Ras Family Research Articles

SCP Phosphatases and Oncogenesis

Small SCP phosphatases CTDSP1, CTDSP2, and CTDSPL specifically dephosphorylate serine and threonine residues in protein molecules. The enzymes are involved in regulating activity of RNA polymerase II at the transition from transcription initiation to elongation, regulating expression of neuron-specific genes, and activating the key cell-cycle protein pRb at the G1/S boundary. In addition, the substrates of SCP phosphatases include SMAD transcription modulators; AKT1 protein kinase, which regulates the cell cycle, apoptosis, and angiogenesis; the TWIST1 and c-MYC transcription factors; Ras family proteins, which are involved in signaling pathways regulating the cell growth and apoptosis; CDCA3, which is associated with cell division; the cyclin-dependent kinase inhibitor p21; and the promyelocytic leukemia protein (PML), which is involved in regulation of the tumor suppressors p53, PTEN, and mTOR. Dysfunction or inactivation of SCP phosphatases leads to various diseases, including cancer. Recently the increase in interest to SCP phosphatases is due to their their tumor growth-inhibiting properties or role in the development of malignant tumors of various etiology and localization. The review discusses the properties of SCP phosphatases and their role in oncogenesis. Understanding the functions of SCP phosphatases and their regulatory mechanisms can be useful in searching for efficient targets for tumor therapy.

Targeting sphingosine kinase 1 (SK1) enhances oncogene-induced senescence through ceramide synthase 2 (CerS2)-mediated generation of very-long-chain ceramides

Senescence is an antiproliferative mechanism that can suppress tumor development and can be induced by oncogenes such as genes of the Ras family. Although studies have implicated bioactive sphingolipids (SL) in senescence, the specific mechanisms remain unclear. Here, using MCF10A mammary epithelial cells, we demonstrate that oncogenic K-Ras (Kirsten rat sarcoma viral oncogene homolog) is sufficient to induce cell transformation as well as cell senescence—as revealed by increases in the percentage of cells in the G1 phase of the cell cycle, p21WAF1/Cip1/CDKN1A (p21) expression, and senescence-associated β-galactosidase activity (SA-β-gal). Furthermore, oncogenic K-Ras altered SL metabolism, with an increase of long-chain (LC) C18, C20 ceramides (Cer), and very-long-chain (VLC) C22:1, C24 Cer, and an increase of sphingosine kinase 1 (SK1) expression. Since Cer and sphingosine-1-phosphate have been shown to exert opposite effects on cellular senescence, we hypothesized that targeting SK1 could enhance oncogenic K-Ras-induced senescence. Indeed, SK1 downregulation or inhibition enhanced p21 expression and SA-β-gal in cells expressing oncogenic K-Ras and impeded cell growth. Moreover, SK1 knockdown further increased LC and VLC Cer species (C18, C20, C22:1, C24, C24:1, C26:1), especially the ones increased by oncogenic K-Ras. Fumonisin B1 (FB1), an inhibitor of ceramide synthases (CerS), reduced p21 expression induced by oncogenic K-Ras both with and without SK1 knockdown. Functionally, FB1 reversed the growth defect induced by oncogenic K-Ras, confirming the importance of Cer generation in the senescent phenotype. More specifically, downregulation of CerS2 by siRNA blocked the increase of VLC Cer (C24, C24:1, and C26:1) induced by SK1 knockdown and phenocopied the effects of FB1 on p21 expression. Taken together, these data show that targeting SK1 is a potential therapeutic strategy in cancer, enhancing oncogene-induced senescence through an increase of VLC Cer downstream of CerS2.

Association analysis between the interaction of RAS family genes mutations and papillary thyroid carcinoma in the Han Chinese population.

Papillary thyroid carcinoma (PTC) is the major subtype of thyroid cancer, accounting for 75%-85% of all thyroid malignancies. This study aimed to identify the association between the interactions of single nucleotide polymorphisms (SNPs) in RAS family genes and PTC in the Han Chinese population, to provide clues to the pathogenesis and potential therapeutic targets for PTC. Hap Map and NCBI-db SNP databases were used to retrieve SNPs. Haploview 4.2 software was used to filter SNPs based on specific parameters, six SNPs of RAS gene (KRAS-rs12427141, KRAS-rs712, KRAS-rs7315339, HRAS-rs12628, NRAS-rs14804 and NRAS-rs2273267) were genotyped by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) in 673 PTC patients and 657 healthy controls, the interactive effect was evaluated by crossover analysis, logistic regression and GMDR software.We found that genetic mutation in rs712 have significant associations with PTC risk after Bonferroni correction (p<0.001). The interaction between KRAS-rs12427141 and HRAS-rs12628 increased the risk of PTC (U=-2.119, p<0.05), the interaction between KRAS-rs2273267 and HRAS-rs7315339 reduced the risk of PTC (U=2.195, p<0.05). GMDR analysis showed that the two-factor model (KRAS-rs712, NRAS-rs2273267) was the best (p=0.0107). Summarily, there are PTC-related interactions between RAS family genes polymorphisms in the Han Chinese population.

