bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract A46: A pan-cancer RAS mutant library elucidates the transformation potential of RAS variants
Authors: Moore Amanda R. , Fortin Jean-Philippe , Costa Michael , Callow Marinella , Haley Benjamin , Manning Gerard , Sokol Ethan S. , Malek Shiva 
Published: June 2021

Abstract
RAS mutations are highly prevalent in tumors, especially those of the lung, colon, skin and pancreas. The tissues in which these tumors arise are enriched for specific RAS isoform and codon preferences. For example, mutations in KRAS are prevalent in lung, colon and pancreas, while NRAS mutations are predominantly found in melanoma. Additionally, mutations in KRAS predominantly occur at the hotspot codons G12 or G13 and NRAS mutations predominantly occur at the hotspot codon Q61. Independent of the RAS isoform, the mutated codon and specifically the amino acid substitution at that codon contribute to distinct biochemical differences. We aim to address if different amino acid substitutions at hotspot codons induce differential transformation, i.e., KRAS G12C versus KRAS G12D. Further, we aim to address if different codons are disposed to differential transformation capabilities and if these are isoform specific, for example, if KRAS Q61 mutations are similar to KRAS G12 mutations and if KRAS Q61 mutations are similar to NRAS Q61 mutations. Additionally, we aim to characterize the transforming capacity of RAS alterations outside of hotspot codons. Hundreds of sporadic insertions and point mutations in RAS have been observed clinically, yet most of these alterations have not been experimentally investigated. To address these questions, we created a pooled library of 184 RAS (KRAS, NRAS and HRAS) alterations identified in 29,000 tumors from a panel of ~100,000 pan-cancer tumors. These alterations include missense, insertion and deletion mutations irrespective of the incidence in which they occur. The library contains the greatest representation of KRAS alterations, 104. Using this pooled library, we measured the relative proliferation effect of these RAS variants using Ba/F3 cells in a competition assay. In interleukin 3 (IL-3)-dependent murine pro B-cells, Ba/F3, expression of well-characterized RAS hotspot mutations led to IL-3-independent proliferation. Due to the robust Ba/F3 cellular transformation we observed upon expression of RAS variants, we sought to use this assay for the pooled-library screen. The Ba/F3 system enables us to interrogate the transformation capacity of the RAS alterations and to identify the relative strength of these mutations while assaying independent of tissue type.
Citation Format: Amanda R. Moore, Jean-Philippe Fortin, Michael Costa, Marinella Callow, Benjamin Haley, Gerard Manning, Ethan S. Sokol, Shiva Malek. A pan-cancer RAS mutant library elucidates the transformation potential of RAS variants [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A46.
Abstract B01: Inhibition of RAS signaling and tumorigenesis through targeting vulnerabilities in RAS biochemistry
Authors: Khan Imran , Spencer-Smith Russell , Teng Kevin , Koide Akiko , Koide Shohei , O'Bryan John P. 
Published: June 2021

Abstract
RAS GTPases are important mediators of oncogenesis in humans. However, pharmacologic inhibition of RAS has proved challenging. We have taken a novel approach to discover vulnerabilities in RAS that can be exploited to inhibit RAS signaling and tumorigenesis. Monobodies are single-domain synthetic binding proteins that achieve levels of affinity and selectivity similar to antibodies but are insensitive to the redox potential of their environment. We have developed a panel of monobodies that target distinct vulnerabilities in RAS. We recently described the activity of the NS1 monobody at inhibiting RAS signaling. NS1 binds to the α4-α5 allosteric lobe of RAS to prevent RAS dimerization and nanoclustering. When introduced into cells as a genetically encoded reagent, NS1 inhibits RAS signaling and oncogenic transformation in vitro through blocking the ability of RAS to self-associate and stimulate the dimerization and activation of RAF. Using a chemically regulated NS1 expression system, we demonstrate that targeting the α4-α5 dimerization interface with NS1 inhibits KRAS-driven tumors in vivo. In addition to NS1, we will discuss our results with monobodies targeting additional aspects of RAS biochemistry. Our results establish the importance of RAS dimerization through the α4-α5 region in mediating RAS signaling and oncogenic transformation of cells both in vitro and in vivo and reveal additional vulnerabilities in RAS that may be targeted to inhibit RAS-driven tumors.
Citation Format: Imran Khan, Russell Spencer-Smith, Kevin Teng, Akiko Koide, Shohei Koide, John P. O'Bryan. Inhibition of RAS signaling and tumorigenesis through targeting vulnerabilities in RAS biochemistry [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B01.
Abstract B03: Biologic and biochemical interactions of NF1 GAP on KRAS G13x mutations
Authors: Rabara Dana , Stephens Robert , Holderfield Matthew 
Published: June 2021

