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Background: Major Depressive Disorder is a leading cause of disability worldwide and one of the most common disorders in the United States. Contributors to an individual’s risk for experiencing depressive disorders include individual and social factors. Although the social determinants of health (SDOH) are conditions that contribute to healthy functioning, health outcomes, and quality of life, it is unclear to what extent adverse SDOH experiences are associated with self-reporting depressive disorder (DD). Methods: Data from the 2017 Behavioral Risk Factor Surveillance System (BRFSS), a population-based telephone survey of noninstitutionalized U.S. adults, was employed in this study. Lifetime diagnosis of DD was self-reported among survey participants who also completed the Social Determinants of Health optional module within 17 participating BRFSS states and entities. Rao-Scott chi-square analysis was used to determine the association of individual and SDOH measures with DD. Results: The prevalence of DD among participating states was 19.85%. Among respondents, significant proportional differences were observed for each SDOH indicator regarding DD status. However, significant differences in health insurance coverage were not observed. Conclusion: Survey respondents with a lifetime diagnosis of DD experienced adverse SDOH conditions in greater proportion than individuals not reporting DD. Individual and community-based approaches to address the contextual influences of depressive disorders should be aggressively implemented.

Tumorigenic and non-neoplastic tissue injury occurs via the ischemic microenvironment defined by low oxygen, pH, and nutrients due to blood supply malfunction. Ischemic conditions exist within regions of pseudopalisading necrosis, a pathological hallmark of glioblastoma (GBM), the most common primary malignant brain tumor in adults. To recapitulate the physiologic microenvironment found in GBM tumors and tissue injury, we developed an in vitro ischemic model and identified chromodomain helicase DNA-binding protein 7 (CHD7) as a novel ischemia-regulated gene. Point mutations in the CHD7 gene are causal in CHARGE syndrome (a developmental disorder causing coloboma, heart defects, atresia choanae, retardation of growth, and genital and ear anomalies) and interrupt the epigenetic functions of CHD7 in regulating neural stem cell maintenance and development. Using our ischemic system, we observed microenvironment-mediated decreases in CHD7 expression in brain tumor-initiating cells and neural stem cells. Validating our approach, CHD7 was suppressed in the perinecrotic niche of GBM patient and xenograft sections, and an interrogation of patient gene expression datasets determined correlations of low CHD7 with increasing glioma grade and worse patient outcomes. Segregation of GBM by molecular subtype revealed a novel observation that CHD7 expression is elevated in proneural versus mesenchymal GBM. Genetic targeting of CHD7 and subsequent gene ontology analysis of RNA sequencing data indicated angiogenesis as a primary biological function affected by CHD7 expression changes. We validated this finding in tube-formation assays and vessel formation in orthotopic GBM models. Together, our data provide further understanding of molecular responses to ischemia and a novel function of CHD7 in regulating angiogenesis in both neoplastic and non-neoplastic systems. Stem Cells 2019;37:453–462

A pathologic hallmark of glioblastoma (GBM), the most common and deadly primary brain tumor in adults, is pseudopalisading necrotic regions in which ischemic conditions are found. Ischemic microenvironments characterized by low oxygen, restricted glucose, and acidic pH due to poor blood supply occurs in both solid tumors and non-neoplastic tissue injury. Modeling these physiologically relevant ischemic conditions within glioblastoma in vitro, we identified chromodomain helicase DNA binding protein 7 (CHD7) as a novel ischemia-regulated gene. CHD7 is an epigenetic reader that controls neural stem cell maintenance and is mutated in CHARGE syndrome, a developmental disorder associated with cranial nerve abnormalities. CHD7 mRNA expression was consistently suppressed in multiple patient-derived xenograft brain tumor lines in response to ischemic conditions, and lower expression of CHD7 correlates with poor GBM patient survival. These microenvironment-mediated decreases in CHD7 protein and mRNA levels observed in glioblastoma were also mirrored in neural progenitor cells in vitro, and CHD7 levels were reduced in the perinecrotic niche of GBM patient and xenograft tissue sections. Genetic targeting of CHD7 increased angiogenesis in vitro in association with differential regulation of angiogenesis associated genes as determined in RNA sequencing analysis. Although targeting CHD7 in orthotopically implanted xenograft brain tumor models did not affect survival outcomes in mice, we observed marked differences in blood vessel architecture and organization, suggesting a major role for CHD7 in regulating vessel formation within these tumors. Together, our data provide insight into the molecular responses to ischemia that regulate angiogenesis.

