bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract B009: TEAD inhibition overcomes YAP1/TAZ-driven resistance to RAS inhibitors in KRASG12C-mutant cancers
Authors: Edwards Alexander C. , Stalnecker Clint A. , Morales Alexis J. , Taylor Khalilah E. , Hallin Jill , Olson Peter , Tang Tracy T. , Engstrom Lars , Post Leonard , Christensen James G. , Cox Adrienne D. , Der Channing J. 
Published: May 2023

Abstract
Activated mutants of KRAS comprise the major oncogenic drivers in lung (LAC), colorectal (CRC), and pancreatic ductal (PDAC) adenocarcinoma. Recent success in covalently targeting one KRAS mutant (KRASG12C) led to FDA approval of the first anti-KRAS therapy (G12Ci). However, both primary and treatment-induced acquired resistance to G12Ci have been observed. While analyses of relapsed patients have identified reactivation of the key KRAS effector signaling network as a driver of resistance, the mechanisms in ~50% of patients are not known. To identify potential resistance mechanisms, we applied a CRISPR-Cas9 loss-of-function screen targeting the druggable genome. In addition to genes discovered recently in relapsed patients (e.g., PTEN, NF1), we also identified loss of multiple components of the Hippo tumor suppressor pathway as drivers of G12Ci resistance (NF2, LATS1/2, TAOK1/2 and STK3/4). We therefore determined if activation of the functionally related transcriptional co-activators, YAP1 and TAZ, normally inhibited by Hippo signaling, can drive resistance to G12Ci. We first determined that ectopic expression of constitutively activated YAP1/TAZ was sufficient to impair the anti-proliferative and pro-apoptotic effects of G12Ci treatment in KRASG12C-mutant LAC, CRC, and PDAC cell lines. Conversely, genetic suppression of YAP1/TAZ enhanced G12Ci sensitivity. YAP1/TAZ requires association with TEAD and other transcription factors to regulate transcription. We determined that YAP1/TAZ mutants deficient in TEAD binding failed to drive resistance to G12Ci treatment. Further supporting a role for TEAD, both overexpression of a TEAD dominant negative mutant and treatment with pan-TEAD pharmacological inhibitors phenocopied the effects of YAP1/TAZ genetic suppression and sensitized KRASG12C mutant cancer cells to G12Ci. Finally, transcriptional analyses support a model where YAP1/TAZ-TEAD overcomes KRASG12C addiction by restoring a subset of KRAS-dependent gene transcription. In summary, our observations support YAP1/TAZ-TEAD signaling as a novel driver of resistance to KRAS inhibition and support the use of TEAD inhibitors to enhance the anti-tumor efficacy of KRAS-targeted therapies.
Citation Format: Alexander C. Edwards, Clint A. Stalnecker, Alexis J. Morales, Khalilah E. Taylor, Jill Hallin, Peter Olson, Tracy T. Tang, Lars Engstrom, Leonard Post, James G. Christensen, Adrienne D. Cox, Channing J. Der. TEAD inhibition overcomes YAP1/TAZ-driven resistance to RAS inhibitors in KRASG12C-mutant cancers [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B009.
Abstract B013: Exploring the switch II pocket of KRAS(G12D) with mutant-selective monobody inhibitors
Authors: Hattori Takamitsu , Akkapeddi Padma , Khan Imran , Koide Akiko , Glasser Eliezra , Ketavarapu Gayatri , Whaby Michael , Zuberi Mariyam , Teng Kai Wen , Lefler Julia , Maso Lorenzo , Bang Injin , Ostrowski Michael C. , O’Bryan John P. , Koide Shohei 
Published: May 2023

