JMJD3 Plays Essential Roles in the Maintenance of Hematopoietic Stem Cells and Leukemic Stem Cells through the Regulation of p16^INK4a

Hematopoiesis is a complex process that involves the interplay between lineage-specific transcription and epigenetic regulation, including histone modifications. Tri-methylation of histone H3 at Lys27 (H3K27me3) is an epigenetic mark for transcriptional repression. Jumonji domain-containing 3 (JMJD3) acts as a histone demethylase for H3K27 and contributes to various cellular processes including senescence and differentiation through transcriptional regulation. In the hematopoietic system, JMJD3 has been reported to be required for M2 macrophage development and terminal thymocyte differentiation. However, the roles of JMJD3 in normal hematopoiesis and leukemogenesis are still largely elusive.

To address this issue, we generated pIpC-inducible Jmjd3 conditional KO (cKO) mice. Jmjd3-deficient (Jmjd3^Δ/Δ) mice grew healthy and did not show obvious hematopoietic abnormalities, except a slight decrease of myeloid cells. To investigate the role of JMJD3in hematopoietic stem cell (HSC) function, a competitive repopulation assay was performed using control and Jmjd3^Δ/Δ HSCs. The results showed that the chimerism of Jmjd3^Δ/Δ cells was significantly decreased compared with that of control cells in all the hematopoietic lineages, indicating that JMJD3 is essential for long-term repopulating ability of HSCs. To further investigate the effect of Jmjd3 deletion in leukemogenesis, c-kit⁺ bone marrow (BM) cells from control and Jmjd3 cKO mice were transduced with MLL-AF9 fusion protein that rapidly induces acute leukemia. L-GMPs (the fraction containing leukemic stem cells (LSCs)) were sorted from MLL-AF9-transduced BM cells and subjected to colony replating and bone marrow transplantation (BMT) assays. In contrast control L-GMPs that continued to form colonies after multiple rounds of replating, Jmjd3^Δ/Δ L-GMPs ceased to proliferate after third rounds of replating. In addition, recipients transplanted with Jmjd3^Δ/Δ L-GMPs exhibited a significant delay in the onset of leukemia compared with those transplanted with controlL-GMPs. These results indicate that JMJD3 plays essential roles in maintaining stem cell properties not only in normal HSCs but also in LSCs.

We next investigated underlying molecular mechanisms. Previous studies demonstrated the INK4a/ARF locus, a key executor of cellular senescence, is regulated by JMJD3. Thus, we examined whether JMJD3 regulates INK4a/ARF locus in hematopoietic cells under proliferative and oncogenic stresses. We found that enforced expression of Jmjd3 in in vitro-cultured and cytokine-stimulated hematopoietic stem-progenitor cells (HSPCs) significantly upregulated the expression of p16^INK4a compared with control cells. In addition, transformation of HSPCs by MLL-AF9 induced expression of Jmjd3, but not other H3K27me3-related genes, such as Utx and EZH2, which was accompanied by the upregulation of p16^INK4a. In contrast, no obvious expressional change was observed in p19^ARF in both cases. In Jmjd3^Δ/Δ HSPCs, no upregulation of p16^INK4a was detected in HSPCs by cytokine-induced proliferation or MLL-AF9-induced transformation, where H3K27me3 was tightly associated with promoter region of p16^INK4a locus. These results strongly suggest that proliferative and oncogenic stresses induces the expression of Jmjd3 in HSPCs, which subsequently upregulates p16^INK4a through demethylating H3K27me3 on the p16^INK4a promoter and consequently maintains stem cell potential by inhibiting excessive entry into cell cycle. Deficiency of Jmjd3 fails upregulation of p16^INK4a, which induces continuous and excessive cell proliferation and finally causes exhaustion of stem cell pool. In conclusion, we propose the idea that JMJD3-p16^INK4a axis plays essential roles in maintaining HSC and LSC pool size under proliferative and oncogenic stresses.