Targeting Aurka Effectively Suppresses Kras-Mutant Tumorigenesis Through Regulating P70S6K Phosphorylation in Gastrointestinal Cancers

panel of KRAS mutant or amplified cancer cell lines including gastric (AGS, SNU-1, SNU601), esophageal (ESO26) and colon (HCT116, SW480, SW620). Western blot analysis data demonstrated that MLN8237 treatment or knockdown of AURKA decreased the cell viability and increased the protein expression of BIM, p21, p27, Cleaved-Caspase3, and Cleaved-PARP. Of note, we also found downregulation of p70S6K protein phosphorylation at Thr-389 following knockdown or inhibition of AURKA. The knockdown of mutant KRAS recapitulated the signaling effects of AURKA inhibition leading to a remarkable reduction of phospho-P70S6K and AURKA. Surprisingly, knockdown of wild-type KRAS did not produce these signaling effects, suggesting that AURKA is an important downstream effector of mutant KRAS that regulates P70S6K. Of note, we found that knockdown of mutant KRAS significantly decreased the mRNA and protein levels of AURKA, a finding that was not produced in cells with wild-type KRAS. To confirm the pro-survival role of P70S6K, the knockdown of P70S6K proteins by siRNA suppressed the cell growth of KRAS-mutant GI cancer cell lines, which was followed by increased protein level of pro-apoptotic markers such as cleaved-caspase 3 and BIM. Using immunofluorescence, we found that AURKA colocalizes with p70S6K. In addition, using Immunoprecipitation analysis, AURKA was in the same protein complex as P76S6K both in vivo and in vitro; this interaction was abolished by pharmacological inhibition of AURKA using MLN8237. Using tumor xenograft mouse models of four KRAS mutant cancer cell lines, we detected a significant response to MLN8237 showing significant tumor regression in all models. Conclusion: Mechanistically, our results demonstrate a previously unknown mutKRAS-AURKA-P70S6K signaling axis in GI cancers. We also suggest that AURKA is a downstream druggable effector of mutant KRAS. Targeting AURKA is a novel therapeutic strategy for treating KRAS-mutant GI cancers, including those refractory to currently available therapeutic options.

146 SELECTIVE ERADICATION OF K-RAS MUTATED CANCER CELLS BY DELIVERY OF BACTERIAL TOXINS Ilana Boustanai, Dina Kazanov, Shiran Shapira, Nadir Arber Introduction: Inactivation of TP53 is the most frequent genetic damage in human cancer. In addition, hyperactivation of the RAS pathway is common in many human malignancies (Lung (LC)~40%, pancreatic (PC)>95% and colorectal cancer (CRC)~50%). Despite multiple attempts, targeting these pathways for the treatment of cancer, for example through the development of RAS pathway inhibitors has not proven to be effective thus far. Herein, we propose to exploits the hyperactive RAS pathway and TP53 mutation status of human cancer to deliver targeted anti-tumor therapy. We had previously reported that a recombinant adenovirus, carrying a pro-apoptotic gene (PUMA) under the regulation of RAS-responsive elements (PY4) effectively suppressed the growth of human cancer cells harboring hyperactive RAS (Giladi et al, 2007). Furthermore, we had shown, both in vitro and in vivo, that replacing the pro-apoptotic gene with a bacterial toxin can improve the efficacy of this system (Shapira et al, 2015). Aim:To establish a tight regulated dual system by expressing a toxin under PY4 elements in cancer cells, while sparing normal cells by expressing the anti-toxin under p53 responsive elements (RGC) specifically in non-malignant cells.Methods: Adenoviral vectors carrying the toxin (PY4-MazF-mcherry) and the antitoxin (RGC-MazE-GFP) were designed under the regulation of RAS and p53 responsive elements, respectively. A virus carrying the toxin but lacking the PY4 serves as a control virus. Those three constructs were cloned into a "first generation" ∆E1/∆E3 human type5 adenoviral vector. Virus particles were produced, their titer was calculated by the End-Point Dilution Assay and their potency was tested in vitro. Cell death was measured qualitatively by using the fluorescent microscopy and was quantified by the enzymatic MTT assay. HCT116+/+;Rasmut/p53wt and HCT116-/;Rasmut/p53mut CRC cell lines, as well as, A549;Rasmut/p53wt and H2030 Rasmut/p53wt LC cells were used for testing the potency and safety of the system.Results: Massive cell death was induced in a dose-dependent manner (~60%; 7.5 MOI) in CRC cells harboring mutated Ras after co-infection with the toxin and the antitoxin viruses. The antidote was able to protect ~70% of the cells that were affected by background low level expression of the toxin construct, due to promoter leakage [Fig1]. Similar results were obtained in a colony formation assay and FACS analysis of co-infected LC cells. A549 harboring mutated Ras showed greater sensitivity (~82% early apoptotic cells) compared to H1650 cells harboring WT Ras (~27% early apoptotic cells) [Fig2]. These results indicate that the antitoxin indeed actively protects cells with WT p53.Conclusion: Exploiting the activated RAS pathway and mutation in p53 holds promise for effective and safe therapy that could specifically target tumor cells while sparing normal tissues.

