Abstract PR13: Polymerase-mediated ultramutagenesis: A new approach for modeling the high mutational load of human cancer
Authors: Li Hao-Dong , Lu Changzheng , Zhang He , Cuevas Ileana C. , Sahoo Subhransu S. , Fu Yang-Xin , Castrillon Diego H. 
Published: June 2020

Abstract
Cancer is characterized by increased mutation rate, and recent work has led to a deeper understanding of mutator phenotypes and underlying molecular mechanisms. Differences in the mutational landscape of individual cancers underlie key aspects of clinical behavior. For example, overall base substitution rate is the best predictor of immune therapy response. Most genetically engineered mouse models of cancer (GEMMs) reiterate a few driver events but fail to recapitulate the high mutational loads that are the ultimate cause of most human cancer, making GEMMs inadequate for many aspects of tumor behavior, such as immune responses. The highest base substitution rates in cancers (≥100/Mb) result from specific heterozygous single amino acid substitutions (usually P286R) in the proofreading domain of DNA polymerase epsilon (POLE), rendering POLE highly error prone. POLE mutations are most common in endometrial cancer but occur with lower incidence in a wide range of cancers. We hypothesized that GEMMs could be generated by recapitulating PoleP286R via conditional knockin. Previously, we showed that constitutive expression of PoleP286R throughout the body triggered malignancies of diverse lineages. Mice harboring LSL-PoleP286R and the endometrial BAC-Sprr2f-Cre developed endometrial cancers starting at 1 year. Endometrial cancers exhibited histologic features previously reported in human POLE tumors, and metastasized in all mice (p&lt;0.00001, n=36 vs n=28 controls). Of note, 100% penetrance was achieved through a single (monoallelic) amino acid change, with experimental animals efficiently generated in a single generation. Whole-genome and targeted cancer gene panel sequencing of primary tumors (n=12) and cell lines (n=6) revealed distinctive mutational signatures and the highest nucleotide substitution rates described in murine cancers, with some tumors exceeding 100 base substitutions/Mb. Since most DNA replication errors are repaired by mismatch repair (MMR), and some human tumors harbor POLE mutations and defective MMR (dMMR), we introduced dMMR via conditional Msh2 knockout. This further accelerated tumor progression and mortality (p&lt;0.00001, n=36 Pole alone vs. n=28 Pole+Msh2), revealing clear synergism. In both models, there were robust antitumor immune responses, as evidenced by increased T-cell infiltrates, accelerated tumor growth following T-cell depletion by anti-CD4 antibody, and clinical regression following anti-CTLA4/PDL1 checkpoint therapy. In summary, this is a robust approach to model the high mutational loads intrinsic to human cancer. Expression of PoleP286R in a specific lineage is sufficient to result in cancers with greater ease than previous strategies such as dMMR alone, and with mutational loads in the range of human cancers. The approach may be useful for diverse models and investigations.
This abstract is also being presented as Poster A34.
Citation Format: Hao-Dong Li, Changzheng Lu, He Zhang, Ileana C. Cuevas, Subhransu S. Sahoo, Yang-Xin Fu, Diego H. Castrillon. Polymerase-mediated ultramutagenesis: A new approach for modeling the high mutational load of human cancer [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr PR13.