Abstract
KRAS mutations are common among 30% of cancers and promote tumor growth by constitutively activating the MAPK pathway independent of mitogenic stimuli. KRAS mutations at codons 12, 13, or 61 stabilize the KRAS-GTP complex by preventing GAP protein-stimulated GTP hydrolysis. There are two major RAS-GAP proteins, NF1 and p120, and genetic deletions or inactivating mutations of NF1 GAP also constitutively activate RAS proteins and the MAPK pathway by increasing cellular RAS-GTP levels, but KRAS and NF1 mutations are thought to be functionally redundant and rarely occur in the same tumor. Surprisingly, results from clinical trials in mCRC patients with KRAS G13D mutations suggest that inhibition of upstream receptor tyrosine kinases may be effective in a subset of patients, but the underlying mechanism has never been explained. Here, we show that approximately 50% of cell lines with KRAS codon 13 mutations also carry NF1 mutations, but NF1 mutations are rarely observed in cell lines with KRAS codon 12 or 61 mutations. We hypothesize that presence or lack of a functional NF1 determines the response to EGFR inhibitors. Here we used SW48 isogenic KRAS-mutated cell lines to show that presence of NF1 sensitizes KRAS wild-type and KRAS G13D mutated cells to EGFR inhibition, and NF1 knockdown rescues proliferation in KRAS G13D but not KRAS G12D cells. Furthermore, introducing NF1 into G13D mutated cancer cells impairs KRAS signal transduction, suggesting that NF1 may be active in KRAS G13x mutated cells. To test this directly, we used biochemical assays to study G13D KRAS mutants with NF1. The intrinsic GTPase activity of KRAS is very low, and NF1 or p120 GAP accelerates GTP hydrolysis of KRAS wild-type significantly. NF1 and p120 GAP are inactive on KRAS G12D. However, NF1, but not p120 GAP, notably stimulates GTP hydrolysis of KRAS G13D. These results demonstrate that KRAS G13D is partially responsive to NF1-GAP stimulated hydrolysis and suggest that KRAS G13x cancers that have a functional NF1 may respond to EGFR inhibition.
Citation Format: Dana Rabara, Robert Stephens, Matthew Holderfield. Biologic and biochemical interactions of NF1 GAP on KRAS G13x mutations [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B03.
Abstract B08: ETC inhibitors alter oncogenic KRAS signal transduction
Authors: Sharma Kanika , Yi Ming , Grandinetti Giovanna , Chougule Ashish B. , Liaw Philip , Yanofsky Stephen , Ungashe Solomon , Thomas Jeffery H. , Garland William , Holderfield Matthew 
Published: June 2021

Abstract
KRAS proteins regulate many cellular processes and gain-of-function KRAS mutations constitutively activate effector signal transduction pathways independent of mitogenic stimuli. Oncogenic KRAS-driven cancers also frequently have high glycolytic rates and may have increased sensitivity to metabolic inhibitors. In this study, we characterize a small molecule found to be active in a drug screen that uses transgenic Drosophila melanogaster expressing human KRASG12V in the wing to assess kRAS suppressing activity. The compound partially restored normal wing development in the KRASG12V flies and inhibited growth and signal transduction in multiple oncogenic KRAS-driven cell lines. It also inhibited Complex 1 of the electron transport chain (ETC). Increased glycolysis or addition of exogenous aspartate was sufficient to rescue KRAS function and cellular growth after ETC inhibition. These data indicate that oncogenic KRAS signal transduction requires high cellular ATP and amino acid biosynthesis and suggest a synthetic lethal interaction between KRAS and metabolic targets. We also describe co-mutations that sensitize KRAS-driven cell lines to OxPhos Inhibition and show how differential OxPhos inhibitor sensitivity in lung and pancreatic cancer cell lines can correlate with low and high glycolysis ssGSEA gene signatures.
Citation Format: Kanika Sharma, Ming Yi, Giovanna Grandinetti, Ashish B. Chougule, Philip Liaw, Stephen Yanofsky, Solomon Ungashe, Jeffery H. Thomas, William Garland, Matthew Holderfield. ETC inhibitors alter oncogenic KRAS signal transduction [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B08.
Abstract B13: Parallel targeting of RAF/MEK/ERK pathway in RAS-mutant embryonal rhabdomyosarcoma
Authors: Vaseva Angelina V. , Bandyopadhyay Abhik , Pozzo Vanessa Del , Goodwin Craig M. , Gautam Prson , Wennerberg Krister , Wood Kris C. , Der Channing J. , Houghton Peter J. 
Published: June 2021

