bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract B44: GTPase/GAP/GEF-Glo™: A bioluminescent system to measure GTPase, GAP, and GEF activities
Authors: Mondal Subhanjan , Goueli Said A. 
Published: May 2015

Abstract
It is well known that GTPases of the Ras superfamily play major roles in cell functions such as cell signaling, cell proliferation, cell differentiation, cytoskeleton modulation and cell motility. Deregulation or mutation of these proteins has considerable consequences resulting in multiple pathological conditions. Targeting GTPases and its regulators have been challenging due to lack of convenient assays.
We have developed a homogenous bioluminescent assay for monitoring the activities of GTPase and its immediate regulators: GAP and GEF. Mg2+ plays a critical role in influencing affinity of GTPases with GTP/GDP. By manipulating Mg2+ concentrations in the reaction buffer allows continuous progression of the GTPase cycle and hydrolysis of GTP. The assay relies on the conversion of remaining GTP to ATP and detection of the latter using luciferin/luciferase combination. The GTPase/GAP/GEF-Glo™ assay system enables monitoring of GTPase-, GAP-stimulated GTPase-, GAP- and GEF- activities. The assay system has a high dynamic range and signal-to-background ratio; it is in a convenient “add-mix-read” format and applicable to high throughput screening.
Citation Format: Subhanjan Mondal, Said A. Goueli. GTPase/GAP/GEF-Glo™: A bioluminescent system to measure GTPase, GAP, and GEF activities. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B44. doi: 10.1158/1557-3125.RASONC14-B44
Abstract B57: Distinct interactions of Ras superfamily members with common protein targets
Authors: Smith Russell Spencer , Wong Katy A. , Russo Angela , O'Bryan John P. 
Published: May 2015

Abstract
Ras superfamily members act as molecular switches to many key signaling pathways, including proliferation cytoskeletal integrity, differentiation and cellular cargo transport. The Ras subfamily, consisting of KRas, NRas and HRas are the most widely studied Ras-family members due to the high instance of Ras activating mutation in human cancers. Rab subfamily members are important regulators of endocytosis, consisting of Rab5 (early endosome), Rab7 (recycling endosome) and Rab11 (late endosome). We have discovered that common binding partners exist between these Ras subfamilies, resulting in distinct interactions and regulatory effects. Two such commonalities are scaffold protein ITSN1 and lipid kinase PI3KC2β. There is evidence that the learning disabilities associated with Down syndrome may be somewhat attributed to deregulated vesicular transport in the brain. DS patients also have a 10-20 fold increased risk of developing acute myeloid leukemia (AML). ITSN1, a regulator of endocytosis and activator of the oncogenic AKT pathway (1), is overexpressed in Down syndrome patients. PI3KC2β is a member of the PI3K family of lipid kinases, which phosphorylate phosphatidylinositol leading to AKT activation. Treatment of neuroblastoma and AML cell lines with PI3KC2β-specific inhibitors greatly decreases proliferation (2) and silencing PI3KC2β in neuroblastoma cell lines results in a 50% reduction in tumor volume in xenograft models. Our previous work demonstrated that nucleotide-free HRas preferentially binds to PI3KC2β, inhibiting its activity (see poster by JP O'Bryan; 3). The addition of ITSN1 disrupts this interaction, presumably restoring the active state of both proteins. In contrast, constitutively activated Rab5 and KRas preferentially interact with PI3KC2β. Interestingly KRas sequesters PI3KC2β to the plasma membrane. The current dogma states that Ras proteins must be GTP-loaded and associated with the plasma membrane to activate their classic effectors suggesting that KRas would be active in this capacity and may therefore positively regulate PI3KC2β activity. Here we present evidence of three Ras superfamily members interacting in distinct ways with the same target protein and provide support for their related yet contrasting roles in disease states.
Citation Format: Russell Spencer Smith, Katy A. Wong, Angela Russo, John P. O'Bryan. Distinct interactions of Ras superfamily members with common protein targets. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B57. doi: 10.1158/1557-3125.RASONC14-B57
Abstract B58: EGFR and IR/IGFR activation sustains ERK1/2 and AKT signaling independently of mutant Kras in pancreatic cancer cells lines
Authors: Takeuchi Kenneth K. , Crawford Howard C. 
Published: May 2015