Background For glioblastoma (GBM) treatments to be effective in vivo, understanding the effects of the tumor microenvironment is imperative. In traditional cell culture conditions, glucose concentrations do not model physiologic levels, nor the diminished concentrations found in tumor niches. We therefore sought to profile the differences in kinase activity in GBM cells cultured in restricted glucose to identify pathways that could be targeted with small molecule inhibitors. Methods Using the PamStation12 platform, we examined the ability of GBM lysates from cells cultured in standard or low glucose conditions to phosphorylate 144 tyrosine and 144 serine/threonine peptides that correspond to known protein phosphorylation sites. Potential kinase targets were identified and validated using small molecule kinase inhibitors in GBM spheroid cultures. Results Using results from two GBM patient-derived xenografts, we determined common changes to peptides derived from Phospholipase C, Gamma 1 (PLCG1) and Raf-1. Using PLC and Raf inhibitors, we found a significantly stronger growth inhibitory effect of the PLC inhibitor U73122 under restricted glucose conditions. In contrast, Raf inhibitors were significantly growth inhibitory regardless of the nutrient level tested. Conclusions Together, our data demonstrate that kinase activity is altered in low glucose conditions and that kinomic profiling can assist with the identification of effective strategies to target GBM growth. Our data further suggest the importance of accurately modeling the tumor microenvironment to reproduce cancer cell signaling and develop drug screens for anti-cancer agents.

Background Depending on the level, differentiation state, and tumor stage, reactive nitrogen and oxygen species inhibit or increase cancer growth and tumor initiating cell maintenance. The rate-limiting enzyme in a pathway that can regulate reactive species production but has not been thoroughly investigated in glioblastoma (GBM; grade IV astrocytoma) is guanosine triphosphate (GTP) cyclohydrolase 1 (GCH1). We sought to define the role of GCH1 in the regulation of GBM growth and brain tumor initiating cell (BTIC) maintenance. Methods We examined GCH1 mRNA and protein expression in patient-derived xenografts, clinical samples, and glioma gene expression datasets. GCH1 levels were modulated using lentiviral expression systems, and effects on cell growth, self-renewal, reactive species production, and survival in orthotopic patient-derived xenograft models were determined. Results GCH1 was expressed in GBMs with elevated but not exclusive RNA and protein levels in BTICs in comparison to non-BTICs. Overexpression of GCH1 in GBM cells increased cell growth in vitro and decreased survival in an intracranial GBM mouse model. In converse experiments, GCH1 knockdown with short hairpin RNA led to GBM cell growth inhibition and reduced self-renewal in association with decreased CD44 expression. GCH1 was critical for controlling reactive species balance, including suppressing reactive oxygen species production, which mediated GCH1 cell growth effects. In silico analyses demonstrated that higher GCH1 levels in glioma patients correlate with higher glioma grade, recurrence, and worse survival. Conclusions GCH1 expression in established GBMs is pro-tumorigenic, causing increased growth due, in part, to promotion of BTIC maintenance and suppression of reactive oxygen species.

Glioblastoma (GBM), or grade IV astrocytoma, is a deadly disease due in part to the high degree of intratumoral heterogeneity that contributes to treatment failures. One cellular subset enriched for the capacity to propagate tumor growth and promote therapeutic resistance is brain tumor initiating cells (BTICs). The aim of the study was to define the roles of GTP cyclohydrolase 1 (GCH1) in regulation of BTIC phenotypes via reactive species. We examined GCH1 mRNA and protein expression in patient-derived xenografts, clinical samples and glioma gene expression datasets. GCH1 levels were modulated using lentiviral expression systems, and effects on cell growth, self-renewal, reactive species production and in vitro animal survival were determined. We found that GCH1 RNA and protein expression were increased in BTICs in comparison to non-BTICs, but that GCH1 was not exclusive to the BTIC fraction. Indeed, GCH1 was elevated in GBM in comparison to normal brain. Overexpression of GCH1 in GBM cells increased cell growth in vitro and decreased survival in an intracranial GBM mouse model. In contrast, GCH1 knockdown with short hairpin RNA in GBM cells led to growth inhibition in vitro as well as increased survival in animal models. Mechanistically, GCH1 increased CD44 expression and was critical for controlling reactive species balance, including suppressing reactive oxygen species production. In silico analyses demonstrated that higher GCH1 levels in glioma patients correlate with higher glioma grade, recurrence and worse survival. Together, our data suggest that upregulation of GCH1 in BTICs promotes tumor maintenance and is a key regulator of reactive oxygen species in GBM.

