bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract A37: Mapping KRAS signaling pathways using the Mammalian-Membrane Two-Hybrid (MaMTH) assay to elucidate novel therapeutic targets
Authors: Grozavu Ingrid Claudia , Snider Jamie , Lyakisheva Anna , Stagljar Igor 
Published: June 2021

Abstract
Background: KRAS is a well-established cancer driver. Clinically successful therapeutics have not yet been developed despite its disease burden, suggesting that KRAS-driven cancers are more complicated than previously thought.
Aim: Our goal is to identify the global changes in KRAS interaction patterns that occur in disease states. This is accomplished by mapping the dynamic interactome of both WT and oncogenic KRAS isoforms using the Mammalian Membrane Two-Hybrid (MaMTH) assay, which is suitable for identification of protein interactors of virtually any integral membrane or membrane-associated protein.
Methods: To probe the protein-protein interactions (PPIs) of KRAS, we used the Mammalian Membrane Two-Hybrid (MaMTH) assay, a split-ubiquitin-based two-hybrid system that can be applied to any cell line. This assay is suited for PPI detection using both low- to-medium throughput array and high-throughput large-scale PPI screening. We generated stably expressing “bait”-tagged KRAS constructs in HEK293T cells that contained the MaMTH reporter system. KRAS baits, namely KRAS-WT, -G12D, -G12V, and -Q61H, were characterized using signaling assays as well as within the MaMTH system using “prey”-tagged CRAF interaction control, Western blotting, and immunofluorescence techniques. Currently, we are performing unbiased screening for PPIs of KRAS using the human ORFeome (hORFeome) library consisting of approximately 13,000 fully sequenced human ORFs.
Results: We first validated compatibility of KRAS in the MaMTH assay through several means. Oncogenic KRAS variants showed increased interaction signal with CRAF (a known KRAS binding partner) compared to KRAS-WT. This corresponded with upregulated MAPK pathway activation by oncogenic KRAS, as determined from increased pERK levels via Western blotting. KRAS bait expression levels were similar across all variants, suggesting that these findings were not due to differential expression of KRAS isotypes. Additionally, we confirmed that the KRAS bait correctly localizes to the plasma membrane, consistent with previous literature. After establishing KRAS compatibility with the MaMTH assay, we have begun unbiased screening of the hORFeome against KRAS baits in the WT, G12D, G12V, and Q61H isoforms.
Conclusions: We have characterized KRAS compatibility with the MaMTH assay. Currently, we are performing large screening protocols for high-throughput detection of PPIs of KRAS using MaMTH.
Citation Format: Ingrid Claudia Grozavu, Jamie Snider, Anna Lyakisheva, Igor Stagljar. Mapping KRAS signaling pathways using the Mammalian-Membrane Two-Hybrid (MaMTH) assay to elucidate novel therapeutic targets [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 A37.
Abstract A43: Investigating novel inhibitors of the IMP-1-KRAS mRNA interaction
Authors: Liu Victor , Wang Chuyi , Lee Chow 
Published: June 2021

Abstract
K-Ras functionality is integral for cell growth and differentiation due its canonical role in signaling the PI3K and MAPK pathways. Genetic analysis on various cancers revealed that mutations in the K-Ras gene are responsible for 21% of all cases, which include lung, pancreatic and colorectal cancer. Prior efforts at directly targeting mutant K-Ras have been largely unsuccessful, thus creating a demand for novel therapeutic approaches. Common to the aforementioned cancers is expression of the Insulin-Like Growth Factor 2 mRNA Binding Protein 1 (IMP-1). IMP-1 is part of the VICKZ family of RNA-binding proteins known for post-transcriptional regulation, including its ability to shield its mRNA substrates from decay. One notable substrate of IMP-1 is the K-Ras mRNA, which was identified using UV-crosslinking experiments. Downregulation of IMP-1 through siRNA treatment led to a reduction of K-Ras protein by 60% in SW480 colorectal cancer cells and consequently reduced cell proliferation. The studies suggest a relationship between IMP-1 binding, mRNA turnover and the resultant protein expression of target substrates. To study the IMP-1-mRNA relationship and to help find inhibitors of such interaction, we developed a fluorescence polarization (FP) method that employed 45-nt K-Ras and 39-nt CD44 fluorescein-labeled RNA probes. Using full-length IMP1, truncated and point-mutated IMP1, we showed that the FP method faithfully recapitulated the relative affinity of recombinant proteins for both K-Ras and CD44 RNAs. The FP method was then used to find inhibitors of the IMP-1-K-Ras mRNA interaction and to elucidate potential domain target of inhibitors. We found that oligonucleotides SM6 and SM7, but not SM2, could significantly reduce the FP units, consistent with the results from electrophoretic mobility shift assay. In addition, the molecule UNBC143 was found to decrease FP units in a dose-dependent manner, suggesting its ability to inhibit IMP-1-K-Ras RNA interaction. The SM oligonucleotides and UNBC143 were also assessed on K-Ras expression in SW480 human colon cancer cells. These experiments and their results will be discussed in further detail upon presentation.
Citation Format: Victor Liu, Chuyi Wang, Chow Lee. Investigating novel inhibitors of the IMP-1-KRAS mRNA interaction [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 A43.
Abstract A45: Protective autophagy elicited by RAF→MEK→ERK inhibition suggests a treatment strategy for RAS-driven cancers
Authors: Kinsey Conan G. , Camolotto Soledad A. , Boespflug Amelie M. , Gullien Katrin P. , Foth Mona , Shea Jill E. , Seipp Michael T. , Yap Jeffrey T. , Burrell Lance D. , Lum David H. , Whisenant Jonathan R. , Gilcrease Weldon , Cavalieri Courtney C. , Rehbein Kaitrin M. , Cutler Stephanie L. , Affolter Kajsa E. , Welm Alana L. , Welm Bryan E. , Scaife Courtney L. , Snyder Eric L. , McMahon Martin 
Published: June 2021

