RAS-mutant Cancer Cells Research Articles

LncRNA GABPB1-AS1 and GABPB1 regulate oxidative stress during erastin-induced ferroptosis in HepG2 hepatocellular carcinoma cells

Ferroptosis is a non-apoptotic, iron-dependent oxidative form of cell death that is specifically induced by erastin in RAS mutant cancer cells. Ferroptotic cell death is the result of membrane lipid peroxide damage caused by the accumulation of hydroxyl radicals derived from H2O2 by the Fenton reaction. Peroxidases are key cellular antioxidant enzymes that block such damaging processes. Few studies have examined the roles of long non-coding RNAs (lncRNAs) in the regulation of cellular oxidative stress, especially in ferroptosis. Here, we demonstrated that erastin upregulated the lncRNA GABPB1-AS1, which downregulated GABPB1 protein levels by blocking GABPB1 translation, leading to the downregulation of the gene encoding Peroxiredoxin-5 (PRDX5) peroxidase and the eventual suppression of the cellular antioxidant capacity. Such effects critically inhibited the cellular antioxidant capacity and cell viability. Additionally, high expression levels of GABPB1 were correlated with poor prognosis of hepatocellular carcinoma (HCC) Patients, while high GABPB1-AS1 levels in HCC patients correlated with improved overall survival. Collectively, these data demonstrate a mechanistic link between GABPB1 and its antisense lncRNA GABPB1-AS1 in erastin-induced ferroptosis and establish GABPB1 and GABPB1-AS1 as attractive therapeutic targets for HCC.

Dual Inhibition of GLUT1 and the ATR/CHK1 Kinase Axis Displays Synergistic Cytotoxicity in KRAS-Mutant Cancer Cells.

The advent of molecularly targeted therapeutic agents has opened a new era in cancer therapy. However, many tumors rely on nondruggable cancer-driving lesions. In addition, long-lasting clinical benefits from single-agent therapies rarely occur, as most of the tumors acquire resistance over time. The identification of targeted combination regimens interfering with signaling through oncogenically rewired pathways provides a promising approach to enhance efficacy of single-agent-targeted treatments. Moreover, combination drug therapies might overcome the emergence of drug resistance. Here, we performed a focused flow cytometry-based drug synergy screen and identified a novel synergistic interaction between GLUT1-mediated glucose transport and the cell-cycle checkpoint kinases ATR and CHK1. Combined inhibition of CHK1/GLUT1 or ATR/GLUT1 robustly induced apoptosis, particularly in RAS-mutant cancer cells. Mechanistically, combined inhibition of ATR/CHK1 and GLUT1 arrested sensitive cells in S-phase and led to the accumulation of genotoxic damage, particularly in S-phase. In vivo, simultaneous inhibition of ATR and GLUT1 significantly reduced tumor volume gain in an autochthonous mouse model of KrasG12D -driven soft tissue sarcoma. Taken together, these findings pave the way for combined inhibition of GLUT1 and ATR/CHK1 as a therapeutic approach for KRAS-driven cancers. SIGNIFICANCE: Dual targeting of the DNA damage response and glucose transport synergistically induces apoptosis in KRAS-mutant cancer, suggesting this combination treatment for clinical validation in KRAS-stratified tumor patients.

Innovative dual system for selective eradication of cancer cells using exosomes carrying natural bacterial toxin-antitoxin (TA).

e14635 Background: Inactivation of P53 and activation of ras are frequent genetic alterations in cancer. We have shown in vitro and in vivo, that the TA system can selectively and effectively eradicate RAS-mutated cancer cells. Aim: Selective killing of cancer cells while sparing the normal cells based on tumor genetic signature. Methods: A “first generation” ΔE1/ΔE3 human type-5 adenoviral-vectors for gene delivery were designed and constructed to specifically target cancer cells. They are designated as "PY4-mazF-mCherry" (PY4, ras responsive element), "ΔPY4-mazF-mCherry" (control viruses) and "RGC-mazE-IRES-GFP" (RGC, P53 responsive element). Their potency was tested in vitro, by the enzymatic MTT assay, microscopic observation, colony formation assay and FACS analysis, and in a xenograft model of CRC. Next, we generated, small natural vesicles, exosomes, that directly targeted cancer through specific small antibody fragments against CD24 that is expressed in most cancer cells and rarely on normal cells. Results: The TA system ("PY4-mazF-mCherry"+"RGC-mazE-IRES-GFP") induced a massive cell death, in a dose-dependent manner in vitro, 69% as compared to 19% in control co-infected ("ΔPY4-mazF-mCherry"+"RGC-mazE-IRES-GFP") HCT116 CRC cells (mutated RAS and p53). In vivo, growth of HCT116-/- ( KRASmutand P53mut) and HCT116+/+ ( KRASmut and P53wt) tumors were significantly inhibited (70% and 65%, respectively). Conclusions: 1. Abusing the P53 genetic status and the activated Ras pathway holds promising effective and safe strategy to target tumor cells while sparing normal tissues. 2. It is a proof of concept for personalized cancer therapy based on the tumor genetic profile.