Brain tumor microenvironments characterized by hypoxia, restricted glucose, and acidic stress contain a subset of neoplastic cells that thrive in these environments, identified as brain tumor initiating cells (BTICs). BTICs are resistant to chemo- and radiotherapy, providing a reservoir for tumor recurrence and a desirable target for glioma treatments. Standard of care for glioblastoma (GBM) includes surgical resection, radiation therapy, and the chemotherapeutic agent temozolomide, which prolongs life expectancy by months and is not curative. Prior studies suggested the efficacy of chemotherapies including temozolomide was increased by reducing expression of carbonic anhydrase 9 (CA9). CA9 is a hypoxia responsive gene elevated in tumors that is important for regulating intracellular pH and contributing to the acidic extracellular microenvironment. After confirming basal and hypoxia-induced expression of CA9 in GBM BTICs, we targeted CA9 activity with the small molecule inhibitor SLC-0111 alone or in combination with temozolomide. In multiple GBM BTIC lines, SLC-0111 reduced cell growth in vitro and showed additional benefit when used concurrently with temozolomide. Mechanistically, SLC-0111 treatment increased temozolomide DNA damage and resulted in changes to cellular metabolism and pH. SLC-0111 decreased the enrichment of BTICs after temozolomide treatment as determined via BTIC marker expression and functionally with neurosphere formation. SLC-0111 was detected in the brain after oral gavage administration of a formulation in Phase I clinical trials for breast cancer patients. SLC-0111 and temozolomide also significantly reduced the growth of subcutaneous and orthotopic models of a recurrent GBM greater than either drug alone. Together, our data suggest that SLC-0111 can sensitize GBM BTICs to the chemotherapy temozolomide and extend animal survival.

Gliomas are central nervous system tumors that primarily occur in the brain and arise from glial cells. Gliomas include the most common malignant brain tumor in adults known as grade IV astrocytoma, or glioblastoma (GBM). GBM is a deadly disease for which the most significant advances in treatment offer an improvement in survival of only ∼2 months.To develop novel treatments and improve patient outcomes, we and others have sought to determine the role of molecular signals in gliomas. Recent Advances: One signaling molecule that mediates important biologies in glioma is the free radical nitric oxide (NO). In glioma cells and the tumor microenvironment, NO is produced by three isoforms of nitric oxide synthase (NOS), NOS1, NOS2, and NOS3. NO and NOS affect glioma growth, invasion, angiogenesis, immunosuppression, differentiation state, and therapeutic resistance.These multifaceted effects of NO and NOS on gliomas both in vitro and in vivo suggest the potential of modulating the pathway for antiglioma patient therapies. Antioxid. Redox Signal. 26, 986-999.

Tumor microenvironments can promote stem cell maintenance, tumor growth, and therapeutic resistance, findings linked by the tumor-initiating cell hypothesis. Standard of care for glioblastoma (GBM) includes temozolomide chemotherapy, which is not curative, due, in part, to residual therapy-resistant brain tumor-initiating cells (BTICs). Temozolomide efficacy may be increased by targeting carbonic anhydrase 9 (CA9), a hypoxia-responsive gene important for maintaining the altered pH gradient of tumor cells. Using patient-derived GBM xenograft cells, we explored whether CA9 and CA12 inhibitor SLC-0111 could decrease GBM growth in combination with temozolomide or influence percentages of BTICs after chemotherapy. In multiple GBMs, SLC-0111 used concurrently with temozolomide reduced cell growth and induced cell cycle arrest via DNA damage in vitro. In addition, this treatment shifted tumor metabolism to a suppressed bioenergetic state in vivo. SLC-0111 also inhibited the enrichment of BTICs after temozolomide treatment determined via CD133 expression and neurosphere formation capacity. GBM xenografts treated with SLC-0111 in combination with temozolomide regressed significantly, and this effect was greater than that of temozolomide or SLC-0111 alone. We determined that SLC-0111 improves the efficacy of temozolomide to extend survival of GBM-bearing mice and should be explored as a treatment strategy in combination with current standard of care.