Abstract
Pancreatic ductal adenocarcinoma was responsible for ~43,000 deaths in the USA in 2017 and is the epitome of a recalcitrant cancer driven by a pharmacologically intractable oncoprotein, KRAS. Although the clinical picture remains grim, the mechanisms by which key alterations in tumor suppressors and proto-oncogenes contribute to PDA have been dissected. Downstream of KRAS, the RAF→MEK→ERK signaling pathway plays a central role in pancreatic carcinogenesis. However, to date, pharmacologic inhibition of this pathway has provided no clinical benefit to PDA patients. Here we show that inhibition of KRAS→RAF→MEK→ERK signaling in PDA cell lines elicits autophagy, a process of cellular recycling that protects pancreatic cancer cells from the potentially cytotoxic effects of KRAS pathway inhibition. Furthermore, combined inhibition of MEK1/2 plus autophagy displays synergistic antiproliferative effects against PDA cell lines in vitro, and promotes regression of xenografted patient-derived PDA tumors in mice. Finally, treatment of a KRAS-mutated PDA patient on a compassionate basis with the combination of trametinib plus hydroxychloroquine resulted in a partial but nonetheless striking disease response. These data suggest that this combination therapy may represent a new strategy to target RAS-driven cancers such as PDA.
Citation Format: Conan G. Kinsey, Soledad A. Camolotto, Amelie M. Boespflug, Katrin P. Gullien, Mona Foth, Jill E. Shea, Michael T. Seipp, Jeffrey T. Yap, Lance D. Burrell, David H. Lum, Jonathan R. Whisenant, Weldon Gilcrease, Courtney C. Cavalieri, Kaitrin M. Rehbein, Stephanie L. Cutler, Kajsa E. Affolter, Alana L. Welm, Bryan E. Welm, Courtney L. Scaife, Eric L. Snyder, Martin McMahon. Protective autophagy elicited by RAF→MEK→ERK inhibition suggests a treatment strategy for RAS-driven cancers [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 A45.
Abstract B07: Inhibition of Ras signalling by targeting Son of Sevenless with Affimers
Authors: Saunders Sophie E. , Tiede Christian , Edwards Thomas , Breeze Alexander , Tomlinson Darren 
Published: June 2021