Anchorage-independent growth conditions reveal a differential SOS2 dependence for transformation and survival in RAS-mutant cancer cells

ABSTRACT The RAS family of genes (HRAS, NRAS, and KRAS) is mutated in around 30% of human tumours. Wild-type RAS isoforms play an important role in mutant RAS-driven oncogenesis, indicating that RasGEFs may play a significant role in mutant RAS-driven transformation. We recently reported a hierarchical requirement for SOS2 in mutant RAS-driven transformation in mouse embryonic fibroblasts, with KRAS>NRAS>HRAS (Sheffels et al., 2018). However, whether SOS2 deletion differentially affects mutant RAS isoform-dependent transformation in human tumour cell lines has not been tested. After validating sgRNAs that efficiently deleted HRAS and NRAS, we showed that the differential requirement for SOS2 to support anchorage-independent (3D) growth, which we previously demonstrated in MEFs, held true in cancer cells. KRAS-mutant cells showed a high dependence on SOS2 for 3D growth, as previously shown, whereas HRAS-mutant cells did not require SOS2 for 3D growth. This differential requirement was not due to differences in RTK-stimulated WT RAS activation, as SOS2 deletion reduced RTK-stimulated WT RAS/PI3K/AKT signalling in both HRAS and KRAS mutated cell lines. Instead, this differential requirement of SOS2 to promote transformation was due to the differential sensitivity of RAS-mutated cancer cells to reductions in WT RAS/PI3K/AKT signalling. KRAS mutated cancer cells required SOS2/PI3K signaling to protect them from anoikis, whereas survival of both HRAS and NRAS mutated cancer cells was not altered by SOS2 deletion. Finally, we present an integrated working model of SOS signaling in the context of mutant KRAS based on our findings and those of others.

KRAS-mutant colon cancer cells respond to combined treatment of ABT263 and axitinib.

Significant challenges to develop selective and effective pharmacological inhibitors for important oncoproteins like RAS continue impeding the success to treat cancers driven by such mutations. In the present study, the ABT263 and axitinib combination imposed synergistic effects on RAS-mutant colon cancer cells. The combination inhibited in vitro and in vivo growth of the cancer cells by enhancing apoptosis. Furthermore, AKT and Wnt/β-catenin signaling pathways were slightly down-regulated by the combination in KRAS-mutant colon cancer cells. The current results indicate that oncogene addiction can be targeted for therapy in colon cancer cells harboring the RAS-mutant. Therefore, targeting oncogene addiction can be a viable strategy for treating refractory cancers driven by important oncogenes, such as KRAS, which are otherwise difficult to be targeted by small molecules.

Autophagy inhibition specifically promotes epithelial-mesenchymal transition and invasion in RAS-mutated cancer cells

ABSTRACTMacroautophagy/autophagy inhibition is a novel anticancer therapeutic strategy, especially for tumors driven by mutant RAS. Here, we demonstrate that autophagy inhibition in RAS-mutated cells induces epithelial-mesenchymal transition (EMT), which is associated with enhanced tumor invasion. This is at least partially achieved by triggering the NFKB/NF-κB pathway via SQSTM1/p62. Knockdown of ATG3 or ATG5 increases oncogenic RAS-induced expression of ZEB1 and SNAI2/Snail2, and activates NFKB activity. Depletion of SQSTM1 abolishes the activation of the NFKB pathway induced by autophagy inhibition in RAS-mutated cells. NFKB pathway inhibition by depletion of RELA/p65 blocks this EMT induction. Finally, accumulation of SQSTM1 protein correlates with loss of CDH1/E-cadherin expression in pancreatic adenocarcinoma. Together, we suggest that combining autophagy inhibition with NFKB inhibitors may therefore be necessary to treat RAS-mutated cancer.Abbreviations: 4-OHT: 4-hydroxytamoxifen; DIC: differential interference contrast; EMT: epithelial-mesenchymal transition; ESR: estrogen receptor; MAPK/ERK: mitogen-activated protein kinase; iBMK: immortalized baby mouse kidney epithelial cells; MET: mesenchymal-epithelial transition; PI3K: phosphoinositide 3-kinase; RNAi: RNA interference; TGFB/TGF-β: transforming growth factor beta; TNF: tumor necrosis factor; TRAF6: TNF receptor associated factor 6.