Validation of Isoform- and Mutation-Specific RAS Antibodies.

Validation of antibody specificity is essential for the accurate evaluation of protein expression. For antibodies that recognize the gene products of the RAS family of oncogenes (HRAS, KRAS, and NRAS), an important challenge is the determination of selectivity for the four nearly identical HRAS, KRAS4A, KRAS4B, and NRAS proteins. With increasing appreciation for the distinct roles of the different RAS proteins in normal and neoplastic cells, there is aneed for well-validated antibodies to evaluate the function and expression of the different RAS isoforms. Here we describe our experimental approaches to characterize RAS antibodies for their isoform- and mutant-specificity for use in immunoblot analyses.

Structural bioinformatics enhances mechanistic interpretation of genomic variation, demonstrated through the analyses of 935 distinct RAS family mutations.

MotivationProtein-coding genetic alterations are frequently observed in Clinical Genetics, but the high yield of variants of uncertain significance remains a limitation in decision making. RAS-family GTPases are cancer drivers, but only 54 variants, across all family members, fall within well-known hotspots. However, extensive sequencing has identified 881 non-hotspot variants for which significance remains to be investigated.ResultsHere, we evaluate 935 missense variants from seven RAS genes, observed in cancer, RASopathies and the healthy adult population. We characterized hotspot variants, previously studied experimentally, using 63 sequence- and 3D structure-based scores, chosen by their breadth of biophysical properties. Applying scores that display best correlation with experimental measures, we report new valuable mechanistic inferences for both hot-spot and non-hotspot variants. Moreover, we demonstrate that 3D scores have little-to-no correlation with those based on DNA sequence, which are commonly used in Clinical Genetics. Thus, combined, these new knowledge bear significant relevance.Availability and implementationAll genomic and 3D scores, and markdown for generating figures, are provided in our supplemental data.Supplementary information Supplementary data are available at Bioinformatics online.

Design, synthesis and pharmacological evaluation of bicyclic and tetracyclic pyridopyrimidinone analogues as new KRASG12C inhibitors.

KRAS is the most commonly altered oncogene of the RAS family, especially the G12C mutant (KRASG12C), which has been a promising drug target for many cancers. On the basis of the bicyclic pyridopyrimidinone framework of the first-in-class clinical KRASG12C inhibitor AMG510, a scaffold hopping strategy was conducted including a F-OH cyclization approach and a pyridinyl N-atom working approach leading to new tetracyclic and bicyclic analogues. Compound 26a was identified possessing binding potency of 1.87μM against KRASG12C and cell growth inhibition of 0.79μM in MIA PaCa-2 pancreatic cancer cells. Treatment of 26a with NCI-H358cells resulted in down-regulation of KRAS-GTP levels and reduction of phosphorylation of downstream ERK and AKT dose-dependently. Molecular docking suggested that the fluorophenol moiety of 26a occupies a hydrophobic pocket region thus forming hydrogen bonding to Arg68. These results will be useful to guide further structural modification.

Influence of Cholesterol on the Orientation of the Farnesylated GTP-Bound KRas-4B Binding with Anionic Model Membranes.

The Ras family of proteins is tethered to the inner leaflet of the cell membranes which plays an essential role in signal transduction pathways that promote cellular proliferation, survival, growth, and differentiation. KRas-4B, the most mutated Ras isoform in different cancers, has been under extensive study for more than two decades. Here we have focused our interest on the influence of cholesterol on the orientations that KRas-4B adopts with respect to the plane of the anionic model membranes. How cholesterol in the bilayer might modulate preferences for specific orientation states is far from clear. Herein, after analyzing data from in total 4000 ns-long molecular dynamics (MD) simulations for four KRas-4B systems, properties such as the area per lipid and thickness of the membrane as well as selected radial distribution functions, penetration of different moieties of KRas-4B, and internal conformational fluctuations of flexible moieties in KRas-4B have been calculated. It has been shown that high cholesterol content in the plasma membrane (PM) favors one orientation state (OS), exposing the effector-binding loop for signal transduction in the cell from the atomic level. We confirm that high cholesterol in the PM helps KRas-4B mutant stay in its constitutively active state, which suggests that high cholesterol intake can increase mortality and may promote cancer progression for cancer patients. We propose that during the treatment of KRas-4B-related cancers, reducing the cholesterol level in the PM and sustaining cancer progression by controlling the plasma cholesterol intake might be taken into account in anti-cancer therapies.