Development of effective novel anti-tumor treatments will require improved in vitro models that incorporate physiologic microenvironments and maintain intratumoral heterogeneity, including tumor initiating cells. Brain tumor initiating cells (BTIC) are a target for cancer therapy, because BTICs are highly tumorigenic and contribute to tumor angiogenesis, invasion, and therapeutic resistance. Current leading studies rely on BTIC isolation from patient-derived xenografts followed by propagation as neurospheres. As this process is expensive and time-consuming, we determined whether three-dimensional microtumors were an alternative in vitro method for modeling tumor growth via BITC maintenance and/or enrichment. Brain tumor cells were grown as neurospheres or as microtumors produced using the human-derived biomatrix HuBiogel™ and maintained with physiologically relevant microenvironments. BITC percentages were determined using cell surface marker expression, label retention, and neurosphere formation capacity. Our data demonstrate that expansion of brain tumor cells as hypoxic and nutrient-restricted microtumors significantly increased the percentage of both CD133+ and CFSEhigh cells. We further demonstrate that BTIC-marker positive cells isolated from microtumors maintained neurosphere formation capacity in the in vitro limiting dilution assay and tumorigenic potential in vivo. These data demonstrate that microtumors can be a useful three-dimensional biological model for the study of BTIC maintenance and targeting.

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.

Ectopic gene expression through introduction of cDNA and gene silencing by RNA interference each facilitate the elucidation of molecular pathways in both normal and pathologic states. As transfection efficiency in some primary and established cells is low, lentivirus based expression systems with high infection rates can improve experimental design. For example, glioblastoma cells and particularly the cancer stem cell (CSC) fraction can be difficult to transfect but are amenable to viral infection. Greater utilization of lentivirus for expression of cDNA and shRNA in CSCs may be limited due to technical challenges, including elimination of pro-differentiating fetal bovine serum (FBS). We therefore generated a subline of 293Ts that can proliferate and efficiently produce virus in CSC media, designated CSC293Ts. We provide detailed protocols for the generation of CSC293Ts and for the production of lentivirus for CSC infection using glioblastoma as a model. Our data demonstrate that serum free media from CSC293Ts consistently produces greater than 80% infection rates without virus concentration. We believe that the detailed protocols provided here can be adapted for multiple cell types for broad utility.

Glioblastoma (GBM), or grade IV astrocytoma, is a deadly disease due in part to the high degree of intratumoral heterogeneity that contributes to treatment failures. Previous studies have shown the importance of reactive species balances, partially controlled by the coupling of nitric oxide synthases (NOS) with their cofactor, in maintenance of glioma stem cell (GSC) phenotype as well as survival of cancer cells in general. In this study, we investigated the roles of GTP cyclohydrolase 1 (GCH1), which is the first and rate-limiting enzyme of the pathway producing of NOS cofactor producing pathway, in GBM stem cell phenotypes via redox balances. We found that GCH1 RNA and protein expression were increased in GSCs in comparison to non-GSCs, but that GCH1 was not exclusive to the GSC fraction. Indeed, GCH1 was elevated in GBM in comparison to normal brain. Overexpression of GCH1 in GBM cells increased cell growth in vitro and neurosphere-forming capability and decreased survival in an intracranial GBM mouse model. In contrast, GCH1 knockdown with short hairpin RNA in GBM cells led to growth inhibition in vitro as well as increased survival in animal models. GCH1 increased CD44 expression and was upregulated in the detrimental mesenchymal GBM subtype in which CD44 served as a marker. Mechanistically, we found that the expression of GCH1 increased BH4 production, as well as augmented multiple antioxidant pathways, including the expression of PARK7, was critical for controlling reactive species balance, including suppressing reactive oxygen species production. In silico analyses demonstrated that higher GCH1 levels in glioma patients correlate with higher glioma grade, recurrence and worse survival. Together, our data suggest that upregulation of GCH1 in GSCs promotes tumor maintenance and is a key regulator of reactive oxygen species in GBM, and GCH1 pathway is a potential target for therapy. Citation Format: Anh N. Tran, Kiera Walker, David G. Harrison, Wei Chen, James Mobley, Lauren Hocevar, James R. Hackney, Randee Sedaka, Jennifer Pollock, Matthew S. Goldberg, Dolores Hambardzumyan, Sara J. Cooper, G Yancey Gillespie, Anita B. Hjelmeland. Glioblastoma, cancer stem cells, and reactive species balances: A case for GTP cyclohydrolase 1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 163.