Abstract
The G12D mutation is among the most common KRAS mutations associated with cancer, in particular pancreatic cancer. Here, we have developed monobodies, small synthetic binding proteins, that are highly selective to KRAS(G12D) over KRAS(wild type) and other oncogenic KRAS mutations, as well as over HRAS(G12D). Crystallographic studies revealed that, similar to other KRAS mutant-selective inhibitors, the initial monobody bound to the S-II pocket, the groove between switch II and alpha 3 helix. Strikingly, the monobody captured a shape of the S-II pocket distinct from previously reported structures. Unlike other G12D-selective polypeptides reported to date, the monobody used its backbone NH group to directly recognize the side chain of RAS Asp12. This feature closely resembles that of a small-molecule KRAS(G12D) inhibitor, MRTX1133. The monobody also directly interacted with H95, a residue not conserved in HRAS. These features rationalize the high selectivity toward the G12D mutant and the KRAS isoform. Structure-guided affinity maturation resulted in monobodies with low nM KD values. Deep mutational scanning of a G12D-specific monobody generated hundreds of functional and nonfunctional single-point mutants, from which we identified crucial residues for binding and those that contributed to the selectivity toward the GTP- and GDP-bound states. When expressed in cells as genetically encoded reagents, these monobodies engaged selectively with KRAS(G12D) and inhibited KRAS(G12D)-mediated signaling and tumorigenesis. These results demonstrate the feasibility of targeting KRAS(G12D) functions using a mutant selective inhibitor and will guide the development of next-generation KRAS(G12D)-selective inhibitors.
Citation Format: Takamitsu Hattori, Padma Akkapeddi, Imran Khan, Akiko Koide, Eliezra Glasser, Gayatri Ketavarapu, Michael Whaby, Mariyam Zuberi, Kai Wen Teng, Julia Lefler, Lorenzo Maso, Injin Bang, Michael C. Ostrowski, John P. O’Bryan, Shohei Koide. Exploring the switch II pocket of KRAS(G12D) with mutant-selective monobody inhibitors [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B013.
Abstract B018: The development of macrocyclic peptide Ras inhibitors
Authors: Josephson Kristopher , Silvestri Anthony , Fribourgh Jennifer L. , Bray Walter M. , David Brian , Hanna Cameron C. , Chakka Sai Kumar , Hyatt Alex , Porcella Monique , Dieppa-Matos Dio , Pye Cameron R. , Schwochert Joshua 
Published: May 2023

Abstract
While there has been tremendous progress developing small molecule covalent K-Ras G12C inhibitors that bind Ras-off (GDP) and inhibit nucleotide exchange, there remains a need for additional K-Ras inhibitors with different Ras isoform/mutant selectivity profiles and Ras-on (GTP) mechanisms of inhibition. Of particular interest are compounds with a pan-K-Ras profile that inhibit multiple oncogenic mutants and wild type K-Ras, but achieve a therapeutic window by sparing H and N-Ras. In addition, addressing cancers driven by K-Ras oncogenic mutants beyond G12C, K-Ras amplification, or upstream dysregulation where K-Ras is maintained in the GTP-bound state will require “Ras-on” compounds that prevent effector binding. We were interested in the potential of peptide macrocycles that bind the Switch II pocket of K-Ras, in a different conformation than the small molecules, to inhibit Ras-on by allosterically blocking Raf and other effector binding. KRpep-2d is a 19-residue disulfide bridged peptide macrocycle discovered by phage display against K-Ras G12D bound to GDP. Due to its high molecular weight (2561 Da), polarity and metabolic instability, KRpep2d is inactive in in vitro cell assays. And while biochemical experiments have shown that it binds the Ras-off GDP-bound conformation of K-Ras with high affinity to inhibit nucleotide exchange, it has low affinity for K-Ras GTP and does not allosterically inhibit Raf binding. Using parallel macrocyclic peptide chemistry, we made 180 analogs of KRpep-2d to explore truncation of the exocyclic residues and replacement of the labile disulfide bond. UNP-6752, the best compound from this exploratory library, had K-Ras affinities similar to KRpep2d with a 50-fold K-Ras GDP/GTP preference, but was only 11 residues and cyclized with a non-reducible linker. Critically this compound lacked the eight arginine residues of KrPep-2d, which both preclude passive-cell permeability and induce mast cell degranulation. A second iteration of 350 compounds designed to explore single or multiple amino acid substitutions in the core of UNP-6752 led to compound UNP-7572 with sub-micromolar affinity for K-Ras GTP and an IC50 in a K-Ras-Raf TR-FRET assay of ~800 nM. Working from the UNP-7572 we are tuning selectivity for K-Ras and our progress further improving allosteric Ras-Raf inhibition, cell permeability and activity will be presented.
Citation Format: Kristopher Josephson, Anthony Silvestri, Jennifer L. Fribourgh, Walter M. Bray, Brian David, Cameron C. Hanna, Sai Kumar Chakka, Alex Hyatt, Monique Porcella, Dio Dieppa-Matos, Cameron R. Pye, Joshua Schwochert. The development of macrocyclic peptide Ras inhibitors [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B018.
Abstract B023: Inhibition of RAS signaling and tumorigenesis through targeting novel vulnerabilities
Authors: Kahn Imran , Koide Akiko , Zuberi Mariyam , Ketavarapu Gayatri , Denbaum Eric , Teng Kai Wen , Rhett J. Matthew , Spencer-Smith Russell , Hobbs G. Aaron , Camp Earnest Ramsay , Koide Shohei , O'Bryan John P. 
Published: May 2023