Abstract
Mutations in Ras result in increased signaling through the MAPK pathway, resulting in increased cell proliferation and survival. These activating mutations are found in 30% of all cancers, making inhibition of Ras a key therapeutic strategy. However, due to its lack of small-molecule binding pockets, Ras-based therapies have been difficult to develop, leading to the assumption it is “undruggable.” A novel method of inhibiting aberrant signaling through the MAPK pathway is to prevent the activation of Ras by inhibiting its guanine nucleotide exchange factor Sos. Our aim is to isolate and characterise Affimer reagents to aid the identification of druggable pockets on Sos. Affimer reagents are a nonantibody binding scaffold designed to mimic the binding properties of antibodies. Affimers present two variable regions for molecular recognition that have been used to probe for hot spot regions on protein surfaces. 57 Affimers have been isolated that bind to the catalytic domains of Sos; of these, 17 have been shown to inhibit Sos catalysed nucleotide exchange in vitro, a selection of which have IC50 values in the nano-molar range. We are currently studying the effects of expressing these Affimer reagents in mammalian cells on MAPK signaling, cell proliferation, survival and migration and have shown the Affimers are capable of inhibiting EGF-stimulated signaling through the MAPK pathway. Structural studies are also ongoing and will be used to determine the binding properties of the Affimers in the hope of identifying novel binding pockets on Sos and possible sites of allosteric regulation as demonstrated with anti-Ras Affimers.
Citation Format: Sophie E. Saunders, Christian Tiede, Thomas Edwards, Alexander Breeze, Darren Tomlinson. Inhibition of Ras signalling by targeting Son of Sevenless with Affimers [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 B07.
Abstract B11: O-GlcNAcylation is required for mutant KRAS-induced lung tumorigenesis
Authors: Shiraishi Takumi , Tran Phuoc T. , Malek Reem , Lafargue Audrey , Barbhuiya Mustafa , Wang Xing , Simons Brian , Ballew Matthew , Nugent Katriana , Groves Jennifer , Williams Russell , Wang Hailun , Verdone James , Yildirir Gokben , Henry Roger , Zhang Bin , Wong John , Wang Ken , Nelkin Barry , Pienta Kenneth , Felsher Dean , Zachara Natasha , Taparra Kekoa 
Published: June 2021

Abstract
Mutant KRAS drives glycolytic flux in lung cancer, potentially impacting aberrant protein glycosylation. Recent evidence suggests aberrant KRAS drives flux of glucose into the hexosamine biosynthetic pathway (HBP). HBP is required for various glycosylation processes, such as protein N- or O-glycosylation and glycolipid synthesis. However, its function during tumorigenesis is poorly understood. One contributor and proposed target of KRAS-driven cancers is a developmentally conserved epithelial plasticity program called epithelial-mesenchymal transition (EMT). Here we showed in novel autochthonous mouse models that EMT accelerated KrasG12D lung tumorigenesis by upregulating expression of key enzymes of the HBP pathway. We demonstrated that HBP was required for suppressing KrasG12D-induced senescence, and targeting HBP significantly delayed KrasG12D lung tumorigenesis. To explore the mechanism, we investigated protein glycosylation downstream of HBP and found elevated levels of O-linked β-N-acetylglucosamine (O-GlcNAcylation) post-translational modification on intracellular proteins. O-GlcNAcylation suppressed KrasG12D oncogene-induced senescence (OIS) and accelerated lung tumorigenesis. Conversely, loss of O-GlcNAcylation delayed lung tumorigenesis. O-GlcNAcylation of proteins SNAI1 and c-MYC correlated with the EMT-HBP axis and accelerated lung tumorigenesis. Our results demonstrated for the first time that O-GlcNAcylation was sufficient and required to accelerate KrasG12D lung tumorigenesis in vivo, which was reinforced by epithelial plasticity programs.
Citation Format: Takumi Shiraishi, Phuoc T. Tran, Reem Malek, Audrey Lafargue, Mustafa Barbhuiya, Xing Wang, Brian Simons, Matthew Ballew, Katriana Nugent, Jennifer Groves, Russell Williams, Hailun Wang, James Verdone, Gokben Yildirir, Roger Henry, Bin Zhang, John Wong, Ken Wang, Barry Nelkin, Kenneth Pienta, Dean Felsher, Natasha Zachara, Kekoa Taparra. O-GlcNAcylation is required for mutant KRAS-induced lung tumorigenesis [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 B11.
Abstract B18: Clostridium perfringens lethal toxin specifically targets RAS and disrupts RAS signaling pathway
Authors: Abreu-Blanco Maria , Yi Ming , Denson Jean-Paul , Holderfield Matthew 
Published: June 2021

