KDM6B Overexpression and TET2 Deficiency Cooperatively Drive Development of Myelodysplastic Syndrome and Chronic Myelomonocytic Leukemia-like Phenotype in Mice

Ten-eleven translocation 2 (TET2) dioxygenase mutations resulting in enzymatic deficiency are among the most frequently reported molecular aberrations in myeloid neoplasms. Accumulating evidence indicates that biological interactions between TET2 deficiency and other molecular lesions are important drivers of myeloid disorder. For example, microbial-mediated innate immune signaling was demonstrated to play a critical role in promoting myeloproliferation in mice with TET2 deficiency. Histone demethylase KDM6B is an innate immune signal activator downstream of Toll like receptors (TLR) and is overexpressed in bone marrow (BM) hematopoietic stem and progenitor cells (HSPC) from patients with myelodysplastic syndrome (MDS) and chronic myelomonocytic leukemia (CMML). We previously demonstrated that overexpression of KDM6B in the hematopoietic compartment of mice led to a MDS-like phenotype and hyperactivation of innate immune signals (Wei Blood Advances 2018). These observations suggest that KDM6B overexpression and consequent innate immune deregulation may cooperatively interact with TET2 deficiency to drive myeloid disorders. In support of this hypothesis, transcriptomic analysis in the BM HSPCs of patients with MDS and CMML revealed that patients with concurrent KDM6B overexpression and TET2 deficiency had significantly increased activation of innate immune genes and signals.

To investigate the interaction between KDM6B overexpression and TET2 deficiency, we developed a "double lesion" mouse model with both KDM6B overexpression and TET2 deficiency by crossing Vav-KDM6B mice with TET2^flox/flox/Vav-Cre mice. Double lesion mice exhibited significant monocytosis compared to Vav-Cre or TET2 deficient mice (p=0.02 and p=0.03). Double lesion mice also experience reduced hemoglobin (p=0.01 and p=0.01) and splenomegaly (p=0.004 and p=0.01). Myeloid skewing was observed in the BM of double lesion mice as large expansile aggregates of immature myelomonocytic precursors, increase of Gr-1+ myeloid cells (p=0.001 and p=0.01), and decrease of Ter119+ erythroid cells (p=0.01 and p=0.02). An increase in the BM Lin-/Sca1+/cKit+ (LSK) population was also observed in double lesion mice (p=0.004 and p=0.05) particularly in aged mice (50-60 week old). Moreover, the BM of double lesion mice exhibited increased repopulating function, illustrated by higher chimerism of CD45.2 donor cells in CD45.1 recipient mice 6 months following competitive transplantation (p=0.004 and p=0.04). Overall, the MDS/CMML-like hematopoietic phenotype observed in double lesion mice, which was more significant than that observed in TET2 deficient single lesion mice, indicated that overexpression of KDM6B and TET2 deficiency cooperatively altered hematopoiesis and promoted development of myeloid disorders.

To identify the biological mechanisms underlying the cooperative impact of KDM6B overexpression and TET2 deficiency in BM HSPCs, we performed transcriptomic analysis on LSKs collected from double lesion, TET2 deficient, Vav-KDM6B, and Vav-Cre mice. RNA-Seq illustrated hyper-activation of multiple innate immune pathways in the LSK of double-lesion mice compared to the other groups. Consistently, peripheral blood cytokine levels of TNF, IL-1, and IL-6 were significantly elevated in double lesion mice. RNA-Seq also revealed that the LSKs of double-lesion mice displayed significant down-regulation of cell cycle checkpoint and DNA repair signals. This finding was confirmed by the observation of increased phosphorylated γH2AX and aneuploidy in hematopoietic cells from double lesion mice.

Finally, we applied GSK-J4, an inhibitor KDM6 proteins, to the BM LSKs from each mouse cohort and performed methylcellulose medium supported colony formation assays. Significantly reduced colony formation was observed in both the double lesion and TET2 deficiency groups (p<0.01 and p<0.05).

Taken together, biological and molecular studies of the KDM6B-TET2 double lesion model demonstrate that KDM6B overexpression and TET2 deficiency cooperatively cause significant hematopoietic impacts and drive development of myeloid disorders mediated by profound dysregulation of innate immune and genomic stability regulatory signaling in BM HSPCs. Our study also suggests that targeting KDM6B and its associated innate immune signaling has therapeutic potential in myeloid disorders with TET2 deficiency.