Abstract 5154: Modeling cancer driver-like events in barrier bypass-clonal expansion in vitro assays
Authors: Korenjak Michael , Huskova Hana , Ardin Maude , Zhivagui Maria , Guyton Kathryn , Barupal Dinesh K. , Straif Kurt , Herceg Zdenko , Olivier Magali , Hollstein Monica , Zavadil Jiri 
Published: July 2016

BACKGROUND Cancer genomes harbor mutational spectra that document exposures to external factors and endogenous events underlying tumor development. Information on candidate cancer driver alterations is accessible from public compendia of somatic mutations, yet much of this knowledge remains descriptive and of limited mechanistic insight. Simple, robust and rapid systems are thus needed for well-controlled experimental investigations of functional impact of carcinogenic exposures on the genome and on cancer cell growth.

METHODS We use barrier bypass-clonal expansion (BBCE) assays based on primary human and murine cell cultures, in which mutations are introduced by mutagenic carcinogens and examined by deep sequencing, after the exposed cells have bypassed a selective pressure barrier and have clonally immortalized. A customized deep sequencing data analysis pipeline is used to decipher both the mutational signatures and the putative functional driver events selected and enriched for during the clonal outgrowth phase.

RESULTS Using the BBCE assays, we tested the global mutagenic effects of a number of known human carcinogens. We obtained 25 independently arising clones, altogether harboring 15,200 acquired mutations, with varying numbers per clone, of which ∼7,600 were non-synonymous. These affected 250 genes currently listed in the COSMIC Cancer Gene Census. Eighty-four genes were recurrently mutated across the BBCE clone panel, including well-established oncogenes (HRAS, ABL1, EGFR, MYC, PIK3CG) and tumor suppressors (APC, ATM, BRCA2, PTCH1, TP53). A number of epigenetic and chromatin regulators also acquired recurrent mutations, among them ASH1L, BAZ1A, BAZ1B, EP400, HDAC6, and members of histone lysine demethylase and methyltransferase families. Collectively, the recurrent alterations affected pathways regulating DNA damage response, DNA repair, cell cycle, cell death, transcription and chromatin structure, and developmental pathways of TGF-beta, Notch, WNT and ERBB signaling. Thus, as in human cancers, mutations driving critical steps of cellular stress bypass and clonal immortalization arise and become selected for when these processes are modeled in vitro.

CONCLUSIONS The BBCE assays constitute a unique resource amenable to follow-up functional studies of particular mutations in cancer genes. Data will be presented describing systematic genome editing and pharmacological manipulation of select mutated genes, followed by assessment of resulting phenotypic and molecular traits. In summary, our BBCE approach may yield new mechanistic insights into driver-like events underlying cancer development.

ACKNOWLEDGMENTS Funding from International Agency for Research on Cancer; ITMO CANCER-INSERM Plan Cancer 2015 grant to J.Z.

Citation Format: Michael Korenjak, Hana Huskova, Maude Ardin, Maria Zhivagui, Kathryn Guyton, Dinesh K. Barupal, Kurt Straif, Zdenko Herceg, Magali Olivier, Monica Hollstein, Jiri Zavadil. Modeling cancer driver-like events in barrier bypass-clonal expansion in vitro assays. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5154.