Ras Degradation Research Articles

Sequential Azidation/Azolation of Prenylated Derivatives and a Click Reaction Enable Selective Labeling and Degradation of RAS Protein.

We propose the introduction of the azido and azo-functionalities into prenylated derivatives under mild conditions in a selective and efficient way. Upon protocol establishment and substrate scope determination, we apply this method to prenylated protein (citronellol-BSA) labeling, chemical pulldown, and enrichment. Eventually, we achieve the degradation of RAS on MCF-7 and HeLa cell lines by employing the well-designed probe von Hippel-Lindau derivatives C4 through the sequential azidation/azolation and click-reaction (SACR) pathway targeting the prenyl functionality attached to the Caax motif of the tested RAS protein. This method displays great potential in regulation of prenylated molecules.

Targeting Ras with protein engineering.

Ras proteins are small GTPases that regulate cell growth and division. Mutations in Ras genes are associated with many types of cancer, making them attractive targets for cancer therapy. Despite extensive efforts, targeting Ras proteins with small molecules has been extremely challenging due to Ras's mostly flat surface and lack of small molecule-binding cavities. These challenges were recently overcome by the development of the first covalent small-molecule anti-Ras drug, sotorasib, highlighting the efficacy of Ras inhibition as a therapeutic strategy. However, this drug exclusively inhibits the Ras G12C mutant, which is not a prevalent mutation in most cancer types. Unlike the G12C variant, other Ras oncogenic mutants lack reactive cysteines, rendering them unsuitable for targeting via the same strategy. Protein engineering has emerged as a promising method to target Ras, as engineered proteins have the ability to recognize various surfaces with high affinity and specificity. Over the past few years, scientists have engineered antibodies, natural Ras effectors, and novel binding domains to bind to Ras and counteract its carcinogenic activities via a variety of strategies. These include inhibiting Ras-effector interactions, disrupting Ras dimerization, interrupting Ras nucleotide exchange, stimulating Ras interaction with tumor suppressor genes, and promoting Ras degradation. In parallel, significant advancements have been made in intracellular protein delivery, enabling the delivery of the engineered anti-Ras agents into the cellular cytoplasm. These advances offer a promising path for targeting Ras proteins and other challenging drug targets, opening up new opportunities for drug discovery and development.

Potent and selective eradication of tumor cells by an EpCAM-targeted Ras-degrading enzyme

Despite decades of efforts, an urgent need remains to develop tumor cell-selective rat sarcoma (Ras)-targeting therapies that can treat patients with Ras-driven tumors. Here we report modular engineered proteins that degrade Ras selectively in tumor cells that overexpress the tumor cell marker epithelial cell adhesion molecule (EpCAM) by fusing the Ras degrader Ras-Rap1-specific endopeptidase with the translocation domain of the Pseudomonas aeruginosa exotoxin A (ETA) or diphtheria toxin (DT). Redirection to EpCAM is achieved by a designed ankyrin repeat protein. In two-dimensional tumor cell cultures, complete degradation of Ras proteins after 24h was observed with EpCAM-targeted Ras degraders fused to ETA or DT in EpCAM-overexpressing MCF7 and HCT116 cells, with median inhibition concentration values at sub-nanomolar levels. The viability of EpCAM-low non-cancerous fibroblasts remained unaffected. In a three-dimensional (3D) tumor-on-a-chip system that mimics the natural tumor microenvironment, effective Ras degradation and selective toxicity toward tumor cells, particularly with the ETA-fused constructs, was determined on-chip. To conclude, we demonstrate the potential of modular engineered proteins to kill tumor cells highly selectively by simultaneously exploiting EpCAM as a tumor-specific cell surface molecule as well as Ras as an intracellular oncotarget in a 3D system mimicking the natural tumor microenvironment.

Intracellular delivery of bacterial effectors for cancer therapy using biodegradable lipid nanoparticles.

