Abstract 4525: Modulating chemoresistance: Uncovering the role of mutant SMAD4R361H in colorectal cancer using PDO and PDX models
Authors: Pfohl Ulrike , Silvestri Alessandra , Schumacher Dirk , Boehnke Karsten , Haybaeck Johannes , Bashir Sanam , Kühn Ralf , Keil Marlen , Schäfer Reinhold , Walther Wolfgang , Regenbrecht Christian 
Published: August 2019

Abstract
Mutations in the transforming growth factor-β (TGF-β) pathway occur frequently in colorectal cancers (CRC). TGF-β is integral for key cellular processes, including proliferation, migration, differentiation and apoptosis. TGF-β/SMAD4 controls the activation of the pathway. A previously described R361H mutation in SMAD4 has been correlated with decreased overall survival and is suspected to modulate chemoresistance. The aim of the present study is to generate CRISPR-engineered SMAD4R361H CRC organoids and to utilize them as in vitro and in vivo models to investigate the effect of SMAD4R361H mutation on drug response. We have established independent organoid (PDO) and matching patient-derived xenograft (PDX) models from 5 different regions of a chemo-naïve CRC. By targeted amplicon sequencing using a cancer hot spot gene panel, we identified the SMAD4R361H mutation in two of the 5 subpopulations, whereas three of the PDOs were SMAD4wt. Semi-automated compound screening revealed a strong resistance phenotype to afatinib, sapitinib and gefitinib as compared to the wild-type models in vitro. Similar drug response was observed in in vivo testing. To investigate the mechanism of drug resistance, we have engineered isogenic organoids (SMAD4R361H/CRISPR) from SMAD4wt organoids using CRISPR-Cas9 genome-editing. In brief, SMAD4wt organoids were transfected with an all-in-one spCas9-sgRNA-vector and a ssODN as repair template bearing the R361H point mutation. To increase homologous-directed repair, we co-transfected the organoids with a plasmid vector containing i53-bpA, an inhibitor of 53BP1. Single clones were then isolated and genotyped by locus specific PCR to confirm the particular R361H point mutation. We were able to achieve up to 50 % knock-in efficiency in these organoids. Utilizing CRISPR-Cas9, engineered SMAD4R361H/CRISPR organoids were established. These PDOs were subjected with both, native SMAD4wt and SMAD4R361H PDOs, to an in vitro drug screening, allowing for a direct comparison of their drug sensitivity. Based on these results, confirmatory in vivo experiments were designed to investigate how SMAD4 may be involved in modulating resistance in CRC organoids. These models have a prominent role in discovering the connection between genotype and phenotype in complex PDO and PDX models. While this approach is not limited to studying the role of the R361H mutation, it has a broad range of applications in understanding cancer biology, intra-tumor heterogeneity, drug response and may eventually lead to new clinical insights, including design of new therapeutic strategies.
Citation Format: Ulrike Pfohl, Alessandra Silvestri, Dirk Schumacher, Karsten Boehnke, Johannes Haybaeck, Sanam Bashir, Ralf Kühn, Marlen Keil, Reinhold Schäfer, Wolfgang Walther, Christian Regenbrecht. Modulating chemoresistance: Uncovering the role of mutant SMAD4R361H in colorectal cancer using PDO and PDX models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4525.