Defining the role of RUNX1T1 in "combined" small cell lung cancer.

Abstract

e20101 Background: The RUNX1T1 gene, also known as ETO, was first identified in the fusion transcript AML1/ETO, generated by the translocation between chromosome 8 and 21, which occurs in 12%-15% of AML. We recently discovered that RUNX1T1 was specifically amplified only in the small cell lung cancer (SCLC) but not the non-small cell lung cancer (NSCLC) component of combined SCLC tumors. Here, we investigated if RUNX1T1 expression plays a role in transforming NSCLC to SCLC in combined SCLC tumor formation. Methods: We examined tumors from 92 of our SCLC patients by targeted exome and CNV analyses. RUNX1T1 mRNA expression was determined in various cancers by analysis of the CCLE and TCGA databases. RUNX1T1 mRNA expression was detected by RNAscope analysis of another 22 patient SCLC tumors. Stable overexpression of RUNX1T1 in NSCLC and SCLC cell lines used commercial RUNX1T1 lentiviral constructs. The resulting stable cell lines were analyzed by RT-qPCR, western blotting, Affymetrix gene array, and E2F-luciferase assays. Results: We found that 2 of 2 patients with combined SCLC demonstrated RUNX1T1 amplification in the SCLC component. Notably, only 2 of the remaining 90 patients with pure SCLC demonstrated RUNX1T1 amplification. RUNX1T1 mRNA is highly expressed in SCLC compared to the majority of other cancers, including NSCLC, using either CCLE or TCGA databases. Western blots confirmed higher RUNX1T1 protein expression in SCLC vs NSCLC cell lines, and RNAscope analysis confirmed greater RUNX1T1 mRNA expression in SCLC vs NSCLC patient tumors. When we stably overexpressed RUNX1T1 in NSCLC cell lines, there was no increase in neuroendocrine protein expression, however we did find an increased sensitivity to etoposide. Bioinformatic analysis of microarray data demonstrated significantly altered E2F pathway activity, and we could show a significant increase in E2F-luciferase activity after RUNX1T1 overexpression. Interestingly, this was associated with decreased p21 expression, which is a putative suppressor of E2F activity. Conclusions: RUNX1T1 expression is specifically upregulated in SCLC compared to NSCLC. E2F pathway activity is elevated after RUNX1T1 overexpression, consistent with an increased SCLC phenotype.