MAP kinase and autophagy pathways cooperate to maintain RAS mutant cancer cell survival

Oncogenic mutations in the small GTPase KRAS are frequently found in human cancers, and, currently, there are no effective targeted therapies for these tumors. Using a combinatorial siRNA approach, we analyzed a panel of KRAS mutant colorectal and pancreatic cancer cell lines for their dependency on 28 gene nodes that represent canonical RAS effector pathways and selected stress response pathways. We found that RAF node knockdown best differentiated KRAS mutant and KRAS WT cancer cells, suggesting RAF kinases are key oncoeffectors for KRAS addiction. By analyzing all 376 pairwise combination of these gene nodes, we found that cotargeting the RAF, RAC, and autophagy pathways can improve the capture of KRAS dependency better than targeting RAF alone. In particular, codepletion of the oncoeffector kinases BRAF and CRAF, together with the autophagy E1 ligase ATG7, gives the best therapeutic window between KRAS mutant cells and normal, untransformed cells. Distinct patterns of RAS effector dependency were observed across KRAS mutant cell lines, indicative of heterogeneous utilization of effector and stress response pathways in supporting KRAS addiction. Our findings revealed previously unappreciated complexity in the signaling network downstream of the KRAS oncogene and suggest rational target combinations for more effective therapeutic intervention.

Resistance to Src inhibition alters the BRAF-mutant tumor secretome to promote an invasive phenotype and therapeutic escape through a FAK>p130Cas>c-Jun signaling axis.

Few therapy options exist for patients with advanced papillary and anaplastic thyroid cancer. We and others have previously identified c-Src as a key mediator of thyroid cancer pro-tumorigenic processes and a promising therapeutic target for thyroid cancer. To increase the efficacy of targeting Src in the clinic, we sought to define mechanisms of resistance to the Src inhibitor, dasatinib, to identify key pathways to target in combination. Using a panel of thyroid cancer cell lines expressing clinically relevant mutations in BRAF or RAS, which were previously developed to be resistant to dasatinib, we identified a switch to a more invasive phenotype in the BRAF-mutant cells as a potential therapy escape mechanism. This phenotype switch is driven by FAK kinase activity, and signaling through the p130Cas>c-Jun signaling axis. We have further shown this more invasive phenotype is accompanied by alterations in the secretome through the increased expression of pro-inflammatory cytokines, including IL-1β, and the pro-invasive metalloprotease, MMP-9. Furthermore, IL-1β signals via a feedforward autocrine loop to promote invasion through a FAK>p130Cas>c-Jun>MMP-9 signaling axis. We further demonstrate that upfront combined inhibition of FAK and Src synergistically inhibits growth and invasion, and induces apoptosis in a panel of BRAF- and RAS-mutant thyroid cancer cell lines. Together our data demonstrate that acquired resistance to single-agent Src inhibition promotes a more invasive phenotype through an IL-1β>FAK>p130Cas>c-Jun>MMP signaling axis, and that combined inhibition of FAK and Src has the potential to block this inhibitor-induced phenotype switch.

Combined treatment of ABT199 and irinotecan suppresses KRAS-mutant lung cancer cells

Lung cancer has become the most prevalent neoplasm throughout the world with 1,2 million deaths per year. Molecular genetic analyses have suggested that KRAS mutation is much more frequency in NSCLC. Significant challenges are to develop selective pharmacological inhibitors for RAS mutation to treat cancers driven. In our study, we used the combinatorial strategy to target oncogene addiction for RAS-mutant cells and the data showed that ABT199 and irinotecan leads to RAS-mutant lung cancer cell growth inhibition and enhanced apoptosis in vitro and in vivo. Furthermore, PI3K/AKT signaling was down-regulated by the combination in KRAS-mutant lung cancer cells. Importantly, the effects of ABT199 and irinotecan combination are synergistic on the RAS-mutant lung cancer cells. Therefore, the combination suggests a strong synergy in vivo and a potential avenue for therapeutic treatment of KRAS-mutant cancers which are otherwise difficult targeted by small molecules.