Abstract
Recent sequencing of childhood rhabdomyosarcoma identified the presence of RAS pathway mutations in nearly 75% of high-risk embryonal rhabdomyosarcoma (ERMS). While RAS oncoproteins (HRAS, NRAS and KRAS) are well-established therapeutic targets for many adult human cancers, still very little is known about the role of RAS mutations in the development and maintenance of ERMS. We sequenced ERMS cell lines and PDX tumors and identified 4 cell lines harboring activating mutations in H- or NRAS and two cell lines with wild-type RAS. By siRNA mediated knockdown, we showed that mutant H- or NRAS was critical for the growth of all RAS-mutant ERMS cell lines and that RAF/MEK/ERK signaling pathway, but not PI3K/AKT, was mediator of RAS dependency in these cells. However, in vivo treatment of RAS-mutant ERMS xenografts with the MEK inhibitor trametinib showed modest response as compared to BRAF-mutant astrocytoma xenografts. We reasoned that similarly to other RAS-driven cancers, ERMS tumors are able to acquire resistance to inhibitors of the RAF/MEK/ERK pathway. We performed CRISPR screen and identified that inhibition of ERK2 potentiated trametinib treatment. We show that combining trametinib with ERK1/2 inhibitor leads to potent synergistic MEK/ERK pathway inhibition and ERMS tumor growth suppression.
Citation Format: Angelina V. Vaseva, Abhik Bandyopadhyay, Vanessa Del Pozzo, Craig M. Goodwin, Prson Gautam, Krister Wennerberg, Kris C. Wood, Channing J. Der, Peter J. Houghton. Parallel targeting of RAF/MEK/ERK pathway in RAS-mutant embryonal rhabdomyosarcoma [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B13.
Abstract B16: Role of RasGRF2 in AnxA6-mediated growth of TNBC cells
Authors: Whalen Diva S. , Widatalla Sarrah E. , Korolkova Olga Y. , Williams Stephen D. , Beasley Heather K. , Sakwe Amos M. 
Published: June 2021

Abstract
Introduction: The mechanisms by which annexin A6 (AnxA6) inhibits anchorage-independent tumor cell growth but promotes tumor cell motility remain unclear. Using whole-genome gene expression profiling, we identified RasGRF2 (GRF2), a Ca2+-activated RasGEF, as one of the most upregulated genes following downregulation of AnxA6 in breast cancer (BC) cells. Here, we examined the role of GRF2 as a critical RasGEF in AnxA6-meditated BC cell growth.
Methods and Results: We show that the cellular levels of GRF2 are relatively low in AnxA6-high mesenchymal-like BC cells but higher in AnxA6-low basal-like BC cells. We also show that loss of AnxA6 in AnxA6-high BC cells is associated with early onset and rapid growth of xenograft tumors, while upregulation of AnxA6 in AnxA6-low BC cells is associated with a decrease in growth of xenograft tumors. To explore the reciprocal expression of GRF2 and AnxA6, we demonstrate that ionomycin-induced surge in intracellular Ca2+ led to GRF2 downregulation and that AnxA6, GRF2 and Ras proteins are within the same complex. We demonstrate that upon activation of EGFR, GRF2 is rapidly degraded and that the basal GRF2 levels were restored as the receptor activity decreased. The activation/degradation of GRF2 was followed by the activation of ERK1/2, was not affected by chelation of intracellular Ca2+ with BAPTA, but GRF2 was stabilized by chelation of extracellular Ca2+ with EGTA. Lapatinib treatment efficiently blocked the activation of EGFR but did not block the degradation of GRF2. Finally, we show that downregulation of GRF2 and inhibition of EGFR led to a greater inhibition of tumor cell growth.
Conclusion: Together, these data identify RasGRF2 as a major effector of AnxA6-dependent breast tumor growth and that reduced cellular levels of GRF2 imply active RasGEF. Combined inhibition of GRF2 and EGFR may be a viable alternative treatment for triple-negative BC.
Citation Format: Diva S. Whalen, Sarrah E. Widatalla, Olga Y. Korolkova, Stephen D. Williams, Heather K. Beasley, Amos M. Sakwe. Role of RasGRF2 in AnxA6-mediated growth of TNBC cells [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B16.
Abstract B19: New tools to study the role of RAS/CRAF interaction in RAS-driven lung cancer
Authors: Baer Romain , Rosewell Ian , Hancock David , Molina-Arcas Miriam , Downward Julian 
Published: June 2021