Bacterial effector proteins are virulence factors that are secreted and mediate orthogonal post-translational modifications of proteins that are not found naturally in mammalian systems. They hold great promise for developing biotherapeutics by regulating malignant cell signaling in a specific and targeted manner. However, delivering bacterial effectors into disease cells poses a significant challenge to their therapeutic potential. In this study, we report on the design of a combinatorial library of bioreducible lipid nanoparticles containing disulfide bonds for highly efficient bacterial effector delivery and potential cancer therapy. A leading lipid, PPPDA-O16B, identified from the library, can encapsulate and deliver DNA plasmids into cells. The gene cargo is released in response to the reductive cellular environment that is upregulated in cancer cells, leading to enhanced gene delivery and protein expression efficiency. Furthermore, we demonstrate that PPPDA-O16B can deliver the bacterial effector protein, DUF5, to degrade mutant RAS and inactivate downstream MAPK signaling cascades to suppress cancer cell growth in vitro and in tumor-bearing mouse xenografts. This strategy of delivering bacterial effectors using biodegradable lipid nanoparticles can be expanded for cancer cell signaling regulation and antitumor studies.

Structural optimization of novel Ras modulator for treatment of Colorectal cancer by promoting β-catenin and Ras degradation

Ras protein has been considered a fascinating target for anticancer therapy because its malfunction is closely related to cancer. However, Ras has been considered undruggable because of the failure to regulate its malfunction by controlling the Ras activation mechanism. Recently, Lumakras targeting the G12C mutation was approved, and therapeutic interest in Ras for anticancer therapy has been rejuvenated. Here, we present a series of compounds that inhibit Ras via a unique mechanism of action that exploits the relationship between the Wnt/β-catenin pathway and Ras. KYA1797K (1) binds to axin to stabilize the β-catenin destruction complex that causes the phosphorylation and subsequent degradation of Ras, similar to canonical β-catenin regulation. Based on the chemical structure of 1, we performed a structural optimization and identified 3-(2-hydroxyethyl)-5-((6-(4-nitrophenyl)pyridin-2-yl)methylene)thiazolidine-2,4-dione (13d) as the most potent compound. 13d displayed antitumor effects in a colorectal cancer model with enhanced inhibition activity on Ras. The results of this study suggest that the further development of 13d could contribute to the development of Ras inhibitors with novel mechanisms of action.

Selected Articles from This Issue

Despite the proven benefits of antibody-based therapies for HER2-amplified cancers, there remains an urgent need for novel therapeutic approaches to combat acquired and innate drug resistance. Here, O'Brien and colleagues demonstrate that tucatinib, a small molecule inhibitor of HER2, has selective activity in HER2-driven cancers. Tucatinib showed single agent and combined efficacy with trastuzumab and inhibitors of CDK4/6 in cell line xenograft models. The selective nature of tucatinib make it an ideal combination partner for HER2-based therapies. Data presented here provide insight into the optimal patient selection and combination strategies for the expanded clinical development of tucatinib.Head and neck squamous cell carcinoma (HNSCC) is a common cancer with limited treatment options. Recent clinical trials in patients with HRAS-mutant HNSCC have shown the efficacy of tipifarnib, a farnesyl transferase inhibitor (FTI) that blocks a required posttranslational modification of HRAS and other proteins. Here, Javaid and colleagues have validated HRAS as a key therapeutic target of tipifarnib and identified multiple signaling pathways that could cause treatment resistance. Combining FTI with an ERK inhibitor blocked compensatory signaling changes, enhancing the efficacy of FTI and showing that this combination could be a promising treatment strategy for patients with HRAS-mutant HNSCC.Pancreatic ductal adenocarcinoma (PDAC) is the most RAS-addicted of all cancers and development of anti-RAS therapies is a major priority. RRSP-DTB is an engineered biologic with specific RAS proteolytic activity. Vidimar and colleagues conducted a study to assess in vitro RAS degradation and in vivo activity of RRSP-DTB in RAS-driven PDAC models. As a single agent, RRSP-DTB effectively degraded RAS in cells and engaged RAS in mice leading to nearly complete regression of PDAC patient-derived xenografts without development of resistance. These results support further study of RRSP-DTB as an unprecedented RAS biodegrader for RAS-addicted tumors.Clinical outcomes in FLT3-mutant acute myeloid leukemia (AML) remain exceedingly poor due to FLT3 tyrosine kinase inhibitor (TKI) resistance. FF-10101 is the first covalent FLT3 inhibitor and recently completed phase I clinical evaluation. Here, Ferng and colleagues profile FF-10101 against a panel of FLT3 TKI resistance-mutations and off-target resistance mechanisms. FF-10101 suppresses all clinically described on-target mutations but not those that compromise covalent binding to FLT3. FF-10101 also exhibits activity in an in vitro model of cytokine-driven TKI resistance. Thus, FF-10101 shows the potential to benefit AML patients already treated with existing FLT3 TKIs or patients harboring certain FLT3 mutations.

