Abstract 2006: Sequencing of mismatch repair deficient tumors identifies a synthetic lethal interaction with other DNA repair pathways.
Authors: Yesilyurt Betül T. , Zhao Hui , Sagaert Xavier , Coenegrachts Lieve , Kalender Zeynep , Garcia Diego , De Keersmaker Kim , Matthijs Gert , Thienpont Bernard , Aerts Stein , Cools Jan , Amant Frederic , Lambrechts Diether 
Published: August 2014

Abstract
DNA replication errors that persist as mismatch mutations make up the molecular fingerprint of mismatch repair (MMR) deficient tumors and convey them with different prognostic and therapeutic outcome upon chemotherapy. Using whole-genome and -exome sequencing, we here characterize the somatic mutation patterns underlying MMR-deficient tumors. In particular, we show that mutation frequencies are &gt;30-fold increased relative to MMR-proficient tumors: insertion-deletion (indel) mutations are largely confined to homopolymers, whereas substitutions mainly consist of single basepair transitions located near indels. Since exonic indels result in loss-of-function frameshift mutations, indels were less frequent in the exome due to negative selection. However, a small fraction of exonic indels was positively selected and occurred as hotspot mutations, preferentially affecting the ‘DNA double-strand break (DSB) repair by homologous recombination (HR)’ pathway (on average, each MMR-deficient tumor contained 2.6 indels in this pathway). We quantified DSB repair by immunofluorescent staining of RAD51 foci in 8 MMR-deficient and 4 MMR-proficient tumor cultures in the presence of the PARP inhibitor olaparib. We did not observe a difference in RAD51 foci formation between MMR-deficient and MMR-proficient tumor cultures in the absence of olaparib (10±2% of cells versus 13±2% showed RAD51 foci formation, P=0.74). Upon 10μM olaparib exposure, MMR-deficient tumor cultures accumulated less RAD51-positive foci (19±3% in MMR-deficient versus 37±4% in MMR-proficient cells; P=0.02), thereby confirming that these tumors were at least partly deficient in DNA DSB repair by HR. We also investigated the degree of unrepaired DNA damage by H2AX immunofluorescence and olaparib drastically increased the number of H2AX foci in all cells regardless of their MMR status. Furthermore, MMR-deficient cultures exhibited a dose-dependent reduction in proliferation under olaparib, whereas MMR-proficient cells did not respond (P&lt;0.001 by repeated measurement). On average, 1, 3 and 10μM of olaparib decreased proliferation of MMR-deficient cells by respectively 15%, 20% and 42% (P=0.02, P&lt;0.001 and P&lt;0.001, respectively versus untreated cells), whereas no effect was seen in MMR-proficient tumors (P=NS for all concentrations of olaparib). Finally, high-throughput profiling of hotspot mutations also more accurately detected microsatellite instability (MSI) in several cancer types compared to standard diagnostic methods. Sequencing of MMR-deficient tumors thus identified an unexpected synthetically lethal interaction between the major DNA repair pathways, offering novel treatment opportunities for MMR-deficient tumors.
Citation Format: Betül T. Yesilyurt, Hui Zhao, Xavier Sagaert, Lieve Coenegrachts, Zeynep Kalender, Diego Garcia, Kim De Keersmaker, Gert Matthijs, Bernard Thienpont, Stein Aerts, Jan Cools, Frederic Amant, Diether Lambrechts. Sequencing of mismatch repair deficient tumors identifies a synthetic lethal interaction with other DNA repair pathways. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2006. doi:10.1158/1538-7445.AM2013-2006