Kiera Walker

Structure‐based approach to pharmacophore identification, in silico screening, and three‐dimensional quantitative structure–activity relationship studies for inhibitors of Trypanosoma cruzi dihydrofolate reductase function

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. Proteins 2008.

Mutant tristetraprolin: a potent inhibitor of malignant glioma cell growth

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 of the 3′ untranslated region 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.

NOS Expression and NO Function in Glioma and Implications for Patient Therapies.

SIGNIFICANCE 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. CRITICAL ISSUES 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. FUTURE DIRECTIONS 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.

Development of a Sox2 reporter system modeling cellular heterogeneity in glioma

BACKGROUND 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. METHODS 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. RESULTS 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. CONCLUSIONS 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.

Addition of carbonic anhydrase 9 inhibitor SLC-0111 to temozolomide treatment delays glioblastoma growth in vivo

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.

Modeling Physiologic Microenvironments in Three-Dimensional Microtumors Maintains Brain Tumor Initiating Cells

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.

Reactive species balance via GTP cyclohydrolase I regulates glioblastoma growth and tumor initiating cell maintenance

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.

Abstract 1925: Modeling physiologic microenvironments in three-dimensional microtumors facilitates brain tumor initiating cell maintenance

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

Abstract C77: Tristetraprolin suppression is associated with advanced stage colorectal cancer

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