Mulberry polyphenol extracts attenuated senescence through inhibition of Ras/ERK via promoting Ras degradation in VSMC.

Ageing is one of the major risk factors of human diseases, including cancer, diabetes, and cardiovascular disease. Mulberry exhibits a wide range of functions, such as anti-oxidant, anti-inflammation, and anti-diabetes. In this study, we investigated the role of mulberry polyphenol extract (MPE) in K-Ras-induced senescence of smooth muscle cells. Forced expression of K-Ras enhanced senescence of smooth muscle A7r5 cells as shown by the elevation of β-galactosidase activity. Treatment with MPE significantly repressed the Ras, phosphorylated ERK, and β-galactosidase level. MPE triggered the association of cyclins with their corresponding cyclin-dependent protein kinases and hyperphosphorylated retinoblastoma (RB). MPE also down-regulated the levels of K-Ras-induced CDK inhibitors. MPE enhanced the phosphorylated AMP-dependent protein kinase (AMPK) and inducible nitric oxide synthase (iNOS) level in the presence of K-Ras. Pretreatment with either L-NAME or AMPK inhibitor reversed the effects of MPE. In addition, L-NAME and AMPK inhibitor repressed the MPE-induced total and phosphorylated 3-hydroxy-3-methylglutaryl coenzyme A (HMG-Co A) level. MPE repressed K-Ras-induced G0/G1 arrest, whereas L-NAME and AMPK inhibitor blocked the effects of MPE. Our results indicated that MPE recovered the K-Ras-induced senescence of vascular smooth muscle cells through iNOS and AMPK-dependent pathway. Our findings suggested that MPE may prevent ageing-induced atherosclerosis.

Inhibition of Acute Leukemia By a Small Molecule KYA1797K Which Destabilize RAS and β-Catenin

Introduction: Signaling pathways in acute leukemia are aberrantly activated to cause leukemogenesis and relapse after treatment. Like in many other malignancies, upregulation of WNT/beta-catenin pathway and hyperactive RAS is known to be associated with treatment resistance in leukemia. However, there are little studies about RAS singaling pathyway and leukemia, and it is a field of study that needs to be revealed. KYA1797K is a recently developed small molecule, binds directly to RGS domain of axin and enhances the beta-catenin destruction complex which activates GSK3beta and results in degradation of beta-catenin and RAS. In the current study, we tried to find the role of RAS inhibition by KYA1797K in leukemic cell lines and in patient's BM samples. Moreover, other small molecule PCK412 (Midosaturin) was also used for comparison. Materials & Methods: Leukemic cells (MOLT-4, THP-1, MOLT-4, Jurka, KG-1, MV4-11, RS4-11) were cultured in RPMI1640 media under various concentration (0.1-10µM for KYA1797K, 0.5-500nM for PKC412) for 48h and with Erlotinib (1µM) for comparison. Cell proliferation assay on each leukemic cell was done and immunoblotting for β-catenin, GSK3β, Pan-RAS, N-RAS was checked. Downstream targets of Wnt pathway (c-Myc, CD44, LEF1, Met, TCF1/TCF7) were studied by immunoblotting. MOLT-4 was stimulated with Wnt3a (200ng/mL, 4h) and changes in Wnt pathway were observed. Bone marrow samples of AML and ALL patients were evaluated for β-catenin and RAS. KYA1797K (Nat Chem Biol 2016, 12:593) was kindly provided by Prof. Kang-Yell Choi. Results: Suppression of leukemic cells by KYA1797K was evident starting from the concentration of 5 microM. (fig.1) Beta-catenin was down regulated in all cell lines by KYA1797K. Pan-RAS decreased in MOLT-4 and THP-1. All the downstream targets evaluated were down regulated by KYA1797K in MOLT-4 culture, and was evident at the concentration of 5microM. (fig.2) Stimulation of Wnt pathway by Wnt3a was inhibited by KYA1797K. (fig.3) Bone marrow samples from 7 ALL patients showed various status of β-catenin and RAS expression. Two high-risk patients showed suppression of β-cateinin and N-RAS by KYA1797K. (fig.4) In MV4-11 (FLT3 mutant) and RS4-11 (FLT3 wild type), IC50 for PKC412 was higher in RS4-11 compared to MV4-11 while KYA1797K showed same IC50 in both cell lines. β-catenin and RAS downregulation was observed by KYA1797K. Effects of KYA1797K analyzed by qRT-PCR and immunoblot for FLT3, N-RAS, MEK, ERK, ETS2 showed that KYA1797K downregulates FLT3 in MV4-11 even though FLT3 is not the main target of action. It was less effective on RS4-11. (fig.5) In bone marrow samples of ALL patient with FLT3 mutation, KYA1797K 1µM showed effect in reducing leukemia cells. (fig.6) Conclusion: This preclinical study suggests that KYA1797K may be an option for patients with acute leukemia. KYA1797K effectively destabilized β-catenin and RAS in acute leukemia even under Wnt pathway activation. FLT3, N-RAS, MEK, ERK, ETS2 were down regulated by KYA1797K, hence KYA1797K has a potential application for acute leukemia with FLT3 mutation. Extended studies including further in vivo study are needed to build up a strategy in small molecule therapy to target RAS in acute leukemia. Although with limitation, we suggest RAS inhibitor as a potential drug for leukemia. Disclosures No relevant conflicts of interest to declare.

