences in GBM.

HGG-25. Targeting KLF5 reduces tumorigenic phenotype in a murine glioblastoma (GBM) model

Sex differences are evident in the incidence, therapeutic response and survival of patients with various cancers including the most common, aggressive and incurable GBM. We generated a murine GBM model of male and female astrocytes with dual loss of NF1 and P53 that yielded a sex-biased transformation of the astrocytes with male cells being significantly more tumorigenic and therapeutically resistant compared to female cells. In our current work, we aimed to delineate the molecular mechanisms driving this sex-biased tumorigenic phenotype in order to deliver therapies that are more effective to patients with GBM. We examined the inhibition of KLF5 on tumorigenic phenotypes using cellular bio-functional assays. We used barcoded transposon calling cards to determine the genomic localization of KLF5 and the differentially induced gene expression in male versus female GBM cells. Chemical inhibition or shRNA knock-down of KLF5 significantly reduced proliferation, migration, clonogenic stem-cell frequency and survival, but increase apoptosis in male and female GBM cells. Interestingly, male, but not female, GBM cells exhibited an increased migratory phenotype after radiation that inhibition of KLF5 significantly reduced. Moreover, KLF5 inhibition significantly reduced the protein expression of tumorigenic PDGFRB, AKT, ERK and the stem marker Sox-2. Transposon calling cards mapped unique KLF5 genomic localization in male versus female GBM cells with significantly differential gene expression profiles between the two. The top genes induced by KLF5 in male cells were primarily affiliated with poorer prognosis and reduced survival, whereas in female cells they were affiliated with better prognosis and improved survival in patients with cancer. Our findings provide a promising exploratory avenue for KLF5 as a therapeutic target in GBM and warrants further investigation in order to delineate the precise molecular mechanisms driving this antitumor response so that targeted therapy would be more effective taking into consideration the sex-differences in patients with GBM.

Nuclear respiratory factor 1 transcriptomic signatures as prognostic indicators of recurring aggressive mesenchymal glioblastoma and resistance to therapy in White American females.

The mechanisms contributing to recurrence of glioblastoma (GBM), an aggressive neuroepithelial brain tumor, remain unknown. We have recently shown that nuclear respiratory factor 1 (NRF1) is an oncogenic transcription factor and its transcriptional activity is associated with the progression and prognosis of GBM. Herein, we extend our efforts to (1) identify influential NRF1-driven gene and microRNA (miRNA) expression for the aggressiveness of mesenchymal GBM; and (2) understand the molecular basis for its poor response to therapy. Clinical data and RNA-Seq from four independent GBM cohorts were analyzed by Bayesian Network Inference with Java Objects (BANJO) and Markov chain Monte Carlo (MCMC)-based gene order to identify molecular drivers of mesenchymal GBM as well as prognostic indicators of poor response to radiation and chemotherapy. We are the first to report sex-specific NRF1 motif enriched gene signatures showing increased susceptibility to GBM. Risk estimates for GBM were increased by greater than 100-fold with the joint effect of NRF1-driven gene signatures-CDK4, DUSP6, MSH2, NRF1, and PARK7 in female GBM patients and CDK4, CASP2, H6PD, and NRF1 in male GBM patients. NRF1-driven causal Bayesian network genes were predictive of poor survival and resistance to chemoradiation in IDH1 wild-type mesenchymal GBM patients. NRF1-regulatable miRNAs were also associated with poor response to chemoradiation therapy in female IDH1 wild-type mesenchymal GBM. Stable overexpression of NRF1 reprogramed human astrocytes into neural stem cell-like cells expressing SOX2 and nestin. These cells differentiated into neurons and form tumorospheroids. In summary, our novel discovery shows that NRF1-driven causal genes and miRNAs involved in cancer cell stemness and mesenchymal features contribute to cancer aggressiveness and recurrence of aggressive therapy-resistant glioblastoma.

