Abstract 4101: Targeting the EGFR/STAT3 axis in NSCLC with resistance to EGFR tyrosine kinase inhibitors using an oligonucleotide-based decoy

Abstract

Abstract EGFR (Epidermal Growth Factor Receptor) is a major target for the treatment of Non-Small Cell Lung Cancer (NSCLC), but patients lacking EGFR mutation are insensitive to EGFR inhibition. Patients with EGFR mutations initially respond to EGFR tyrosine kinase inhibitors (TKIs), but eventually develop acquired resistance as a result of secondary mutations in the EGFR tyrosine kinase domain. Upregulation of Signal Transducer and Activator of Transcription (STAT3), a key oncogenic molecule in NSCLC that is downstream of EGFR signaling, contributes to intrinsic and acquired resistance to EGFR TKIs. In the clinic, agents targeting STAT3 have not been satisfactory to date. To address the limitations of available STAT3 inhibitors, we are currently testing a cyclic oligonucleotide molecule with a novel mechanism that acts as a STAT3 “decoy” (Cyclic STAT3 decoy, CS3D). CS3D mimics the DNA consensus sequence in the promoter region of STAT3-responsive genes, causing the binding of STAT3 dimers and preventing transcriptional regulation of STAT3-target genes. The effects of CS3D are compared to mutant cyclic STAT3 decoy (CS3M), which differs from CS3D by one base-pair. We have tested the ability of CS3D to produce anti-tumor effects in NSCLC cells that are EGFR WT (201T) and in EGFR mutant cells that also carry the T790M resistant mutation (H1975). Initial in vitro studies showed that CS3D caused a 50% inhibition in cell proliferation in 201T and H1975 relative to CS3M using MTS assays. Flow cytometry studies also demonstrated that CS3D caused a 2-fold increase in the percent of apoptotic cells as compared to CS3M. CS3D also caused a 2-fold reduction in expression of STAT3-target genes c-Myc, Bcl-xL and IL-6. Compared to CS3M, CS3D inhibited colony formation by 70%. Using an in vivo mouse xenograft model of 201T and H1975, CS3D caused a 96.5% reduction in tumor growth in 201T (P<0.007) compared to CS3M, while an 81.7% inhibition was seen in H1975 (P<.0001). Utilizing IHC, analysis of residual tumors also illustrated that CS3D induced more caspase3 cleavage relative to CS3M. Additionally, western blot analysis showed 70% reduction in c-Myc protein level in response to CS3D. These results suggest CS3D can be effective as a single therapeutic agent. Combining CS3D with EGFR inhibitors such as afatinib in vitro significantly suppressed cell viability by 85.2% and 80% in 201T and H1975 respectively, as compared to single treatment (CS3D alone reduced cell viability by 53%, and 51.5%, and afatinib alone reduced viability by 31.4% and 34.03% in 201T and H1975, respectively). The combination of CS3D and afatinib warrants further testing in vivo. These data suggest that CS3D alone or in combination with EGFR tyrosine kinase inhibitors produces anti-tumor effects in NSCLC with intrinsic and acquired resistance to EGFR TKIs. Supported by funding to the Masonic Cancer Center from the Minnesota 5th Order of Eagles. Citation Format: Christian Njatcha, Mariya Farooqui, Jennifer R. Grandis, Jill M. Siegfried. Targeting the EGFR/STAT3 axis in NSCLC with resistance to EGFR tyrosine kinase inhibitors using an oligonucleotide-based decoy [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 4101. doi:10.1158/1538-7445.AM2017-4101