The AMPK inhibitor overcomes the paradoxical effect of RAF inhibitors through blocking phospho–Ser-621 in the C terminus of CRAF

The dimerization-driven paradoxical activation of RAF proto-oncogene Ser/Thr kinase (RAF) is the predominant cause of drug resistance and toxicity in cancer therapies with RAF inhibitors. The scaffold protein 14-3-3, which binds to the RAF C terminus, is essential for RAF activation under physiological conditions, but the molecular basis is unclear. Here we investigated whether and how 14-3-3 regulates the dimerization-driven transactivation of the RAF isoform CRAF by RAF inhibitors and affects drug resistance and toxicity by virtue of the dominant role of CRAF in these processes. We demonstrated that 14-3-3 enhances the dimerization-driven transactivation of CRAF by stabilizing CRAF dimers. Further, we identified AMP-activated protein kinase (AMPK) and CRAF itself as two putative kinases that redundantly phosphorylate CRAF's C terminus and thereby control its association with 14-3-3. Next, we determined whether the combinatory inhibition of AMPK and CRAF could overcome the paradoxical effect of RAF inhibitors. We found that the AMPK inhibitor (AMPKi) not only blocked the RAF inhibitor–driven paradoxical activation of ERK signaling and cellular overgrowth in Ras-mutated cancer cells by blocking phosphorylation of Ser-621 in CRAF but also reduced the formation of drug-resistant clones of BRAFV600E-mutated cancer cells. Last, we investigated whether 14-3-3 binding to the C terminus of CRAF is required for CRAF catalytic activity and observed that it was dispensable in vivo. Altogether, our study unravels the molecular mechanism by which 14-3-3 regulates dimerization-driven RAF activation and identified AMPKi as a potential agent to counteract drug resistance and adverse effects of RAF inhibitors in cancer therapies.

Abstract 865: Small molecule-mediated modulation of Ras elicits inhibition of phospho ERK signaling through negative feedback on SOS1

Abstract Oncogenic mutation of Ras is responsible for more than 30% of all human tumors. Therefore, pharmacological modulation of Ras has attracted great interest as a therapeutic strategy. Our laboratory has recently discovered small molecules that activate Son of Sevenless (SOS)-catalyzed nucleotide exchange on Ras and paradoxically inhibit downstream signaling. Here we describe how pharmacologically targeting SOS1 induces biphasic modulation of Ras-GTP and downstream ERK levels. We consistently observed this phenotype in a variety of cell lines expressing different Ras mutant isoforms, and using multiple chemotypes of compound. In elucidating the molecular mechanism, we observed that compound treatment caused an increase in phosphorylation at ERK consensus motifs on SOS1 that was not observed with the expression of a non-phosphorylatable S1178A SOS1 mutant, or after pre-treatment with an ERK inhibitor. Phosphorylation at S1178 on SOS1 is known to inhibit the association between SOS1 and GRB2 and disrupt SOS1 membrane localization. Consistent with this, we show that wild-type SOS1 and GRB2 dissociated in a time dependent fashion in response to compound treatment, and conversely, this interaction was enhanced with the expression of a S1178A SOS1 mutant. Pre-treatment with an ERK inhibitor prevented compound-induced inhibition of the association between SOS1 and GRB2. Furthermore, in cells expressing either S1178A SOS1 or a constitutively membrane-bound CAAX box tagged SOS1 mutant, we observed elevated Ras-GTP levels over time in response to compound, as compared to the biphasic changes in Ras-GTP exhibited in cells expressing wild-type SOS1. These results show that small molecule agonists of SOS1 cause paradoxical inhibition of Ras-GTP and MAPK signaling in Ras mutant cancer cells, through negative feedback on SOS1. Overall, these compounds provide us with the unique opportunity to better understand the biological functions of SOS and Ras in cancer cells, and may aid in the discovery of small molecules to treat Ras-driven tumors. Citation Format: Jennifer E. Howes, Denis T. Akan, Michael C. Burns, Qi Sun, Andrew J. Little, DeMarco V. Camper, Jason R. Abbott, Jason Phan, Taekyu Lee, Olivia W. Rossanese, Alex G. Waterson, Stephen W. Fesik. Small molecule-mediated modulation of Ras elicits inhibition of phospho ERK signaling through negative feedback on SOS1 [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 865.

