bluuebunny/crossref_metadata_2025 huggingface dataset:
Terminal Sliding‐Mode Control of Uncertain Robotic Manipulator System with Predefined Convergence Time
Authors: Wang Yang , Chen Mingshu , Song Yu
Published: June 2021

This paper concentrates on the predefined‐time trajectory tracking for an uncertain robotic manipulator system. First, a modified predefined‐time control (PTC) algorithm is proposed. Subsequently, with the help of proposed modified PTC algorithm and the nonsingular design method of terminal sliding mode, a novel nonsingular terminal sliding‐mode control (NTSMC) scheme is proposed for ensuring the predefined‐time convergence of tracking errors. The advantages of the newly proposed control scheme are as follows. (i) Unlike the conventional predefined‐time sliding‐mode control (SMC) which only guarantees the predefined‐time convergence of sliding‐mode surface, the proposed scheme can guarantee the predefined‐time convergence of tracking errors. (ii) Compared with the conventional PTC algorithm, the proposed modified PTC algorithm can reduce the initial control peaking and enhance the precision of convergence time. The performance and effectiveness of the proposed control scheme are illustrated by comparing with the existing methods.
IL-17 Is a Key Regulator of Mucin-Galectin-3 Interactions in Asthma
Authors: Mammen Manoj J. , Ali Jamil , Aurora Amita , Sharma Umesh C. , Aalinkeel Ravikumar , Mahajan Supriya D. , Sands Mark , Schwartz Stanley A.
Published: June 2021

Mucus hypersecretion and chronic airway inflammation are standard characteristics of several airway diseases, such as chronic obstructive pulmonary disease and asthma. Increased mucus secretion from increased mucin gene expression in the airway epithelium is associated with poor prognosis and mortality. We previously showed that the absence of tissue inhibitor of metalloproteinase 1 (TIMP-1) enhances lung inflammation, airway hyperreactivity, and lung remodeling in asthma in an ovalbumin (OVA) asthma model of TIMP-1 knockout (TIMPKO) mice as compared to wild-type (WT) controls and mediated by increased galectin-3 (Gal-3) levels. Additionally, we have shown that in the lung epithelial cell line A549, Gal-3 inhibition increases interleukin-17 (IL-17) levels, leading to increased mucin expression in the airway epithelium. Therefore, in the current study, we further examined the relationship between Gal-3 and the production of IL-17-axis cytokines and critical members of the mucin family in the murine TIMPKO asthma model and the lung epithelium cell line A549. While Gal-3 may regulate a Th1/Th2 response, IL-17 could stimulate the mucin genes, MUC5B and MUC5AC. Gal-3 and IL-17 interactions induce mucus expression in OVA-sensitized mice. We conclude that Gal-3 may play an essential role in the pathogenesis of asthma, and modulation of Gal-3 may prove helpful in the treatment of this disease.
Pyrotinib Treatment in Patients With HER2-positive Metastatic Breast Cancer and Brain Metastasis: Exploratory Final Analysis of Real-World, Multicenter Data
Authors: Anwar Munawar , Chen Qitong , Ouyang Dengjie , Wang Shouman , Xie Ning , Ouyang Quchang , Fan Peizhi , Qian Liyuan , Chen Gannong , Zhou Enxiang , Guo Lei , Gu Xiaowen , Ding Boning , Yang Xiaohong , Liu Liping , Deng Chao , Xiao Zhi , Li Jing , Wang Yunqi , Zeng Shan , Hu Jinhui , Zhou Wei , Qiu Bo , Wang Zhongming , Weng Jie , Liu Mingwen , Li Yi , Tang Tiegang , Wang Jianguo , Zhang Hui , Dai Bin , Tang Wuping , Wu Tao , Xiao Maoliang , Li Xiantao , Liu Hailong , Li Lai , Yi Wenjun
Published: June 2021

Purpose:

Patients with HER2-positive (HER2+) metastatic breast cancer (MBC) have poor prognoses. Pyrotinib has shown promising antitumor activity in MBC to improve progression-free survival (PFS). However, findings based on real-world data to analyze whether pyrotinib affects overall survival (OS) remain scarce.