Abstract
Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer related death in the United States. Roughly 45,000 patients are diagnosed with the disease each year and of these patients 94% will not survive past 5 years. The most common genetic alteration associated with PDAC is an oncogenic mutation in the KRAS gene, which is believed to initiate the disease. Mutant Kras is generally thought to signal constitutively to its downstream effector proteins, such as Raf/MEK/Erk and PI3K/AKT. However, recently we and others have demonstrated that Epidermal Growth Factor Receptor (EGFR) signaling upstream of mutant Kras is necessary for unlocking its oncogenic potential in the pancreas, distinct from Kras driven tumorigenesis in the lung or colon. Unfortunately, while EGFR inhibition is effective in blocking pancreatic tumorigenesis in mice, it has limited benefit in treating human PDAC patients. Furthermore, it has been shown that wild-type Nras and Hras have indispensable signaling roles in cells expressing oncogenic Kras, and that both the mutant and WT Ras signals are required for cell growth. Here we show that pancreatic cancer cell lines containing oncogenic Kras still require upstream signaling to maintain high levels of both active Erk and AKT although these signals are not necessarily EGFR derived. We have identified a subset of pancreatic cancer cell lines that rely upon IR/IGFR signaling to maintain the high levels of Erk and AKT signaling required for continued cell growth. Additionally, this signaling appears to be independent of oncogenic Kras and instead correlates with the levels of active wild-type Nras and Hras. This data indicates that patients may benefit from the simultaneous inhibition of both the EGFR and IR/IGFR signaling pathways.
Citation Format: Kenneth K. Takeuchi, Howard C. Crawford. EGFR and IR/IGFR activation sustains ERK1/2 and AKT signaling independently of mutant Kras in pancreatic cancer cells lines. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B58. doi: 10.1158/1557-3125.RASONC14-B58
Abstract IA01: Ras membrane localization, ciliary trafficking and anti-Ras drug candidates
Authors: Wittinghofer Alfred 
Published: May 2015

Abstract
GTP-binding (G) proteins are molecular switches that cycle between a GDP-bound OFF and a GTP-bound ON state and are regulated by Guanine nucleotide exchange factors (GEFs) and GTPase Activating Proteins (GAPs). The Ras superfamily of G proteins are 20-25 kDa proteins involved in almost every aspect of cell function. Members of the Ras subfamily such as the oncoprotein Ras itself or RheB, but also ciliary proteins such as the inositol phosphatase INPP5E or the α-subunit of phosphodiesterase PDE6, have a C-terminal CaaX motif, the cysteine residue of which is modified with the lipophilic farnesyl moiety. The farnesyl group and further modifications are required for membrane attachment and function. Spatiotemporal regulation of Ras proteins relies on a highly dynamic shuttling between different membrane compartments. Transport between membrane compartments in the cell is mediated by factors such as PDEδ, the delta subunit of phosphodiesterase and possibly similar proteins.
Arl3 is a member of the Arf subfamily of the Ras superfamily. In the GTP-bound form Arl3 interacts with a variety of proteins called downstream effectors. These are the delta subunit of phosphodiesterase PDEδ, HRG4 (human retina gene 4) also called Unc119 and various other proteins such as BART (Binder of Arl2). Arl3 regulates the binding of ciliary and other cargo to PDEδ and HRG4.
We will discuss how spatiotemporal regulation of Ras proteins is mediated by the Arl2 and 3 conformational switch cycle and how this has suggested a new avenue towards anti-Ras drugs.
Citation Format: Alfred Wittinghofer. Ras membrane localization, ciliary trafficking and anti-Ras drug candidates. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr IA01. doi: 10.1158/1557-3125.RASONC14-IA01
Abstract IA02: Developing therapies for Ras-driven cancers
Authors: Cichowski Karen 
Published: May 2015