TAMI-54. SEXUAL DIMORPHISM IN IRON ACQUISITION IN GLIOBLASTOMA

Abstract Sexual dimorphism in incidence and the clinical outcomes of Glioblastoma (GBM) has been reported, however, our knowledge of contributing biological mechanisms is limited. Iron acquisition is key to robust tumor growth. Upregulation of Transferrin (TF, iron transport protein)/Transferrin receptor (TFR) is critical for found in multiple different cancers, specifically, we have identified H-ferritin (FTH1) as a contributor to iron transport and protection in cancer stem cells. To interrogate brain tumor iron uptake mechanisms,we performed binding studies on homogenized samples of human male and female GBM tissue samples using 125I labeled TF and FTH1. Tumors from males had a ̴ 3.8-fold increased binding of both proteins compared to tumors from females. We interrogated iron uptake in a syngeneic orthotopic mouse model (GL261 cells) using male and female mice. After the tumors were established, radioactive 125I labeled TF and FTH1 proteins were injected retro-orbitally in the mice. After 24 hours, tumors wereremoved, and analyzed for TF and FTH1 uptake. Male tumors showed an increased uptake, of ̴ 3.2-fold, as compared to female tumors. There was no significant difference in TF uptake between male and female tumors nor between tumor and matched non-tumor brain tissue. We next queried role of FTH1 in the context of sexual dimorphism in GBM in a FTH1+/- mouse strain developed in our laboratory. Survival was monitored in the mice which were injected with GL261 cells at 3 months. Male mice that had reduced expression of FTH1 had poorer survival as compared to the male wild type controls whereas wild type and FTH+/- females had no major differences in survival outcomes. In summary, this study demonstrates sexual dimorphism in iron acquisition in GBM and animal models further suggesting a pathophysiological role of iron metabolism in GBM development and its possible role in prognosis.

The Translocator Protein (TSPO) Genetic Polymorphism A147T Is Associated with Worse Survival in Male Glioblastoma Patients.

Simple SummaryThe translocator protein 18 kDa (TSPO) gene is highly expressed in glioblastoma (GBM), the most common primary malignant brain tumor, which remains one of the most difficult tumors to treat. TSPO is located in the outer mitochondrial membrane and binds cholesterol through its C-terminal domain. One frequent single-nucleotide polymorphism (SNP) rs6971, which changes the alanine 147 into threonine (Ala147Thr), has been found in the C-terminal domain of the TSPO region and dramatically alters the affinity with which TSPO binds drug ligands. However, the potential association between the TSPO genetic variants and GBM clinical outcomes is not known. Here, we evaluated the effects of the Ala147Thr SNP localized in this TSPO region on biological, sex-specific, overall, and progression-free GBM survival. Our findings suggest an association between the TSPO rs6971 variant and adverse outcomes in male GBM patients but not in females. These findings also suggest that the TSPO rs6971 SNP could be used as a prognostic marker of survival in GBM patients.Glioblastoma (GBM) is the most common primary brain tumor in adults, with few available therapies and a five-year survival rate of 7.2%. Hence, strategies for improving GBM prognosis are urgently needed. The translocator protein 18kDa (TSPO) plays crucial roles in essential mitochondria-based physiological processes and is a validated biomarker of neuroinflammation, which is implicated in GBM progression. The TSPO gene has a germline single nucleotide polymorphism, rs6971, which is the most common SNP in the Caucasian population. High TSPO gene expression is associated with reduced survival in GBM patients; however, the relation between the most frequent TSPO genetic variant and GBM pathogenesis is not known. The present study retrospectively analyzed the correlation of the TSPO polymorphic variant rs6971 with overall and progression-free survival in GBM patients using three independent cohorts. TSPO rs6971 polymorphism was significantly associated with shorter overall survival and progression-free survival in male GBM patients but not in females in one large cohort of 441 patients. We observed similar trends in two other independent cohorts. These observations suggest that the TSPO rs6971 polymorphism could be a significant predictor of poor prognosis in GBM, with a potential for use as a prognosis biomarker in GBM patients. These results reveal for the first time a biological sex-specific relation between rs6971 TSPO polymorphism and GBM.

Brd4-bound enhancers drive cell-intrinsic sex differences in glioblastoma

Sex can be an important determinant of cancer phenotype, and exploring sex-biased tumor biology holds promise for identifying novel therapeutic targets and new approaches to cancer treatment. In an established isogenic murine model of glioblastoma (GBM), we discovered correlated transcriptome-wide sex differences in gene expression, H3K27ac marks, large Brd4-bound enhancer usage, and Brd4 localization to Myc and p53 genomic binding sites. These sex-biased gene expression patterns were also evident in human glioblastoma stem cells (GSCs). These observations led us to hypothesize that Brd4-bound enhancers might underlie sex differences in stem cell function and tumorigenicity in GBM. We found that male and female GBM cells exhibited sex-specific responses to pharmacological or genetic inhibition of Brd4. Brd4 knockdown or pharmacologic inhibition decreased male GBM cell clonogenicity and in vivo tumorigenesis while increasing both in female GBM cells. These results were validated in male and female patient-derived GBM cell lines. Furthermore, analysis of the Cancer Therapeutic Response Portal of human GBM samples segregated by sex revealed that male GBM cells are significantly more sensitive to BET (bromodomain and extraterminal) inhibitors than are female cells. Thus, Brd4 activity is revealed to drive sex differences in stem cell and tumorigenic phenotypes, which can be abrogated by sex-specific responses to BET inhibition. This has important implications for the clinical evaluation and use of BET inhibitors.