WDR76 degrades RAS and suppresses cancer stem cell activation in colorectal cancer

BackgroundStabilization of RAS is a key event for the hyper-activation of Wnt/β-catenin signaling and activation of cancer stem cell (CSC) in colorectal cancer (CRC). WD Repeat protein 76 (WDR76) mediates the polyubiquitination-dependent degradation of RAS in hepatocellular carcinoma (HCC). We investigated whether WDR76 destabilizes RAS and acts as a tumor suppressor inhibiting CSC activation in CRC.MethodsWe generated mice with deletion of Wdr76 (Wdr76−/−) and crosses of Wdr76−/− with ApcMin/+ (Wdr76−/−; ApcMin/+) and compared them with wildtype mice (Wdr76+/+) and ApcMin/+ mice (Wdr76+/+; ApcMin/+), respectively. Intestinal crypt lengthening, tumorigenesis and CSC activation were analyzed by histology, immunohistochemistry, and immunoblotting. CRC cell line was engineered to stably express or knockdown WDR76 or control vector and was analyzed after spheroid culture.ResultsWdr76−/− mice, with increased Ras level, displayed crypt elongation and hyper-proliferation. Wdr76−/−; ApcMin/+ mice developed more tumors with bigger sizes than ApcMin/+ mice and their tumors showed increased proliferation and CSC activation with elevated RAS and β-catenin levels. In CRC cells, overexpression or knockdown of WDR76 decreased or increased the numbers and sizes of CRC spheroids with inhibition or activation of CSC markers, respectively. In human CRC, lower level of WDR76 was associated with poor patient survival.ConclusionsIn analyses of mice with deletion of Wdr76 and CRC spheroids, we found that RAS stability plays important roles in tumorigenesis by affecting proliferation and CSC activation. Our results suggest that destabilization of RAS by WDR76 is a potential strategy for targeting malignant CRC involving CSC activation.Graphic abstract

LZTR1 facilitates polyubiquitination and degradation of RAS-GTPases

Leucine zipper-like transcriptional regulator 1 (LZTR1) encodes a member of the BTB-Kelch superfamily, which interacts with the Cullin3 (CUL3)-based E3 ubiquitin ligase complex. Mutations in LZTR1 have been identified in glioblastoma, schwannomatosis, and Noonan syndrome. However, the functional role of LZTR1 in carcinogenesis or human development is not fully understood. Here, we demonstrate that LZTR1 facilitates the polyubiquitination and degradation of RAS via the ubiquitin-proteasome pathway, leading to the inhibition of the RAS/MAPK signaling. The polyubiquitination and degradation of RAS was also observed in cells expressing MRAS, HRAS, NRAS, and KRAS as well as oncogenic RAS mutants and inhibited the activation of ERK1/2 and cell growth. In vivo ubiquitination assays showed that MRAS-K127 and HRAS-K170 were ubiquitinated by LZTR1 and that the polyubiquitinated-chains contained mainly Ub-K48, K63, and K33-linked chains, suggesting its possible involvement in autophagy. Immunoprecipitation analyses showed the interaction of LZTR1 and RAS-GTPases with autophagy-related proteins, including LC3B and SQSTM1/p62. Co-expression of LZTR1 and RAS increased the expression of lipidated form of LC3B. However, long-term treatment with chloroquine had little effect on RAS protein levels, suggesting that the contribution of autophagy to LZTR1-mediated RAS degradation is minimal. Taken together, these results show that LZTR1 functions as a “RAS killer protein” mainly via the ubiquitin-proteasome pathway regardless of the type of RAS GTPase, controlling downstream signal transduction. Our results also suggest a possible association of LZTR1 and RAS-GTPases with the autophagy. These findings provide clues for the elucidation of the mechanisms of RAS degradation and regulation of the RAS/MAPK signaling cascade.

