bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract A50: Oncogenic K-Ras accelerates cancer stem cell activation via aberrant Wnt/β-catenin signaling
Authors: Moon Byoung-San , Jeong Woo-Jeong , Choi Kang-Yell
Published: May 2015

Oncogenic K-Ras is known to play an important role in tumor growth, but their involvement in the activation of cancer stem cells (CSCs) is not clear. Here, we show the relationship between K-Ras mutation and CSC activation in the tumorigenesis of colorectal cancer (CRC) by systematic in vitro and in vivo studies as well as by clinical evaluations of human specimens. K-Ras mutation is revealed as an important factor for CSC activation through initial APC loss. The critical role of APC mutation in tumorigenesis involving the activation of CSCs by K-Ras mutation was confirmed by genetic studies using APCMin/+ and K-RasG12D(LA2) mice. We therefore propose that CSC activation by both APC and K-Ras mutations was induced via the Wnt/β-catenin signaling pathway, which was initially activated by APC loss and further enhanced through the Ras-ERK pathway.

Citation Format: Byoung-San Moon, Woo-Jeong Jeong, Kang-Yell Choi. Oncogenic K-Ras accelerates cancer stem cell activation via aberrant Wnt/β-catenin signaling. [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 A50. doi: 10.1158/1557-3125.RASONC14-A50
Abstract A52: KRAS: The Rarefied Ras Oncogene
Authors: Pershing Nicole L. , Lampson Benjamin , Counter Christopher
Published: May 2015

The Ras family of small GTPases is comprised of three isoforms, HRAS, NRAS and KRAS, which are mutated in up to a third of all human cancers to promote tumorigenesis. Despite nearly identical amino acid sequence, we discovered divergent codon usage between isoform mRNA sequences. Specifically, KRAS is enriched in underrepresented (rare) codons relative to mammalian transcripts, whereas HRAS is characterized by common codons and NRAS is composed of an intermediate codon mixture. Rare codons can limit protein expression, and we tested and found that successively changing rare to common codons increased KRAS protein expression. To determine the impact of rare codon bias on KRAS-driven carcinogenesis, we converted rare to common codons in exon 3 of the endogenous Kras gene, then treated heterozygous Krasex3op/wt mice with the carcinogen urethane to induce Kras mutation-positive lung tumors. We now report that the multiplicity of tumors in these Krasex3op/wt mice was significantly reduced, and only weaker oncogenic mutations were detected in the Krasex3op allele compared to the Kraswt allele. Since high oncogenic RAS expression can induce a senescent growth arrest instead of transformation to a tumorigenic state, we hypothesized that converting rare to common codons in Kras to increase protein expression may augment senescence in response to activating mutations. Indeed, ectopic expression of oncogenic Krasex3op induced a stronger growth arrest in cultured cells than the oncogenic version of the native Kras. In summary, we have identified a novel regulatory mechanism hard-wired into the RAS genes that has a direct impact on tumorigenesis.

Citation Format: Nicole L. Pershing, Benjamin Lampson, Christopher Counter. KRAS: The Rarefied Ras Oncogene. [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 A52. doi: 10.1158/1557-3125.RASONC14-A52
Abstract B03: Synergistic control of GBM growth by MEK and PI3kinase signaling in a RAS-driven preclinical orthotopic model for human glioblastoma multiforme
Authors: Meskini Rajaa El , Iacovelli Anthony , Kulaga Alan , Gumprecht Michelle , Martin Philip , Baran Maureen , Householder Deborah , Dyke Terry van , Ohler Zoe Weaver
Published: May 2015

PI3K, RTK/RAS, and Rb signaling are commonly altered in human GBM. We utilized a genetically engineered mouse (GEM) model for GBM, designated “TRP,” that expresses GFAP-T121 to suppress Rb (T), the KrasG12D mutation (R), and is heterozygous for a PTEN null allele (P), to develop an efficient and tractable orthotopic mouse model for GBM treatment preclinical evaluation. The orthotopic brain tumors presented linear foci of necrosis with peudopalisading by neoplastic cells described in human GBM, and were highly proliferative, invasive, and vascular. Immunohistochemistry analysis of TRP orthotopic tumors identified markers characteristic of human GBM, and tumor progression was readily examined by serial MRI.