Deletions on Chromosome Y and Downregulation of the SRY Gene in Tumor Tissue Are Associated with Worse Survival of Glioblastoma Patients.

Simple SummaryGlioblastoma (GBM) is one of the most common and most aggressive brain tumors with higher prevalence among men than women. Loss of chromosome Y (LOY) in the peripheral blood cells has been associated with increased risk of developing cancer. However, there is a lack of data about LOY in GBM tumor tissue and the potential impact on patients’ prognosis. Through droplet digital PCR (ddPCR) analysis of 10 markers spread throughout chromosome Y in 105 male GBM patients, we were able to identify deletion of SRY gene as a factor strongly correlating with reduced overall survival. This finding was later corroborated by the analysis of GBM gene expression data collected in TCGA, showing correlation between decreased SRY expression and shortened overall survival.Background: Biological causes of sex disparity seen in the prevalence of cancer, including glioblastoma (GBM), remain poorly understood. One of the considered aspects is the involvement of the sex chromosomes, especially loss of chromosome Y (LOY). Methods: Tumors from 105 isocitrate dehydrogenase (IDH) wild type male GBM patients were tested with droplet digital PCR for copy number changes of ten genes on chromosome Y. Decreased gene expression, a proxy of gene loss, was then analyzed in 225 IDH wild type GBM derived from TCGA and overall survival in both cohorts was tested with Kaplan–Meier log-rank analysis and maximally selected rank statistics for cut-off determination. Results: LOY was associated with significantly shorter overall survival (7 vs. 14.6 months, p = 0.0016), and among investigated individual genes survival correlated most prominently with loss of the sex-determining region Y gene (SRY) (10.8 vs. 14.8 months, p = 0.0031). Gene set enrichment analysis revealed that epidermal growth factor receptor, platelet-derived growth factor receptor, and MYC proto-oncogene signaling pathways are associated with low SRY expression. Conclusion: Our data show that deletions and reduced gene expression of chromosome Y genes, especially SRY, are associated with reduced survival of male GBM patients and connected to major susceptibility pathways of gliomagenesis.

TAMI-17. RELATIONSHIP BETWEEN IRON METABOLISM, IMMUNE CELL INFILTRATION AND SEX-BASED SURVIVAL DIFFERENCES IN GLIOMAS

Abstract Iron plays a central role in cellular metabolism, both in normal cellular functioning and in tumorigenesis. Recent evidence has shown sex-based survival differences in glioblastoma (GBM) may be related to differential expression of metabolism genes. We previously reported the iron regulating gene, HFE, was shown to have a sex-based survival impact in both low-grade gliomas and GBM. We additionally found that females with low HFE expressing tumors have significantly higher survival than males in GBM. To evaluate the relationship between iron gene expression and sex-based survival differences in GBM, we analyzed TCGA GBM gene expression and clinical data. We first analyzed the impact of iron genes on sex-based survival. In addition to HFE, FTL, TFRC, TF, and SLC39A8 (ZIP8), also showed sex-based survival differences. We then compared correlations of HFE and other iron genes to identify whether male and female GBMs differ in iron regulation and metabolism. HFE expression is significantly positively correlated with HMOX1, SLC25A28, SLC11A2, FTH1, HAMP, and TFR2 only in females. Alternatively, HFE expression is negatively correlated with ACO2 (mitochondrial aconitase) in males and ACO1 (cytoplasmic aconitase) in females. We noted that the expression of certain iron genes was highly associated with immune cell infiltration based on sex. TFR2, LRP1, and XIST expression were negatively correlated with low immune cell infiltration in females, but not males. Alternatively, in males, SLC11A2, ACO2, FOXO1, HIF1a, and HAMP genes were negatively correlated with immune infiltration. This suggests that differences in iron regulation between males and females may be contributing to differences in immune function and subsequent survival in GBM. These data suggest that the iron signature of a tumor reflects and possibly drives the metabolic and immune landscape of the tumor microenvironment thereby directly impacting survival differences between male and female GBMs.