WDR76 is a RAS binding protein that functions as a tumor suppressor via RAS degradation

Stability regulation of RAS that can affect its activity, in addition to the oncogenic mutations, occurs in human cancer. However, the mechanisms for stability regulation of RAS involved in their activity and its roles in tumorigenesis are poorly explored. Here, we identify WD40-repeat protein 76 (WDR76) as one of the HRAS binding proteins using proteomic analyses of hepatocellular carcinomas (HCC) tissue. WDR76 plays a role as an E3 linker protein and mediates the polyubiquitination-dependent degradation of RAS. WDR76-mediated RAS destabilization results in the inhibition of proliferation, transformation, and invasion of liver cancer cells. WDR76−/− mice are more susceptible to diethylnitrosamine-induced liver carcinogenesis. Liver-specific WDR76 induction destabilizes Ras and markedly reduces tumorigenesis in HRasG12V mouse livers. The clinical relevance of RAS regulation by WDR76 is indicated by the inverse correlation of their expressions in HCC tissues. Our study demonstrates that WDR76 functions as a tumor suppressor via RAS degradation.

Neratinib and entinostat combine to rapidly reduce the expression of K-RAS, N-RAS, Gαq and Gα11 and kill uveal melanoma cells

ABSTRACTThere is no efficacious standard of care therapy for uveal melanoma. Unlike cutaneous disease, uveal melanoma does not exhibit RAS mutations but instead contains mutations with ~90% penetrance in either Gαq or Gα11. Previously we demonstrated that neratinib caused ERBB1/2/4 and RAS internalization into autolysosomes which resulted in their proteolytic degradation. In PDX isolates of uveal melanoma, neratinib caused the internalization and degradation of Gαq and Gα11 in parallel with ERBB1 breakdown. These effects were enhanced by the HDAC inhibitor entinostat. Similar data were obtained using GFP/RFP tagged forms of K-RAS V12. Down regulation of Gαq and Gα11 expression and RAS-GFP/RFP fluorescence required Beclin1 and ATG5. The [neratinib + entinostat] combination engaged multiple pathways to mediate killing. One was from ROS-dependent activation of ATM via AMPK-ULK1-ATG13-Beclin1/ATG5. Another pathway was from CD95 via caspase 8-RIP1/RIP3. A third was from reduced expression of HSP70, HSP90, HDAC6 and phosphorylation of eIF2α. Downstream of the mitochondrion both caspase 9 and AIF played roles in tumor cell execution. Knock down of ATM/AMPK/ULK-1 prevented ATG13 phosphorylation and degradation of RAS and Gα proteins. Over-expression of activated mTOR prevented ATG13 phosphorylation and suppressed killing. Knock down of eIF2α maintained BCL-XL and MCL-1 expression. Within 6h, [neratinib + entinostat] reduced the expression of the immunology biomarkers PD-L1, ODC, IDO-1 and enhanced MHCA levels. Our data demonstrate that [neratinib + entinostat] down-regulates oncogenic RAS and the two key oncogenic drivers present in most uveal melanoma patients and causes a multifactorial form of killing via mitochondrial dysfunction and toxic autophagy.Abbreviations: ERK: extracellular regulated kinase; PI3K: phosphatidyl inositol 3 kinase; ca: constitutively active; dn: dominant negative; ER: endoplasmic reticulum; AIF: apoptosis inducing factor; AMPK: AMP-dependent protein kinase; mTOR: mammalian target of rapamycin; JAK: Janus Kinase; STAT: Signal Transducers and Activators of Transcription; MAPK: mitogen activated protein kinase; PTEN: phosphatase and tensin homologue on chromosome ten; ROS: reactive oxygen species; CMV: empty vector plasmid or virus; si: small interfering; SCR: scrambled; IP: immunoprecipitation; VEH: vehicle; HDAC: histone deacetylase.

