Tet2 Loss Accelerates The Myeloproliferative Neoplasm (MPN) Phenotype Of Jak2V617F Knockin Mice But Is Insufficient To Cause Leukemic Transformation

TET2 mutations are early somatic events in the pathogenesis of acute myeloid leukemia (AML), myelodysplastic syndrome (MDS) and myeloproliferative neoplasms (MPN) and are one of the most common genetic lesions found in these diseases. In MPN, TET2 mutations are enriched within more advanced disease phenotypes such as myelofibrosis and leukemic transformation and often co-occur with the JAK2V617F mutation, which is present in the majority of MPN patients.

We have developed and characterized a Jak2V617F conditional knockin mouse (Jak2^VF/+), the phenotype of which closely recapitulates the features of human MPN. To determine the impact of Tet2 loss on Jak2V617F-mediated MPN, we crossed Tet2 conditional knockout mice with Jak2^VF/+ knockin and Vav-Cre transgenic mice and backcrossed the compound mutant animals. We then characterized the effects of heterozygous and homozygous loss of Tet2 on the phenotype of Jak2^VF/+ mice. We assessed peripheral blood counts, histopathology, hematopoietic differentiation using flow cytometry, colony formation and re-plating capacity. We also evaluated the effects of Tet2 loss on the transcriptome of the HSC compartment using gene expression microarrays and on HSC function using competitive bone marrow transplantation assays.

Similar to Jak2^VF/+/VavCre⁺ mice, Tet2^+/-/Jak2^VF/+/VavCre⁺ and Tet2^-/-/Jak2^VF/+/VavCre⁺ mice develop leukocytosis, elevated hematocrits (HCT) and thrombocytosis. Tet2^-/-/Jak2^VF/+/VavCre⁺ mice demonstrate enhanced leukocytosis and splenomegaly compared to the other groups. All groups demonstrate myeloid expansion, erythroid hyperplasia and megakaryocytic abnormalities consistent with MPN in the bone marrow and spleen, while more prominent myeloid expansion and megakaryocytic morphological abnormalities are observed in Tet2^-/-/Jak2^VF/+/VavCre⁺ mice as compared to the other groups. Notably, we do not see the development of acute myelogenous leukemia (AML) in Tet2^-/-/Jak2^VF/+/VavCre⁺ mice at 6 months. We see enhanced expansion of lineage^lowSca1⁺cKit^high (LSK) cells (enriched for HSC) most prominently in the spleens of Tet2^+/-/Jak2^VF/+/VavCre⁺ and Tet2^-/-/Jak2^VF/+/VavCre⁺ mice as compared to Jak2^VF/+/VavCre⁺ mice.

In colony forming assays, we find that Tet2^-/-/Jak2^VF/+/VavCre⁺ LSK cells have enhanced re-plating activity compared to Jak2^VF/+/VavCre⁺ LSK cells and that Tet2^-/-/Jak2^VF/+/VavCre⁺ LSK cells form more colonies that Tet2^-/-/Jak2^+/+/VavCre⁺ cells. Gene expression analysis demonstrates enrichment of a HSC self-renewal signature inTet2^-/-/Jak2^VF/+/VavCre⁺ LSK cells. Concordant with this, we find that Tet2^-/-/Jak2^VF/+/VavCre⁺ LSK cells have enhanced competitive repopulation at 16 weeks as compared to Jak2^VF/+/VavCre⁺ and Tet2^+/-/Jak2^VF/+/VavCre⁺ LSK cells.

In aggregate these findings demonstrate that Tet2 loss promotes disease progression in MPN but is insufficient to drive full leukemic transformation.