Abstract 978: In vivo shRNA screening identifies synthetic cytotoxicity in CREBBP/EP300 mutant head and neck cancer
Authors: Kumar Manish , Bridges Kathleen , Molkentine David , Yang Liangpeng , Sheth Aakash , Meyn Raymond , Frederick Mitchell , Myers Jeffrey , Pickering Curtis , Skinner Heath D. 
Published: August 2018

The p300-CBP1 coactivator family members (CREB-binding protein (CREBBP) and E1A binding protein p300 (EP300)) share significant homology and act as transcriptional co-activators for a large number of target genes. These genes are collectively mutated in approximately 14% of all head and neck squamous cell cancers carcinomas (HNSCC) and are targeted by multiple agents in clinical development. Because of this the p300-CBP1 coactivator family presents a promising target in HNSCC. Recently, our group performed in-vivo screening utilizing a barcoded shRNA library of targets of known anti-neoplastic agents. A total of 5 HNSCC cell lines of varying HPV status (HPV (-): Cal27, HN31, UMSCC22A; HPV (+): UMSCC47, UPCISCC152) were transduced with the shRNA library and allowed to form subcutaneous, flank tumors, which were then treated with IR (2 Gy/day) to achieve an approximate 20% reduction in tumor volume. The tumors were then harvested and barcodes were sequenced. Interestingly, this screen revealed that CREBBP or EP300 depletion led to dramatic sensitization to IR, but only in tumors harboring a mutation in either CREBBP or EP300, independent of HPV status. To investigate this phenomenon further, we utilized chemical inhibition of CREBBP as well as shRNA to CREBBP in multiple HNSCC cell lines of varying HPV and CREBBP mutational statuses. In those cell lines harboring a CREBBP mutation, we found significant in vitro sensitization to IR on clonogenic assay following CREBBP inhibition, however this phenomenon was not observed in CREBBP wild type cells. All cell lines tested were generally resistant to IR-induced apoptosis, as measured by TUNEL assay and PARP cleavage. However, IR combined with inhibition of CREBBP led to dramatically increased IR-associated apoptosis as well as increased γ-H2AX and decreased BRCA1 foci following radiation. We next examined CREBBP inhibition using two shRNA clones specific for CREBBP in an in vivo UMSCC47 xenograft model. In this model, inhibition of CREBBP alone or IR alone (2 Gy x 8 d) led to minimal tumor volume reduction (tumor growth delay (TGD) 6 and 2 days respectively). However, the combination of each shCREBBP clone and IR led to a TGD of 24 and 23 days (p&lt;0.0001 for both). Analysis of these tumors following radiation identified an increase in PARP cleavage and decrease in BRCA1 in the combined treatment group. Finally, we examined clinical outcomes in a cohort of 75 HPV (-) HNSCC patients treated with surgery and radiation. Mutations in CREBBP or EP300 were significantly associated with poorer survival (median survival 18 mos vs. 62.8 mos in wild type; p=0.015). Our results suggest the synthetic cytotoxicity of combining CREBBP inhibition and IR in tumors harboring mutations in the p300-CBP1 coactivator family. As mutations in this family are relatively common in HNSCC, and are associated with worse survival, this strategy could dramatically improve patient outcome.

Citation Format: Manish Kumar, Kathleen Bridges, David Molkentine, Liangpeng Yang, Aakash Sheth, Raymond Meyn, Mitchell Frederick, Jeffrey Myers, Curtis Pickering, Heath D. Skinner. In vivo shRNA screening identifies synthetic cytotoxicity in CREBBP/EP300 mutant 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 978.