We have employed a structure-based three-dimensional quantitative structure-activity relationship (3D-QSAR) approach to predict the biochemical activity for inhibitors of T. cruzi dihydrofolate reductase-thymidylate synthase (DHFR-TS). Crystal structures of complexes of the enzyme with eight different inhibitors of the DHFR activity together with the structure in the substrate-free state (DHFR domain) were used to validate and refine docking poses of ligands that constitute likely active conformations. Structural information from these complexes formed the basis for the structure-based alignment used as input for the QSAR study. Contrary to indirect ligand-based approaches the strategy described here employs a direct receptor-based approach. The goal is to generate a library of selective lead inhibitors for further development as antiparasitic agents. 3D-QSAR models were obtained for T. cruzi DHFR-TS (30 inhibitors in learning set) and human DHFR (36 inhibitors in learning set) that show a very good agreement between experimental and predicted enzyme inhibition data. For crossvalidation of the QSAR model(s), we have used the 10% leave-one-out method. The derived 3D-QSAR models were tested against a few selected compounds (a small test set of six inhibitors for each enzyme) with known activity, which were not part of the learning set, and the quality of prediction of the initial 3D-QSAR models demonstrated that such studies are feasible. Further refinement of the models through integration of additional activity data and optimization of reliable docking poses is expected to lead to an improved predictive ability.

The ischemic microenvironment characterized by low oxygen and glucose due to poor blood supply occurs in both solid tumors and non-neoplastic tissue injury. Ischemia occurs in regions of pseudopalisading necrosis, a hallmark of the most common and deadly primary brain tumor in adults, glioblastoma. Modeling physiologic low oxygen and glucose in glioblastoma in vitro, we identified chromodomain helicase DNA binding protein 7 (CHD7) as a novel ischemia-regulated gene. CHD7 is an epigenetic modifier regulating neural stem cell maintenance and mutated in CHARGE syndrome, a developmental disorder associated with cranial nerve abnormalities. Microenvironment-mediated decreases in CHD7 protein and mRNA levels were observed in glioblastoma and neural progenitor cells in vitro, and CHD7 levels were reduced in the perinecrotic niche of GBM patient and xenograft sections. Genetic targeting of CHD7 increased angiogenesis in vitro in association with differential regulation of angiogenesis associated genes as determined in RNA sequencing analysis. Further suggesting the importance of CHD7 levels in glioblastoma, patient gene expression datasets show a correlation between down regulation of CHD7 and increasing glioma grade and worse patient outcomes. Together, our data provide insight into the molecular responses to ischemia that regulate angiogenesis.

Glioblastoma (GBM), or grade IV astrocytoma, is a deadly disease due in part to the high degree of intratumoral heterogeneity that contributes to treatment failures. One cellular subset enriched for the capacity to propagate tumor growth and promote therapeutic resistance is brain tumor initiating cells (BTICs). The aim of the study was to define the roles of GTP cyclohydrolase 1 (GCH1) in regulation of BTIC phenotypes via reactive species. We examined GCH1 mRNA and protein expression in patient-derived xenografts, clinical samples and glioma gene expression datasets. GCH1 levels were modulated using lentiviral expression systems, and effects on cell growth, self-renewal, reactive species production and in vitro animal survival were determined. We found that GCH1 RNA and protein expression were increased in BTICs in comparison to non-BTICs, but that GCH1 was not exclusive to the BTIC fraction. Indeed, GCH1 was elevated in GBM in comparison to normal brain. Overexpression of GCH1 in GBM cells increased cell growth in vitro and decreased survival in an intracranial GBM mouse model. In contrast, GCH1 knockdown with short hairpin RNA in GBM cells led to growth inhibition in vitro as well as increased survival in animal models. Mechanistically, GCH1 increased CD44 expression and was critical for controlling reactive species balance, including suppressing reactive oxygen species production. In silico analyses demonstrated that higher GCH1 levels in glioma patients correlate with higher glioma grade, recurrence and worse survival. Together, our data suggest that upregulation of GCH1 in BTICs promotes tumor maintenance and is a key regulator of reactive oxygen species in GBM.