Experimental Design:

This real-world study is an exploratory analysis of brain metastasis (BM) and the final update of our preceding study of 168 patients with HER2+ MBC. PFS, OS, tumor mutation burden (TMB), clinical benefit rate (CBR), and overall response rate (ORR) were analyzed.

Results:

Pyrotinib treatment led to a median PFS time of 8.00 months and a median OS of 19.07 months in the 168 participants. High TMB was associated with poor OS (P = 0.0072) and PFS (P = 0.0028). In the 39 patients with BM, the median PFS and OS were 8.67 and 13.93 months, respectively. The surgery/radiation (S/R) group of patients with BM had prolonged survival (PFS: 9.97 vs. 7.73 months P = 0.19; OS: 20.67 vs. 12.43 months P = 0.021) compared with the no surgery/no radiation group (NS/NR). The CBR was 58.6% (S/R) vs. 41.4% (NS/NR), while the ORR was 24.1% (S/R) vs. 31.0% (NS/NR).

Conclusions:

Pyrotinib shows promise as a novel pan-HER2 tyrosine kinase inhibitor (TKI) for the treatment of BM and should be evaluated further. Surgical or radiotherapy in combination with pyrotinib was found to statistically improve OS in our cohort. TMB could be an exploratory biomarker for predicting PFS and OS, but its clinical application still needs further verification.
Sulindac, a Nonselective NSAID, Reduces Breast Density in Postmenopausal Women with Breast Cancer Treated with Aromatase Inhibitors
Authors: Thompson Patricia A. , Huang Chuan , Yang Jie , Wertheim Betsy C. , Roe Denise , Zhang Xiaoyue , Ding Jie , Chalasani Pavani , Preece Christina , Martinez Jessica , Chow H.-H. Sherry , Stopeck Alison T.
Published: June 2021

Purpose:

To evaluate the effect of sulindac, a nonselective anti-inflammatory drug (NSAID), for activity to reduce breast density (BD), a risk factor for breast cancer.

Experimental Design:

An open-label phase II study was conducted to test the effect of 12 months' daily sulindac at 150 mg twice daily on change in percent BD in postmenopausal hormone receptor–positive breast cancer patients on aromatase inhibitor (AI) therapy. Change in percent BD in the contralateral, unaffected breast was measured by noncontrast magnetic resonance imaging (MRI) and reported as change in MRI percent BD (MRPD). A nonrandomized patient population on AI therapy (observation group) with comparable baseline BD was also followed for 12 months. Changes in tissue collagen after 6 months of sulindac treatment were explored using second-harmonic generated microscopy in a subset of women in the sulindac group who agreed to repeat breast biopsy.

Results:

In 43 women who completed 1 year of sulindac (86% of those accrued), relative MRPD significantly decreased by 9.8% [95% confidence interval (CI), −14.6 to −4.7] at 12 months, an absolute decrease of −1.4% (95% CI, −2.5 to −0.3). A significant decrease in mean breast tissue collagen fiber straightness (P = 0.032), an investigational biomarker of tissue inflammation, was also observed. MRPD (relative or absolute) did not change in the AI-only observation group (N = 40).

Conclusions:

This is the first study to indicate that the NSAID sulindac may reduce BD. Additional studies are needed to verify these findings and determine if prostaglandin E2 inhibition by NSAIDs is important for BD or collagen modulation.
Neoadjuvant Selicrelumab, an Agonist CD40 Antibody, Induces Changes in the Tumor Microenvironment in Patients with Resectable Pancreatic Cancer
Authors: Byrne Katelyn T. , Betts Courtney B. , Mick Rosemarie , Sivagnanam Shamilene , Bajor David L. , Laheru Daniel A. , Chiorean E. Gabriela , O'Hara Mark H. , Liudahl Shannon M. , Newcomb Craig , Alanio Cécile , Ferreira Ana P. , Park Byung S. , Ohtani Takuya , Huffman Austin P. , Väyrynen Sara A. , Dias Costa Andressa , Kaiser Judith C. , Lacroix Andreanne M. , Redlinger Colleen , Stern Martin , Nowak Jonathan A. , Wherry E. John , Cheever Martin A. , Wolpin Brian M. , Furth Emma E. , Jaffee Elizabeth M. , Coussens Lisa M. , Vonderheide Robert H.
Published: June 2021

