PRC2 Mutaties in T-cel Acute Lymfatische Leukemie: Suz12 inactivatie coöpereert met JAK3 Mutant Signaling in T-cel Acute Lymfatische Leukemie

T-cell acute lymphoblastic leukaemia (T-ALL) is characterized by deregulation of transcriptional control and impairment of epigenetic homeostasis. PRC2 is the epigenetic complex responsible for writing the H3K27me3 mark associated with gene repression and maintenance of cell identity. Up to 25% of T-ALL patients harbour inactivating mutations in SUZ12, EZH2 and EED components of the PRC2 complex. A targeted re-sequencing on 155 T-ALL patients performed in our lab, showed that PRC2 mutations, and SUZ12 mutations in particular, frequently co-occur with mutations in the IL7R-JAK-STAT pathway. To assess whether loss of PRC2 activity cooperates with mutant JAK3 signalling during T-ALL development, we expressed the JAK3(M511I) mutant in mouse bone marrow cells in vivo and tested the effect of Cas9-mediated inactivation of Suz12 on leukaemia development. Hematopoietic stem/progenitor cells (HSPCs) from transgenic mice constitutively expressing Cas9 were co-transduced with a retroviral vector containing Suz12 guide RNA (also expressing BFP) and a vector containing JAK3(M511I) (also expressing GFP). After injection of the transduced cells into recipients, the animals were followed by serial blood sampling. The mice developed a T-cell leukaemia that infiltrated all haematological organs. Initially, the pre-dominant leukemic population in the blood was only GFP (JAK3 M511I) positive, but over time a rapid expansion of GFP/BFP double positive cells was observed. At time of sacrifice the majority of cells in the blood and hematopoietic organs was GFP/BFP positive. Surprisingly, mice injected with Suz12 gRNA only cells also developed T-cell leukaemia, although at a longer latency (112 days) compared to JAK3(M511I) + Suz12 gRNA mice (71 days). These data strongly indicate that inactivation of Suz12 cooperates with JAK3(M511I) during T-ALL development. Currently, the mechanism of cooperation is under investigation. Differential gene expression profiling showed that the majority of genes are upregulated upon Suz12 inactivation. Thus, 2300 (96%) genes were upregulated in contrast to 107 (4%) downregulated genes comparing JAK3(M511I) + Suz12 gRNA to JAK3(M511I)-only leukaemia. In Suz12 gRNA-only leukaemia versus wildtype thymocyte, 1510 (75%) genes were up- and 514 (25%) were downregulated. ChIP-seq analysis revealed that H3K27me3 levels were drastically reduced genome widely upon Suz12 inactivation, in accordance with intra-nuclear FACS staining for this histone mark. We also checked individual loci that exhibited reduced binding of Suz12, reduced levels of H3K27me3 and increased promoter marks (H3K4me3). Among these loci we found Hoxa genes (Hoxa1, Hoxa3, Hoxa5) and Notch1 target genes (Hes1, Hes2, Hes7, Hey2, Heyl, Dtx1, Dtx4), the latter pathway being important in T-cell development and T-ALL. Our data show that Suz12 inactivation cooperates with mutant JAK3 signalling to drive leukaemia development in vivo and demonstrates the use of Cas9 transgenic mice to study the role of tumour suppressor genes in T-ALL mouse models.status: publishe