A Simply Modified Lymphocyte for Systematic Cancer Therapy.

Cytotherapy has received considerable attention in the field of cancer therapy, and various chemical or genetic methods have been applied to remold natural cells for improved therapeutic outcome of cytotherapy. A simple method to modify lymphocytes for cancer treatment by using a clinically used molecule, δ-aminolevulinic acid (δ-ALA), is reported here. After incubation with this molecule, tumor-targeted lymphocytes spontaneously synthesize anti-neoplastic drug protoporphyrin X (PpIX), and specifically accumulate in cancer tissue. Under periodic 630 nm laser irradiation, lymphocytes generate vesicle-like apoptotic body (Ab) containing the above-produced PpIX, and the facilitated delivery of PpIX from Ab makes an excellent therapeutic effect for Ras-mutated cancer cells under a second irradiation. Importantly, a microfluidic device is further fabricated to simplify cell sorting and drug synthesis with a one-step operation, which could promote generalization of this strategy. In vitro and in vivo studies confirm the success of such an easy-operated and global-regulated strategy for cancer therapy.

Knockdown of ferroportin accelerates erastin-induced ferroptosis in neuroblastoma cells.

Ferroptosis is a new-found iron-dependent form of non-apoptotic regulated cell death (RCD), which is activated on therapy with several antitumor agents, but the potential mechanism remains unclear. Erastin, exhibiting selectivity for RAS-mutated cancer cells, induces ferroptosis by increasing iron and lipid reactive oxygen species (ROS) levels in cell. Ferroportin (Fpn), the sole iron export protein, participates in the regulation of intracellular iron concentration. In this study, we investigated the role of Fpn on ferroptosis induced by erastin in SH-SY5Y cells. The cell viability was determined by CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay kit. The activity of caspase-3 was measured by ELISA kit. qRT-PCR was performed to examine the mRNA expression of Fpn. Western blot assay was conducted to examine the expression level of marker proteins. Specific commercial kits were used to examine the levels of MDA, ROS and iron in cells, respectively. Ferroptosis was evaluated by intracellular lipid ROS level and iron concentration. Hepcidin could prevent erastin-induced ferroptosis by degrading Fpn. Erastin (5 μg/mL) was observed to induce ferroptosis in neuroblastoma cells at 6 hours, which was promoted by knockdown of Fpn. The expression of Fpn gene and protein was decreased in SH-SY5Y cells treated with erastin. After treatment with erastin, Fpn siRNA transfection in SH-SY5Y cells was able to accelerate ferroptosis-associated phenotypic changes. Fpn acted as a negative regulator of ferroptosis by reducing intracellular iron concentration. Knockdown of Fpn enhanced anticancer activity of erastin. These results suggested that knockdown of Fpn accelerated erastin-induced ferroptosis by increasing iron-dependent lipid ROS accumulation, highlighting Fpn as a potential therapeutic target site for neuroblastoma. Thus, Fpn inhibitors may provide new access for chemosensitization of neuroblastoma.

\u03b3-Tocotrienol induces apoptosis in pancreatic cancer cells by upregulation of ceramide synthesis and modulation of sphingolipid transport