Abstract
Clostridium perfringens lethal toxin (TpeL) belongs to the family of large clostridial glucosylating cytotoxins. Clostridial toxins are glucosyltransferases that modify and deactivate small GTPases of the RHO and RAS subfamily. TpeL mono-glucosylates in the switch I domain (Thr35) of RAC and RAS small GTPases. RAS family proteins function as key regulators of cell proliferation, differentiation, survival and gene expression. Moreover, mutations in RAS proteins are highly prevalent in human cancers. Considering the specificity of TpeL for RAS, we decided to investigate the biochemical interaction between TpeL and RAS, and potentially develop TpeL as a tool to disrupt the RAS signaling pathway in vivo. First, we used RAS-dependent MEFs expressing different KRAS mutations and BRAF V600E to assess the specificity of TpeL in vivo. We expected that proliferation should be inhibited in MEF cells expressing RAS isoforms, but MEF cells expressing BRAF V600E should not be affected. We found that TpeL treatment did not affect the viability of the BRAF V600E cells and only induced toxicity in RAS expressing cells. Consistent with this result, TpeL treatment inhibited MAPK signal transduction in the RAS expressing cells, but not in the BRAF V600E cells. Surprisingly, both cell viability as well as pERK levels remained unaffected in the KRAS Q61R MEF cells, suggesting that KRAS Q61R is resistant to TpeL. Furthermore, TpeL treatment of cancer cell lines shows significant differences in potency, with KRAS G12C cell lines being more sensitive to the toxin. Our cellular data suggest a correlation between TpeL sensitivity and intrinsic hydrolysis rate of the KRAS alleles. Biochemical assays corroborate our hypothesis that TpeL preferentially glucosylates KRAS GDP loaded in vitro. Moreover, TpeL glucosylation inhibits GEF-mediated nucleotide exchange, effectively blocking KRAS on the GDP inactive state. To further follow up the correlation between KRAS GDP levels and TpeL sensitivity, we used a combination treatment of TpeL and EGFR/FGFR inhibitors. Combination screen shows a synergistic effect; we observed increased TpeL sensitivity in combination with the inhibitors. By elucidating the mechanism of substrate binding and substrate specificity, we have gained insight into the TpeL-RAS interaction and we are working on developing the toxin as a therapeutic tool.
Citation Format: Maria Abreu-Blanco, Ming Yi, Jean-Paul Denson, Matthew Holderfield. Clostridium perfringens lethal toxin specifically targets RAS and disrupts RAS signaling pathway [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 B18.
Abstract B28: Direct targeting oncogenic Ras mutants by IgG-format cytosol-penetrating antibody
Authors: Shin Seung-Min , Kim Ji-Sun , Hong Jin-Sun , Park Seong-wook , Jun Sei-Yong , Kweon Hye-Jin , Choi Dong-Ki , Kim Yong-Sung 
Published: June 2021

Abstract
Oncogenic Ras mutants, and most frequently KRas mutants (86% of Ras-driven cancers), are found in approximately 25% of human cancers and are high-priority anticancer drug targets. Despite 30 years of effort to develop drugs that directly target oncogenic Ras mutants, no effective pharmacologic inhibitors for these mutants are clinically available, mainly because of the lack of suitable surface binding pockets for small molecules. More than 50 therapeutic antibodies have been clinically approved against many extracellular proteins. However, such antibodies do not have the capacity to localize in intracellular cytosolic regions after receptor-mediated endocytosis, restricting their therapeutic application for targeting cytosolic proteins. Our group recently developed a platform technology of cytosol-penetrating antibody, which in the IgG format can reach the cytosolic space of living cells owing to its endosomal escaping ability after receptor-mediated endocytosis. Exploiting the cytosol-penetrating antibody technology, we have engineered a human IgG1 format antibody, named iMab (internalizing and protein-protein interaction [PPI] interfering monoclonal antibody), which internalizes into the cytosol of living cells and selectively binds to the activated GTP-bound form of oncogenic Ras mutants. iMab specifically binds to the PPI interfaces of activated Ras with effector proteins to block the associations, thereby inhibiting the Ras downstream oncogenic signaling and exerting antiproliferation effects on oncogenic Ras mutant tumor cells. For in vivo antitumor efficacy assessment, we further engineer iMab to have tumor tissue-homing ability by fusion of tumor-associated integrin αvβ3/αvβ5 binding cyclic peptide to the N-terminus of light chain. When systemically administered, the iMab variant significantly inhibited the in vivo growth of oncogenic Ras-mutated tumor xenografts in mice, but not wild-type Ras-harboring tumors. Our results demonstrate the feasibility of developing antibody therapeutics that directly target cytosolic proteins involved in disease-associated PPIs, such as oncogenic Ras mutants, by systemic administration, similar to conventional therapeutic antibody regimens. Because the oncogenic Ras targeting antibody holds many desirable features of the conventional IgG antibody, it shows great potential for development as a first-in-class anticancer antibody.
Citation Format: Seung-Min Shin, Ji-Sun Kim, Jin-Sun Hong, Seong-wook Park, Sei-Yong Jun, Hye-Jin Kweon, Dong-Ki Choi, Yong-Sung Kim. Direct targeting oncogenic Ras mutants by IgG-format cytosol-penetrating antibody [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 B28.
Abstract B40: Role of mutant HRAS in growth and drug sensitivity of head and neck squamous cell cancers (HNSCC)
Authors: Javaid Sehrish , Waters Andrew M. , Nguyen Victoria V. , Der Channing J. , Cox Adrienne D. 
Published: June 2021

