Unraveling the heterogeneous mutational signature of spontaneously developing tumors in MLH1-/-mice
Authors: Gladbach Yvonne Saara , Wiegele Leonie , Hamed Mohamed , Merkenschlager Anna-Marie , Fuellen Georg , Junghanss Christian , Maletzki Claudia 
Published: August 2019

MLH1 knock out mice represent a preclinical model that resembles features of the human counterpart. As these mice develop mismatch repair deficient (MMR-D) neoplasias in a sequential twin-peaked manner (first lymphomas, then gastrointestinal tumors) we aimed at identification of the underlying molecular mechanisms. Using whole-exome sequencing, we focused on (I) shared and (II) mutually exclusive mutations and described the processes of ongoing mutational events in tumor-derived cultures.

A heterogeneous genetic landscape was found, with few mutations shared among different neoplasias (ARID1AandIDH2). Mutations in tumor suppressor genesSMAD4andPOLEwere mutually exclusive in lymphomas, most likely contributing to a more aggressivein vivophenotype. Comparing the mutational profile of selected primary tumors and their corresponding cell line uponin vitroculture revealed continuous increased numbers of somatic gene mutations. The same was true for coding microsatellite mutations in selected MMR-D target genes, showing a gradual increase duringin vitropassage. With respect to this latter type of mutations, partial overlap was detectable, yet recognizing shared vaccination antigens. The two most promising candidates areAKT3, a RAC-gamma serine/threonine-protein kinase with relevance in maintenance of cellular homeostasis and the endonucleaseERCC5(Excision Repair 5), involved in DNA excision repair.

Novel results of a comparison between spontaneously developing lymphomas and gastrointestinal tumors as models for MMR-D driven tumorigenesis are reported. In addition to identification ofARID1Aas a potentially causative mutation hotspot, our comprehensive characterization of the mutational signature is a starting point for immune-based approaches to therapy.

Author Summary

This study describes the mutational spectrum of MLH1-/--associated tumors, spontaneously developing in mice. While these tumors arise at the bottom of the same germline mutation, the clinical presentations as well as resulting molecular alterations are heterogeneous, and thus likely being directly linked. Highly aggressive lymphomas, developing early in life are ultra-hypermutated and harbor mutations in tumor suppressor genesSMAD4andPOLE.Gastrointestinal tumors develop later in life and show different mutations. By performing in-depth whole exome sequencing analysis, we here identified for the first time a common mutational hotspot.ARID1Aconstitutes a potentially causative mutation, shared among different MLH1-/--associated tumors and thus irrespective of the origin. Additional interesting and identified candidate genes include AKT3, a RAC-gamma serine/threonine-protein kinase and the endonuclease ERCC5. Both genes are bona fide tumor suppressors with significant relevance in DNA excision repair and maintenance of cellular homeostasis. This finding is of particular relevance for subsequent therapeutic and - even more important - prophylactic vaccination approaches aiming at entity-overlapping treatment of MLH1-/--related tumors.