144 SR-18662: A POTENT COLORECTAL CANCER GROWTH INHIBITOR Julie M. Kim, Timothy J. Huang, Ainara Ruiz de Sabando, Vincent W. Yang, Agnieszka Bialkowska, Thomas D. Bannister, Chao Wang Background: Krüppel-like factor 5 (KLF5) is a member of the SP/KLF family of zinc finger transcription factors and is highly expressed in the proliferative regions of the intestinal epithelium. KLF5 is upregulated in human colorectal cancer specimens and is also associated with cancer progression. In addition, haploinsufficiency of KLF5 in mice harboring a germline mutation in Apc, a colon cancer suppressor gene, also significantly reduces intestinal tumor formation. These findings suggest that an inhibitor of KLF5 expression would be useful as a therapeutic agent to suppress intestinal tumor progression. In a collaborative effort with The Scripps Research Institute, we developed ML264, a small molecule KLF5 inhibitor, which restricts the growth of colorectal cancer (CRC) by inhibiting cell cycle progression as well as the expression of KLF5 and EGR-1 (1). In ongoing optimization efforts we have identified a fourth generation analogue, SR-18662, that has so far shown superior efficacy. Aim: To investigate the mechanism whereby SR-18662 inhibits CRC proliferation and to evaluate its efficacy in vivo. Methods: In vitro: All experiments were done in DLD-1 and HCT116 human CRC cell lines. We analyzed cell viability using the Cell-Titer Glo system, performed cell cycle and cell death analysis using flow cytometry, and investigated protein levels of various components of the EGFR and WNT signaling pathways as well as cyclins using western blotting techniques. Cells were treated with DMSO (control), ML264, SR15006 (a 3rd-generation analogue), and SR-18662 at 24, 48, and 72 hours. In vivo: We subcutaneously injected DLD-1 cells into athymic mice and monitored tumor growth. Then we intraperitoneally injected 5mg/kg or 10 mg/kg of SR-18662 vs. vehicle control for ten days once or twice daily, with a two-day mid-treatment break. Results: In vitro: The cell viability assay found that SR-18662 inhibited cellular proliferation more effectively than did ML264 within 72 hours. Unlike ML264, SR-18662 can initiate cellular death. Flow cytometry analysis showed an increase in cells attenuated in S phase as well as a significant increase in apoptosis after SR-18662 treatment. Western blot analysis showed a decrease in EGR-1, KLF5, β-catenin, cyclin E, cyclin D1, as well as an increase in cleaved caspase 3. In vivo: We observed a significant dose-dependent decrease in xenograft growth at a lower dose of SR-18662 than was used for a previous ML264 study. Conclusion: In vitro and in vivo, our most recent analogue, SR-18662 strongly inhibits the growth of colorectal cancer cells, moreso than did ML264, and has the potential to be an effective drug for colorectal cancer therapy.

145 TARGETING AURKA EFFECTIVELY SUPPRESSES KRAS-MUTANT TUMORIGENESIS THROUGH REGULATING P70S6K PHOSPHORYLATION IN GASTROINTESTINAL CANCERS Lihong Bishop, Zheng Chen, Omar Omar, Abbes Belkhiri, Wael M. El-Rifai Background: KRAS is the most frequently mutated isoform of RAS protein family in cancer cells. Mutations and amplifications of KRAS, clinically undruggable, are found in various human cancers, including upper gastrointestinal (UGI) cancers and colorectal cancer (CRC). We have previously shown overexpression of Aurora Kinase A (AURKA) in gastrointestinal cancers. In this study, we investigated whether targeting AURKA can be an effective therapeutic approach in KRAS-mutant cancers of the gastrointestinal tract. Methods and Results: To investigate the role of AURKA in KRAS mutant cancer cells, ATP-GLO cell viability and clonogenic cell survival assays were utilized to show that pharmacological inhibition of AURKA by MLN8237 or the knockdown of AURKA by siRNA reduced cell viability of a

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AGA Abstracts

AGA Abstracts

linear regression. Spearman correlations were performed between filtered and scaled microbial species (relative abundances) and miRNAs for multi-omics analysis. Results: Alpha and beta diversities were not different between mucosal microbiome of IBS patients and HCs (p>0.05). Three clusters were identified using hierarchical clustering of unweighted unifrac distances using all subjects. Cluster 1 (62.5% of samples) was enriched in Bacteroidetes and Firmicutes, Cluster 2 (29.5% of samples) was predominantly enriched in Bacteroidetes and Cluster 3 (8% of samples) was enriched in Proteobacteria. We found 24 species associated with the cluster differences (FDR p <0.05). Clusters 1 and 2 were enriched in women compared to Cluster 3 (p<0.05). IBS symptom severity, bowel habit subtype and other clinical traits did not differ between clusters when adjusted for sex. Multi-omics analysis identified 7 significantly correlated miRNA-bacteria pairs in IBS that were not seen in HCs (FDR p<0.1). Hsa-miR-200a/ Parabacteroides distasonis showed highest correlation (rho= -0.67, FDR p=0.03, Figure). MiR-200a targets transcription factors ZEB1 and ZEB2 which regulate E-cadherin expression, affecting the initiation and stabilization of cell-cell adhesion. Parabacteroides distasonis has been found to be associated with reduced E-cadherin in celiac disease. Conclusions: Distinct clusters of microbiota were identified in mucosal biopsies in IBS. Distinct miRNAs may play a role in shaping gut microbial composition in IBS.