Development of effective novel anti-tumor treatments will require improved in vitro models that incorporate physiologic microenvironments and maintain intratumoral heterogeneity including tumor initiating cells. Brain tumor initiating cells (BTIC) are a target for cancer therapy because they are highly tumorigenic and contribute to tumor angiogenesis, invasion, and therapeutic resistance. Current leading studies rely on BTIC isolation from patient-derived xenografts followed by propagation as neurospheres. As this process is expensive and time-consuming, we determined whether three-dimensional microtumors were an alternative in vitro method for modeling tumor growth via BITC maintenance and/or enrichment. Brain tumor cells were grown as neurospheres or as microtumors produced using a human-derived biomatrix HuBiogelTM and maintained with physiologically relevant microenvironments. Percentages of BITCs were determined based on cell surface marker expression (CD133), label retention (carboxyfluorescein succinimidyl ester; CFSE), and tumorsphere formation capacity. Our data demonstrate that expansion of brain tumor cells as hypoxic and nutrient restricted microtumors significantly increased the percentage of both CD133+ and CFSE+ cells. We further demonstrate that BTIC-marker positive cells isolated from microtumors maintain neurosphere formation capacity in the in vitro limiting dilution assay and tumorigenic potential in vivo. These data demonstrate that microtumors can be a useful three-dimensional biological model for the study of BTIC maintenance and targeting. Citation Format: Ashley Gilbert, Kiera Walker, Anh Tran, Yancey Gillespie, Raj Singh, Anita B. Hjelmeland. Modeling physiologic microenvironments in three-dimensional microtumors facilitates brain tumor initiating cell maintenance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1925. doi:10.1158/1538-7445.AM2017-1925

Brain tumor initiating cells (BTICs) are resistant to chemo- and radiotherapy, providing a reservoir for tumor recurrence and a desirable target for glioma treatments. Standard of care for glioblastoma (GBM; grade IV astrocytoma) includes the chemotherapeutic agent temozolomide, which prolongs life expectancy by months and is not curative. Prior studies suggested the efficacy of chemotherapies including temozolomide was increased by reducing expression of carbonic anhydrase 9 (CA9). CA9 is a hypoxia responsive gene elevated in tumors that is important for regulating intracellular pH and contributing to the acidic extracellular microenvironment. After confirming basal and hypoxia-induced expression of CA9 in GBM BTICs, we targeted CA9 activity with the small molecule inhibitor SLC-0111 alone or in combination with temozolomide. In multiple GBM BTIC lines, SLC-0111 reduced cell growth in vitro and showed additional benefit when used concurrently with temozolomide. Importantly, SLC-0111 inhibited the enrichment of BTICs after temozolomide treatment as determined via BTIC marker expression and neurosphere formation capacity. These data suggested the potential of SLC-0111 as a chemosensitizer, which we next evaluated in preclinical studies using a subcutaneous recurrent GBM model. GBMs treated with SLC-0111 in combination with temozolomide significantly regressed and the resulting in vivo growth delay was greater than that of temozolomide or SLC-0111 alone. Together, our data suggest that SLC-0111 can sensitize GBM BTICs to the chemotherapy temozolomide and significantly delay disease progression. Citation Format: Nathaniel H. Boyd, Kiera Walker, Paul C. McDonald, Mark O. Bevensee, Yancey G. Gillespie, Burt Nabors, Shoukat Dedhar, Anita B. Hjelmeland. Targeting the tumor microenvironment: inhibition of carbonic anhydrase 9 impedes brain tumor initiating cell chemoresistance and delays glioblastoma growth in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 171. doi:10.1158/1538-7445.AM2017-171

Glioblastomas (GBMs) are the most common primary brain tumors in adults and one of the most aggressive cancers with high rates of recurrence and therapeutic resistance. In GBMs, subpopulations of highly tumorigenic cells called brain tumor initiating cells (BTICs) have been characterized by their unique capacity to promote tumor maintenance, therapeutic resistance, and angiogenesis. BTIC maintenance is known to be regulated by reactive oxygen and nitrogen species. GTP cyclohydrolase, or GCH1, is a critical molecule regulating reactive species levels. We found that expression of GCH1 RNA and protein were upregulated in BTICs in comparison to non-BTICs. Overexpression of GCH1 in glioma cells increased cell growth in vitro and increased tumor formation and decreased survival in an intracranial GBM mouse model. In contrast, GCH1 depletion with short hairpin RNA in GBM cells led to growth inhibition in vitro as well as increased survival in animal orthotopic models. Furthermore, genetic modulation of GCH1 led to altered ROS levels in GBM xenolines. In silico analyses demonstrate that higher GCH1 levels in glioma patients correlate with higher glioma grade, recurrence and worse survival. Together, our data suggest that upregulation of GCH1 in BTICs promotes tumor maintenance and is a key regulator of reactive oxygen species in glioblastoma. Citation Format: Anh N. Tran, Kiera Walker, David Harisson, Wei Chen, James Mobley, Lauren Hocevar, James R. Hackney, Randee S. Sedaka, Jennifer S. Pollock, Sara J. Cooper, George Y. Gillespie, Anita B. Hjelmeland. GTP cyclohydrolase in brain tumor stem cells is implicated in glioblastoma growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5462. doi:10.1158/1538-7445.AM2017-5462