Purpose:

CD40 activation is a novel clinical opportunity for cancer immunotherapy. Despite numerous active clinical trials with agonistic CD40 monoclonal antibodies (mAb), biological effects and treatment-related modulation of the tumor microenvironment (TME) remain poorly understood.

Patients and Methods:

Here, we performed a neoadjuvant clinical trial of agonistic CD40 mAb (selicrelumab) administered intravenously with or without chemotherapy to 16 patients with resectable pancreatic ductal adenocarcinoma (PDAC) before surgery followed by adjuvant chemotherapy and CD40 mAb.

Results:

The toxicity profile was acceptable, and overall survival was 23.4 months (95% confidence interval, 18.0–28.8 months). Based on a novel multiplexed immunohistochemistry platform, we report evidence that neoadjuvant selicrelumab leads to major differences in the TME compared with resection specimens from treatment-naïve PDAC patients or patients given neoadjuvant chemotherapy/chemoradiotherapy only. For selicrelumab-treated tumors, 82% were T-cell enriched, compared with 37% of untreated tumors (P = 0.004) and 23% of chemotherapy/chemoradiation-treated tumors (P = 0.012). T cells in both the TME and circulation were more active and proliferative after selicrelumab. Tumor fibrosis was reduced, M2-like tumor-associated macrophages were fewer, and intratumoral dendritic cells were more mature. Inflammatory cytokines/sec CXCL10 and CCL22 increased systemically after selicrelumab.

Conclusions:

This unparalleled examination of CD40 mAb therapeutic mechanisms in patients provides insights for design of subsequent clinical trials targeting CD40 in cancer.
Abstract A10: Quantitative biophysical analysis defining key components modulating KRAS recruitment to the plasma membrane
Authors: Lakshman Bindu , Messing Simon , Schmid Eva M. , Clogston Jeffrey D. , Gillette William K. , Esposito Dominic , Kessing Bailey , Fletcher Daniel A. , Nissley Dwight V. , McCormick Frank , Stephen Andrew G. , Jean-Francois Frantz L.
Published: June 2021

The KRAS fraction in the plasma membrane (PM) correlates with activation of the MAPK pathway and subsequent cellular proliferation. Understanding KRAS interaction with the PM constitutes a challenge given the complexity of the cellular environment. To gain insight on key components necessary for KRAS signal transduction at the membrane, we make use of reconstituted liposomes and giant unilamellar vesicles as synthetic membranes. Using surface plasmon resonance (SPR) spectroscopy, we demonstrate that KRAS and RAF1 domains interact with these membranes primarily through electrostatic interactions with negatively charged lipids reinforced by additional interactions involving phosphatidyl ethanolamine and cholesterol. RAF1 52-188 (RBDCRD) interacts with the membrane significantly more strongly than the isolated RBD or CRD domains and synergizes KRAS membrane partitioning. Calmodulin and PDE6 delta, but not galectin3, passively sequester KRAS and prevent it from binding to the PM. RAF1 RBDCRD interacts with membranes preferentially at non-raft lipids domains. The carboxyterminal O-methylation is crucial for KRAS membrane localization. These results contribute to a better understanding of how KRAS-membrane interaction can be tuned by multiple factors.