Abstract
The Ras pathway is one of the most commonly deregulated pathways in cancer. However Ras-driven tumors are often refractory to conventional therapies and a clinically effective targeted therapy has not yet been developed. Moreover, in some cancers KRAS mutations are used to exclude patients from being treated with specific targeted agents. As such, developing an effective targeted therapy for Ras-driven tumors is an important endeavor. We have been focusing on developing therapies for distinct Ras-driven cancers: Nf1-mutant cancers (nervous system malignancies and melanoma) as well KRas/p53-mutant non-small cell lung cancer (NSCLC). We have been using PI3K/mTOR inhibitors as a platform to develop novel combination therapies and have identified several new combinations, a subset of which are being evaluated in the clinic. We have also been deconstructing the mechanism by which these agents cooperate, which is providing insight into developing useful biomarkers and additional drug combinations.
Citation Format: Karen Cichowski. Developing therapies for Ras-driven cancers. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr IA02. doi: 10.1158/1557-3125.RASONC14-IA02
Abstract IA03: Regulation of K-Ras function by lysine acetylation
Authors: Haigis Kevin M. 
Published: May 2015

Abstract
Activating point mutations in K-Ras are common in cancers of the lung, colon, and pancreas and are highly predictive of poor therapeutic response. To date, there are no effective therapies to combat the extreme oncogenicity of mutant K-Ras. One potential strategy for overcoming the deleterious effects of mutant K-Ras is to alter its post-translational modification. While therapies targeting farnesylation have been explored, and ultimately failed, the therapeutic potential of targeting other modifications remains to be seen. In this regard, we have found that K-Ras is acetylated on lysine 104 (K104), a residue that is conserved in all Ras homologs from humans to budding yeast. Acetylation on K104 does not affect K-Ras localization, like many of the post-translational modifications of Ras, but instead confers resistance to GEF-induced nucleotide exchange by destabilizes the alpha helical structure of the Switch II domain. Mutation of K104 to glutamine, which mimics constitutive acetylation, attenuates GTP-loading and the transforming activity of K-Ras. These results indicate that acetylation is a negative regulatory modification on K-Ras and provide a new angle in the search for cancer therapies that target K-Ras post-translational modification. In order to translate this knowledge into a therapeutic strategy, however, we must identify the enzymes that regulate K-Ras acetylation. Ideally, we would identify the deacetylases, as inhibiting these enzymes would lock K-Ras into its hyper-acetylated, inactive state. Recently, we identified HDAC6 and SIRT2 as deacetylases that regulate the acetylation state of K-Ras in cancer cells. Genetic or chemical inhibition of either of these enzymes dramatically affects the growth properties of cancer cell lines expressing mutationally activated K-Ras, suggest that therapeutic targeting of HDAC6 and/or SIRT2 may represent a new way to treat cancers expressing mutant forms of K-Ras.
Citation Format: Kevin M. Haigis. Regulation of K-Ras function by lysine acetylation. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr IA03. doi: 10.1158/1557-3125.RASONC14-IA03
Abstract IA05: Next-generation strategies to target RAF
Authors: Bollag Gideon 
Published: May 2015

Abstract
RAF is a well-studied effector of RAS. Oncogenic activation of BRAF fuels cancer growth by constitutively promoting MAPK pathway signaling independent of RAS activation. Efforts to develop agents to block mutated BRAF have brought substantial therapeutic improvement in personalized treatment of metastatic melanoma. Nonetheless, many patients relapse and relapsed tumors generally display MAPK pathway re-activation. The development of these agents also revealed an unexpected consequence of these targeted therapies: stimulated growth of certain cancers. Structurally-diverse ATP-competitive RAF kinase inhibitors can either inhibit or paradoxically activate the MAPK pathway depending whether the pathway is activated by a BRAF mutation or by an upstream event, such as RAS mutation or receptor tyrosine kinase activation. We have identified next generation RAF inhibitors (dubbed “paradox breakers”) that inhibit mutant RAF cells without activating the MAPK pathway in cells containing an upstream activation event. These new inhibitors maintain efficacy in BRAF-driven cell and animal models. In a cell model that expresses the same HRAS mutation prevalent in squamous cell tumors from patients treated with RAF inhibitors, first-generation RAF inhibitors stimulate the in vitro and in vivo growth of the cells and induce the expression of MAPK pathway response genes including EGFR ligands; by contrast paradox breakers such as PLX8394 had no effect. In addition, PLX8394 is able to overcome several known mechanisms of resistance to first generation RAF inhibitors. By dissociating MAPK pathway inhibition from pathway activation, the next generation RAF inhibitors may yield both improved safety and more durable efficacy than first generation RAF inhibitors.
Citation Format: Gideon Bollag. Next-generation strategies to target RAF. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr IA05. doi: 10.1158/1557-3125.RASONC14-IA05
Abstract IA11: Progress in pancreatic cancer modeling
Authors: Tuveson David 
Published: May 2015