Both PI3K and MAPK pathway inhibitors inhibited growth of TRP GEM-derived primary tumor cells, but did not result in significant apoptosis. However, when GBM derived cells were treated with a combination of pathway inhibitors such as BKM120 (a pan-PI3K inhibitor currently in clinical trials for solid tumors) and PD0325901 (a MEK inhibitor), potency was enhanced with a substantial increase in cell death. Analysis of downstream targets revealed a synergistic effect on target downregulation in the PI3K pathway. In the orthotopic GBM model, the PI3K/MAPK targeted combination delayed tumor growth by increasing GBM cell apoptosis and resulted in significantly increased survival. Taken together, our results validate this new orthotopic model for the assessment of targeted therapeutic regimens for human GBM, and show that the feedback loops and incomplete pathway suppression, observed in vivo with targeted single agents, can be overcome with a combination treatment strategy.

Citation Format: Rajaa El Meskini, Anthony Iacovelli, Alan Kulaga, Michelle Gumprecht, Philip Martin, Maureen Baran, Deborah Householder, Terry van Dyke, Zoe Weaver Ohler. Synergistic control of GBM growth by MEK and PI3kinase signaling in a RAS-driven preclinical orthotopic model for human glioblastoma multiforme. [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 B03. doi: 10.1158/1557-3125.RASONC14-B03
Abstract B15: NRASG12V oncogene mediates self-renewal in a murine model of acute myelogenous leukemia
Authors: Sachs Zohar , LaRue Rebecca S. , Nguyen Hanh T. , Sachs Karen , Hassan Nurul Azyan Mohd , Diaz-Flores Ernesto , Rathe Susan K. , Sarver Aaron L. , Bendall Sean C. , Ha Ngoc A. , Diers Miechaleen D. , Nolan Garry P. , Shannon Kevin M. , Largaespada David A.
Published: May 2015

Mutant RAS oncoproteins activate signaling molecules that drive oncogenesis in multiple human tumors including acute myelogenous leukemia (AML). However, the specific functions of these pathways in AML are unclear, thwarting the rational application of targeted therapeutics. To elucidate the downstream functions of activated NRAS in AML, we employed a murine model that harbors Mll-AF9 and a tetracycline repressible, activated NRAS (NRASG12V). We performed gene expression microarray and RNA sequencing of our AML cells in the presence and absence of NRASG12V. By employing computational approaches to explore NRASG12V-responsive genes in our model, we found that NRASG12V enforced the leukemia self-renewal gene expression signature and was required to maintain an MLL-AF9 and Myb-dependent leukemia self-renewal gene expression program. In functional assays, NRASG12V was required for leukemia self-renewal independently of its effects on growth and survival. We used CyTOF (mass cytometry) for a multiplexed analysis of RAS-dependent signaling intermediates, and found that Mac-1Low cells, which harbor leukemia stem cells, were preferentially sensitive to NRASG12V withdrawal. Using RAS-pathway inhibitors, we found NRASG12V maintained leukemia self-renewal through mTor and Mek pathway activation, implicating these pathways as potential targets for cancer stem cell-specific therapies. Together, these experimental results define a RAS oncogene-driven function that is critical for leukemia maintenance and represents a novel mechanism of oncogene addiction. Recent work has shown that NRASG12V has bimodal effects in hematopoietic stem cells (Li et al. Nature 2013). To understand the mechanism of these bimodal effects, we have performed single cell RNA sequencing on our AML model. We expect that these analyses will reveal the cell-type specific NRASG12V –mediated mechanisms of leukemia self renewal.

Citation Format: Zohar Sachs, Rebecca S. LaRue, Hanh T. Nguyen, Karen Sachs, Nurul Azyan Mohd Hassan, Ernesto Diaz-Flores, Susan K. Rathe, Aaron L. Sarver, Sean C. Bendall, Ngoc A. Ha, Miechaleen D. Diers, Garry P. Nolan, Kevin M. Shannon, David A. Largaespada. NRASG12V oncogene mediates self-renewal in a murine model of acute myelogenous leukemia. [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 B15. doi: 10.1158/1557-3125.RASONC14-B15
Abstract B22: Combined MEK and JAK inhibition rescues mutant hematopoietic stem cell function and provides long-term survival in NrasG12D/G12D mice
Authors: Kong Guangyao , Mark Wunderlich , Yang David , Ranheim Erik A. , Young Ken H. , Wang Jinyong , Chang Yuan-I , Du Juan , Liu Yangang , Tey Sin Ruow , Zhang Xinmin , Juckett Mark , Mattison Ryan , Damnernsawad Alisa , Zhang Jingfang , Mulloy James C. , Zhang Jing
Published: May 2015