Mutation bias within oncogene families is related to proliferation-specific codon usage

It is well known that in cancer gene families some members are more frequently mutated in tumor samples than their family counterparts. A paradigmatic case of this phenomenon is KRAS from the RAS family. Different explanations have been proposed ranging from differential interaction with other proteins to preferential expression or localization. Interestingly, it has been described that despite the high amino acid identity between RAS family members, KRAS employs an intriguing differential codon usage. Here, we found that this phenomenon is not exclusive to the RAS family. Indeed, in the RAS family and other oncogene families with two or three members, the most prevalently mutated gene in tumor samples employs a differential codon usage that is characteristic of genes involved in proliferation. Prompted by these observations, we chose the RAS family to experimentally demonstrate that the translation efficiency of oncogenes that are preferentially mutated in tumor samples is increased in proliferative cells compared to quiescent cells. These results were further validated by assessing the translation efficiency of KRAS in cell lines that differ in their tRNA expression profile. These differences are related to the cell division rate of the studied cells and thus suggest an important role in context-specific oncogene expression regulation. Altogether, our study demonstrates that dynamic translation programs contribute to shaping the expression profiles of oncogenes. Therefore, we propose this codon bias as a regulatory layer to control cell context-specific expression and explain the differential prevalence of mutations in certain members of oncogene families.

BIOM-26. MOLECULAR PREDICTORS OF RESPONSE TO SELINEXOR IN RECURRENT GLIOBLASTOMA (GBM)

Abstract The nuclear export protein exportin 1 (XPO1) is overexpressed in many cancers, including GBM. Selinexor is an inhibitor of XPO1 that crosses the blood-brain-barrier and targets cancer cells by sequestering tumor suppressor proteins and oncoprotein mRNAs in the nucleus, inducing cancer cell apoptosis. We previously reported encouraging results from a phase II trial of selinexor for molecularly unselected patients with recurrent GBM (ASCO 2019). Pre-treatment tumors from 57 patients were exome and RNA sequenced to explore molecular correlates of response, in a hypothesis generating, post-hoc, exploratory analysis. We compared overall survival (OS) and progression-free survival (PFS) between mutated and wild-type patients. Two mutated genes were associated with longer survival in selinexor treated patients: DOCK8 (n=7; PFS, P=0.013, hazard ratio [HR]=3.75 [1.32–10.62]; overall survival, P=0.009, HR=15.39 [2.00–118.34]) and PDX1 (n=5, PFS, P=0.014, HR=4.468 [1.361–14.670]). Other commonly mutated genes in glioma, including IDH1 (n=9) were observed but not associated with survival. RNAseq data were used to infer differential protein activities, which were input into a machine learning model that compared patients with selinexor sensitive disease (best response of partial or complete response, n=7) and resistant disease (best response of progressive disease, n=23). We found the inferred activities of three proteins emerged as the most associated with response and could be combined in a model to accurately predict benefit from selinexor treatment (area under the ROC curve from leave one out cross validation = 0.89 permutation test P&amp;lt; 0.04). The three proteins were ZC3H12A (also called MCPIP-1), a negative regulator of inflammation; RAB43, a member of the RAS family that binds GTP and regulates vesicle trafficking, and SOCS3, a suppressor of cytokine signaling that can antagonize JAK/STAT signaling. Together these data identified mutations and proteins activities for identifying patients most likely to benefit from selinexor treatment. Further studies are required for validation. ClinicalTrials.gov:NCT01986348.

Anti-Tumor Activity of ASTX029, a Dual Mechanism Inhibitor of ERK1/2, in Preclinical AML Models

Oncogenic mutations in genes such as the RAS family (KRAS, NRAS or HRAS) or receptor tyrosine kinases (RTKs) drive tumor growth through aberrant activation of the mitogen activated protein kinase (MAPK) signaling pathway. Acute myeloid leukemia (AML) patients frequently exhibit activating mutations in MAPK pathway members, such as NRAS and KRAS, suggesting that these malignancies may be driven by aberrant activation of the MAPK pathway. Targeting of the MAPK pathway has been clinically validated in solid tumors, with agents targeting BRAF and MEK approved for the treatment of BRAF-mutant melanoma. However, there is currently no approved therapy directly targeting activated RAS family members and resistance to MAPK pathway inhibitors is frequently associated with reactivation of MAPK signaling. ERK1/2 (ERK) is a downstream node in the MAPK pathway and therefore represents an attractive therapeutic target for inhibition of MAPK signaling in these settings. We have recently described in vivo anti-tumor activity in MAPK-activated solid tumor models following treatment with ASTX029, a highly potent ERK inhibitor developed using fragment-based drug design. ASTX029 has a distinctive ERK binding mode which confers dual mechanism inhibition of ERK, inhibiting both the catalytic activity of ERK and its phosphorylation by MEK. Here, we demonstrate that ASTX029 is also active in AML models and potently inhibits in vitro and in vivo MAPK signaling and growth in these models. Using a panel of 15 AML cell lines, we investigated sensitivity to ASTX029 in vitro. We observed that 8 cell lines bearing mutations leading to increased MAPK pathway signaling were sensitive to treatment with ASTX029 with an average IC50 value of 47 nM, in contrast to an average IC50 value of 1800 nM for cell lines without activating mutations. The phosphorylation of RSK, a direct substrate of ERK, was suppressed for up to 24 h following treatment with ASTX029 in vitro. We have previously demonstrated good oral bioavailability for ASTX029 and once daily dosing resulted in significant tumor growth inhibition in AML cell line xenograft models. To confirm target engagement in vivo, we examined MAPK signaling in xenograft tissue and observed inhibition of the phosphorylation of RSK and of ERK itself, consistent with the dual mechanism of action proposed for ASTX029. In summary, the ERK inhibitor, ASTX029, has potent activity against MAPK-activated tumor models, including AML models, and is now being tested in a Phase 1/2 clinical trial in advanced solid tumors (NCT03520075). These data highlight its therapeutic potential for the treatment of AML in patients with mutations leading to MAPK pathway activation and support further investigation in these patient populations. Disclosures Hindley: Astex Pharmaceuticals: Current Employment. Fazal:Astex Pharmaceuticals: Current Employment. Munck:Astex Pharmaceuticals: Current Employment. Martins:Astex Pharmaceuticals: Current Employment. Shah:Astex Pharmaceuticals: Current Employment. Wilsher:Astex Pharmaceuticals: Current Employment. Wallis:Astex Pharmaceuticals: Current Employment. Keer:Astex Pharmaceuticals, Inc.: Current Employment. Lyons:Astex Pharmaceuticals: Current Employment.