Abstract
RAS GTPases are important mediators of oncogenesis with nearly 30% of human tumors harboring mutant RAS proteins. However, pharmacological inhibition of RAS has proven challenging. We have employed Monobody technology to discover novel 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. In contrast to antibodies, Monobodies are fully functional in the reducing environment of the cytoplasm and thus are particularly suitable as genetically encoded “tool biologics”. We previously developed the NS1 Monobody that inhibited RAS by targeting the α4-α5 allosteric lobe to prevent RAS self-association and nanoclustering, and NS1 has become a widely used tool in the RAS research community. Following on this success, we sought to identify additional vulnerabilities in RAS. Based on our discovery that nucleotide-free RAS (apoRAS) inhibits PIK3C2B function, we assessed the feasibility of selectively targeting this state of RAS as an approach to inhibit oncogenic RAS function. Here, we have developed several Monobodies and extensively characterized one of them, R15. Although NS1 was agnostic to the nucleotide state of RAS, R15 bound exclusively to the apo state of all three RAS isoforms but did not interact with nucleotide-loaded RAS. When expressed in cells, R15 selectively inhibited the signaling and transforming activity of RAS mutants with elevated intrinsic nucleotide exchange rates (i.e., “fast exchange mutants”), such as G13D and Q61L. Surprisingly, R15 bound and inhibited RAS(G12D) mutants which are not reported to be fast exchange. Biochemical studies demonstrated that RAS captured with R15 from cell lysates was indeed nucleotide free, suggesting that R15 traps apoRAS and prevents nucleotide reloading. Finally, intracellularly expressed R15 selectively inhibited the tumor forming capacity of human cell lines driven by fast exchange RAS mutants but not RAS(G12V) in mouse xenografts. Thus, in contrast to conventional wisdom, our approach has established a new opportunity to selectively inhibit certain RAS mutants by targeting the apo state of RAS with drug-like molecules.
Citation Format: Imran Kahn, Akiko Koide, Mariyam Zuberi, Gayatri Ketavarapu, Eric Denbaum, Kai Wen Teng, J. Matthew Rhett, Russell Spencer-Smith, G. Aaron Hobbs, Earnest Ramsay Camp, Shohei Koide, John P. O'Bryan. Inhibition of RAS signaling and tumorigenesis through targeting novel vulnerabilities [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B023.
Abstract B024: Combined inhibition of farnesyltransferase and MEK is effective in fusion-negative HRAS-mutant rhabdomyosarcoma
Authors: Odeniyide Patience , Lisok Alla , Fertig Elana J. , Skaist Alyza , Pratilas Christine A. 
Published: May 2023