Overactive Ras signaling is prevalent in human juvenile and chronic myelomonocytic leukemias (JMML/CMML) that are refractory to conventional chemotherapy. Conditional activation of endogenous NrasG12D/G12D in hematopoietic cells of mice leads to an acute myeloproliferative disease (MPN) that recapitulates many features of JMML and myeloproliferative variant of CMML. We found that as JMML/CMML initiating cells, NrasG12D/G12D hematopoietic stem cells (HSCs) show strong ERK1/2 hyperactivation, hyperproliferation, and depletion with concomitant expansion of downstream progenitors. MEK pathway inhibition alone prolongs the presence of mutant HSCs, but fails to restore their proper function. Consequently, ∼60% of NrasG12D/G12D mice treated with MEK inhibitor alone died within 20 weeks and the remaining animals also displayed significant JMML/CMML-like phenotypes. In contrast, simultaneous inhibition of JAK/STAT signaling, a commonly hyperactivated pathway in human and mouse CMML, more potently inhibits human and mouse CMML cell growth in vitro, sustainably rescues mutant HSC function in vivo, and promotes long-term survival without evident disease manifestation in NrasG12D/G12D mice. Our results provide a strong rationale for combined targeting of MEK/ERK and JAK/STAT in treating JMML and CMML patients.

Citation Format: Guangyao Kong, Wunderlich Mark, David Yang, Erik A. Ranheim, Ken H. Young, Jinyong Wang, Yuan-I Chang, Juan Du, Yangang Liu, Sin Ruow Tey, Xinmin Zhang, Mark Juckett, Ryan Mattison, Alisa Damnernsawad, Jingfang Zhang, James C. Mulloy, Jing Zhang. Combined MEK and JAK inhibition rescues mutant hematopoietic stem cell function and provides long-term survival in NrasG12D/G12D mice. [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 B22. doi: 10.1158/1557-3125.RASONC14-B22
Abstract B27: SERPINE1 expression is required for HDACi-induced proliferative arrest in ras-transformed epithelial cells
Authors: Higgins Paul J.
Published: May 2015

Malignant transformation of mammalian cells with ras family oncogenes results in dramatic changes in cellular architecture and growth traits. The generation of flat revertants of v-K-ras-transformed epithelial cells by exposure to the histone deacetylase inhibitor sodium butyrate (NaB) was found to be dependent on transcriptional activation of the SERPINE1 gene (encoding the type-1 inhibitor of urokinase and tissue-type plasminogen activators; PAI-1). NaB-initiated SERPINE1 expression preceded induced cell spreading and G1 arrest. To assess the relevance of SERPINE1 induction to proliferative blockade in this cell system more critically, several complementary approaches were used. The addition of a stable, long half-life, recombinant SERPINE1 mutant to SERPINE1-deficient v-K-ras-/c-Ha-ras-transformants or to SERPINE1 functionally null, NaB-resistant, 4HH cells (engineered by antisense knockdown of SERPINE1 mRNA transcripts) resulted in marked cytostasis in the absence of NaB. Transfection of ras-transformed cells with the Rc/CMVSERPINE1 expression vector or infection with an adenoviral SERPINE1 construct, moreover, significantly elevated constitutive SERPINE1 synthesis (10- to 20-fold) with a concomitant reduction in proliferative rate. These data suggest that high-level SERPINE1 expression suppresses growth of chronic ras-oncogene transformed cells and is likely a major cytostatic effector of NaB exposure. Supported by NIH grant GM057242.

Note: This abstract was not presented at the conference.

Citation Format: Paul J. Higgins. SERPINE1 expression is required for HDACi-induced proliferative arrest in ras-transformed epithelial cells. [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 B27. doi: 10.1158/1557-3125.RASONC14-B27
Abstract B29: A critical role of the SUMO pathway in KRAS-driven oncogenesis
Authors: Yu Bing , Swatkoski Steve , Holly Alesia , Lee Liam Changwoo , Giroux Valentin , Lee Chih-Shia , Carver Joseph , Creighton Chad , Ann David K. , Figg William Douglas , Gucek Marjan , Luo Ji
Published: May 2015

The SUMOylation pathway modulates the activity of many cellular proteins and plays an important role in cellular stress protection. We identified the SUMO E2 ligase Ubc9 to be critical for KRAS-driven transformation in colorectal cancer cells. Ubc9 knockdown severely impairs clonogenic and anchorage-independent growth of KRAS mutant cells in vitro and attenuates tumor growth in xenograft models. Ubc9 mutant studies indicate that Ubc9 enzymatic activity is required for KRAS-driven transformation, thus Ubc9 inhibitor could be potentially useful in this setting. SILAC mass-spectrometry reveals that the SUMOylation of multiple proteins are elevated in KRAS mutant cells. Over-expression of some of these SUMO target proteins partially rescued transformation growth of KRAS mutant cells upon Ubc9 depletion. Our findings suggest a model where the SUMO pathway is required for relieving the oncogenic stress associated with KRAS transformation to maintain cell viability.