Beyond the Mevalonate Pathway: Control of Post-Prenylation Processing by Mutant p53.

Missense mutations in the TP53 gene are among the most frequent alterations in human cancer. Consequently, many tumors show high expression of p53 point mutants, which may acquire novel activities that contribute to develop aggressive tumors. An unexpected aspect of mutant p53 function was uncovered by showing that some mutants can increase the malignant phenotype of tumor cells through alteration of the mevalonate pathway. Among metabolites generated through this pathway, isoprenoids are of particular interest, since they participate in a complex process of posttranslational modification known as prenylation. Recent evidence proposes that mutant p53 also enhances this process through transcriptional activation of ICMT, the gene encoding the methyl transferase responsible for the last step of protein prenylation. In this way, mutant p53 may act at different levels to promote prenylation of key proteins in tumorigenesis, including several members of the RAS and RHO families. Instead, wild type p53 acts in the opposite way, downregulating mevalonate pathway genes and ICMT. This oncogenic circuit also allows to establish potential connections with other metabolic pathways. The demand of acetyl-CoA for the mevalonate pathway may pose limitations in cell metabolism. Likewise, the dependence on S-adenosyl methionine for carboxymethylation, may expose cells to methionine stress. The involvement of protein prenylation in tumor progression offers a novel perspective to understand the antitumoral effects of mevalonate pathway inhibitors, such as statins, and to explore novel therapeutic strategies.

Double Genetic Hits and Subclonal Mosaicism in the Ras Signaling Pathway in Myeloid Neoplasia