Abstract
Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood, and RAS pathway mutations are the known driver mutations in the majority of fusion-negative (FN) RMS. Studies have demonstrated that HRAS mutations are enriched in infants with FN-RMS and can be associated with inferior outcomes. Using HRAS-mutant RMS models, we have demonstrated that tipifarnib (farnesyl transferase inhibitor, FTI) decreases ERK signaling and decreases in vitro and in vivo tumor growth. The effects of tipifarnib can be incomplete, and limitations may be due to adaptive or acquired resistance, or the presence of concurrent mutations that limit the response to single agents. These features suggest that HRAS-mutated FN-RMS may be sensitive to inhibition with combination therapy that prevents or delays the emergence of resistance. We examined the transcriptional effects of tipifarnib in FN-RMS cell lines using bulk RNA-sequencing to identify therapeutic vulnerabilities that may be exploited by RAS-directed therapies. Analysis of RNA sequencing data revealed downregulation of ERK transcriptional output genes upon treatment with tipifarnib, confirming the role of the MEK-ERK pathway in mediating the response to FTI. Trametinib (MEKi) inhibits tumor growth in xenograft models of FN-RMS but has only modest activity as a single agent. The efficacy of inhibition with FTI and MEKi has not been previously explored in RAS-driven FN-RMS. We therefore tested tipifarnib in combination with trametinib in HRAS-mutant FN-RMS cell lines and utilized in vitro assays to evaluate the effects of the combination on cell growth, differentiation and signaling via RAS effector pathways. In HRAS-mutant cells, we observed additive dose-dependent 2D and 3D growth inhibition in response to the combination. Further, tipifarnib and trametinib more potently reduced ERK phosphorylation than either drug individually, indicating effective RAS pathway inhibition. Additionally, we found that the combination of tipifarnib and trametinib induced myosin heavy chain expression, suggesting both inhibition of proliferation and promotion of myogenic differentiation. In an in vivo model, the combination of tipifarnib and trametinib led to tumor regression which was not seen with either drug alone. Our data suggest that the combination of tipifarnib and trametinib is active in models of HRAS-driven FN-RMS and may represent an effective therapeutic strategy for a genomically-defined subset of patients with FN-RMS.
Citation Format: Patience Odeniyide, Alla Lisok, Elana J. Fertig, Alyza Skaist, Christine A. Pratilas. Combined inhibition of farnesyltransferase and MEK is effective in fusion-negative HRAS-mutant rhabdomyosarcoma [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B024.
Abstract B031: Inhibition of mutant RAS via myotubularin related protein 7-mimicking peptide
Authors: Saar Daniel , Weidner Philip , Burgermeister Elke , Kragelund Birthe B. 
Published: May 2023

Abstract
The coiled coil domain of Myotubularin Related Protein 7 (MTMR7) and a peptide of the same sequence (M7CC) have been shown to have an anti-cancer effect via direct interaction with PPARγ (Weidner et al. 2020). The same studies also found an inhibition of ERK-mediated inactivation of PPARγ by the same peptide. In this work we have followed up on our previous findings and show a direct effect on mutant RAS activity, mediated by MTMR7 and M7CC. In the presence of MTMR7 or M7CC, a drop of active RAS-levels along with a delocalization from the plasma membrane can be observed. This goes along with a significant drop in the levels of phosphorylated ERK and the expression of RAS-controlled genes. This led to a decrease of cell proliferation in cell culture in different cancer cell lines and of tumor volume in mouse models. The effect of MTMR7 or M7CC on RAS levels was shown to be mediated by a direct interaction with RAS. Using biophysical studies including solution-state NMR spectroscopy, the interaction was in vitro shown to happen with the globular domain of K-Ras4B. Our results support a model for MTMR7 as part of a novel RAS-regulation machinery, and highlights MTMR7 as promising scaffold to be exploited for peptide-based cancer treatment. References: Weidner, Philip; Söhn, Michaela; Schroeder, Torsten; Helm, Laura; Hauber, Veronika; Gutting, Tobias et al. (2020): Myotubularin-related protein 7 activates peroxisome proliferator-activated receptor-gamma. In: Oncogenesis 9 (6), S. 59. DOI: 10.1038/s41389-020-0238-8.
Citation Format: Daniel Saar, Philip Weidner, Elke Burgermeister, Birthe B. Kragelund. Inhibition of mutant RAS via myotubularin related protein 7-mimicking peptide [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B031.
Abstract IA01: Targeting KRAS: Light at the end of the tunnel
Authors: Barbacid Mariano 
Published: May 2023

