Role of KDM2B and Non-Canonical PRC1.1 As a Tumor Suppressor in T-ALL
Authors: Isshiki Yusuke , Nakajima-Takagi Yaeko , Oshima Motohiko , Aoyama Kazumasa , Saraya Atsunori , Sakaida Emiko , Nakaseko Chiaki , Yokote Koutaro , Koseki Haruhiko , Iwama Atsushi 
Published: October 2019

Introduction: Polycomb repressive complexes (PRCs) play an important role for the transcriptional repression of their target genes through histone modification. KDM2B is a component of non-canonical PRC1.1 and has a role for the recruitment of the complex to the target gene loci. It has a zinc finger-CxxC (ZF-CxxC) domain which specifically binds to unmethylated sequences in CpG islands (CGIs) and deletion of the CxxC domain induces complete loss of KDM2B occupancy and removal of other PRC1.1 components from CGIs. Recent studies revealed that loss of function mutations of several PRC component genes such as EZH2, EED, SUZ12 and BCOR were frequently detected in human T-cell acute lymphoblastic leukemia (T-ALL), which suggested PRCs have a tumor suppressive role in T cell development. Our group have reported conditional knock out of Bcor, encoding a component protein of non-canonical PRC1.1, induced T-ALL in mice. However, it is still unknown how KDM2B and non-canonical PRC1.1 regulate T cell leukemogenesis. Therefore, we performed detailed analysis of mice deficient for Kdm2b ZF-CxxC domain (Kdm2bΔCxxC/ΔCxxC) specifically in hematopoietic cells.

Methods: We used the conditional Kdm2b allele (Kdm2bfl) mice, which contains LoxP sites flanking Kdm2b exon 13 encoding the ZF-CxxC domain. To generate hematopoietic cell specific Kdm2b KO mice, we transplanted Kdm2bfl/fl;Cre-ERT total BM cells into lethally irradiated CD45.1+ recipient mice and deleted Kdm2b 4 weeks after transplantation by intraperitoneally injecting of tamoxifen.

Results: During the observation period of 300 days, almost all of the Kdm2b KO mice developed lethal T-ALL. They showed thymomegaly and splenomegaly and presented infiltration of donor-derived leukemic cells into the bone marrow, spleen, thymus and peripheral blood. Flow cytometric analysis revealed that T-ALL cells were mainly CD4 and CD8-double positive (DP). Notch1 active mutations in exons 26, 27, 28 or 34 were found in over a half of the T-ALL cases, indicating the Notch1 activation could be a driver for leukemic transformation in this mouse model. RNA sequence analysis of the DP cells revealed activation of Myc, which plays a key role in the development of T-ALL, and their downstream target genes in Kdm2b-deficient T-ALL. ChIP sequence analysis of DP thymocytes expressing 3xFLAG-KDM2B confirmed the binding peaks of KDM2B at the promotor of Myc. Peak calling analysis of the ChIP sequence data revealed that KDM2B was mainly located at transcript start sites (TSS), where KDM2B was co-localized with H2AK119ub1 and H3K27me3 histone marks. In addition, ChIP sequencing of H3K4me3 revealed that the KDM2B target genes include more bivalent genes than non-target genes. We next compared histone modification status around TSS in WT and Kdm2b KO DP cells and Kdm2b-deficient T-ALL cells. Global levels of H2AK119ub1 were significantly decreased in T-ALL cells and the reduction was mainly observed at the promoters of KDM2B target genes. Direct target genes of NOTCH1 including Myc also lost H2AK119ub1 at their promotors in T-ALL cells. Peak calling analysis of KDM2B, BCOR and NOTCH1 ChIP sequence data revealed that their target genes were closely overlapped at the promotor region. Moreover, EZH2 binding peaks were also overlapped with those of KDM2B and NOTCH1, suggesting that non-canonical PRC1.1 and PRC2 cooperatively antagonize NOTCH1-mediated gene activation. KEGG pathway analysis of the genes with overlapping binding peaks among KDM2B, EZH2 and NOTCH1 showed significant enrichment of T cell receptor signaling, Notch1 signaling and cell cycle pathway, all of which play an important role for the development of T-ALL. Of interest, H3K27me3 levels of the common target genes of EZH2, KDM2B, and NOTCH1 were much lower than the EZH2-specific target genes, indicating that H2AK119ub1 plays a key role in the repression of NOTCH1 targets.

Conclusions: Our findings suggest that KDM2B recruits non-canonical PRC1.1 at the promotor regions of NOTCH1 targets to restraint thymocytes from transformation in concert with PRC2.

Disclosures

No relevant conflicts of interest to declare.