Unraveling the heterogeneous mutational signature of spontaneously developing tumors in MLH1^-/-mice

Abstract

28 29 Abbreviations: CMMR-D -constitutional mismatch repair deficiency, cMS -coding 30 microsatellite, MB -Megabase, MMR -mismatch repair, MMR-D -mismatch repair 31 deficiency, TMB -tumor mutational burden, GIT -gastrointestinal tumor, SNV -single 32 nucleotide variant, WES -whole exome sequencing 33 34 Keywords: Tumor mutational burden, exclusive/ shared mutations, predicted tumor antigens Mutations in MLH1 -/tumors 3 35Abstract 36 MLH1 knock out mice represent a preclinical model that resembles features of the human 37 counterpart. As these mice develop mismatch repair deficient (MMR-D) neoplasias in a 38 sequential twin-peaked manner (first lymphomas, then gastrointestinal tumors) we aimed at 39 identification of the underlying molecular mechanisms. Using whole-exome sequencing, we 40 focused on (I) shared and (II) mutually exclusive mutations and described the processes of 41 ongoing mutational events in tumor-derived cultures. 42 A heterogeneous genetic landscape was found, with few mutations shared among different 43 neoplasias (ARID1A and IDH2). Mutations in tumor suppressor genes SMAD4 and POLE 44 were mutually exclusive in lymphomas, most likely contributing to a more aggressive in vivo 45 phenotype. Comparing the mutational profile of selected primary tumors and their 46 corresponding cell line upon in vitro culture revealed continuous increased numbers of 47 somatic gene mutations. The same was true for coding microsatellite mutations in selected 48 MMR-D target genes, showing a gradual increase during in vitro passage. With respect to this 49 latter type of mutations, partial overlap was detectable, yet recognizing shared vaccination 50 antigens. The two most promising candidates are AKT3, a RAC-gamma serine/threonine-51 protein kinase with relevance in maintenance of cellular homeostasis and the endonuclease 52 ERCC5 (Excision Repair 5), involved in DNA excision repair. 53 Novel results of a comparison between spontaneously developing lymphomas and 54 gastrointestinal tumors as models for MMR-D driven tumorigenesis are reported. In addition 55 to identification of ARID1A as a potentially causative mutation hotspot, our comprehensive 56 characterization of the mutational signature is a starting point for immune-based approaches 57 to therapy. 58 59 Mutations in MLH1 -/tumors 4 60 Author Summary 61 This study describes the mutational spectrum of MLH1 -/--associated tumors, spontaneously 62 developing in mice. While these tumors arise at the bottom of the same germline mutation, 63 the clinical presentations as well as resulting molecular alterations are heterogeneous, and 64 thus likely being directly linked. Highly aggressive lymphomas, developing early in life are 65 ultra-hypermutated and harbor mutations in tumor suppressor genes SMAD4 and POLE. 66 Gastrointestinal tumors develop later in life and show different mutations. By performing in-67 depth whole exome sequencing analysis, we here identified for the first time a common 68 mutational hotspot. ARID1A constitutes a potentially causative mutati...

In Vivo Vaccination with Cell Line-Derived Whole Tumor Lysates – Neoantigen Quality, Not Quantity Matters

“…Then, we moved to the therapeutic approach (Figure 1). The survival bene t of mice treated with a lysate from the Mlh1 -/-A7450 cells compared to the 328 lysate was shown before [21] and ( Figure 6A). Here, the median overall survival was 11 weeks.…”

“…The cell lines 328 and A7450 T1 M1 were processed likewise [20,21] for the visualization. With the complex Heatmap [25] R package, their patterns and correlations were revealed in oncoprint.…”

“…The selected genes of the oncoprint are known for the relevance for tumor initiation, progression, apoptosis, and suppressors functions ( Figure 3A). Mlh1 -/tumors harbor mutations in Pik3ca, Msh3, Braf, and/or Kras, and Erbb3 [21]. The A7450 T1 M1 cell line harbors nonsense and missense single nucleotide variants (SNVs) in the Wnt signaling pathway regulator Apc gene.…”

“…To test the immunogenicity of whole cancer vaccines on a more global level, Mlh1 -/mice received two independent tumor lysates, either harboring high (= 328, 167 mutations/Mb) or moderate (= A7450 T1 M1, 27 mutations/Mb) TMB [21] (Figure 1).…”

“…Hence, we addressed the question of whether cell-line derived tumor lysates might provide an alternative source of highly immunogenic tumor Ags. In a pilot study, we identi ed different outcomes upon vaccination with two individual cell line-derived lysates in the therapeutic setting [21]. We hypothesized that the mutational signature predicts response.…”