Abstract
Activating mutations in the HRAS oncogene are present in 5-11% of head and neck squamous cell cancers (HNSCC). We set out to determine if HRAS is a driver of HNSCC growth and whether pharmacologic inhibitors of HRAS membrane association and/or RAS effector signaling may have therapeutic value in these cancers. To determine the importance of HRAS, we depleted it genetically from HRAS-mutant HNSCC cell lines by shRNA and evaluated the effects on cell growth and survival. To determine the potential therapeutic value of blocking HRAS membrane association and/or signaling through the key RAS effector, ERK MAPK, we treated HNSCC cells with inhibitors of farnesyltransferase (FTIs, tipifarnib and lonafarnib) or ERK1/2 (ERKi, SCH772984), respectively. We monitored target inhibition by aqueous/detergent fractionation, gel shift and immunoblotting; quantitated cell viability by Alamar blue assay; and assessed cell cycle distribution and apoptosis by flow cytometry. We found that genetic depletion of mutant HRAS inhibited the growth of HRAS-mutant HNSCC cell lines in vitro, caused a modest G1 cell cycle arrest and robustly induced apoptosis. Since genetic depletion is not clinically feasible, we wished to identify targeted therapies suitable for this patient population. Several cancer types driven by mutant RAS are sensitive to inhibitors of the ERK pathway downstream of RAS, and HRAS is the sole RAS isoform whose activity is blocked by FTIs. We found that FTIs and ERKi each inhibited their respective targets at nanomolar range but only FTIs inhibited the growth of HRAS-mutant HNSCC in a target-dependent manner. In cell lines where FTI treatment upregulated ERK signaling, ERKi enhanced sensitivity to FTI. We established that genetic suppression of mutant HRAS blocks HNSCC growth, validating HRAS as a potential therapeutic target. HRAS-mutant HNSCC cell lines are sensitive to FTI but, surprisingly, less sensitive to ERKi. The combination of FTI and ERKi may further sensitize a subset of HRAS-mutant HNSCC. Other targeted combinations will also be discussed.
Citation Format: Sehrish Javaid, Andrew M. Waters, Victoria V. Nguyen, Channing J. Der, Adrienne D. Cox. Role of mutant HRAS in growth and drug sensitivity of head and neck squamous cell cancers (HNSCC) [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 B40.
Abstract B46: Systems modeling of Ras reveals systems mechanisms that dictate response to treatment
Authors: Stites Edward C. 
Published: June 2021

Abstract
A relative abundance of biochemical and mechanistic data makes the Ras signaling network an ideal “model system” for the application of systems biology approaches to cancer biology. We here describe our application of systems modeling to the previously unexplained situation where colorectal cancer (CRC) patients with a KRAS G13D mutations are more responsive to anti-EGFR agents than patients with other KRAS mutations. Our computational model of Ras signaling was developed in accordance with the well-established network architecture of RasGTP signal regulation. Measured biochemical and biophysical properties comprise the parameters of the model. The previous biochemical characterization of specific oncogenic mutants allows different oncogenic alleles to be modeled. We use the model to find the behaviors that logically follow from what is known, but which the scope and complexity of the network prevent from otherwise being inferred. We simulated anti-EGFR dose responses for the three most common Ras mutants in CRC: G12D, G12V, and G13D. Our simulations unexpectedly found KRAS G13D was considerably more responsive to anti-EGFR agents. This implies that the known biochemical differences between these mutants are sufficient to explain why KRAS G13D CRC is more sensitive to EGFR inhibition. Our model further predicted that wild-type RasGTP levels within KRAS-mutant cancers should decrease much more for KRAS G13D cancers than KRAS G12V and KRAS G12D cancers. Our analysis predicted that these variations in wild-type RasGTP levels follow from mutant-specific differences in their interaction with Ras GAPs like the tumor suppressor neurofibromin (NF1). This mechanism explains why KRAS G13D CRC, but not KRAS G12D and G12V CRC, retains a dependency upon EGFR. We have now completed experiments that confirm this systems-level mechanism that governs responsiveness to treatment.
Citation Format: Edward C. Stites. Systems modeling of Ras reveals systems mechanisms that dictate response to treatment [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 B46.