Citation Format: Bindu Lakshman, Simon Messing, Eva M. Schmid, Jeffrey D. Clogston, William K. Gillette, Dominic Esposito, Bailey Kessing, Daniel A. Fletcher, Dwight V. Nissley, Frank McCormick, Andrew G. Stephen, Frantz L. Jean-Francois. Quantitative biophysical analysis defining key components modulating KRAS recruitment to the plasma membrane [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 A10.
Abstract A11: Screening the Ras proteome microenvironment using APEX2
Authors: Mo Stephanie P. , Coulson Judy M. , Prior Ian A.
Published: June 2021

Ras proteins are small GTPases that regulate cellular growth and proliferation. The three RAS genes KRAS, HRAS and NRAS are ubiquitously expressed and share almost complete sequence homology; despite this they exhibit isoform-specific signaling in vitro and in vivo. Differences in localization, including nanoclustering on the cell surface, have been suggested to determine isoform-specific signaling. Currently, over 20 Ras effectors have been identified, although most work has focused on the Raf-MAP kinase and PtdIns 3-kinase (PI3K) pathways downstream of Ras. It has been shown that Ras isoform-specific signaling is highly context-dependent with significant cell type-dependent differences in coupling to canonical effector pathways. While the main Ras signaling network is understood, proteins regulating localization and interactions within the Ras network are still being identified. Here, we have performed a comprehensive analysis of the Ras isoform-specific interactome with particular interest in the role of cell surface nanoclustering and plasma membrane organization. We have screened the proteome microenvironment in living mammalian cells via APEX2, an engineered promiscuous peroxidase, which biotinylates vicinal proteins to the protein of interest. Here, APEX2-tagged Ras was used to identify proximal proteins to KRAS, HRAS and NRAS using mass spectrometry. Our preliminary data have shown that APEX2 is a suitable screening tool for the Ras proteome microenvironment. This method has allowed us to observe differences between the Ras isoforms, which are undergoing validation.

Citation Format: Stephanie P. Mo, Judy M. Coulson, Ian A. Prior. Screening the Ras proteome microenvironment using APEX2 [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 A11.
Abstract A21: Loss of Argonaute 2 leads to oncogene-induced senescence in mutant RAS-driven cancer
Authors: Siebenaler Ronald F. , Shankar Sunita , Tien Jean C. , Dommeti Vijaya L. , Zelenka-Wang Sylvia , Waninger Jessica , Mody Malay , Chugh Seema , Kumar-Sinha Chandan , Chinnaiyan Arul M.
Published: June 2021

The RAS gene family is among the most commonly mutated genes within cancer, but little progress has been made in successfully targeting RAS mutations. Targeting binding partners of mutated RAS, however, presents a promising alternative therapeutic strategy. With the goal of uncovering novel interactors of RAS, we recently identified Argonaute 2 (AGO2) of the RNA-induced silencing complex (RISC) as a novel partner of the Switch II domain of KRAS. In order to assess the role of AGO2 in KRAS-G12D driven disease, we developed a mouse model of pancreatic cancer with conditional loss of AGO2. While AGO2 knockout did not prevent development of early precursor pancreatic intraepithelial (PanIN) lesions, loss of AGO2 prevented progression to late-stage PanINs, pancreactic ductal adenocarcinoma (PDAC), and metastatic disease. AGO2 null lesions displayed increased activation of the EGFR-RAS signaling axis during PanIN development. This signaling resulted in an increase in WT RAS-GTP activation, pEGFR-Y1068, and pERK levels leading to the development of oncogene-induced senescence in these PanIN lesions. Furthermore, we observed that EGFR-mediated phosphorylation of AGO2-Y393 disrupted the interaction between WT RAS and AGO2. This regulation by EGFR, however, was blocked in cells expressing mutant KRAS. These results suggested that the interaction of mutant RAS and AGO2 was vital to tumor development. To better assess the role of AGO2 loss in mutant RAS driven cancer, we performed AGO2 knockdown in multiple cell lines expressing mutations in either NRAS or HRAS isoforms. In each cell line, AGO2 directly interacted with KRAS, NRAS, and KRAS. In addition to suppressing growth in mutant RAS-driven cells (T24: HRAS-G12V, SK-MEL-2: NRAS-Q61H), loss of AGO2 produced marked increases in beta-galactosidase and p16 expression, as well as a decrease in cyclin D1, suggesting development of oncogene-induced senescence. Interestingly, upon AGO2 loss, cells displayed induction of pEGFR and pERK similar to what was observed in our pancreatic mouse model, and despite decreased expression of mutant RAS, WT RAS-GTP loading upon AGO2 loss was strongly induced. Together these results suggest a unique EGFR-AGO2-RAS signaling axis that requires AGO2-RAS interaction to prevent induction of oncogene-induced senescence in mutant RAS-driven cancers.