Abstract
Although ductal pancreatic cancer remains highly lethal, recent laboratory progress in demystifying this disease have provided several new therapeutic approaches. Somatically mutant KRAS is present in most cases of pancreatic cancer, and mouse models have clarified the role of oncogenic Kras in neoplastic initiation and progression. Surprisingly, methods that target canonical MAPK/PI3K Ras effector pathways have been only partially successful in mouse models.
Therefore, we have also investigated additional aspects of pancreatic tumor biology that contribute to disease pathogenesis. Chief contributors to the unusual desmoplastic structure and drug-refractory nature of pancreatic tumors are activated pancreatic stellate cells. PSCs assume a fibroblastic morphology and function during tumor evolution, resulting in a collagen- and glycosaminoglycan-rich extracellular matrix that prevents angiogenesis and compresses the resident vasculature such that drug delivery is impeded. Additionally, PSCs secrete a variety of ligands, such as Connective Tissue Growth Factor, that promote neoplastic cell survival and thereby suppress therapeutic responses to classical and targeted agents. The role of the ECM and paracrine/autocrine signals in supporting oncogenic Kras signaling are currently being explored in mouse models and additional cell culture systems.
Citation Format: David Tuveson. Progress in pancreatic cancer modeling. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr IA11. doi: 10.1158/1557-3125.RASONC14-IA11
Abstract IA15: Membrane targeting of Ras
Authors: Tsai Frederick , Fehernbacher Nicole , Sung Pamela , Court Helen , Philips Mark R. 
Published: May 2015

Abstract
Ras proteins are targeted to membranes by virtue of post-translational modifications of their C-terminal hypervariable regions (HVRs). Because Ras proteins signal only when associated with cellular membranes, the Ras trafficking pathway is considered an attractive area for anti-cancer drug discovery. The three mammalian Ras genes, hras, nras and kras, give rise to four Ras proteins because the transcript of the kras locus is alternatively spliced to encode K-Ras4A and K-Ras4B. The kras locus is most often mutated in human cancer and when mutated gives rise to oncogenic forms of both K-Ras4A and K-Ras4B. The four Ras protein differ only in their membrane targeting HVRs. Each Ras protein ends in a CAAX sequence, which signals for a three-step modification that includes farnesylation, AAX proteolysis and carboxyl methylation. N-Ras, H-Ras and K-Ras4A are further modified by palmitoylation of one or two cysteines upstream of the CAAX sequence. Palmitoylation of N-Ras and H-Ras occurs on the cytoplasmic face of the Golgi apparatus. Whereas farensylation affords relatively weak membrane affinity, palmitoylation of the farnesylated protein provides stable association with membranes. Palmitoylation is readily reversible and a palmitoylation/depalmitoylation cycle drives a cycle of trafficking to and from the Golgi. K-Ras4B is unique in utilizing a polybasic sequence upstream of the CAAX motif to associate with the plasma membrane through an electrostatic interaction. We have shown that K-Ras4B can be phosphorylated by PKC at serine 181 in the HVR and this modification partially neutralizes the electrostatic charge such that K-Ras4B loses affinity for the plasma membrane and is relocated on endomembranes in a process we have called the farnesyl-electrostatic switch. Because phosphorylation of K-Ras4B is associated with diminished cell viability we have explored the possibility that induction of phosphorylation of oncogenic K-Ras4B may be one way to target K-Ras transformed cells. Other aspects of K-Ras biology that will be discussed are the relative expression of K-Ras4A and K-Ras4B and the unique membrane trafficking of K-Ras4A. A K-Ras4B membrane release assay suitable for siRNA and small compound screening will also be discussed.
Citation Format: Frederick Tsai, Nicole Fehernbacher, Pamela Sung, Helen Court, Mark R. Philips. Membrane targeting of Ras. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr IA15. doi: 10.1158/1557-3125.RASONC14-IA15
Abstract IA17: Discovery of K-Ras inhibitors for the treatment of cancer
Authors: Fesik Stephen W. 
Published: May 2015