BackgroundCeramide synthesis and metabolism is a promising target in cancer drug development. γ-tocotrienol (GT3), a member of the vitamin E family, orchestrates multiple effects that ensure the induction of apoptosis in both, wild-type and RAS-mutated pancreatic cancer cells. Here, we investigated whether these effects involve changes in ceramide synthesis and transport.MethodsThe effects of GT3 on the synthesis of ceramide via the de novo pathway, and the hydrolysis of sphingomyelin were analyzed by the expression levels of the enzymes serine palmitoyl transferase, ceramide synthase-6, and dihydroceramide desaturase, and acid sphingomyelinase in wild-type RAS BxPC3, and RAS-mutated MIA PaCa-2 and Panc 1 pancreatic cancer cells. Quantitative changes in ceramides, dihydroceramides, and sphingomyelin at the cell membrane were detected by LCMS. Modulation of ceramide transport by GT3 was studied by immunochemistry of CERT and ARV-1, and the subsequent effects at the cell membrane was analyzed via immunofluorescence of ceramide, caveolin, and DR5.ResultsGT3 favors the upregulation of ceramide by stimulating synthesis at the ER and the plasma membrane. Additionally, the conversion of newly synthesized ceramide to sphingomyelin and glucosylceramide at the Golgi is prevented by the inhibition of CERT. Modulation ARV1 and previously observed inhibition of the HMG-CoA pathway, contribute to changes in membrane structure and signaling functions, allows the clustering of DR5, effectively initiating apoptosis.ConclusionsOur results suggest that GT3 targets ceramide synthesis and transport, and that the upregulation of ceramide and modulation of transporters CERT and ARV1 are important contributors to the apoptotic properties demonstrated by GT3 in pancreatic cancer cells.

Histone Deacetylase Inhibition Enhances the Antitumor Activity of a MEK Inhibitor in Lung Cancer Cells Harboring RAS Mutations.

Non-small cell lung cancer (NSCLC) can be identified by precise molecular subsets based on genomic alterations that drive tumorigenesis and include mutations in EGFR, KRAS, and various ALK fusions. However, despite effective treatments for EGFR and ALK, promising therapeutics have not been developed for patients with KRAS mutations. It has been reported that one way the RAS-ERK pathway contributes to tumorigenesis is by affecting stability and localization of FOXO3a protein, an important regulator of cell death and the cell cycle. This is through regulation of apoptotic proteins BIM and FASL and cell-cycle regulators p21Cip1 and p27Kip1 We now show that an HDAC inhibitor affects the expression and localization of FOXO proteins and wanted to determine whether the combination of a MEK inhibitor with an HDAC inhibitor would increase the sensitivity of NSCLC with KRAS mutation. Combined treatment with a MEK inhibitor and an HDAC inhibitor showed synergistic effects on cell metabolic activity of RAS-mutated lung cancer cells through activation of FOXOs, with a subsequent increase in BIM and cell-cycle inhibitors. Moreover, in a mouse xenograft model, the combination of belinostat and trametinib significantly decreases tumor formation through FOXOs by increasing BIM and the cell-cycle inhibitors p21Cip1 and p27Kip1 These results demonstrate that control of FOXOs localization and expression is critical in RAS-driven lung cancer cells, suggesting that the dual molecular-targeted therapy for MEK and HDACs may be promising as novel therapeutic strategy in NSCLC with specific populations of RAS mutations. Mol Cancer Ther; 17(1); 17-25. ©2017 AACR.

Abstract B156: Discovery and characterization of AZ6197, a potent and selective ERK1/2 inhibitor

Abstract The RAS/MAPK pathway is a major driver in oncogenesis and is dysregulated in approximately 30% of human cancers, primarily by mutations in BRAF or RAS genes. The extracellular-signal-regulated kinases (ERK1 and ERK2) serve as key central nodes within this pathway. The feasibility of targeting the RAS/MAPK pathway has been demonstrated by the initial positive clinical responses observed to BRAF and MEK inhibitors in BRAF V600E/K metastatic melanoma; however, resistance frequently develops by reactivation of the RAS/MAPK pathway. Direct targeting of ERK1/2 may provide another therapeutic option in tumors with mutations in BRAF or RAS genes and, importantly, may overcome acquired resistance to RAF and MEK inhibitors where RAS/MAPK pathway reactivation has occurred, such as relapsed BRAF V600E/K melanoma. Here we describe the discovery and characterization of compound 35 (Ward et al., J Med Chem 2017 27;60), also known as AZ6197. AZ6197 is a highly potent and selective inhibitor of ERK1 and ERK2, with IC50 of <1nM in biochemical assays. AZ6197 reduces cellular phospho-p90RSK1 levels in BRAF and RAS mutant cancer cell lines and inhibits proliferation of cancer cell lines exhibiting dysregulation of RAS/MAPK pathway signaling, including cell lines with mutations in BRAF, NRAS, or KRAS. AZ6197 is also active in multiple BRAF- and MEK- inhibitor resistant A375 melanoma cell lines, which have acquired a range of mutations that reactivate the RAS/MAPK pathway. In tumor xenograft models, AZ6197 reduces the pharmacodynamic (PD) biomarkers phospho-p90RSK1 and phospho-FRA1 in tumors in line with compound exposure levels, and induces significant tumor regression in the KRAS mutant NSCLC Calu 6 xenograft model. AZ6197 can be combined safely and effectively with the MEK1/2 inhibitor selumetinib in KRAS mutant NSCLC xenograft models, and this simultaneous targeting of MEK and ERK leads to synergistic tumor regressions being observed in multiple xenografts. In summary, AZ6197 is a potent and selective ERK1/2 inhibitor with activity in cell lines and tumor xenograft models with RAS/MAPK pathway activation. AZ6197 also has significant activity in BRAF mutant melanoma cell lines in which resistance to RAF and MEK inhibitors is due to the acquisition of NRAS and MEK1 mutations. Combination treatment of AZ6197 and the MEK1/2 inhibitor selumetinib drives synergistic tumor regression in multiple KRAS mutant xenograft models. Citation Format: Vikki Flemington, Iain Simpson, Emma Davies, David Robinson, Nicola Lindsay, Lyndsey Hanson, Philip Hopcroft, Michael Tonge, Karen Roberts, Richard Ward. Discovery and characterization of AZ6197, a potent and selective ERK1/2 inhibitor [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B156.

