TET2 Loss Accelerates Leukemogenesis By Disrupting Mismatch Repair Proteins

TET2 mutations (TET2^MT) occur around 40% of myeloid neoplasms (MN) and often constitute founder hits, as concluded from patient's clonal architecture and subclinical TET2^MT clones in healthy individuals at risk for MN. The mechanisms of TET2^MT in leukemogenesis involve at least two potential mechanisms: i) TET2 mutations could lead to stem cell expansion and altered differentiation and/or ii) they may convey a mutator phenotype with progression due to a higher rate of subsequent genetic hits. Both mechanisms are compatible with the weak driver function of TET2^MT. Here, we studied whether TET2^MT predispose to additional oncogenic mutations through faulty DNA repair,i.e., whether they produce a clonally acquired mutator phenotype. TET2^MT lead to the inhibition of passive demethylation. Alternatively, they impair demethylation via excision repair of 5fC/5caC. Moreover, global 5mC accumulation may increase background C>T mutation rates via 5mC deamination linking TET2 to base excision repair machinery.

We investigated this hypothesis because of the finding of increased mutability found in Tet2^KO and Tet2^kdin the mouse as determined by the numbers of coexisting subclonal hits in Tet2^MTdisease (Pan F et.al., Nature Comm, 2017). Using WES analyses (n=435), we identified 95 cases with TET2^MT stringently selected for high VAF and most damaging effects as confirmed by 2D-UPLC-MS/MS assay showing low levels of the TET2-dependent DNA oxidation products. To test whether these TET2^MT indeed predisposed to additional mutations, we enumerated somatic SNV vs. TET2^WT cases. Sequencing of MSH2, MSH3, MSH6, MLH1, CHEK2, and BRCA2 excluded the potential effects of alteration in these genes on our results. Similarly, the MMR gene expression were not significantly different among 20 TET2^MT MDS, 71 TET2^WT MDS pts, and 17 controls, indicating MMR downregulation was not the culprit.

Consistent with our theory, TET2^MT pts had a 1.5-fold increase in median WES SNV (p<.0001), particularly at 5hmC sites. Pts with a TET2^MT VAF>50% had a 2.1-fold increase in median WES SNV (p=.03), consistent with a gene-dose effect. HeLa TET2^kd cells also had a 24-fold increase in spontaneous mutations, reversed with TET2 cDNA knock-in. In mice, 1.4-fold increased mutagenicity at Tet2-dependent active demethylation sites was found, in a manner also suggestive of a defective MMR. TET2^MT cases (n=5) were shown to be microsatellite-stable at 5 TET2-independent poly-dA microsatellite loci, suggesting that hypermutagenicity was not driven by global MMR dysfunction and may occur only at CpG-containing microsatellites. MOLM-13 TET2^kd increased PARP inhibitor sensitivity by almost 3-fold, suggesting that TET2^kd renders cells vulnerable to DNA damage. In murine cells, by overexpressing Flag/V5-tagged TET2 in MEL cells and subjecting them to protein affinity purification, we identified MSH6 as a novel TET2 binding partner. Using HPRT mutability assay, we measured mutational frequency in the HPRT1 gene in control, TET2^kd, MSH6^kd,and TET2^kd/MSH6^kd HeLa cells. Moreover, HPRT1 mutational frequency in TET2^kd HeLa cells increased 24-fold compared to parental controls while MSH6^kd HeLa cells had a further 10-fold increase in HPRT1, likely reflecting the presence of TET2-independent mechanisms for MSH6-mediated MMR. Intriguingly, the dual TET2^kd/MSH6^kd HeLa cells had equivalent MSI and mutational frequencies to the MSH6^kd HeLa cells, supporting MSH6-dependence for the mutator phenotype induced by TET2 loss. TET2:MSH6 interactions were validated in co-immunofluorescence experiments in MEL and MOLM-13 cells, and TET2^kd altered MSH6 nuclear co-localization. TET2 immunoprecipitation from human CD34⁺ cells demonstrated abundant MSH2 binding to TET2, although we were unable to co-precipitate MSH6 in the same experiment.

In sum, we uncovered novel connections among TET2, MMR proteins, epigenetic modifications, and genomic instability. Given that MSH2/6 is known to preferentially bind 5hmC, TET2 may target MSH2/6 to TET2-dependent DNA loci. Genomic instability due to TET2 dysfunction may allow therapeutic targeting of DNA repair proteins in the subset of pts with TET2^MT-driven MN.