DNMT3A R882H Can Cooperate with FLT3-ITD to Cause AML in Mice

Somatic mutations in the DNA methyltransferase, DNMT3A, have been identified in approximately 22% of de novo AML cases and in ~10% of patients with MDS. To understand how mutations in DNMT3A lead to hematopoietic abnormalities, we generated transgenic mice capable of overexpressing wild type human DNMT3A, or the most common AML mutation (R882H). The system used allows for the inducible expression of DNMT3A upon the expression of a coactivator and addition of Doxycycline (Dox) in the feed. A single founder line with the WT DNMT3A allele (overexpressed ~4 fold over endogenous WT murine Dnmt3a), and two founder lines with the R882H DNMT3A allele (one line overexpressing at ~16 fold level over endogenous mouse Dnmt3a and the other at ~4.5 fold overexpression) were established in a pure C57Bl6/J background. All three lines have been shown to overexpress the transgene in bone marrow when crossed to coactivator transgenic mice carrying the Rosa26-rtTA allele, and eating Dox chow. There is significant DNA hypomethylation in the bone marrow cells of the high expressing R882H line after the mice have been on Dox chow for 3 months.

To determine the effect of R882H DNMT3A on hematopoiesis, we performed competitive transplantation studies where we mixed bone marrow derived from R882H DNMT3A Tg x rtTA doubly transgenic mice (Ly5.2+) with bone marrow from wild type mice (Ly5.1/5.2+) at a 1:1 ratio, and transplanted it into lethally irradiated recipients. After a 1 month engraftment period, mice were placed on Dox chow and monitored for 1 year. All control genotypes were included. After 6 months of dox chow administration, neither R882H Tg x rtTA line displayed a competitive advantage in the peripheral blood. However, there was a trend towards myeloid skewing in R882H DNMT3A expressing cells. At one year post-transplant, mice were sacrificed. In the spleen and peripheral blood, there were trends towards a myeloid differentiation bias in low-expressing R882H Tg x rtTA mice, but a significant myeloid bias in the bone marrow of high expressing R882H Tg x rtTA mice. This demonstrated that R882H DNMT3A expression leads to a myeloid differentiation bias over time, providing a possible explanation for the observation that R882H mutations are enriched for myeloid leukemias.

A tumor watch of mice with all genotype combinations demonstrated that mice expressing R882H DNMT3A do not develop AML, even after 1.5 years on Dox chow. To address whether a second mutation is required to cooperate with R882H DNMT3A to cause AML, we performed a study using donor mice derived from the above competitive transplantation experiments (aged for one year) that were chimeric for R882H Tg x rtTA marrow and wild type marrow at a ~50:50 ratio. This chimeric marrow sample was transduced with a retroviral vector that expresses a human FLT3-ITD allele, one of the most common cooperating mutations with R882H DNMT3A. Ten recipient mice have been analyzed for a minimum of 4 months. Between 6 and 9 weeks after transplantation, two mice developed AML derived entirely from Ly5.2 expressing cells, demonstrating that the leukemic clone arose and rapidly expanded in R882H expressing bone marrow cells. Peripheral blood analysis in these mice and five others (7/10 total) demonstrated that FLT3-ITD expressing cells preferentially expand in the R882H expressing cells, while 1/10 mice showed no preference. We were able to analyze a single cohort (n=4) of mice at 3 months post engraftment, and found >25% enrichment of FLT3-ITD positive cells in R882H expressing cells. Two of ten mice did not display peripheral blood expansion of cells expressing FLT3-ITD. An additional two mice in this study developed Ly5.2/R882H-derived hematopoietic diseases with a longer latency, which are currently being assessed for leukemic properties by transplantation. Irrespective of the presence of FLT3-ITD expression, R882H expressing cells showed either stable engraftment or expansion over time. Six of ten mice displayed an expansion of R882H expressing cells (no FLT3-ITD allele) demonstrating a clonal advantage, 3/10 mice displayed stable engraftment, and only 1/10 showed a loss. Taken together, our results show that expression of the R882H allele can confer a competitive advantage independently after serial transplantation, but when combined with the common cooperating FLT3-ITD mutation, the FLT3-ITD expressing cells preferentially expand in R882H expressing cells, and can synergize to cause AML.