Autophagy acts through TRAF3 and RELB to regulate gene expression via antagonism of SMAD proteins

Macroautophagy can regulate cell signalling and tumorigenesis via elusive molecular mechanisms. We establish a RAS mutant cancer cell model where the autophagy gene ATG5 is dispensable in A549 cells in vitro, yet promotes tumorigenesis in mice. ATG5 represses transcriptional activation by the TGFβ-SMAD gene regulatory pathway. However, autophagy does not terminate cytosolic signal transduction by TGFβ. Instead, we use proteomics to identify selective degradation of the signalling scaffold TRAF3. TRAF3 autophagy is driven by RAS and results in activation of the NF-κB family member RELB. We show that RELB represses TGFβ target promoters independently of DNA binding at NF-κB recognition sequences, instead binding with SMAD family member(s) at SMAD-response elements. Thus, autophagy antagonises TGFβ gene expression. Finally, autophagy-deficient A549 cells regain tumorigenicity upon SMAD4 knockdown. Thus, at least in this setting, a physiologic function for autophagic regulation of gene expression is tumour growth.

Oncogenic RAS-induced CK1\u03b1 drives nuclear FOXO proteolysis

Evasion of forkhead box O (FOXO) family of longevity-related transcription factors-mediated growth suppression is necessary to promote cancer development. Since somatic alterations or mutations and transcriptional dysregulation of the FOXO genes are infrequent in human cancers, it remains unclear how these tumour suppressors are eliminated from cancer cells. The protein stability of FOXO3A is regulated by Casein Kinase 1 alpha (CK1α) in an oncogenic RAS-specific manner, but whether this mode of regulation extends to related FOXO family members is unknown. Here we report that CK1α similarly destabilizes FOXO4 in RAS-mutant cells by phosphorylation at serines 265/268. The CK1α-dependent phosphoregulation of FOXO4 is primed, in part, by the PI3K/AKT effector axis of oncogenic RAS signalling. In addition, mutant RAS coordinately elevates proteasome subunit expression and proteolytic activity to eradicate nuclear FOXO4 proteins from RAS-mutant cancer cells. Importantly, dual inhibition of CK1α and the proteasome synergistically inhibited the growth of multiple RAS-mutant human cancer cell lines of diverse tissue origin by blockade of nuclear FOXO4 degradation and induction of caspase-dependent apoptosis. Our findings challenge the current paradigm that nuclear export regulates the proteolysis of FOXO3A/4 tumour suppressors in the context of cancer and illustrates how oncogenic RAS-mediated degradation of FOXOs, via post-translational mechanisms, blocks these important tumour suppressors.