β-Catenin-RAS interaction serves as a molecular switch for RAS degradation via GSK3β.

RAS proteins play critical roles in various cellular processes, including growth and transformation. RAS proteins are subjected to protein stability regulation via the Wnt/β-catenin pathway, and glycogen synthase kinase 3 beta (GSK3β) is a key player for the phosphorylation-dependent RAS degradation through proteasomes. GSK3β-mediated RAS degradation does not occur in cells that express a nondegradable mutant (MT) β-catenin. Here, we show that β-catenin directly interacts with RAS at the α-interface region that contains the GSK3β phosphorylation sites, threonine 144 and threonine 148 residues. Exposure of these sites by prior β-catenin degradation is required for RAS degradation. The introduction of a peptide that blocks the β-catenin-RAS interaction by binding to β-catenin rescues the GSK3β-mediated RAS degradation in colorectal cancer (CRC) cells that express MT β-catenin. The coregulation of β-catenin and RAS stabilities by the modulation of their interaction provides a mechanism for Wnt/β-catenin and RAS-ERK pathway cross-talk and the synergistic transformation of CRC by both APC and KRAS mutations.

Abstract 1673: Ras degrading small molecules inhibit the transformation of colorectal cancer cells independently of beta-catenin signaling

Abstract Development of drugs controlling Ras is an emerging issue in cancer therapy; however, no clinically applicable drug is currently available. On the basis of the mechanism of Ras protein stability regulation via the Wnt/β-catenin signaling pathway, we previously identified small molecules inhibiting the proliferation and transformation of various colorectal cancer (CRC) cells via degradation of both β-catenin and Ras. Due to the absence of Ras degradation in cells expressing non-degradable mutation in β-catenin, and the need to determine an alternative mechanism for Ras degradation, we established a cell-based system to screen compounds that degrade Ras independently of the Wnt/β-catenin signaling pathway. A cell-based high-content screening (HCS) system monitoring the level of EGFP-K-RasG12V was established using HCT-116 cells harboring a non-degradable mutant β-catenin (ΔS45). Through HCS of a chemical library composed of 10,000 compounds, and by subsequent characterization of candidate compounds, we identified several compounds that degraded Ras without affecting β-catenin levels. KY7749, the most effective compound, inhibited the proliferation and transformation of CRC cells, including those resistant to the EGFR monoclonal antibody, cetuximab, due to KRAS mutations. Small molecules that degrade Ras independently of β-catenin would be a potential treatments for cancers caused by aberrant EGFR, Ras, and β-catenin. Citation Format: Jeong-Ha Hwang, Eun Ji Ro, Wookjin Shin, Kang-Yell Choi. Ras degrading small molecules inhibit the transformation of colorectal cancer cells independently of beta-catenin signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1673.

Identification of Ras-degrading small molecules that inhibit the transformation of colorectal cancer cells independent of \u03b2-catenin signaling

Although the development of drugs that control Ras is an emerging topic in cancer therapy, no clinically applicable drug is currently available. We have previously utilized knowledge of the Wnt/β-catenin signaling-dependent mechanism of Ras protein stability regulation to identify small molecules that inhibit the proliferation and transformation of various colorectal cancer (CRC) cells via degradation of both β-catenin and Ras. Due to the absence of Ras degradation in cells expressing a nondegradable mutant form of β-catenin and the need to determine an alternative mechanism of Ras degradation, we designed a cell-based system to screen compounds that degrade Ras independent of the Wnt/β-catenin signaling pathway. A cell-based high-content screening (HCS) system that monitors the levels of EGFP-K-RasG12V was established using HCT-116 cells harboring a nondegradable mutant CTNNB1 (ΔS45). Through HCS of a chemical library composed of 10,000 compounds and subsequent characterization of hits, we identified several compounds that degrade Ras without affecting the β-catenin levels. KY7749, one of the most effective compounds, inhibited the proliferation and transformation of CRC cells, especially KRAS-mutant cells that are resistant to the EGFR monoclonal antibody cetuximab. Small molecules that degrade Ras independent of β-catenin may able to be used in treatments for cancers caused by aberrant EGFR and Ras.