Abstract
Aberrant activation of the small GTPase Ras by oncogenic mutation or constitutively active receptor tyrosine kinases (RTKs) results in the deregulation of cellular signals governing growth and survival in cancer. The guanine nucleotide exchange factor Son of Sevenless (SOS) catalyzes the rate-limiting step in the activation of Ras by exchanging GDP for GTP. SOS is therefore a key control point for the propagation of RTK and Ras signaling. Here we report the discovery of small molecules that bind to a unique pocket on the Ras:SOS:Ras complex, increase SOScat-catalyzed nucleotide exchange, and perturb Ras signaling pathways in cells. X-ray crystallographic studies of Ras:SOS:Ras complexed with these small molecules reveal that they bind in a hydrophobic pocket in the CDC25 domain of SOS adjacent to the Switch II region of Ras. The structure-activity relationships exhibited by these compounds can be rationalized on the basis of the x-ray structures of multiple co-complexes. In addition, structure-based mutational analyses indicate that this newly identified pocket is essential for compound activity. As predicted, these molecules increase Ras-GTP levels in cells. However, they unexpectedly inhibit MAPK and PI3K signaling. Our studies suggest a novel way to target K-Ras and offer possible starting points for the discovery of compounds that could be used to treat Ras-driven tumors.
Citation Format: Stephen W. Fesik. Discovery of K-Ras inhibitors for the treatment of cancer. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr IA17. doi: 10.1158/1557-3125.RASONC14-IA17
Abstract A11: Adipocytes promote ovarian cancer cell survival in the presence of chemotherapeutic drugs
Authors: Yang Jiang , Sachdev Monisha , Moses Marsha 
Published: May 2015

Abstract
Ovarian cancer cells, both primary and metastatic, reside in an environment rich in adipocytes. Adipocytes have been shown to promote ovarian cancer metastasis by secreting chemoattractant cytokines and to stimulate growth by directly transferring fatty acids into cancer cells. In addition to metastasis, another key reason for the high mortality rate among ovarian cancer patients is chemoresistance. Whether adipocytes have any effects on ovarian cancer chemoresistance remains unknown. To address this question, we isolated conditioned media from human adipocytes and found that the conditioned media increased ovarian cancer cell survival in the presence of chemotherapeutic drugs. We confirmed the protective effects of adipocytes using different human ovarian cancer cell lines (CAOV3, OVCAR5, OVCAR8 and SKOV3) and multiple chemotherapy drugs (cisplatin, paclitaxel and doxorubicin). Conditioned media dialyzed using a 10kDa cut-off membrane displayed the same protection against chemotherapy drugs, suggesting that proteins, not lipids, are more likely to mediate the pro-survival effects of adipocytes. Adiponectin and Leptin are two of the most upregulated proteins during adipocyte differentiation. However, inhibition of these two proteins, either through siRNA silencing or antibody neutralization, did not diminish the pro-survival effects of adipocytes. We also investigated the downstream signaling pathways and found that Akt was activated by adipocyte-conditioned media and was critical for adipocyte-induced effects given that treating ovarian cancer cells with Akt pathway inhibitor LY294002 abolished the pro-survival effects of the adipocytes. Taken together, these data demonstrate that secreted factors from adipocytes enhance the survival of ovarian cancer cells treated with chemotherapeutic drugs by activating the Akt pathway and suggest that adipocytes and associated factors may be important mediators of ovarian cancer chemoresistance. (This work is supported by the Weitzman Family Fund.)
Citation Format: Jiang Yang, Monisha Sachdev, Marsha Moses. Adipocytes promote ovarian cancer cell survival in the presence of chemotherapeutic drugs. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr A11.