Abstract 4973: Discovery of LY3214996, a selective and novel ERK1/2 inhibitor with potent antitumor activities in cancer models with MAPK pathway alterations

Abstract The RAS/MAPK pathway is dysregulated in approximately 30% of human cancers, and the extracellular-signal-regulated kinases (ERK1 and ERK2) serves as key central nodes within this pathway. The feasibility and clinical impact of targeting the RAS/MAPK pathway has been demonstrated by the therapeutic success of BRAF and MEK inhibitors in BRAF V600E/K metastatic melanoma. However, resistance develops frequently through reactivation of the pathway. Therefore, simultaneous targeting of multiple effectors such as RAF, MEK and ERK in this pathway, offers a potential for enhanced efficacy while delaying and overcoming resistance. LY3214996 is a highly selective inhibitor of ERK1 and ERK2, with IC50 of 5 nM for both enzymes in biochemical assays. It potently inhibits cellular phospho-RSK1 in BRAF and RAS mutant cancer cell lines. In an unbiased tumor cell panel sensitivity profiling for inhibition of cell proliferation, tumor cells with MAPK pathway alterations including BRAF, NRAS or KRAS mutation are generally sensitivity to LY3214996. In tumor xenograft models, LY3214996 inhibits PD biomarker phospho-p90RSK1 in tumors and the PD effects are correlated with compound exposures and anti-tumor activities. LY3214996 shows either similar or superior anti-tumor activity as compared to other published ERK inhibitors in BRAF or RAS mutant cell lines and xenograft models. Oral administration of single-agent LY3214996 significantly inhibits tumor growth in vivo and is well tolerated in BRAF or NRAS mutant melanoma, BRAF or KRAS mutant colorectal, lung and pancreatic cancer xenografts or PDX models. Therefore, LY3214996 can be tailored for treatment of cancers with MAPK pathway alteration. In addition, LY3214996 has anti-tumor activity in a vemurafenib-resistant A375 melanoma xenograft model due to MAPK reactivation, may have potential for treatment of melanoma patients who have failed BRAF therapies. More importantly, LY3214996 can be combined with investigational and approved agents in preclinical models, particularly KRAS mutant models. Combination treatment of LY3214996 and CDK4/6 inhibitor abemaciclib was well tolerated and results in potent tumor growth inhibition or regression in multiple in vivo cancer models, including KRAS mutant colorectal and non-small cell lung cancers. Here, we first report the preclinical characterization of LY3214996, a novel small molecule ERK1/2 inhibitor currently in Phase I clinical trials in patients with advanced and metastatic cancers (NCT02857270). Citation Format: Shripad V. Bhagwat, William T. McMillen, Shufen Cai, Baohui Zhao, Matthew Whitesell, Lisa Kindler, Robert S. Flack, Wenjuan Wu, Karen Huss, Bryan Anderson, Xiu-Juan Yuan, Susan Jaken, Denis McCann, Brian Mathes, Andrew J. Dropsey, Jason Manro, Jennie Walgren, Eunice Yuen, Xueqian Gong, Michael J. Rodriguez, Jianping Huang, Ramon V. Tiu, Sajan Joseph, Sheng-Bin Peng. Discovery of LY3214996, a selective and novel ERK1/2 inhibitor with potent antitumor activities in cancer models with MAPK pathway alterations [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 4973. doi:10.1158/1538-7445.AM2017-4973

The Atypical Kinase RIOK1 Promotes Tumor Growth and Invasive Behavior

Despite being overexpressed in different tumor entities, RIO kinases are hardly characterized in mammalian cells. We investigated the role of these atypical kinases in different cancer cells. Using isogenic colon-, breast- and lung cancer cell lines, we demonstrate that knockdown of RIOK1, but not of RIOK2 or RIOK3, strongly impairs proliferation and invasiveness in conventional and 3D culture systems. Interestingly, these effects were mainly observed in RAS mutant cancer cells. In contrast, growth of RAS wildtype Caco-2 and Bcr-Abl-driven K562 cells is not affected by RIOK1 knockdown, suggesting a specific requirement for RIOK1 in the context of oncogenic RAS signaling. Furthermore, we show that RIOK1 activates NF-κB signaling and promotes cell cycle progression. Using proteomics, we identified the pro-invasive proteins Metadherin and Stathmin1 to be regulated by RIOK1. Additionally, we demonstrate that RIOK1 promotes lung colonization in vivo and that RIOK1 is overexpressed in different subtypes of human lung- and breast cancer. Altogether, our data suggest RIOK1 as a potential therapeutic target, especially in RAS-driven cancers.