Citation Format: Bing Yu, Steve Swatkoski, Alesia Holly, Liam Changwoo Lee, Valentin Giroux, Chih-Shia Lee, Joseph Carver, Chad Creighton, David K. Ann, William Douglas Figg, Marjan Gucek, Ji Luo. A critical role of the SUMO pathway in KRAS-driven oncogenesis. [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 B29. doi: 10.1158/1557-3125.RASONC14-B29
Abstract B30: Differential network reprogramming by distinct activating K-Ras mutations
Authors: Hammond Dean E. , Mageean Craig J. , Rusilowicz Emma V. , Clague Michael J. , Prior Ian A.
Published: May 2015

In colon cancer, KRAS is most frequently mutated at codon 12, but a significant fraction of patients have mutations at codon 13. Clinical data suggest different outcomes and responsiveness to treatment between these two groups. To investigate the differential effects upon cell status associated with KRAS mutations we performed a quantitative analysis of the proteome and phospho-proteome of isogenic SW48 colon cancer cell lines. Parental cells were compared with cells in which one allele of the endogenous gene has been edited to harbour specific KRAS mutations (G12V, G12D or G13D). Each mutation generates a distinct signature, although the codon 12 mutants overlap significantly whereas G13D is highly divergent and shows more potent up-regulation of phosphopeptide responses. Codon-specific responses within our dataset will be highlighted. Our work raises fundamental questions about basic Ras biology with implications for the design and interpretation of large-scale comparative studies analysing oncogenic Ras signalling. Furthermore, the application of SILAC based quantitative proteomics within a panel of isogenic cell lines represents a promising pipeline for target and biomarker discovery.

Citation Format: Dean E. Hammond, Craig J. Mageean, Emma V. Rusilowicz, Michael J. Clague, Ian A. Prior. Differential network reprogramming by distinct activating K-Ras mutations. [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 B30. doi: 10.1158/1557-3125.RASONC14-B30
Abstract B35: Dual inhibitors of FT and GGT-1 as novel therapeutic agents for K-Ras-dependent tumors
Authors: Aslamuzzaman Kazi , Zhang Xiaolei , Luo Yunting , Patel Ronil , Fletcher Steven , Cummings Christopher , Lawrence Harshani , Hamilton Andrew , Sebti Said M.
Published: May 2015

Inhibition of mutant H-Ras farnesylation with farnesyltransferase inhibitors (FTIs) blocks its binding to membranes and its ability to activate oncogenic signaling. In contrast, inhibition of K-Ras farnesylation with FTIs leads to its prenylation by geranylgeranyltransferase I (GGT-1), and therefore, inhibition of K-Ras prenylation and oncogenic function requires blocking both FT and GGT-1. Furthermore, several proteins downstream of K-Ras that mediate its malignant transforming activity also require farnesylation (e.g.Rheb) or gernaylgeranylation (e.g. Ral). In addition, the ability of mutant K-Ras to induce lung cancer in mouse models is severely hampered when both FT and GGT-1 are conditionally deficient. These observations prompted us to design small molecule dual FT and GGT-1 inhibitors. In this presentation, the development of FGTI-2734 as a novel therapeutic for K-Ras dependent cancers will be described. The presentation will focus on the effect of this dual inhibitor as compared to the selective FTI-2148 and GGTI-2418 on K-ras prenylation, oncogenic signaling and malignant transformation in cancer cells that depend on K-Ras.

Citation Format: Kazi Aslamuzzaman, Xiaolei Zhang, Yunting Luo, Ronil Patel, Steven Fletcher, Christopher Cummings, Harshani Lawrence, Andrew Hamilton, Said M. Sebti. Dual inhibitors of FT and GGT-1 as novel therapeutic agents for K-Ras-dependent tumors. [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 B35. doi: 10.1158/1557-3125.RASONC14-B35
Abstract B41: Direct inhibition of oncogenic KRAS by hydrocarbon-stapled SOS1 helices
Authors: Leshchiner Elizaveta S. , Bellairs Joseph , Bird Gregory H. , Opoku-Nsiah Kwadwo , Godes Marina , Walensky Loren D.
Published: May 2015