TMOD-25. IN VIVO MODEL OF TREATMENT-RESISTANT GLIOBLASTOMA HIGHLIGHTS SEX DIFFERENCES IN SURVIVAL

Abstract BACKGROUND Sex differences independent from acute hormone action are evident in the incidence and tumorigenesis of several cancers, including glioblastoma (GBM). Malignant brain tumors with mesenchymal, proneural, and neural GBM subtypes occur twice as frequently in males than in females, implying a molecular basis for the sex disparities observed. Accordingly, response to therapy and overall survival, may also differ between male and female GBM patients. Further elucidation of these differences would benefit from a robust preclinical testing system, such as the one described here. METHODS Adult (U87-MG) GBM cells were exposed to a total radiation dose of 10 Gy, effecting an epithelial to mesenchymal transition. The resultant irradiated U87-10Gy cells were tested for cell viability and allowed to reach confluence prior to stereotactic implantation into the right striatum of male and female athymic rats. In vivo advanced MR imaging at 9.4T was carried out weekly starting two weeks after implantation. Advanced MRI parameters were processed for enhancing tumor ROIs in OsiriX 8.5.1 (lite) with Imaging Biometrics™ Software (IQ-AI, Ltd.). RESULTS Tumor take was 90% in males and 60% in females, indicating an ‘incidence’ ratio of 1.5. Overall survival was significantly higher in females (median = 48, range = 40-72 days) than in males (median = 28, range = 27-30 days) (p = 0.024). Immunohistochemical and imaging analyses are pending and will be discussed. CONCLUSION The outcomes noted in this pilot study mirror incidence and survival outcomes noted in GBM patients undergoing standard care. Our model of treatment-resistant glioma provides a robust testing system to study underlying sex differences in GBM biology and treatment response in parallel analyses of male and female data.

TMIC-34. SENESCENT ASTROCYTE INDUCED INFLAMMATION MAY UNDERLIE SEX DIFFERENCES IN THE AGE-DEPENDENT RISE IN GLIOBLASTOMA INCIDENCE

Abstract Sex differences in the rates of glioblastoma increase with age. There are likely to be multiple biological mechanisms underlying these sex differences. Here, we focused on the changes that occur with aging in the secretomes of male and female astrocytes to determine whether sex differences in chemokine and cytokine secretion, and inflammatory cell recruitment, could be one of those mechanisms. We focused on glioblastoma (GBM) and the role that senescent astrocytes might play in the age-dependent widening of the gap between male and female GBM cases. We found that the senescence associated secretory phenotype of male and female astrocytes significantly differed, notably in the enrichment of Fractalkine (1.33:1 (F:M)), the primary chemoattractant for microglia, in female compared to male SASP. This was in contrast to the greater abundance of tumorigenic growth factors like bFGF (1.25:1 (M:F)), in the male SASP. Implantation of either male or female senescent astrocytes into the brains of mice resulted in the recruitment of microglia to the injection site. Regardless of the recipient mouse sex, female astrocyte implantation resulted in significantly larger microglial infiltrates (2-fold) and greater astrocyte activation, as compared to the injection of equal numbers of male senescent cells. We propose a model for how sex differences in astrocyte SASP could result in lower rates of GBM with age in females: greater microglial attraction to senescent cells results in their enhanced clearance and consequently a reduction in senescence-associated GBM promotion.