Abstract
The RAF/MAPK pathway is a major RAS effector pathway implicated in RAS oncogenic properties, regulating a diversity of cellular processes. As RAF kinases (A-, B-, CRAF) are thought to be attractive therapeutic targets for RAS-driven cancers, pan-RAF inhibitors are currently under development. However, preclinical studies focusing on targeting only the CRAF kinase by genetic inactivation revealed effective inhibition of tumor development without inducing significant systemic toxicities in a model of KRAS-driven lung tumor initiation. Recently, Sanclemente et al. further demonstrated that the complete ablation of CRAF in established advanced lung tumors (KrasG12V/Trp53) triggers sustainable tumor regression. Although this approach validates CRAF as a powerful therapeutic target for KRAS-driven lung cancer, specific CRAF inhibitors are not available yet. It was described more than 20 years ago that RAS GTPases strongly interact with CRAF kinase through its Ras Binding domain. This interaction is required to activate CRAF. Many efforts are being made to try to disrupt this interaction with small compounds, but currently none of those drugs are potent enough for further testing in vivo. Surprisingly, this therapeutic approach has never been proven to be efficient in vivo. Using CRISPR/Cas9 technology, we engineered a new mouse model in which the Ras binding domain of CRAF is constitutively mutated to completely abolish its interaction with RAS GTPases (CRAFR89L). We also engineered two murine lung cancer cell lines (KrasG12D/Trp53) that are heterozygous for this mutation (CRAFR89L/KO). Preliminary data confirm that CRAF interaction with RAS is minimally involved in 2D cell proliferation, as well as KRAS-driven MAPK pathway activation, but important for anchorage-independent cell growth.
Citation Format: Romain Baer, Ian Rosewell, David Hancock, Miriam Molina-Arcas, Julian Downward. New tools to study the role of RAS/CRAF interaction in RAS-driven lung cancer [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B19.
Abstract B22: Identifying protein interactors of oncogenic Kras in pancreatic cancer cells via proximity labelling
Authors: Cheng Derek K. , Tuveson David A. 
Published: June 2021

Abstract
Kras is mutated in over 90% of pancreatic adenocarcinoma (PDAC), the most common type of pancreatic cancer. Under normal conditions, Kras is a tightly regulated signaling protein that can toggle between active and inactive states to control cell proliferation. Mutated Kras (G12D) predominantly remains active, thus constitutively promoting proliferation and tumorigenesis. Studies that target downstream pathways of Kras, Raf/ERK and PI3K/AKT, have had some preclinical success but minimal clinical impact on pancreatic cancer. Therefore, it remains possible that in the pancreatic cancer context other undiscovered or underappreciated protein-protein interactions exist, including transient ones. In order to identify such mutant specific interactions in mouse tumor cells, we compare the biotinylated proteins from BirA biotin ligase fusions to wild-type and mutant Kras, as well as localization controls. Conditions have been optimized such that enrichment of biotinylated endogenous Raf1 and B-raf in BirA-KrasG12D compared to BirA-KrasWT can be detected by both Western blot and mass spectrometry analysis. Mass spectrometry has also identified other candidate interactors of KrasG12D, which are being validated biochemically with functional genetic experiments to follow.
Citation Format: Derek K. Cheng, David A. Tuveson. Identifying protein interactors of oncogenic Kras in pancreatic cancer cells via proximity labelling [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B22.
Abstract B24: Development of small-molecule RAS inhibitors using Affimer reagents
Authors: Haza Katarzyna , Martin Heather , Tiede Christian , Tipping Kevin , Trinh Chi , Foster Holly , Trowbridge Rachel , Foster Richard , Edwards Thomas , Breeze Alexander , McPherson Michael , Tomlinson Darren 
Published: June 2021

