Abstract 2812: Status of TGFbeta signaling determines PARP inhibitor sensitivity in head and neck cancer
Authors: Liu Qi , Ma Lin , Palomero Luis , Pujana Miquel Àngel , Jones Trevor , Ha Patrick , Murnane John , Barcellos-Hoff Mary Helen 
Published: August 2018

Abstract
Although human papilloma virus (HPV) status is the most informative prognostic marker in head and neck squamous cell carcinoma (HNSCC), the mechanisms that provide the survival advantage are poorly understood. Here we demonstrate that defects in transforming growth factor β (TGFβ) signaling confer a profound vulnerability in DNA repair for HPV+ HNSCC. We interrogated HNSCC profiled by The Cancer Genome Atlas (TCGA) with a 77 gene chronic TGFβ signature, defined as those genes that were significantly and reciprocally altered in MCF10A cultured for 7 days with TGFβ or a small molecule inhibitor of TGFβ type 1 receptor kinase (TBRIi). The 50 TGFβ-upregulated genes and 27 TGFβ-downregulated genes were negatively correlated (P&lt;0.0001; R2=-0.68) in single-sample gene set enrichment analysis confirmed that the signature is informative in HNSCC. Unsupervised clustering of 294 HNSCC TCGA patient specimens using the TGFβ signature showed that HPV+ cancers were clustered by loss of TGFβ regulation. Patients whose cancers exhibit this profile had significantly better overall survival (P=0.002), independent of HPV status. We previously found that TGFβ signaling suppresses miR-182, which is a reported inhibitor of homologous recombination (HR). HPV+ cell lines increased expression of miR-182 compared to HPV- cell lines. Thus we evaluated radiation induced RAD51 foci formation, a biomarker of HR, in viable explants of patient-derived xenografts (PDX) treated with or without TBRIi. HPV+ PDX showed significantly fewer RAD51 foci than HPV- samples (P&lt;0.005). HPV- PDX specimens treated with TBRIi also reduced RAD51 foci formation. We then established an HPV- cell line containing reporters to measure the frequency of DNA double strand break (DSB) repair with HR or alternative end-joining (Alt-EJ) following DSB induction with I-SceI endonuclease. TBRIi decreased HR events. In contrast, Alt-EJ was significantly increased (P&lt;0.05). Poly (ADP-ribose) polymerase 1 (PARP1) is required for DSB repair with Alt-EJ. TCGA data shows dramatically increased expression of PARP1 in HPV+ HNSCC (P&lt;0.0001). Hence, we speculated that loss of TGFβ signaling increase reliance on PARP1 mediated Alt-EJ to cope with DSB. Consistent with this, HPV+ cell lines were more sensitive to olaparib; while olaparib in combination with TGFβ inhibitor significantly increased cell death in HPV- cell lines by flow cytometry analysis of annexin V positive cells, but not in HPV+ cell lines. Notably, antagonizing miR-182 in HPV+ cells significantly reduced olaparib sensitivity (P&lt;0.05). Together these data indicate HPV suppression of TGFβ signaling in turn shifts DNA repair from error-free HR to hazardous Alt-EJ through upregulated miR-182. Importantly, TGFβ inhibitors can recapitulate this DNA repair deficiency in hard to treat HPV- HNSCC.
Citation Format: Qi Liu, Lin Ma, Luis Palomero, Miquel Àngel Pujana, Trevor Jones, Patrick Ha, John Murnane, Mary Helen Barcellos-Hoff. Status of TGFbeta signaling determines PARP inhibitor sensitivity in head and neck cancer [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 2812.