IMMU-01. MYELOID-DERIVED SUPPRESSOR CELL HETEROGENEITY DRIVES GLIOBLASTOMA PROGRESSION IN A SEX-DEPENDENT MANNER

Abstract An immunosuppressive tumor microenvironment is a major factor facilitating glioblastoma (GBM) progression and therapeutic resistance. Immunotherapies have had variable success in improving the outcome of GBM patients, suggesting that there is a need to gain insight into the mechanisms of immunosuppression. Our group previously demonstrated that myeloid-derived suppressor cells (MDSCs) expand in GBM patients and infiltrate tumors, where they suppress the activity of cytotoxic cells. However, the mechanisms by which individual MDSC subsets promote tumorigenesis remain understudied. Using the syngeneic mouse glioma models GL261, CT-2A and SB28, we show that monocytic MDSCs (mMDSCs) are prevalent in tumors and that their frequency is significantly higher in males, who constitute 60% of GBM patients and have a worse prognosis than females. mMDSC abundance was further associated with poor survival, and male mice reached morbidity endpoint earlier. Consistent with preclinical observations, male GBM patient specimens had significantly more IBA+CD204+ immunosuppressive myeloid cells compared to female GBM tissue. In contrast, female tumor-bearing mice had a two-fold increase in circulating granulocytic MDSC (gMDSC) frequency, while this population remained unchanged in males. Female-to-male bone marrow chimeras demonstrated that intrinsic discrepancies in immune cell characteristics drive the sex differences in survival. Consistent with the differential MDSC accumulation pattern, targeting gMDSCs with anti-Ly6G neutralizing antibodies extended the lifespan of female mice without providing a survival advantage to males. However, mMDSCs were protected from the anti-Ly6C depletion strategy due to their systemic and local proliferation, as indicated by ex vivo Ki-67 staining and subsequently confirmed by gene expression analysis. Drug-prediction algorithms using the differential RNA sequencing profiles demonstrated that mMDSCs can be targeted by chemotherapeutics, while immunomodulatory drugs are effective against gMDSCs. Collectively, these findings indicate that MDSC subset variation might represent a therapeutic opportunity for improved therapeutic efficacy of immunotherapies while accounting for sex as a biological variable.

Role of monoamine-oxidase-A-gene variation in the development of glioblastoma in males: a case control study

BackgroundThe Mono-amine oxidase-A (MAO-A) enzyme is involved in the degradation and regulation of catecholamines such as serotonin, dopamine, epinephrine and nor-epinephrine. Preclinical studies suggest that this enzyme may contribute to an environment favorable for growth of malignant glioma. The MAO-A gene is located on the X-chromosome and has at least one functional genetic polymorphism. The aim of the present study was to explore possible effects of MAO-A genotype on development of glioblastoma in males.MethodsGenotypes for 437 glioma cases and 876 population-based controls from the Swedish Glioma International Case–Control study (GICC) were compared. We analyzed the germline DNA using the Illumina Oncoarray. We selected seven single nucleotide polymorphisms (SNPs) located in the MAO-A gene, and imputed genotypes based on data from the 1000 genomes project. We used 1579 male glioblastoma cases and 1875 controls comprising the whole GICC cohort for subsequent validation of findings.ResultsThe rs144551722 SNP was a significant predictor of development of glioblastoma in males (p-value = 0.0056) but not in females even after correction for multiple testing. We conducted haplotype analysis to confirm an association between MAO-A gene and risk of glioblastoma (p-value = 0.016). We found similar results in the validation sample.ConclusionsThese results suggest the possibility of a role for the MAO-A enzyme and the MAO-A gene in the development of glioblastoma in males.

P11.43 Deletions on Y chromosome are associated with shorter survival in glioblastoma

Abstract BACKGROUND Apart from few exceptions, men are more likely to be diagnosed with cancer during their lifetime, including glioblastoma (GBM), but the gender-related differences in GBM are poorly understood. Loss of chromosome Y (LOY) in peripheral blood cells is associated with physiological ageing, but also with disorders like cardiovascular disease, Alzheimer’s disease and different cancer types. In this study, we determined deletions on Y chromosome in tumor tissue of male patients with GBM, and studied the impact of LOY on survival. MATERIAL AND METHODS Ten genes, serving as markers, were selected on both arms of chromosome Y for copy number analysis with droplet digital PCR (ddPCR), enabling detection of loss of a marker in a fraction of the tumor cells used for DNA extraction. A total of 114 tumor samples from male patients were used, derived from a cohort of IDH wild type GBM patients treated with standard radio-chemo therapy. For 61 of these 114 patients, corresponding blood samples were available and analyzed. Different cut-off values were tested for each marker and Kaplan-Meier log-rank analysis was used to estimate overall survival. The mRNA expression for nine of the ten tested genes was available in TCGA, and 225 IDH wild type male GBM were included in a separate survival analysis, where median value of expression was used as group separator. RESULTS Fractional loss, as well as gain of markers was detected. Decreased copy number of the following markers was associated with significantly shorter survival; amelogenin Y-linked (AMELY) (13.5 vs. 19.3 months, p=0.017), neuroligin 4 Y-linked (NLGN4Y) (11.8 vs. 18.9 months, p=0.03) and sex determining region Y (SRY) (10.3 vs. 18.7 months, p=0.002). Additional analysis of SRY in the blood samples verified that copy number alterations were predominantly present in tumors. Survival analysis using mRNA expression data from TCGA showed that reduced expression of SRY was associated with significantly shorter OS (13.8 vs. 19.8 months, p=0.008), but no significant correlation with OS for any of the other markers. CONCLUSION Our data suggests a clonal or at least sporadic occurrence of fractional loss of Y chromosome markers in GBM, as detected with ddPCR. Interestingly, such dosage changes may contribute to shorter survival in men and explain some of the sex disparity seen in GBM. More research is needed to elucidate the molecular mechanisms of LOY and the role of specific Y-linked genes in GBM but also other diseases.