Malignant gliomas rely on the production of certain critical growth factors including VEGF, interleukin (IL)-6 and IL-8, to fuel rapid tumor growth, angiogenesis, and treatment resistance. Post-transcriptional regulation through adenine and uridine-rich elements (ARE) of the 3′ untranslated region (UTR) is one mechanism for upregulating these and other growth factors. In glioma cells, we have shown that the post-transcriptional machinery is optimized for growth factor upregulation secondary to overexpression of the mRNA stabilizer, HuR. The negative regulator, tristetraprolin (TTP), on the other hand, may be suppressed because of extensive phosphorylation. Here we test that possibility by analyzing the phenotypic effects of a mutated form of TTP (mt-TTP) in which 8 phosphoserine residues were converted to alanines. We observed a significantly enhanced negative effect on growth factor expression in glioma cells at the post-transcriptional and transcriptional levels. The protein became stabilized and displayed significantly increased antiproliferative effects compared to wild-type TTP. Macroautophagy was induced with both forms of TTP, but inhibition of autophagy did not affect cell viability. We conclude that glioma cells suppress TTP function through phosphorylation of critical serine residues which in turn contributes to growth factor upregulation and tumor progression.

Dihydrofolate reductase (DHFR) of the parasite Trypanosoma cruzi (T. cruzi) is a potential target for developing drugs to treat Chagas' disease. We have undertaken a detailed structure-activity study of this enzyme. We report here synthesis and characterization of six potent inhibitors of the parasitic enzyme. Inhibitory activity of each compound was determined against T. cruzi and human DHFR. One of these compounds, ethyl 4-(5-[(2,4-diamino-6-quinazolinyl)methyl]amino-2-methoxyphenoxy)butanoate (6b) was co-crystallized with the bifunctional dihydrofolate reductase-thymidylate synthase enzyme of T. cruzi and the crystal structure of the ternary enzyme:cofactor:inhibitor complex was determined. Molecular docking was used to analyze the potential interactions of all inhibitors with T. cruzi DHFR and human DHFR. Inhibitory activities of these compounds are discussed in the light of enzyme-ligand interactions. Binding affinities of each inhibitor for the respective enzymes were calculated based on the experimental or docked binding mode. An estimated 60-70% of the total binding energy is contributed by the 2,4-diaminoquinazoline scaffold.

Introduction: Colorectal cancer (CRC) and other solid tumors rely on production of pro-inflammatory cytokines and growth factors to support their rapid growth. Posttranscriptional control of these factors involves interplay between RNA stabilizing and destabilizing factors. We have observed that the protein tristetraprolin (TTP), a RNA destabilizer, is down-regulated in CRCs and that interleukin (IL)-8, a proinflammatory cytokine, is up-regulated. We hypothesized that loss of negative regulation by TTP selectively favors expression of growth factors and protooncogenes that promote tumor progression. Furthermore, new evidence suggests that TTP is associated estrogen receptor-α (ERα) and functions as a co-repressor of ERα activation interaction with histone deacetylases on the gene promoter; nuclear TTP is linked to this tumor suppressor function. Since CRCs of African Americans (AAs) often present worse outcomes relative to those of Caucasian Americans (CAs), we analyzed the expression of TTP and its targets and assessed its subcellular localization in primary CRCs of AA and CA patients and in cultured colon cancer cells. Its effects on disease progression were also determined. Methods: Paired normal and CRC tissues from 45 patients (AA = 26 and CA = 19), were evaluated by qPCR for mRNA expression levels of TTP, IL-8, and VEGF. Tissue sections were also assayed for immunophenotypic expression by immunohistochemistry, and the staining patterns (nuclear and cytoplasmic) were assessed. To determine TTP effects on the NF-kB pathway, colon cancer cells (CCL235, HCT116 and LoVo) were stimulated with TNF-α, and total RNA was analyzed for TTP, IL-8, VEGF, and cIAP2 expression; interaction of TTP and ERα was also assessed. Results: In 31 of 45 CRCs, there was down-regulated expression of TTP mRNA, and low TTP levels correlated with advanced tumor stage. Low levels of TTP were present in 21 of 26 AAs and in 12 of 19 CAs. There was an inverse correlation between TTP and IL-8 expression in relation to tumor stage. There was an agreement between mRNA expression and immunophenotypic profiles for TTP and IL-8. Furthermore, there was reduced or a lack of nuclear staining of TTP in malignant cells relative to normal/benign epithelial cells. In cultured colon cancer cells, TTP mRNA levels inversely correlated with levels of IL-8, VEGF, and cIAP2 mRNAs, suggesting interactions of TTP with these cell survival factors. Conclusions: Reduced expression of TTP was noted in tumors, especially in CRCs of AAs, and a loss or decreased nuclear expression of TTP was observed in malignant cells. Lower TTP levels correlated with advanced tumor stage. These preliminary findings suggest that TTP negatively regulates the expression of cell survival factors in the NF-kB pathway. This work was supported by funds from the NIH/NCI (a career development supplement to grant U54-CA118948). Citation Format: Esther A. Suswam, Balananda-Dhurjati K. Putcha, Amit K. Tiwari, Trafina Jadhav, Kiera D. Walker, Lualhati Harkins, Samir Amer, Isam-Eldin Eltoum, Sejong Bae, Upender Manne. Molecular and cellular localization profiles of tristetraprolin in colorectal cancer: Implications for tumor progression in diverse patient populations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4953. doi:10.1158/1538-7445.AM2015-4953

