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308 Ezh2 Loss Promotes the Transformation of Early T Cell Precursor Leukemia Via Suppressing Critical Genes for T-Cell Differentiation

Oncogenes and Tumor Suppressors
Program: Oral and Poster Abstracts
Type: Oral
Session: 603. Oncogenes and Tumor Suppressors: Epigenetic Transformation in Myeloid and Lymphoid Malignancies
Sunday, December 6, 2015: 4:45 PM
W308, Level 3 (Orange County Convention Center)

Goro Sashida, MD, PhD1,2, Changshan Wang, MD, PhD2*, Daisuke Sato, MS2*, Motohiko Oshima, PhD2 and Atsushi Iwama, MD, PhD2

1International Research Center for Medical Sciences, Kumamoto University, Kumamoto, Japan
2Department of Cellular and Molecular Medicine, Chiba University Graduate School of Medicine, Chiba, Japan

Early T cell precursor (ETP) acute lymphoblastic leukemia (ALL) has been identified as a new pathologic entity with poor outcome in patients with T-ALL.  In contrast to cortical T-ALL, ETP-ALL has been characterized by the activating mutations in genes regulating cytokine signaling and the inactivating mutations in the polycomb repressor complex 2 (PRC2) component genes including EZH2.  EZH2 catalyzes trimethylation of histone H3 at lysine 27 (H3K27me3) that mediates silencing of critical genes for cell proliferation and differentiation.  Given that loss-of-function mutations in EZH2 are often found in ETP-ALL, EZH2 may play a tumor suppressor role in the pathogenesis of ETP-ALL.  To determine how EZH2 dysfunction promotes the development of ETP-ALL in vivo, we generated a novel mouse model of ETP-ALL by utilizing Ezh2 and p53 conditional knockout mice.  We harvested bone marrow cells from Cre-ERT (WT), Cre-ERT;Ezh2flox/flox (Ezh2 KO), Cre-ERT;Trp53 flox/flox (p53 KO) and Cre-ERT;Trp53 flox/flox;Ezh2flox/flox (DKO) mice, and transplanted them into lethally-irradiated Ly5.1+ recipient mice.  We deleted p53 and Ezh2 via administration of tamoxifen 4 weeks post-transplantation.  We observed that p53 KO mice died dominantly due to CD3+CD8+ cortical T-ALL (median survival; 137 days), as previously reported.  While 2 out of 8 DKO mice developed CD8+ cortical T-ALL, we found that 6 out of 8 DKO mice developed CD4-CD8- (DN) T-ALL with a longer latency (median survival; 189 days).  At the time of sacrifice, DKO mice showed significantly enlarged thymus due to the expansion of c-Kit+CD44+CD25- DN1 and c-Kit+CD44+CD25+ DN2 cells, which were markedly compromised in differentiation into DN3 and further down to CD4+CD8+ (DP) cells.  Histological analysis demonstrated the expansion of cytoplasmic CD3+ (cyCD3) tumor cells in the thymus of DKO mice with DN T-ALL.  Thus, DKO mice faithfully recapitulated the phenotypic features of human ETP-ALL, indicating that the loss of Ezh2 is prerequisite to the development of c-Kit+CD44+CD25+cyCD3+DN ALL in mice lacking p53.

To understand the underlying molecular mechanism in the pathogenesis of Ezh2 loss-induced ETP-ALL, we performed gene expression analysis in DN1, DN2 and DN3 cells isolated from WT and DKO mice at pre-disease and ETP-ALL stages.  Since NOTCH1 activating mutations are not frequently found in patients with ETP-ALL, we observed no significant difference in activation of Notch1-target genes between WT and DKO ETP-ALL cells.  In contrast, Ras-target genes were significantly activated in DKO ETP-ALL DN2 cells relative to WT DN2 cells.  Thus, our ETP-ALL mouse model shows active Ras signaling but lacks Notch1 activation, consistent with the molecular features of human ETP-ALL.  Furthermore, we found that the genes critical for T-cell commitment, including Tcf7 or Bcl11b, were transcriptionally repressed in DKO ETP-ALL DN2 cells compared to WT DN2 cells, while the signature genes of both HSCs and myeloid cells were retained in DKO ETP-ALL DN2 cells.  Taken together, Ezh2 and p53 deletions cooperate to activate the function of HSCs and impede the transcriptional program of T-cell differentiation at the DN2 stage with sustaining myeloid potential.  To determine how H3K27me3 modification contributed to induce ETP-ALL in the absence of Ezh2, we performed H3K27me3-chromatin immunoprecipitation (ChIP) sequencing in WT and DKO ETP-ALL DN1/2 cells.  We found that H3K27me3 marks were lost or kept at low levels at the promoter regions of T-cell differentiation regulators in ETP-ALL cells.  Thus, we sought to determine whether altered DNA hypermethylation contributed to silencing the expression of T-cell differentiation regulators.  The transduction of either Tcf7 or Bcl11b expression alone was not sufficient to induce differentiation of DKO DN1/2 cells in vitro, however, we found that treatment of decitabine, a demethylating agent, clearly induced the differentiation of DKO DN1/2 cells beyond the DN3 stage in vitro, implying that Ezh2 loss and p53 loss cooperatively induced aberrant DNA hypermethylation, thereby impeding the differentiation of DN1/2 cells.  In conclusion, we demonstrated that combined deletion of Ezh2 and p53 altered the epigenetic regulation to an extent not seen in either deletion alone, and induced highly penetrant ETP-ALL characterized by the molecular profile similar to that in patients with ETP-ALL harboring mutations in the PRC2 components.

Disclosures: No relevant conflicts of interest to declare.

*signifies non-member of ASH