Nearly a Third of Clonal Hematopoiesis-Associated DNMT3A Mutations Reduce Protein Stability and May be Associated with Poorer Prognosis

Mutations in three functional domains of DNA methyltransferase 3A (DNMT3A) have been found in individuals with clonal hematopoiesis of indeterminate potential (CHIP), hematological malignancies, and cohorts of patients with overgrowth syndrome (Tatton-Brown Rahman Syndrome, or TBRS). In CHIP, the majority of DNMT3A mutations (~85%) are not at the Arginine 882 residue that is most frequently seen in AML (around 58% of DNMT3A-mutant cases). While most studies to date have focused on DNMT3A mutations in the hotspot residue R882, frameshift or premature stop codon mutations resulting in haploinsufficiency of DNMT3A also have been shown to predispose toward myeloid malignancies. However, the functional impact of these non-R882 missense mutations, and the implications for prognosis, have not yet been examined.

To address this question, we generated a murine model with a single amino acid deletion of tryptophan 293 (corresponding to aa 297 in human) in the PWWP domain of DNMT3A (Dnmt3a^W293Del model). We found that, similar to Dnmt3a-null mice, Dnmt3a^{W293Del/W293Del} mice do not survive beyond postnatal day 24, suggesting that Dnmt3a^W293Del is a hypomorphic mutation. In addition, Dnmt3a^W293Del/+ mice recapitulated many of the features of human TBRS syndrome including obesity and neurological defects. Using methylation-deficient mouse embryonic stem cells, we re-introduced doxycycline-inducible mutant DNMT3A^W297Del and observed only a negligible increase in DNA methylation, while intact DNMT3A^WT protein showed a global 60% increase of DNA methylation, as measured by whole genome bisulfite sequencing.

To determine the molecular mechanisms through which DNMT3A^W293Del acted as DNMT3A-null mutation, we first examined both RNA and protein expression in the Dnmt3a^W293Del murine model. Quantitative PCR revealed that both Dnmt3a^WT and Dnmt3a^W293Del mRNA were expressed at normal levels in the Dnmt3a^W293Del/+ mice. Unexpectedly, however, we found that DNMT3A^W293Del mutant protein could not be detected. Using bicistronic vectors to measure protein stability in human embryonic kidney 293 cells (HEK293T), we demonstrated that loss of DNMT3A^W297Del protein is not due to a translational defect but to impaired protein stability. Additionally, we found application of a proteasome inhibitor could rescue expression of mutant DNMT3A^W297Del expression. Furthermore, using lymphoblastoid cell lines (LCLs) derived from patients, we found DNMT3A protein expression was lower in DNMT3A^W297Del/+ LCLs and that exposure to a proteasome inhibitor could increase protein levels, reinforcing the concept that deletion of aa 297 reduced DNMT3A protein stability.

To determine whether reduced protein stability was a common feature of DNMT3A mutations, we examined stability of 105 additional missense mutations across three functional domains frequently mutated in CHIP, hematological malignancies, and TBRS. Surprisingly, 45 out of 105 missense DNMT3A mutations (42.9%) examined had impaired protein stability, the majority of which were located in the PWWP and catalytic domains. Using variant allelic frequencies (VAFs) from a publicly available dataset of patients with non-hematological malignancies, we found that mutants with reduced protein stability were associated with significantly higher VAFs compared to mutants with unknown function. Notably, R882 mutants had significantly higher VAFs compared to the mutants with impaired protein stability. These data indicate that individuals with R882 mutations may have the highest likelihood of conversion to malignancies, with mutants with impaired stability having moderate likelihoods, and mutants in domains with unknown function may have lowest chance to convert to malignancies and best prognosis.

In this study, we show that mice with a mutation in the PWWP domain of Dnmt3a recapitulate the phenotypes of human overgrowth patients. We further discovered that a large portion of missense DNMT3A mutations have impaired protein stability. This study highlights the importance of understanding how DNMT3A protein expression is regulated and suggests proteasome inhibition may be a potential treatment for patients with particular mutations in DNMT3A, found in overgrowth syndrome, CHIP, and hematological malignancies.