Abstract 911: Destabilizing β-catenin and Ras could be an ideal therapy for treating metastatic colorectal cancer

Abstract INTRODUCTION Aberrant activations of Wnt/β-catenin and Ras signaling pathways by APC (90%) and K-Ras (40-50%) mutations are closely interacted and accelerate CRC tumorigenesis. Protein level of β-catenin as well as Ras is increased by APC loss via inhibiting GSK3B-mediated poly-ubiquitination dependent Ras degradation through recruitment of β-TrCP E3 linker. Moreover, additional K-Ras mutation results in the liver metastasis by secondary activation of the Wnt/β-catenin signaling pathways via Ras-ERK and -AKT pathways in addition to the initial activation by APC loss. Consequently, both β-catenin and Ras were highly increased in human CRC patient tissues, especially in metastatic tumor. EXPERIMENT PROCEDURES To investigate the synergistic effects of aberrant Wnt/β-catenin and Ras signaling pathways on tumor development to metastasis, we established the APCMin/+/K-RasG12DLA2 compound mouse that harbor APC and K-Ras mutations in animal model. We also analyzed Ras protein level using human CRC patient tissues of normal mucosa and adenocarcinoma (N=24) and metastatic adenocarcinoma (N=26) and tumor budding (N=10). To recapitulate the metastatic tumor environment which have enriched receptor tyrosine kinases (RTKs), we maintained and experimented with EGF (20ng/ml) in every in vitro studies. RESULTS We observed that both β-catenin and Ras were increased in adenocarcinoma and metastatic adenocarcinoma of human CRC compared with normal mucosa. Interestingly, both β-catenin and Ras were most significantly increased in tumor budding regions which histopathologically represent epithelial mesenchymal transition (EMT). In murine model, the EMT phenomenon was effectively induced and invasion of small intestinal tumors were occurred in APCMin/+/K-RasG12DLA2 with increased level of β-catenin and Ras compared with APCMin/+. However, EMT phenomenon and its following events such as migration and invasion of CRC cells induced by APC and K-Ras mutations were abolished by destabilization of β-catenin and Ras. CONCLUSION Our results show that destabilizing β-catenin and Ras could be effective therapeutic targets for inhibiting metastasis in the early stage of CRC development Citation Format: Yong-Hee Cho, Jeong-Soo Yoon, Kang-Yell Choi. Destabilizing β-catenin and Ras could be an ideal therapy for treating metastatic colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 911. doi:10.1158/1538-7445.AM2017-911

Small-molecule binding of the axin RGS domain promotes β-catenin and Ras degradation

Both the Wnt/β-catenin and Ras pathways are aberrantly activated in most human colorectal cancers (CRCs) and interact cooperatively in tumor promotion. Inhibition of these signaling may therefore be an ideal strategy for treating CRC. We identified KY1220, a compound that destabilizes both β-catenin and Ras, via targeting the Wnt/β-catenin pathway, and synthesized its derivative KYA1797K. KYA1797K bound directly to the regulators of G-protein signaling domain of axin, initiating β-catenin and Ras degradation through enhancement of the β-catenin destruction complex activating GSK3β. KYA1797K effectively suppressed the growth of CRCs harboring APC and KRAS mutations, as shown by various in vitro studies and by in vivo studies using xenograft and transgenic mouse models of tumors induced by APC and KRAS mutations. Destabilization of both β-catenin and Ras via targeting axin is a potential therapeutic strategy for treatment of CRC and other type cancers activated Wnt/β-catenin and Ras pathways.

A full scale anaerobic–anoxic–aerobic process coupled with low-dose ozonation for performance improvement

The effectiveness of RAS degradation under low dose ozone and effect of ozonated sludge on a full scale anaerobic-anoxic-aerobic (A/A/O) process were examined. The ratios of chemical oxygen demand (COD) to nitrogen and COD to phosphorus of ozonated sludge were 23 and 91 respectively on average, much higher than in influent. Compared with the performance of A/A/O process alone, COD removal efficiency slightly decreased after insertion of the ozonation system. Ammonia removal became more stable, but total nitrogen and total phosphorus removal efficiency was not improved. Sludge volume index (SVI) of activated sludge reached to an optimal value of 80-120 mL/g quickly due to ozonation combined with A/A/O process which reduced the danger of sludge bulking. Overall, the combination of biological process with ozonation at a low dose rate shows promise. Compared with higher dose ozonation, low-dose ozonation can improve the performance of A/A/O effectively at a smaller cost.