Sex-specific clinicopathological significance of novel (Frizzled-7) and established (MGMT, IDH1) biomarkers in glioblastoma.

BackgroundThe Wnt receptor Frizzled-7 (FZD7) promotes tumor progression and can be currently targeted by monoclonal antibody therapy. Here, we determined the prognostic value of FZD7 for the overall survival of glioblastoma (GBM) patients, both as individual marker and taken in combination with the previously-described markers MGMT and IDH1. Additionally, we tested whether these markers (alone or in combination) exhibited sex-specific differences.ResultsHigh levels of FZD7 (FZD7high) associated with shorter survival in GBM patients; however, FZD7high was a significant predictor of poor survival only in male patients. Mutation of IDH1 significantly associated with longer survival in male but not female patients. Methylated MGMT promoter significantly associated with longer survival only in female patients. Combination of FZD7 with MGMT enhanced the prognostic accuracy and abrogated the sex differences observed upon single marker analysis. Combination of FZD7 with IDH1 was a significant predictor of survival in male GBM patients only.Materials and MethodsThree independent cohorts of patients with primary GBM (n=120, n=108 and n=105, respectively) were included in this study. FZD7 and IDH1 were assessed by immunohistochemistry in tissue microarrays. MGMT promoter methylation was determined by methylation-specific polymerase chain reaction. Survival analysis was performed by Kaplan-Meier estimate, log-rank test and Cox proportional hazard regression.ConclusionsOur study identifies novel individual and combination markers with prognostic and, possibly, therapeutic relevance in GBM. Furthermore, our findings substantiate the importance of sexual dimorphism in this type of cancer.

MiR-224 expression increases radiation sensitivity of glioblastoma cells

Glioblastoma (GBM) is the most common and highly aggressive primary malignant brain tumor. The intrinsic resistance of this brain tumor limits the efficacy of administered treatment like radiation therapy. In the present study, effect of miR-224 expression on growth characteristics of established GBM cell lines was analyzed. MiR-224 expression in the cell lines as well as in primary GBM tumor tissues was found to be low. Exogenous transient expression of miR-224 using either synthetic mimics or stable inducible expression using doxycycline inducible lentiviral vector carrying miR-224 gene, was found to bring about 30–55% reduction in clonogenic potential of U87 MG cells. MiR-224 expression reduced clonogenic potential of U87 MG cells by 85–90% on irradiation at a dose of 6Gy, a dose that brought about 50% reduction in clonogenic potential in the absence of miR-224 expression. MiR-224 expression in glioblastoma cells resulted in 55–65% reduction in the expression levels of API5 gene, a known target of miR-224. Further, siRNA mediated down-regulation of API5 was also found to have radiation sensitizing effect on glioblastoma cell lines. Analysis of the Cancer Genome Atlas data showed lower miR-224 expression levels in male GBM patients to correlate with poorer survival. Higher expression levels of miR-224 target API5 also showed significant correlation with poorer survival of GBM patients. Up-regulation of miR-224 or down-regulation of its target API5 in combination with radiation therapy, therefore appear as promising options for the treatment of glioblastoma, which is refractory to the existing treatment strategies.

The C3435T Polymorphism of MDR1 and Susceptibility to Adult Glioma

Introduction: The multidrug resistance-1 (MDR1) gene encodes a pump that prevents potentially carcinogenic substances from crossing the blood-brain barrier. We compare adult glioma cases and controls for the C3435T polymorphism that has been associated with reduced MDR1 expression. Methods: Adult glioma in the San Francisco Bay area and population-based controls were identified between 1991–1994 and 1997–1999. Genotyped cases (n = 458) and controls (n = 528) were compared using logistic regression controlling for age, gender and ethnicity, with later stratification by ethnicity, gender and histology. Results: With CC as the referent, the TT genotype was nonsignificantly less frequent among cases compared to controls (OR, 0.87; 95% CI: 0.6, 1.2). After stratification, only male glioblastoma was associated with TT genotype (OR, 0.51; 95% CI: 0.3, 1.0). Conclusions: Although the C3435T polymorphism does not appear to be associated with other types of glioma, we cannot rule out that this MDR1 polymorphism may be associated with glioblastoma among men.