The high prevalence of oncogenic mutations in the RAS family genes including their down or upstream partners (e.g., NRAS, KRAS, PTPN11, RIT1α) has strong implication in abnormal Ras signaling in the pathogenesis of myeloid neoplasia (MN). For many years RAS mutations have been considered non-targetable. However, recently multiple targeted agents have been introduced as possible future therapies. While common in JMML, RAS mutations are rarely seen in adult MN as the ancestral event. Instead they serve as subclonal hits. To that end, the clinical impact of RAS mutations has been controversial: whereas some studies did not find prognostic impact, others demonstrated that these mutations may confer a poor prognosis. Nevertheless, molecular analysis of core binding factor AML patients showed the coexistence of RAS clones harboring independent hits targeting the same pathway. We stipulate that occurrence of RAS mutations is not random and reasoned that studying cases with multiple RAS mutant subclones may reveal distinct molecular or etio-pathologic background attracting the emergence of RAS mutations as a way to augment MYC activity as a ubiquitous pathway of clonal progression. In a review of a molecular collection of data of a cohort of MN patients (n=1876) including MDS (n=692), MDS/MPN (n=282), AML (pAML, 710; sAML, 192) we found RAS mutations in 21% (403/1876) of the patients. Among them we encountered a subgroup of patients (9%; 38/403) harboring multiple RAS mutations. These multiple hits affected PTPN11, NRAS, KRAS and RIT1-α. Most of these multiple RAS mutants represented a subclonal mosaicism rather than biallelic subclones as indicated by single cell DNA sequencing. Three patients were assessed serially (2 patients/ 2 time points and 1 patient/ 5 time points). In one case NRAS (VAF-24%) and PTPN11 (VAF-28%) were acquired at the time of AML evolution. In an MDS/MPN-U, NRAS/KRAS were both detected at diagnosis with a VAF of 1% and 12% each. Both clones increased over 2 years' time span with NRAS ramping to 43% and reaching KRAS (50%). In the 3rd case, samples were collected over a period of 5 years with NRAS and PTPN11 being detected only at the later time point (VAF of 5% and 15%, respectively). In contrast to competing RAS subclones in adults, our historical cohort of JMML showed that at least one RAS mutation was found in 89% (82/92) of the patients as ancestral events and in mutually exclusive manner while biallelic RAS mutations occurred in 15% (12/82) of the cohort. In adult MN, the most common subclonal mosaicism was encountered in NRAS/KRAS (63%; 24/38), while less fraction of patients had competing subclonal mutations in NRAS (26%; 10/38) or in KRAS (8%; 3/38). Most of these mutations were found in canonical sites (NRAS: G12D/S/A/C, G13D/R/V, Q61A/H/R; KRAS: G12D/A/R, G13D/R/V, A146P/T). Patients carrying RAS mutations had significantly higher WBCs (20.5 vs 7.2, P&amp;lt;.001) consistent with the role of RAS in proliferation, possibly via augmentation of MYC. Isolated chromosome 7 abnormalities were more common among RAS mutant carriers who otherwise showed less complex karyotypes. RAS mutations were mostly enriched in MDS/MPN (26%) while absent in sAML suggesting the impact of these lesions on OS but not on PFS. Analysis of clonal architecture showed that accumulation of RAS subclones was most commonly related to the presence TET2 (17% vs. 8%) and EZH2 (6% vs. 1%) compared to WT. At the time of AML evolution, the presence of RAS mutation did not correlate with OS (18 vs. 13 mo., P=0.07). However, median OS was shorter in MDS and MDS/MPN carrying multiple RAS mutations compared to WT (10 vs. 30 mo., P=0.005) again supporting the contention that these clones may be markers of non-progression-related mortality. In sum, our study supports the notion that ancestral RAS hits are common in childhood MN with a hereditary component whereby the progression is related to the acquisition of secondary hits in biallelic configuration. In contrast in adult and elderly MN, specific genetic background predisposes to RAS mutant mosaicism such as ancestral TET2 and EZH2 lesions and MPN features reflecting the direction of selection pressure towards accumulation of multiple RAS pathway hits. Lack of the gene/dose effect on progression indicates, that in contrast to the compound heterozygous RAS childhood diseases, RAS mutant mosaicism reflects branching rather than linear evolution mode. Disclosures Sekeres: Pfizer: Consultancy; Takeda/Millenium: Consultancy; BMS: Consultancy. Carraway:Abbvie: Other: Independent Advisory Committe (IRC); ASTEX: Other: Independent Advisory Committe (IRC); Takeda: Other: Independent Advisory Committe (IRC); Stemline: Consultancy, Speakers Bureau; Jazz: Consultancy, Speakers Bureau; BMS: Consultancy, Other: Research support, Speakers Bureau; Novartis: Consultancy, Speakers Bureau. Saunthararajah:EpiDestiny: Consultancy, Current equity holder in private company, Patents &amp; Royalties: University of Illinois at Chicago. Patel:Alexion: Other: educational speaker. Maciejewski:Alexion, BMS: Speakers Bureau; Novartis, Roche: Consultancy, Honoraria.

A four prognosis-associated lncRNAs (PALnc) based risk score system reflects immune cell infiltration and predicts patient survival in pancreatic cancer

BackgroundPancreatic cancer (PC) is one of the most common cancers and the leading cause of cancer-related death worldwide. Exploring novel predictive biomarkers for PC patients’ prognosis is in urgent need.MethodsIn the present study, we conducted Cox proportional hazards regression to identify critical prognosis-associated lncRNAs (PALncs) in TCGA PC dataset. Based on the results of multivariate analysis, a PALnc-based risk score system was established, and validated in GSE62452 dataset. The validity and reliability of the risk score system for prognosis of PC were evaluated through ROC analysis. And function enrichment analyses for the PALncs were also performed.ResultIn the multivariate analysis, four PALncs (LINC00476, C9orf163, LINC00346 and DSCR9) were screened out to develop a risk score system, which showed a high AUC at 3 and 5 years overall survival (0.785 at 3 year OS, 0.863 at 5 year OS) in TCGA datasets. And the ROC analysis of the risk score system for RFS in TCGA dataset revealed that AUC for RFS was 0.799 at 3 years and 0.909 at 5 years. Further, the AUC for OS in the validation cohort was 0.705 at 3 years and 0.959 at 5 years. Furthermore, the functional enrichment analysis revealed that these PALncs may be involved in various pathways related to cancer, including Ras family activation, autophagy in cancer, MAPK signaling pathway, HIF-1 signaling pathway, PI3K-Akt signaling pathway, etc. And correlation analysis of these tumor infiltrating immune cells and risk score system revealed that the infiltration level of B cell naïve, plasma cells, and CD8+ T cells are negatively correlated to the risk score system, while macrophages M2 positively correlated to the risk score system.ConclusionOur study established a four PALncs based risk score system, which reflects immune cell infiltration and predicts patient survival for PC.

