Residual Wild-Type Tet2/Tet3 Allele Is the Savior Preventing Mouse Hematopoietic Progenitor Cells from Leukemia Development
Authors: Shrestha Raksha , Koichiro Maie , Sakata-Yanagimoto Mamiko , Bernard Olivier , Matsui Hirotaka , Nakajima-Takagi Yaeko , Kato Takayasu , Koseki Haruhiko , Iwama Atsushi , Chiba Shigeru 
Published: October 2019

Abstract
Background: Loss-of-function mutations in ten-eleven-translocation-2 (TET2) have been identified in myeloid and lymphoid malignancies. They have also been found in pre-leukemic hematopoietic stem cells and even in elderly individuals without hematologic malignancies. Taken together, they suggest TET2 mutation may act as a strong initiator in the pathogenesis of leukemia. TET3 mutation, on the other hand, is rare and its level has been found to decline with age. We hypothesized that somatic mutations of TET2 and age-related downregulation of TET3 lead to impairment of TET enzymes giving rise to hematological malignancies.
Methods: Tet2 fl and Tet3fl mice were crossed with mice expressing Mx1-Cre. Floxed alleles were then deleted by intraperitoneal injection with polyinosinic: polycytidilic acid to generate Tet2fl/flTet3fl/flMx-Cre+, Tet2fl/wtTet3fl/flMx-Cre+ and Tet2fl/flTet3fl/wtMx-Cre+. Moribund mice were analyzed by flow cytometry.
Results: All Tet2fl/flTet3fl/flMx-Cre+ (homo/homo) mice developed acute myeloid leukemia (AML) and died within 16 weeks of age. Tet2fl/wtTet3fl/flMx-Cre+ (hetero/homo) and Tet2fl/flTet3fl/wtMx-Cre+ (homo/hetero) mice developed disease and died from 20 weeks onwards (median survival was 27 weeks in both groups). Among 13 hetero/homo mice, flow cytometry confirmed 11 cases of AML, 1 case of T-cell acute lymphoblastic leukemia (T-ALL), and 1 case of composite AML/T-ALL. Among 7 hetero/homo mice, there were 5 cases of AML, 1 T-ALL, and 1 composite AML/T-ALL. Leukemic cells from bone marrow of all genotypes were transplantable and could be established as cell lines. Multiplex PCR of 3 hetero/homo and 3 homo/hetero mice showed additional loss of wild type Tet2 allele in all three hetero/homo mice with AML. One of two homo/hetero mice with AML showed additional loss of wild-type Tet3 allele. Homo/hetero mouse with T-ALL showed presence of wild-type Tet3 allele. Exome sequencing revealed additional single nucleotide variants in these mice. Exome sequencing of chromosome 3 housing the Tet2 gene, showed uniparental disomy in two and deletion in one hetero homo mice respectively. Exome sequencing of chromosome 6 housing the Tet3 gene showed deletion in one mouse with AML; uniparental disomy was not observed.
Conclusion: Four allele deletion of Tet2 and Tet3 led to aggressive development of AML in mice, suggesting that Tet2 and Tet3 interact to function as tumor suppressors. However, three allele deletion of Tet2/Tet3 resulted in both myeloid and T-cell malignancies after a longer latency. Additional loss of residual Tet2 or Tet3 in these mice showed an AML phenotype similar to that seen in Tet2/Tet3 double knockout mice. These results suggest that loss of both Tet2 and Tet3 are strongly associated with the pathogenesis of myeloid and lymphoid malignancies.

Disclosures
Chiba: Bristol Myers Squibb, Astellas Pharma, Kyowa Hakko Kirin: Research Funding.