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Single-cell CRISPR screening characterizes transcriptional deregulation in T-cell acute lymphoblastic leukemia

ABSTRACT: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive leukemia caused by accumulation of genetic alterations in T-cell progenitors. For many genes it remains unknown how loss-of-function mutations contribute to disease development. Single-cell CRISPR screening in ex vivo pro-T cells allowed us to study the transcriptomic impact of such alterations. A first CRISPR screen targeted 17 well-studied regulators and defined the core transcriptional signatures, such as NOTCH1, MYC, STAT5 and E2F. Additionally, Spi1 was identified as an essential gene associated with the MYC regulon. A second screen targeted 42 poorly characterized genes and identified gene clusters with E2F/MYC and STAT/NOTCH1 signatures having opposing roles. Bcl11b inactivation conferred the strongest growth advantage and was associated with JAK/STAT upregulation, corresponding with publicly available T-ALL patient data. Bcl11b inactivation cooperated with mutant JAK3 in transforming pro-T cells to cytokine-independent growth. With this data, we characterized tumor suppressors and oncogenes in T-ALL, thereby providing insight in the mechanisms of leukemia development.

ORGANISM(S): Mus musculus

PROVIDER: GSE222378 | GEO | 2023/09/30

REPOSITORIES: GEO

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Project description:T-ALL is characterized by deregulation of transcriptional control and impairment of epigenetic homeostasis. We observed that T-ALL cases with mutations in the IL7R-JAK-STAT pathway frequently harbor inactivating mutations in the PRC2 complex, in particular SUZ12 mutations. PRC2 is an epigenetic complex responsible for writing the H3K27me3 mark associated with gene repression. To assess whether loss of SUZ12 cooperates with mutant JAK3 signaling 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 leukemia development. The mice developed a T-cell leukemia that infiltrated most hematological organs (bone marrow, thymus, spleen, lymph nodes, blood). At time of sacrifice the majority of cells in the blood and hematopoietic organs was JAK3(M511I) positive and Suz12 negative. Reduced Suz12 protein and H3K27me3 levels were confirmed with flow cytometry. Moreover, JAK3(M511I) also cooperates with Suz12 gRNA-mediated inactivation in vitro. Pro-T cells carrying both JAK3(M511I) and Suz12 gRNA were able to transform to IL7 independent growth, whereas single mutant (JAK3(M511I) only or Suz12 gRNA only) pro-T cells could not. In order to get insight in the mechanism of cooperation between JAK3 mutant signaling and Suz12 inactivation, we performed and integrated RNA- and ChIP-sequencing (Suz12, H3K27me3, H3K27Ac, H3K4me1, H3K4me3) on leukemic cells from the mice. This showed a global upregulation of many genes, including known PRC2 target genes upon loss of Suz12. Moreover, Suz12 was bound to promoters of Wnt signaling genes (Fzd7, Lrp4) and Wnt target genes (Axin2, Jag2, Mmp9), concomitant with high H3K27me3 levels. Loss of Suz12 led to drastic reduction of H3K27me3 at these loci and upregulation of these Wnt genes. As Wnt signaling is ectopically activated, we wanted to test whether JAK3(M511I) + Suz12 gRNA cells are more sensitive to Wnt inhibitor. JAK3(M511I) + Suz12 pro-T cells and leukemic spleen cells exhibited increased sensitivity to Wnt inhibitor compared to JAK3 mutant control cells, indicating that Wnt signaling contributes to the cooperation between Suz12 loss and JAK3 mutant signaling. In conclusion, our data show that Suz12 inactivation cooperates with mutant JAK3 signaling to drive leukemia development in vivo and demonstrates the use of Cas9 transgenic mice to study the role of tumor suppressor genes in T-ALL mouse models. Moreover, Suz12 loss leads to ectopic activation of Wnt signaling, a pathway contributing to the cooperation of Suz12 loss and JAK3 mutant signaling that can also be targeted with inhibitors.

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Single-cell CRISPR screening characterizes transcriptional deregulation in T-cell acute lymphoblastic leukemia

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