Abstract 3950: A novel combination therapy targeting BCL6 and phospho-STAT3 defeats intratumor heterogeneity in a subset of non-small cell lung cancers
Authors: Deb Dhruba , Rajaram Satwik , Larsen Jill E. , Dospoy Patrick P. , Marullo Rossella , Li Longshan , Avila Kimberley , Cerchietti Leandro , Minna John D. , Wu Lani F. , Altschuler Steven J. 
Published: July 2017

Purpose: Lung cancer is a disease of great oncogenotype complexity (oncogenes and tumor suppressor gene alterations). These alterations can appear in different combinations even within histologically defined lung cancer subtypes. The success of targeted therapy has led to a search for oncogenotype-specific therapies. But, no one therapy fits all oncogenotypes. Here, we investigate whether characterization of oncogene-specific alterations in cellular signaling at single cell level indicate heterogeneity even within cells from the same patient with defined oncogenotype, and whether they can suggest new targets to deal with this heterogeneity.

Methods: We compared signaling alterations in single cells for β-CATENIN, SMAD2/3, phospho-STAT3, P65, FOXO1 and phospho-ERK1/2 among a collection of Human Bronchial Epithelial Cells (HBECs) that have been oncogenically transformed with combinations of TP53, K-RAS, and MYC, commonly found alterations in non-small cell lung cancer (NSCLC). We studied ~3000 cells/signaling marker/HBEC oncogenotype variant using immunofluorescence assays and single-cell image analysis (&gt;1M data points). For downstream target identification and validation we utilized gene expression, Western blot and siRNA mediated knockdown assays. We utilized inhibitors to STAT3 and BCL6 in MTT drug sensitivity and colony formation assay in a panel of NSCLC lines. We used xenografted subcutaneous tumors for the in vivo validation of our results.

Results: When all three oncogenic changes were present and the HBECs were tumorigenic, we observed STAT3 upregulation and SMAD2/3 downregulation. Interestingly, these STAT3 and SMAD2/3 signaling changes were found to be mutually exclusive in single cells within the transformed HBEC strain. We targeted the STAT3 upregulated subpopulation with the STAT3 inhibitor Stattic. But, Stattic treatment failed to eliminate the SMAD2/3 downregulated subpopulation. To target the SMAD2/3 down-regulated subpopulation, we identified BCL6, a downstream gene of SMAD2/3, as a novel target in transformed HBECs. Next, to test the generality of BCL6 as a target, we studied 5 NSCLC cell lines with various level of BCL6 expression: H1693, H1819, H1993, HCC827 and H2009. Our data suggests that BCL6 can also be a therapeutic target in a subset of NSCLC lines. Then we tested the response of these NSCLC lines to a combination of BBI608 (potent STAT3 inhibitor) and FX1 (BCL6 inhibitor). The combination treatment eliminated more cancer cells than the single treatments alone. Finally, we confirmed the benefit of the combination therapy in H1993 xenografted tumors.

Conclusions: We conclude that BCL6 is a new therapeutic target in NSCLC and combination therapy that targets multiple vulnerabilities (Phospho-STAT3 and BCL6) downstream of common oncogenes and tumor suppressors (TP53, K-RAS, and MYC) may provide a potent way to defeat intra-tumor heterogeneity.

Citation Format: Dhruba Deb, Satwik Rajaram, Jill E. Larsen, Patrick P. Dospoy, Rossella Marullo, Longshan Li, Kimberley Avila, Leandro Cerchietti, John D. Minna, Lani F. Wu, Steven J. Altschuler. A novel combination therapy targeting BCL6 and phospho-STAT3 defeats intratumor heterogeneity in a subset of non-small cell lung cancers [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 3950. doi:10.1158/1538-7445.AM2017-3950