Abstract
Ras proteins are small GTPases that are mutationally activated in around 30% of all human cancers. Oncogenic mutations in Ras trigger uncontrolled cellular differentiation and division through uninhibited Ras-GTP signaling. Despite major efforts in developing inhibitors, lack of treatments directly targeting Ras in cancer led to the current assumption that Ras is undruggable. A new approach, which involves use of biologics, has shown great potential for development of Ras inhibitors, as demonstrated by recent increase in the number of antibody mimetic reagents targeting Ras, including single domain antibodies, monobodies and DARPins. We have developed modulators of Ras activity using novel artificial binding proteins, termed Affimers. Affimers are small 91-amino-acid scaffold proteins that constrain one or two randomized nine amino acid loop regions for molecular recognition. Affimers isolated against KRas, the most commonly mutated Ras family member, displayed low nanomolar binding affinities, were shown to be effective at inhibiting nucleotide exchange and blocked interaction between Ras and its effector Raf. When expressed in mammalian cells, Affimers bound with endogenous Ras and inhibited Ras-mediated signaling. Site-directed mutagenesis of Affimer variable regions revealed three residues critical for binding and inhibition. X-ray crystal structure of Affimer in complex with KRas demonstrated that these residues bind into a hydrophobic pocket on Ras, previously described with small molecules. Currently, we are determining whether this interaction gives insight into new modes of therapeutic development using molecular docking in attempt to mimic the Affimer residues with small molecules. This project provides a unique opportunity to further our understanding of Ras biology through the development of reagents that modulate Ras activity. In addition, structural insights into mode of inhibition allow the modelling and design of small-molecule compounds, providing novel therapeutic strategies to slow Ras-addicted tumor growth.
Citation Format: Katarzyna Haza, Heather Martin, Christian Tiede, Kevin Tipping, Chi Trinh, Holly Foster, Rachel Trowbridge, Richard Foster, Thomas Edwards, Alexander Breeze, Michael McPherson, Darren Tomlinson. Development of small-molecule RAS inhibitors using Affimer reagents [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B24.
Abstract B26: The SHOC2 phosphatase complex as a therapeutic target for ERK-pathway inhibition in RAS-driven tumors
Authors: Jones Greg G. , Del Rio Isabel Boned , Sari Sibel , Sekerim Aysen , Young Lucy C. , Hartig Nicole , El-Bahrawy Mona A. , Molina-Arcas Miriam , Downward Julian , Rodriguez-Viciana Pablo 
Published: June 2021

Abstract
Mutual exclusivity of RAS and BRAF mutations in multiple cancer types illustrates the key role of the RAF-MEK-ERK signaling pathway in mediating RAS oncogenesis. Although MEK inhibitors in particular are potent and specific, their on-target toxicity has severely limited their clinical efficacy against RAS-driven tumors and strategies to enhance their therapeutic index are sorely needed. RAF activation is a complex process that involves multiple regulatory steps in addition to RAS binding. Key among them is the dephosphorylation of a conserved inhibitory site (S259-RAF1, S365-BRAF). We have shown that a ternary complex comprising SHOC2, MRAS and PP1 functions as a specific “S259” RAF phosphatase holoenzyme. Gain-of-function mutations at this site in RAF, and indeed SHOC2 itself, are found in the RASopathy Noonan syndrome. We have used CRISPR to knock out SHOC2 in a panel of NSCLC cell lines and show that SHOC2 inhibition strongly sensitizes RAS-mutant cells to MEK inhibitors. SHOC2 inhibition lowers the concentration of MEK inhibitor required for cytotoxicity and drives tumor regressions in xenograft models. Biochemically, we show that SHOC2 inhibition impairs the dimerization of RAF upon signaling rebound after MEK inhibitor treatment, and thereby potentiates the duration of ERK-pathway inhibition. In vivo, conditional SHOC2 ablation in the adult mouse is relatively well tolerated, and strikingly SHOC2 inactivation in the lung suppresses tumor initiation in KRAS-driven models to prolong overall survival. Taken together, our results suggest the SHOC2 complex may provide an attractive target for therapeutic intervention—both as a monotherapy and by significantly enhancing the therapeutic index of clinically available MEK inhibitors.
Citation Format: Greg G. Jones, Isabel Boned Del Rio, Sibel Sari, Aysen Sekerim, Lucy C. Young, Nicole Hartig, Mona A. El-Bahrawy, Miriam Molina-Arcas, Julian Downward, Pablo Rodriguez-Viciana. The SHOC2 phosphatase complex as a therapeutic target for ERK-pathway inhibition in RAS-driven tumors [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B26.