Activating mutations in KRAS represent the most frequent oncogenic driving force among the RAS homologs K-, N- and H-RAS, and are associated with poor prognosis and chemoresistance. KRAS mutations are present in approximately 30% of tumors, and at even higher frequencies in cancers of the pancreas, lung, thyroid gland, colon, and liver. In pancreatic ductal adenocarcinomas (PDAC), which carries a 5-year survival rate of less than 5%, activating KRAS mutations are present in over 90% of tumors. Moreover, these mutations have been causally linked to the initiation and progression of PDAC. Despite the mechanistic insights into KRAS-mediated oncogenesis, development of high affinity targeted inhibitors remains a formidable challenge. Here, we report the synthesis and application of hydrocarbon-stapled peptides as prototype therapeutics for blocking wild type and mutant KRAS in vitro and in cancer cells. Stabilized Alpha-Helices of SOS1 (SAH-SOS1) were generated by inserting hydrocarbon staples into SOS1 peptide sequence to recapitulate the alpha-helical structure of the native KRAS-interaction domain. SAH-SOS1 peptides, but not negative control analogs, bound to a variety of KRAS constructs with nanomolar affinity, inhibiting nucleotide exchange. This sequence-specific biochemical activity correlated with impairment of KRAS-driven cancer cell viability and signal transduction. These studies provide proof-of-concept for the utility of SAH-SOS1 peptides in dissecting and targeting the oncogenic KRAS pathway in human cancer.

Citation Format: Elizaveta S. Leshchiner, Joseph Bellairs, Gregory H. Bird, Kwadwo Opoku-Nsiah, Marina Godes, Loren D. Walensky. Direct inhibition of oncogenic KRAS by hydrocarbon-stapled SOS1 helices. [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 B41. doi: 10.1158/1557-3125.RASONC14-B41
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

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

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

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

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

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

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

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

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

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

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

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.
Abstract A18: Genome-wide methylation profiling to identify potential epigenetic biomarkers associated with response to sunitinib in metastatic renal cell cancer patients (pts)
Authors: Kim Jenny , Labots Mariette , Verheul Henk , Carducci Michael , Begum Shahnaz , Netto George , Hoque Mohammad
Published: May 2015

Background: Sunitiinib is a vascular endothelial growth factor receptor tyrosine kinase inhibitor (VEGFR TKI) that is widely used in the front line setting for the treatment of advanced renal cell cancer. As with other targeted agents, there is a significant heterogeneity in the clinical response to sunintib and a biomarker which would predict patients' response up front would be an invaluable tool in the clinical management of patients (pts) afflicted with this disease. In this regard, whole genome methylation array was performed between sunitinib responders and nonresponders to identify differentially methylated genes between these two subsets of pts.

Methods: mRCC pts. who received sunitinib therapy with available frozen nephrectomy tissues and clinical data were identified. Responders (RES) were identified as patients with progression free survival (PFS)of >11 months (mos) and nonresponders (NRES) as those with PFS <3 mos. Whole genome methylation array was performed using Infinium Humanmethylation450 BeadChip Kit. The statistical analysis performed was based on one-way ANOVA and an empirical Bayes approach was used to moderate standard errors. The QC plots showed that all arrays were fine and successful.

Results: Thirteen pts treated with sunitinib for mRCC with available frozen nephrectomy tissues as well as clinical data were identified. Five were selected for each RES and NRES cohort as described above. Three pts had PFS that ranged between 3-11 mos (INT). All pts. In RES had clear cell subtype. Two of the NRES group were of non-clear cell type. For RES vs. NRES, one genomic location, DENND2D, was significantly hypermethylated in the RES group with a false discovery rate (FDR) of <10%. For RES+INT vs NRES, DENND2D hypermethylation was also seen in the RES+INT group but 4 other genes (TTYH3, TRAPPC12, HLA-DRB6 and cg15042082) were shown to be hypermethylated in the NRES group, also with a FDR<10%.

Conclusion: In this study, DENND2D was significantly hypermethylated in sunitinib RES vs. NRES in mRCC pts. Technical and clinical validation of DENND2D and the other 4 differentially methylated genes are planned as is immunohistochemistry study of DENND2D to evaluate downstream effects of methylation.

Citation Format: Jenny Kim, Mariette Labots, Henk Verheul, Michael Carducci, Shahnaz Begum, George Netto, Mohammad Hoque. Genome-wide methylation profiling to identify potential epigenetic biomarkers associated with response to sunitinib in metastatic renal cell cancer patients (pts). [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 A18.