TET2 Deficiency Cooperates with MSH6 Mutation to Promote the Progression of MDS/MPN through Increasing Tumor Stem Cell Frequency

Introduction:

Myelodysplastic syndrome/myeloproliferative neoplasm (MDS/MPN) overlap syndromes are a heterogeneous group of diseases characterized by the co-occurrence of clinical and pathologic features of both MDS and MPN, and frequently progress to AML. Recurrent mutations contribute to the development of MDS/MPN and related myeloid neoplasms, mutations in splicing genes and epigenetic modifiers (e.g., SRSF2, TET2, and ASXL1) are frequent in patients and occur in up to 80% of cases. Among the regulators of DNA methylation, ten-eleven translocation 2 (TET2) is one of the most frequently mutated genes in clonal hematopoiesis of hematological malignancies, underscoring a pivotal role for TET2 in hematopoietic transformation. Coexistence of TET2 and other mutations in particular is highly suggestive of MDS/MPN. The MSH6 gene is involved in one of the systems repairing the errors that arise during DNA replication, and the importance of concomitant alterations of TET2 and MSH6 within distinct hematopoietic compartments and disease progression remains to be elucidated.

Methods:

To further study the impact of MSH6 alteration on disease progression in Tet2-mediated MDS/MPNs, we crossed the Tet2^-/- mice with Msh6^+/− mice and assessed hematopoietic phenotypes through phenotypic experiments and transplantation experiments. We used FACS to compare hematopoietic parameters of Tet2^-/-;Msh6^+/- mice with Tet2^-/- and WT mice in peripheral blood, bone marrow and spleen. LSC frequencies of Tet2^-/-;Msh6^+/- , Tet2^-/-and WT mice were quantified with a limiting dilution BMT assay. We then examined the capability of serial colony formation in vitro.

Results:

In our study, we found that Tet2^-/-;Msh6^+/- mice (n=10) had significantly shorter mean survival rate than Tet2^-/- (n=62) or Msh6^+/− (n=10) mice(Median survival: 265.5 days for Tet2^-/-;Msh6^+/- group, more than 400 days for both Tet2^-/- and Msh6^+/−). To classify the hematopoietic phenotypes in Tet2^-/-;Msh6^+/-mice, we performed a series of analyses including histology, and flow cytometry on peripheral blood (PB), bone marrow (BM), and spleen (SP). The average numbers of WBC, neutrophil were significantly higher in the PB of Tet2^-/-;Msh6^+/-mice compared with the WT group and the Hb levels in Tet2^-/-;Msh6^+/- mice was the lowest . A subset of the compound Tet2^-/-;Msh6^+/- mutants display myelomonocytic hyperplasia, splenomegaly, developed leukocytosis, with an increased CFU colony numbers upon series clongenic assay. To determine the effect of Msh6 alteration on Tet2^-/- HSPCs, we performed flow cytometric analyses and found that the frequencies of LSK was significantly increased in the BM of Tet2^-/-;Msh6^+/- mice, yielding a disease with core characteristics of MDS/MPN(56%). We further assessed the absolute number of LSCs with limiting dilution assay and observed about 6-fold decrease of HSC number in Tet2^-/- mice.

Conclusions:

This study demonstrates that mutant Msh6 cooperated with Tet2^-/- enhance myeloid proliferation and HSC self-renewal to promote myeloid tumor progress through increasing tumor stem cell frequency. Future studies are warranted using the Tet2 and Msh6 co-mutated mice or patient samples to investigate the cooperative effect between Tet2 mutant and Msh6 in the progression of myeloid malignancies, and may facilitate the development of targeted approaches for therapy.

Figure 1: Mutant Msh6 cooperated with Tet2^-/- enhance myeloid proliferation and HSC self-renewal to promote myeloid tumor progress.

• Kaplan-Meier survival curve representing the overall survival of Tet2^-/-;Msh6^+/-(n=10) , Tet2^-/- (n=62) and Msh6^+/- mice (n=10) .

• PB cell counts andspleen weight of Tet2^-/-;Msh6^+/- , Tet2^-/- and Msh6^+/- mice (n = 6 mice per group).

• Representative frequencies of Gr1+Mac-1+ cells and LSK cells in total BM cells of Tet2^-/-;Msh6^+/-, Tet2^-/-and Msh6^+/- mice(n = 6 mice per group).

• CFU colony numbers upon serial clonogenic assay(n = 3 mice per group).

Figure 2: Mutant Msh6 promote Tet2^-/- MDS/MPN through increasing tumor stem cell frequency

• Representative Myeloperoxidase (MPO) staining of liver.

• Disease spectrum in Tet2^-/-;Msh6^+/-and Tet2^-/- mice (n = 32 for Tet2^-/-;Msh6^+/- group, n = 18 for Tet2^-/- group).

• Poisson statistical analysis from the limiting dilution assay.Symbols represent the percentage of negative mice for each dose of cells.

• Frequencies of functional LSCs were calculated according to Poisson statistics.

No relevant conflicts of interest to declare.