Small GTPase RAB6 deficiency promotes alveolar progenitor cell renewal and attenuates PM2.5-induced lung injury and fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive interstitial lung disease characterized by chronic non-specific inflammation of the interstitial lung and extensive deposition of collagen fibers leading to destruction of lung function. Studies have demonstrated that exposure to fine particulate matter (PM2.5) increases the risk of IPF. In order to recover from PM2.5-induced lung injury, alveolar epithelial cells need to be repaired and regenerated to maintain lung function. Type 2 alveolar epithelial cells (AEC2) are stem cells in the adult lung that contribute to the lung repair process through complex signaling. Our previous studies demonstrated that RAB6, a RAS family member lowly expressed in lung cancer, inhibited lung cancer stem cell self-renewal, but it is unclear whether or not and how RAB6 may regulate AEC2 cell proliferation and self-renewal in PM2.5-induced pulmonary fibrosis. Here, we demonstrated that knockout of RAB6 inhibited pulmonary fibrosis, oxidative stress, and AEC2 cell death in PM2.5-injured mice. In addition, knockout of RAB6 decreased Dickkopf 1(DKK1) autocrine and activated proliferation, self-renewal, and wnt/β-catenin signaling of PM2.5-injured AEC2 cells. RAB6 overexpression increased DKK1 autocrine and inhibited proliferation, self-renewal and wnt/β-catenin signaling in AEC2 cells in vitro. Furthermore, DKK1 inhibitors promoted proliferation, self-renewal and wnt/β-catenin signaling of RAB6 overexpressing AEC2 cells, and attenuated PM2.5-induced pulmonary fibrosis in mice. These data establish RAB6 as a regulator of DKK1 autocrine and wnt/β-catenin signal that serves to regulate AEC2 cell proliferation and self-renewal, and suggest a mechanism that RAB6 disruption may promote AEC2 cell proliferation and self-renewal to enhance lung repair following PM2.5 injury.

A Novel KRAS Antibody Highlights a Regulation Mechanism of Post-Translational Modifications of KRAS during Tumorigenesis.

KRAS is a powerful oncogene responsible for the development of many cancers. Despite the great progress in understanding its function during the last decade, the study of KRAS expression, subcellular localization, and post-translational modifications remains technically challenging. Accordingly, many facets of KRAS biology are still unknown. Antibodies could be an effective and easy-to-use tool for in vitro and in vivo research on KRAS. Here, we generated a novel rabbit polyclonal antibody that allows immunolabeling of cells and tissues overexpressing KRAS. Cell transfection experiments with expression vectors for the members of the RAS family revealed a preferential specificity of this antibody for KRAS. In addition, KRAS was sensitively detected in a mouse tissue electroporated with an expression vector. Interestingly, our antibody was able to detect endogenous forms of unprenylated (immature) and prenylated (mature) KRAS in mouse organs. We found that KRAS prenylation was increased ex vivo and in vivo in a model of KRASG12D-driven tumorigenesis, which was concomitant with an induction of expression of essential KRAS prenylation enzymes. Therefore, our tool helped us to put the light on new regulations of KRAS activation during cancer initiation. The use of this tool by the RAS community could contribute to discovering novel aspects of KRAS biology.

Development of Noonan syndrome by deregulation of allosteric SOS autoactivation

Ras family proteins play an essential role in several cellular functions, including growth, differentiation, and survival. The mechanism of action of Ras mutants in Costello syndrome and cancers has been identified, but the contribution of Ras mutants to Noonan syndrome, a genetic disorder that prevents normal development in various parts of the body, is unknown. Son of Sevenless (SOS) is a Ras guanine nucleotide exchange factor. In response to Ras-activating cell signaling, SOS autoinhibition is released and is followed by accelerative allosteric feedback autoactivation. Here, using mutagenesis-based kinetic and pulldown analyses, we show that Noonan syndrome Ras mutants I24N, T50I, V152G, and D153V deregulate the autoactivation of SOS to populate their active form. This previously unknown process has been linked so far only to the development of Noonan syndrome. In contrast, other Noonan syndrome Ras mutants-V14I, T58I, and G60E-populate their active form by deregulation of the previously documented Ras GTPase activities. We propose a novel mechanism responsible for the deregulation of SOS autoactivation, where I24N, T50I, V152G, and D153V Ras mutants evade SOS autoinhibition. Consequently, they are capable of forming a complex with the SOS allosteric site, thus aberrantly promoting SOS autoactivation, resulting in the population of active Ras mutants in cells. The results of this study elucidate the molecular mechanism of the Ras mutant-mediated development of Noonan syndrome.

Small GTPases of the Ras and Rho Families Switch on/off Signaling Pathways in Neurodegenerative Diseases.