Citation Format: Ronald F. Siebenaler, Sunita Shankar, Jean C. Tien, Vijaya L. Dommeti, Sylvia Zelenka-Wang, Jessica Waninger, Malay Mody, Seema Chugh, Chandan Kumar-Sinha, Arul M. Chinnaiyan. Loss of Argonaute 2 leads to oncogene-induced senescence in mutant RAS-driven 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 A21.
Abstract A28: USP21 promotes stemness of pancreatic cancer cells and bypass of KRAS extinction
Authors: Hou Pingping , Ma Xingdi , Ackroyd Jeffery , Zhang Jianhua , Jiang Shan , Wang Alan Y. , DePinho Ronald A.
Published: June 2021

Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer deaths, with an estimated 44,330 deaths per year in the United States alone. Deubiquitinases play fundamental roles in maintenance of cellular homeostasis, and deregulation of ubiquitin-specific proteases (USPs), the largest group of deubiquitinases, has been reported in multiple cancer types. In particular, we found that USP21 is amplified in 22% of PDAC cases in the patient dataset from the University of Texas Southwestern Medical Center, and it is the most frequently deregulated USP. However, little is known about the role of USP21 in PDAC. To examine biologic function of USP21 amplification in PDAC, we employed a novel “iKPC” mouse model, harboring tetO-LSL-KrasG12D (a transgenic allele with doxycycline-controlled tet-ON system regulating transcriptionally activated KRASG12D), ROSA26-LSL-rtTA, p53L/+ and p48Cre. We induced overexpression of USP21 in PDAC cells isolated from iKPC mice and found that USP21 promotes iKPC pancreatic tumor growth in vivo and drives KRAS-independent pancreatic tumor growth after blockage of KRAS expression. USP21 has a nuclear export signal and shuttles between the nucleus and cytoplasm. By modulating its subcellular localization, we discovered different functions of USP21 in supporting pancreatic cancer growth. Specifically, the nuclear function of USP21 supports pancreatic tumor growth by increasing tumor cell stemness via enhanced activation of the Wnt/beta-catenin signaling pathway. We showed that USP21 directly regulates proteasome pathway-dependent degradation of TCF7 protein. The cytoplasmic function of USP21 stimulates tumor cells to bypass KRAS extinction via MARK3, which phosphorylated its substrates and blocked their function by generating 14-3-3 binding sites. We found that MARK3 interacts with and is deubiquitinated by USP21. Knockdown of Ptpn3, a reported substrate of MARK3, increased protein level of PDGFRA, overexpression of which bypassed KRAS extinction. Moreover, we showed that inhibition of PDGF receptor pathway impairs KRAS-independent PDAC tumor growth driven by USP21 in vivo. Thus, our work demonstrated distinct oncogenic functions of USP21 in the presence and absence of KRAS signaling in PDAC, suggesting that KRAS-mutant PDAC patients with USP21 amplification may become resistant to KRAS inhibition.