Abstract 4164: Regulatory mechanism for Ras degradation and its role in the suppression of cellular transformation and tumorigenesis

Abstract Ras is a well-characterized proto-oncogene, and its various mutations contribute to transformation and tumorigenesis in a wide variety of human cancers. Although the GTP/GDP loading switch is known as a major regulatory mechanism of Ras, other mechanisms affecting Ras activity, especially those related to cancer, are poorly understood. Here, we report a novel regulatory mechanism for Ras degradation and its role in suppression of cellular transformation and tumors induced by Ras mutations. We show that the negative regulators of Wnt/β-catenin signaling are associated with polyubiquitin-dependent degradation of Ras following phosphorylation and subsequent recruitment of α-TrCP-E3 ligase. The interaction of Ras with the WD40 domain of α-TrCP targeted Ras for polyubiquitination and degradation. This process stimulated by Axin or adenomatous polyposis coli (Apc) and was inhibited by Wnt3a via modulating the phosphorylation status of Ras. Ras-mediated cellular transformation was also inhibited by the expression of α-TrCP and/or Axin. In vivo regulation of Ras stability by Wnt/β-catenin signaling was indicated via measurements of the status of Ras in the intestines of mice stimulated with recombinant Wnt3a by intravenous tail vein injection. The Ras deregulation by negative Wnt/β-catenin signaling was also revealed in human cancers such as colorectal cancer and HCCs etc. Together, our results indicate an essential role for phosphorylation and degradation of Ras and suppression of Ras-induced transformation via the Wnt/β-catenin pathway and provide insight into the role of this novel regulatory mechanism for Ras in colorectal tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4164. doi:1538-7445.AM2012-4164

β-catenin Activates ERK: Two Pathways for the Price of One

Wnt/β-catenin signaling is important for developmental processes, but this pathway is also associated with the development of certain cancers. Loss-of-function mutations in proteins involved in the negative regulation of Wnt/β-catenin signaling, such as Axin, result in the accumulation of β-catenin in the nucleus. This leads to the increased expression of β-catenin-responsive genes and to cellular proliferation. Dysregulation of the extracellular signal-regulated kinase (ERK) pathway, such as in the case of cells with mutations in ras , also leads to cancers. To investigate a possible link between these two pathways in carcinogenesis, Jeon et al . established an L929 fibroblast cell line in which production of Axin was inducible by the addition of doxycycline (L929-Axin). Western blot analysis revealed that the increased abundance of Axin in L929-Axin cells inhibited the accumulation of β-catenin, as expected, but also inhibited the phosphorylation of components of the ERK pathway, including Raf-1, MEK (mitogen-activated or extracellular signal-regulated protein kinase kinase), and ERK. L929-Axin cells showed a smaller proliferative response to epidermal growth factor (EGF) than did control cells, as measured by phosphorylation of Raf-1, MEK, and ERK. Flow-cytometry studies revealed that Axin also inhibited EGF-stimulated progression from the G1 to the S phase of the cell cycle. Negative regulation by Axin of the ERK pathway did not occur in cells containing a nondegradable mutant form of β-catenin. Ras, a small guanosine triphosphatase that responds to growth factor signals and activates the ERK pathway, was less abundant in L929-Axin cells than in control cells. This reduction in Ras (which was mediated by lysosomal degradation) did not occur in cells containing nondegradable β-catenin. These data suggest that β-catenin signaling can also activate the ERK signaling pathway, thereby enhancing the likelihood of carcinogenesis. S. H. Jeon, J.-Y. Yoon, Y.-N. Park, W.-J. Jeong, S. Kim, E.-H. Jho, Y.-J. Surh, K.-Y. Choi, Axin inhibits extracellular signal-regulated kinase pathway by Ras degradation via β-catenin. J. Biol. Chem. 282 , 14482-14492 (2007). [Abstract] [Full Text]