Small guanosine triphosphatases (GTPases) of the Ras superfamily are key regulators of many key cellular events such as proliferation, differentiation, cell cycle regulation, migration, or apoptosis. To control these biological responses, GTPases activity is regulated by guanine nucleotide exchange factors (GEFs), GTPase activating proteins (GAPs), and in some small GTPases also guanine nucleotide dissociation inhibitors (GDIs). Moreover, small GTPases transduce signals by their downstream effector molecules. Many studies demonstrate that small GTPases of the Ras family are involved in neurodegeneration processes. Here, in this review, we focus on the signaling pathways controlled by these small protein superfamilies that culminate in neurodegenerative pathologies, such as Alzheimer’s disease (AD) and Parkinson’s disease (PD). Specifically, we concentrate on the two most studied families of the Ras superfamily: the Ras and Rho families. We summarize the latest findings of small GTPases of the Ras and Rho families in neurodegeneration in order to highlight these small proteins as potential therapeutic targets capable of slowing down different neurodegenerative diseases.

Abstract 2582: Dose-dependent NRASG12V-mediated signaling controls cell cycle progression and leukemogenic signaling in a CRISPR/Cas9-modified human AML cell line

Abstract Mutations in RAS family are observed in 6-96% of cancer. In acute myeloid leukemia (AML), mutations in NRAS/KRAS are seen in 10-20% of patients. NRAS proteins are a GDP-GTP regulator and have an important role in proliferative signaling networks such as PI3K and MAPK. However, the role of the magnitude of RAS activation in proliferation and cell cycle progression is not fully understood. To define the role of oncogenic NRAS dose in malignant behavior, we used a genetically engineered THP-1 cell line where the expression of NRASG12V is under the control of a tetracycline-responsive element. In this cell line, doxycycline (Dox) treatment induces expression of NRASG12V in a dose-dependent manner. THP-1 is a NRASG12D-dependent human AML cell line. We used CRISPR/Cas9 to knock out the endogenous NRASG12D and introduced a tetracycline-inducible NRASG12V. We established two modified cell lines: B11 and G6. When grown in the absence of Dox, B11 and G6 are both slow-growing and display high levels of G1 arrest. Growth in 100-1,000 ng/ml of Dox induces NRASG12V expression at levels comparable to native NRASG12D expression in unmodified THP-1 (at the protein and RNA level). To decipher the contribution of NRASG12V dose to cell cycle status, we treated B11 and G6 cell lines with three doses of Dox (0, 1,000, and 10,000 ng/ml) and performed mass cytometry (CyTOF) analysis. Native levels of NRASG12V (1,000 ng/ml Dox) led to maximal cell cycle progression with minimal G1 arrest. Interestingly higher levels of NRASG12V (Dox 10,000 ng/ml) led to reduction in cell cycle progression and an increase in G1 arrest. These data suggest that optimal levels of NRASG12V are required to optimally mediate leukemic activity. Using mass cytometry to assess signaling pathway activation, we also found that optimal NRASG12V maximally activated STAT and p38 pathways, suggesting that these may be critical mediators of NRASG12V-mediated cell cycle progression and self-renewal. These data reveal a dose-dependent role of oncogenic NRAS in mediating the proliferation and demonstrate that excessive oncogenic signaling may inhibit leukemogenic activity. To identify the whole downstream of NRAS signals, CRISPR knockout library, and CRISPR activation library were introduced into B11 and removing Dox to collect clone that can proliferate without NRAS signaling. As a result, 21 NRAS signal-related candidate genes were obtained from the knockout library and 28 from the activation library. DOHH is one of the candidates for CRISPR activation library screening. NRAS activation with Dox can induce the mRNA expression for DOHH in B11. DOHH is newly identified as a downstream of NRAS signaling. Taken together, the doxycycline-NRAS-controllable system can be applied for multiple-application to investigate the whole RAS signaling system. Citation Format: Morito Kurata, Marie L. Antony, Klara E. Noble, Susan K. Rathe, Haruka Hirakouchi, Kouhei Yamamoto, Masanobu Kitagawa, Zohar Sachs, David A. Largaespada. Dose-dependent NRASG12V-mediated signaling controls cell cycle progression and leukemogenic signaling in a CRISPR/Cas9-modified human AML cell line [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2582.

Abstract 5133: Optimized RAS top-down proteomic assay reveals expanded proteoform landscape in malignant cells