Citation Format: Pingping Hou, Xingdi Ma, Jeffery Ackroyd, Jianhua Zhang, Shan Jiang, Alan Y. Wang, Ronald A. DePinho. USP21 promotes stemness of pancreatic cancer cells and bypass of KRAS extinction [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 A28.
Abstract A34: Defining KRAS mutation-specific kinome signatures and vulnerabilities in colorectal cancer
Authors: Srivastava Nishi , Kurimchak Alison , Montavez Carlos , Chernoff Jonathan , Duncan James
Published: June 2021

Activating mutations in KRAS occur in about half of colorectal cancers (CRC) and are associated with drug resistance and poor survival. A number of distinct oncogenic mutations in KRAS have been identified in CRC, including mutations in exon 2-producing (G12D, G12V, and G13D) and in exon 4 (A146T and A146V). Importantly, emerging clinical and basic science data indicate that these KRAS mutations are not all equal in their oncogenic properties and sensitivity to targeted therapies. To access and exploit the distinct kinase programs engaged by the different KRAS mutants, we employed an innovative mass spectrometry-based technique that enables the majority of the human kinome to be analyzed simultaneously, providing the means to explore the KRAS-induced kinome and its response to targeted therapy in an unbiased fashion. Using multiplexed inhibitor beads and mass spectrometry (MIB/MS), we measured differences in protein kinase signaling between the distinct KRAS mutants (G12D, G12V, G13D and A146T) at both baseline and in response to targeted MEK inhibitors in isogenic KRAS mutant xenografts. Kinome profiling of the tumors demonstrated that each KRAS mutant exhibits a unique basal activation signature, with G12D and A146T mutations showing the most divergence from wild-type (WT) KRAS kinome(s). Tumors harboring G12D or A146T knockin mutations displayed intrinsic resistance to MEKi trametinib, while G13D remained sensitive to MEK inhibition comparable to KRAS WT tumors. Notably, tumors expressing G12V KRAS mutations showed enhanced tumor regression relative to KRAS WT, suggesting G12V mutations may sensitize CRC cells to MEK inhibition. Analysis of kinome adaptations to trametinib using MIB/MS revealed that each mutant reprogrammed their kinome(s) uniquely, supporting the design of individualized MEK inhibitor combination therapies tailored to specific KRAS mutations. Our kinome profiling studies further support the notion that not all KRAS mutations are created equal in CRC and demonstrate mutation-specific kinome activation signatures that can be therapeutically exploited in kinase inhibitor combination therapies.

Citation Format: Nishi Srivastava, Alison Kurimchak, Carlos Montavez, Jonathan Chernoff, James Duncan. Defining KRAS mutation-specific kinome signatures and vulnerabilities in colorectal 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 A34.
Abstract A36: Neoadjuvant-like Ezh2 inhibition in Kras-driven lung cancer amplifies inflammation and creates new therapeutic vulnerabilities
Authors: Gargiulo Gaetano
Published: June 2021

Kras-driven non-small cell lung cancers (NSCLC) are a leading cause of death with limited therapeutic options. Many NSCLC exhibit high levels of Ezh2, the enzymatic subunit of the Polycomb repressive complex 2 (PRC2). We tested Ezh2 inhibitors as single agent or prior to chemotherapy in mice with orthotopic Kras-driven NSCLC grafts, which homogeneously express Ezh2. These tumors display sensitivity to EZH2 inhibition by GSK126 but also amplify an inflammatory program involving signaling through NF-kB and genes residing in PRC2-regulated chromatin. During this process tumor cells overcome GSK126 antiproliferative effects. We identified oncogenes that may mediate progression through an in vivo RNAi screen aimed at targets of PRC2/NF-kB. An in vitro compound screening linked GSK126-driven inflammation and therapeutic vulnerability in human cells to regulation of RNA synthesis and proteostasis. Interestingly, GSK126-treated NSCLCs in vivo also showed an enhanced response to a combination of nimesulide-bortezomib. Thus, Ezh2 inhibition may restrict cell proliferation and promote defined adaptive responses. Targeting these responses potentially improves outcomes in Kras-driven NSCLC.

Citation Format: Gaetano Gargiulo. Neoadjuvant-like Ezh2 inhibition in Kras-driven lung cancer amplifies inflammation and creates new therapeutic vulnerabilities [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 A36.
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

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

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

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

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

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

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

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

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

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

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.