The PRC2 complex, with core components EZH2, SUZ12 and EED, is responsible for writing the H3K27me3 histone mark that is associated with gene repression. Sequence data analysis of 419 T-cell acute lymphoblastic leukemia (T-ALL) cases demonstrated that 14.3% of the cases harbored mutations in EZH2 (7.2%), SUZ12 (6.2%) and/or EED (3.8%). A significant positive association was found between SUZ12 loss-of function mutations and JAK3 activating mutations.
The goals of our study were to assess whether SUZ12 inactivation cooperates with JAK3 mutations in driving T-ALL development, and to unravel the mechanism of oncogenic cooperation.
We applied CRISPR gene editing technology to mutate endogenous Suz12 in mouse cells. For cell culture experiments, we used primary pro-T-cells derived from Cas9 transgenic mice that were retrovirally transduced with an activating JAK3(M511I) mutant, a Suz12 guide RNA (Suz12gRNA) or the combination of both. To generate mouse models, we performed bone marrow transplantations with hematopoietic stem/progenitor cells from Cas9 transgenic mice that were transduced with JAK3(M511I), Suz12gRNA or both. These cells were injected in wild type recipient mice and leukemia development was followed. RNA-seq, ATAC-seq and ChIP-seq was performed on leukemia cells. We performed a drug screen with 189 inhibitors targeting a variety of epigenetic factors and signaling molecules on the mouse leukemia cells ex vivo.
The combined expression of JAK3(M511I) with Suz12gRNA conferred IL7-independent growth to ex vivo cultured pro-T cells, whereas JAK3(M511I) or Suz12gRNA alone did not, indicating direct cooperation between mutant JAK3 signaling and Suz12 inactivation. Moreover, in mouse bone marrow transplant leukemia models, combined expression of JAK3(M511I) and Suz12gRNA resulted in accelerated leukemia development with a mean latency of 74 days, compared to JAK3(M511I) or Suz12gRNA alone which generated leukemias with longer latency (112 to 150 days, p < 0.001). JAK3(M511I)+Suz12gRNA leukemias were CD8+ or CD4+CD8+ T-cell leukemias and were transplantable. Effective Suz12 gene editing was confirmed by sequence analysis, Western blotting and intracellular flow cytometry documenting very low Suz12 and H3K27me3 levels. Both in vitro and in vivo data thus showed that inactivation of Suz12 cooperates with JAK3(M511I). Integration of gene expression data with ChIP-seq data for Suz12 and H3K27me3, revealed increased expression of Wnt signaling components and Wnt target genes, as well as activation of the PI3K-AKT-mTOR signaling pathway in the JAK3(M511I)+Suz12gRNA leukemia cells compared to JAK3(M511I) leukemia cells. In addition, a drug screen on JAK3(M511I)+Suz12gRNA leukemia cells revealed increased sensitivity for PI3K, mTOR and Wnt signaling inhibitors, which is in line with upregulation of Pik3cb, Akt3 and Wnt signaling genes detected by RNA-seq and ChIP-seq. Surprisingly, this pharmacological approach also uncovered an increased sensitivity of the JAK3(M511I)+Suz12gRNA leukemia cells to HDAC6 inhibitors compared to JAK3(M511I) leukemia cells.
We have shown that Suz12 inactivation cooperates with JAK3 mutant signaling to drive T-ALL through upregulation of Wnt and PI3K/mTOR pathways. Furthermore, we were also able to identify specific vulnerabilities of the JAK3/Suz12 mutant leukemia cells, as they showed increased sensitivity to Wnt, PI3K/mTOR and HDAC6 inhibitors.