Abstract Traditional proteomic methods face an inherent challenge with the RAS family of GTPases (HRAS, NRAS, KRAS4A, KRAS4B). Expressed at low endogenous abundance, these four isoforms possess considerable sequence similarity, rendering the confident proteomic linkage of a given isoform with a specific mutation or post-translational modification (PTM) a formidable proposition outside of isoform-specific model cell lines. Conversely, top-down proteomic analysis (TDP), which measures intact protein molecules, can precisely identify modified protein forms, or proteoforms, and confidently localize both mutations and PTMs on each RAS isoform present. Previously, a method combining immunoprecipitation and subsequent TDP (IP-TDP) led to the identification of eleven KRAS4B proteoforms, including four mutations and two novel PTMs, within colorectal cancer cell lines and tumor samples†. These initial results demonstrated the great potential for proteoform-resolved measurements to further illuminate the role of KRAS4B mutations and modifications in human cancer. More recently, the NCI RAS Initiative has developed an enhanced RAS proteoform assay incorporating IP-TDP analysis on an Orbitrap Fusion Lumos mass spectrometer. By employing alternate antibodies, recombinant protein standards, and well-characterized model or malignant cell lines, we have obtained substantial improvements in both RAS proteoform detection and characterization, thus facilitating the identification and localization of low-abundance PTMs. Moreover, we have applied our optimized IP-TDP method to a diverse panel of malignant and RAS mutant cell lines with the goal of expanding our knowledge of the RAS proteoform landscape. In doing so, we have observed a wealth of novel RAS proteoforms, of far greater number and complexity than previously anticipated. We have also observed isoform-specific proteoform variations, most notably between KRAS4A and KRAS4B, and initial indications of proteoform context dependence, namely within a given mutation or tissue background. Our results further underscore the power of proteoform-resolved measurements and indicate that RAS-dependent signaling pathways in normal and cancer contexts may be far more complex than previously thought. † I Ntai et. al. “Precise characterization of KRAS4b proteoforms in human colorectal cells and tumors reveals mutation/modification crosstalk.” Proc Natl Acad Sci U S A. 2018 Apr 17; 115(16):4140-4145. Citation Format: Caroline DeHart, Kanika Sharma, Dominic Esposito, Anna Maciag, Dwight Nissley, Frank McCormick. Optimized RAS top-down proteomic assay reveals expanded proteoform landscape in malignant cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5133.

Abstract 1095: Comprehensive pan-cancer analyses of RAS genomic diversity

Abstract The RAS family of small GTPases represents the most commonly activated oncogenes and mutations in KRAS, NRAS and HRAS are found in approximately one third of all human cancers. To better understand the prevalence of somatic RAS mutations and the compendium of genes that have RAS-dependent co-mutation frequencies in an unbiased pan-cancer manner, we analyzed targeted next-generation sequence data from the AACR Project GENIE Registry (GENIE version 6.1-public). Our analyses utilized 644,757 mutations from 64,217 tumors in 97 cancer types. A Bayesian hierarchical model was implemented to estimate cancer-specific prevalence of RAS and non-RAS somatic mutations. To evaluate RAS co-mutations, we compared the observed co-mutation frequencies of RAS and non-RAS somatic mutations to posterior predictive distributions of their expected co-mutation frequencies under independence. The prevalence of RAS codon 12, 13 and 61 mutations varied among cancer types, ranging from 77.7% in pancreatic cancer-PAC, 44.8% in colorectal cancer-CRC, 31.2% in non-small cell lung cancer-NSCLC, 1.1% in breast cancer, and 0.5% in mesotheliomas. A cancer type-specific distribution was observed, such that KRAS was frequently mutated in &amp;gt;25% of the tumors, NRAS was frequently mutated in melanoma, hematologic malignancies and thyroid cancer, while HRAS was overall less frequently mutated. KRAS codon 12 mutations predominated in pancreatic cancers (69.8%), GI cancers (30.9-48.5%), NSCLC (26.3%), endometrial cancer (14.7%) and germ cell tumors (11.6%). KRAS G12C mutations were predominantly seen in NSCLC (12.4%, 95% CI: 11.8-13.1%) with lower frequencies in GI cancers (3.2% in CRC). RAS mutations co-occurred with mutations in STK11, ATM, KEAP1, RBM10 and EPHA5 in NSCLC, TERT in melanoma and PIK3CA in CRC. Patterns of mutual exclusivity emerged between RAS mutations and BRAF in melanoma, thyroid and NSCLC, EGFR and RB1 in NSCLC, GNA11 and GNAQ in melanoma, FGFR3 in bladder cancer, MEN1 in PAC, RNF43 in CRC, BAP1 in melanoma and BAP1 and TERT2 in hepatobiliary carcinoma. A cancer type-specific co-occurrence pattern was noted for RAS and TP53 genomic alterations, with a significant enrichment in TP53 mutations in RAS-mutant PAC but a significant under-representation of TP53 mutations in RAS-mutant endometrial cancer, NSCLC, ovarian cancer and CRC. Analysis of codon specific RAS variants revealed additional co-mutation patterns, including an enrichment in NTRK3 mutations in KRAS G12C-mutant NSCLC and mutual exclusivity between KRAS G12C mutations and EGFR and BRAF in NSCLC. The tissue-distinct differential enrichment in RAS mutations and co-mutation patterns may be reflective of selection of oncogenic RAS signaling under the tumor type-specific modifying effect of other driver mutations. The RAS genetic architecture points to tissue-specific therapeutic vulnerabilities that may be translated to targeted treatment strategies. Citation Format: Robert Scharpf, Gregory Riely, Mark Awad, Michele Lenoue-Newton, Biagio Ricciuti, Julia Rudolph, Leon Raskin, Andrew Park, Jocelyn Lee, Christine Lovly, Valsamo Anagnostou. Comprehensive pan-cancer analyses of RAS genomic diversity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1095.