Abstract
KRAS, an oncogene responsible for at least one fourth of all human cancers, was discovered four decades ago. Yet, the first KRAS selective inhibitor could not be approved until 2021. In spite of this long-awaited breakthrough, approved KRAS inhibitors only block one of the multiple oncogenic isoforms, KRASG12C, an isoform primarily present in lung adenocarcinomas. Moreover, tumor resistance to these KRAS inhibitors is becoming a significant issue in the clinic (Awad et al., N Engl J Med, 2021; Zhao et al., Nature 2021). To provide experimental information that might help to improve current KRAS targeting strategies, we have generated two experimental mouse tumor models of Kras-driven lung adenocarcinoma to compare the therapeutic consequences of eliminating, instead of inhibiting, KRAS oncoproteins. In addition, we have interrogated the molecular mechanisms responsible for the induction of resistance in sotorasib treated KrasG12C/p53 mouse lung adenocarcinomas. We have also investigated the potential role of tampering with KRAS signaling pathways to develop alternative therapeutic strategies against KRAS mutant lung and pancreatic cancers. Previous studies have shown that ablation of RAF1, either by itself, or in combination with the expression of a CDK4 kinase dead isoform, induce significant levels of tumor regression in Kras/p53 driven-lung adenocarcinomas without inducing significant toxicities (Sanclemente et al. Cancer Cell, 2018; Esteban-Burgos et al., PNAS 2020). However, Kras/p53 driven-pancreatic ductal adenocarcinomas (PDAC) are completely resistant to RAF1 ablation, unless RAF1 is deleted in combination with EGFR, an upstream KRAS effector (Blasco et al, Cancer Cell 2019). Yet, ablation of RAF1 and EGFR only resulted in the complete regression of a fraction of PDAC tumors. We have now identified activation of STAT3 as the main mechanism responsible for the resistance of PDAC tumors to RAF1/EGFR ablation. Indeed, combined ablation of RAF1/EGFR/STAT3 completely blocked proliferation of all tested mouse tumor cell lines and organoids. Moreover, all tumors tested in orthotopic allograft models regressed completely with no evidence of tumor regression for at least two months of TMX exposure. Finally, I will discuss potential strategies to pharmacologically validate this triple therapeutic strategy as well as plans to combine it with current KRAS inhibitors.
Citation Format: Mariano Barbacid. Targeting KRAS: Light at the end of the tunnel [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr IA01.
Abstract IA15: Coordinated transcriptional and catabolic programs support iron dependent adaptation to RAS-MAPK pathway inhibition in pancreatic cancer
Authors: Perera Rushika M. 
Published: May 2023

Abstract
The mechanisms underlying metabolic adaptation of pancreatic ductal adenocarcinoma (PDA) cells to pharmacological inhibition of RAS-MAPK signaling are largely unknown. Using transcriptome and chromatin immunoprecipitation profiling of PDA cells treated with the MEK inhibitor, Trametinib (MEKi), we identify transcriptional antagonism between c-MYC and the master transcription factors for lysosome gene expression, the MiT/TFE proteins. Under baseline conditions, c-MYC and MiT/TFE factors compete for binding to lysosome gene promoters to fine-tune gene expression. Treatment of PDA cells or patient organoids with MEKi leads to c-MYC downregulation and increased MiT/TFE-dependent lysosome biogenesis. Quantitative proteomics of immunopurified lysosomes uncovered reliance on ferritinophagy, the selective degradation of the iron storage complex ferritin, in MEKi treated cells. Ferritinophagy promotes mitochondrial iron-sulfur cluster protein synthesis and enhanced mitochondrial respiration. Accordingly, suppressing iron utilization sensitizes PDA cells to MEKi, highlighting a critical and targetable reliance on lysosome-dependent iron supply during adaptation to KRAS-MAPK inhibition.
Citation Format: Rushika M. Perera. Coordinated transcriptional and catabolic programs support iron dependent adaptation to RAS-MAPK pathway inhibition in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr IA15.
Abstract IA18: Somatic predisposition to germline mutations in the RTK/RAS/MAPK pathway
Authors: Goriely Anne 
Published: May 2023

Abstract
Somatic and germline mutations are traditionally viewed as separate entities. Yet, it is well established that germline variants (either inherited or occurring spontaneously) can influence cancer risk - i.e. cancer predisposition syndromes. In this presentation, I will discuss the reverse (but complementary) concept and propose that de novo germline mutations in the RTK/RAS/MAPK pathway occur recurrently because they confer a selective advantage to testicular stem cells, leading to the formation of ‘mole-like’ mutant clones in the ageing testis. This universal mechanism called ‘selfish selection’ relies on principles akin to oncogenesis and explains the paternal age-effect and high birth prevalence observed for several Mendelian disorders, including Noonan (PTPN11), Costello (HRAS), Apert (FGFR2) syndromes and achondroplasia (FGFR3). I will describe the strategies we have developed to show that the human testis is a repository of pathogenic de novo mutations and that selfish ‘moles’ are prevalent in older men’s testes. Our data show that the male germline is particularly vulnerable to dysregulation of the RTK/RAS/MAPK pathway. Importantly, because the germline provides a source of heritable material (unlike somatic tissues), this phenomenon has implications that extend far beyond the individual in whom it takes place, and is predicted to affect disease prevalence, genome heterogeneity and evolution of our species. This process illustrates another facet of the fascinating biology of the RAS/MAPK pathway.
Citation Format: Anne Goriely. Somatic predisposition to germline mutations in the RTK/RAS/MAPK pathway [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr IA18.