POLE Mutation Spectra Are Shaped by the Mutant Allele Identity, Its Abundance, and Mismatch Repair Status

Abstract

Human tumors with exonuclease domain mutations in the gene encoding DNA polymerase ε (POLE) have incredibly high mutation burdens. These errors arise in four unique mutation signatures occurring in different relative amounts, the etiologies of which remain poorly understood. We used CRISPR-Cas9 to engineer human cell lines expressing POLE tumor variants, with and without mismatch repair (MMR). Whole-exome sequencing of these cells after defined numbers of population doublings permitted analysis of nascent mutation accumulation. Unlike an exonuclease active site mutant that we previously characterized, POLE cancer mutants readily drive signature mutagenesis in the presence of functional MMR. Comparison of cell line and human patient data suggests that the relative abundance of mutation signatures partitions POLE tumors into distinct subgroups dependent on the nature of the POLE allele, its expression level, and MMR status. These results suggest that different POLE mutants have previously unappreciated differences in replication fidelity and mutagenesis.

Keywords: DNA polymerase; DNA repair; DNA replication; genomic instability; mismatch repair; mutagenesis.

FIGURE 1.. Whole-exome sequencing nascent mutations from engineered POLE mutant human cell lines shows POLE signature mutations.

(A) Schematic for sequencing mutations that occurred during culturing of human cells expressing mutant POLE alleles. (B) Cell populations were subjected to an initial round of whole-exome sequencing (WES) at an initial arbitrary timepoint (PDL = 0) and again after a defined number of population doublings (PDL_x, where x is indicated for each cell line). Nascent mutations in the population were defined as occurring in PDL_x relative to PDL₀. (C) At an intermediate passage (PDL₅₄, red star), the population was again subjected to WES. Single cells were simultaneously isolated from the PDL₅₄ culture by limiting dilution. These subclones were expanded to a confluent well of a 6 well plate followed by WES. Nascent subclone mutations were defined as occurring in each independent subclone relative to bulk PDL₅₄ sequencing. MuTect2 and VarScan2 were used to determine only those mutations arising during passaging or subclone expansion. Nascent mutations for each indicated sample are shown in the 96 possible trinucleotide contexts. The proportion of each base pair substitution (y-axis) in a specific trinucleotide context to the total SNVs (indicated) in a given sample is plotted. Each exome (60 x 10⁶ bp) was sequenced to a mean depth of ~116x (population studies) and ~154x (subclone studies).

Figure 2.. NMF-derived signature reconstruction identifies POLE and POLE/MMR mutation signatures from engineered human POLE cell lines.

Non-negative matrix factorization was used to extract mutation signatures from exomic SNV profiles from POLE mutant tumors (n = 64) and cell lines (n = 5) and POLE wildtype cell lines (n = 2). SNV/PDL (total nascent SNVs/# of PDLs) is shown for MMR-deficient (A) and –proficient (B) cell lines. Each NMF-derived percent signature contribution was multiplied against the SNV/PDL in the sample to estimate signature-specific mutation accumulation. Mutation spectra and de novo NMF ID (boxed) of signatures identified in the sample are shown.

Figure 3.. Sequencing POLE variant mRNA shows variable allelic expression.

(A) HPRT mutant frequencies were measured for parental MMR-deficient HCT116 cells (POLE^wt/wt), cells that had undergone minimal passaging after Cre recombinase-mediated activation of the POLE-S459F (POLE^wt/S459F-Post Cre) and cells that had undergone extensive (> 1 year) passaging after Cre recombinase-mediated activation of the POLE-S459F (POLE^wt/S459F-Passaged). Individual mutant frequency values are plotted, along with mean and 95% CIs for each group; p-values are shown for each comparison (two sample, unpaired t test; ****, p<0.0001) (B) Total RNA was extracted from POLE variant cell lines and used to prepare cDNA. Loci containing S459 and P286 were amplified from cDNA using PCR and sequenced to high depth using Next-Generation Sequencing. Wild type (gray) and mutant (black) POLE allele frequency is shown for clones that had been passaged for at least 54 passages. * indicates a S459F clone that was re-sequenced after being passaged extensively for over one year. (C) Fresh clones, defined as within 22 cell doublings after Cre excision and single cell selection, were prepared and sequenced as in B. MMR status, POLE genotype and total reads are shown. P286R-l and P286R-r indicate independent clones derived from two distinct CRISPR/Cas9 cleavage sites to the left or right of the P286R codon, respectively. Hatched boxes represent fraction of reads incorrectly spliced into intron 9. (D) Example reads from properly spliced and mis-spliced POLE transcripts spanning exons 9 and 10 are shown.

Figure 4.. Relative abundance of POLE and POLE/MMR mutation signatures assigns human POLE tumors to three distinct groups.

(A) Human POLE tumors with TMBs ≥ 10 Mut/Mb from TCGA were assigned to groups based on the relative abundance of POLE (blue) and POLE/MMR (green) mutation signatures, or lack thereof (red), then each group was sorted from low to high TMB. Engineered (this study) and existing (HCC2998) mutant POLE cell lines are also shown (right). MSI status and MS-indel number are shown below individual patients when known. (B) Mean TMB and 95% CI are plotted for each group and p-values shown for each comparison (two sample, unpaired t test). (C) Mean microsatellite indel number and 95% CI is plotted for each group and p-values are shown for each comparison (two sample, unpaired t test).

Figure 5.. POLE mutant allele-dependent distribution of POLE mutation signatures, independent of MMR status.

(A) NMF-derived POLE signatures (this study) were grouped according to their similarity to existing SBS signatures 10a (NMF6/11, light blue), 10b (NMF10/1/2, dark blue) and 28 (NMF3, gray blue). Mean signature contribution as a proportion of POLE signatures only, 95% CI and p-values are shown. Differing distributions of POLE signatures in human POLE tumor subgroups (as in Figure 4) are shown. Statistical comparisons made on SBS 10a contribution (two sample, unpaired t test). (B) Mean POLE signature contributions are shown for MMR+/− engineered POLE mutant cell lines and variant-specific POLE mutant tumors as in A. Statistical comparisons made on SBS 10a contribution (ANOVA with post-hoc test). (C) Signature contributions to individual trinucleotide base pair substitutions are shown for MMR+/− engineered POLE mutant cell lines and select POLE mutant tumors.