Background: Interleukin (IL)-8, vascular endothelial growth factor (VEGF), and IL-6 contribute to the colorectal cancer (CRC) progression by inhibiting apoptosis and by promoting angiogenesis and tumor proliferation. We have found that the tristetraprolin (TTP) gene attenuates these processes [J Neurooncol. 2013; 113(2):195-205]. TTP expression is lost or reduced in many cancers, including CRCs, and loss of TTP is thought to contribute to tumorigenesis. We hypothesized that low TTP levels favor expression of growth factors and correlate with CRC progression. In addition, we suggest that TTP modulates CRC growth through negative regulation on cell survival and/or anti-apoptotic factors in the NF-kB pathway. We tested this hypothesis by analyzing mRNA expression of TTP and its targets in primary CRCs of African American (AA) and Caucasian American (CA) patients. Methods: We analyzed frozen primary tissues from 45 CRC patients (AA=26 and CA=19), each with corresponding normal (benign/control) tissue. cDNAs were reverse-transcribed from total RNA; mRNA levels of TTP and its target genes (IL-8, VEGF, IL-6) were quantified by the qPCR sybr-green method. Expression levels were normalized to GAPDH. To assess TTP effects on the NF-kB pathway, colon cancer cells (CCL235, HCT116, SW480, and LoVo) were stimulated with TNF-α for 0-24 hr, and total RNA was analyzed for TTP, IL-8, IL-6, VEGF, and cIAP2 expression by qRT-PCR. Levels of HuR mRNA in cells were also assessed. Extracts from the cells were immunoblotted with anti-TTP and antiHuR antibodies. Results: We observed down-regulated expression of TTP mRNA in primary CRCs (31 of 45), and decreased TTP levels correlated with advanced tumor stage. Low levels of TTP were found in 21 of 26 AAs and 12 of 19 CAs. In both racial groups, there was an inverse correlation between TTP and IL-8 expression in relation to tumor stage. Studies with cultured colon cancer cells demonstrated that TTP mRNA levels inversely correlated with levels of IL-8, IL-6, VEGF, and cIAP2 mRNAs, suggesting interactions of TTP with cell survival factors. Western blot analyses confirmed TTP expression levels in these cells. Conclusions: For both racial groups, TTP expression was lower in tumor tissues relative to normal tissues; the difference was more pronouced in CRCs of AAs. Further, lower TTP levels correlated with advanced tumor stage; and TTP negatively regulated the expression of IL-8, VEGF, and cIAP2 in cultured cells. These studies were supported by a pre-pilot project of the UAB/TU/MSM Partnership grant of NIH/NCI, U54-CA 118948. Citation Format: Esther A. Suswam, Balanada Dhurjarti Kumar Putcha, Kiera D. Walker, LaJessica Johnson, Jasmine Howard, Edward E. Partridge, Mona N. Fouad, Sejong Bae, Upender Manne. Tristetraprolin suppression is associated with advanced stage colorectal cancer. [abstract]. In: Proceedings of the Sixth AACR Conference: The Science of Cancer Health Disparities; Dec 6–9, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2014;23(11 Suppl):Abstract nr C77. doi:10.1158/1538-7755.DISP13-C77