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

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive type of leukemia caused by accumulation of genetic alterations in T-cell progenitors. However, for many genes it remains unknown how loss-of-function mutations contribute to disease development. Single-cell CRISPR screening in ex vivo cultured primary murine pro-T cells allowed us to study the transcriptomic impact of such alterations and to link this with effects on cell proliferation and survival. We first targeted 17 well-studied T-cell regulators and defined key transcriptional signatures, such as NOTCH, MYC, STAT and E2F. A second screen targeted 42 poorly characterized genes and identified gene clusters with E2F/MYC and STAT/NOTCH signatures having opposing roles. These analyses identified a cluster of genes, including multiple chromatin modifiers, regulating Myc expression. Additionally, Spi1 was identified as an essential gene for cell survival, associated with the MYC regulon. Bcl11b inactivation conferred the strongest growth advantage and was associated with JAK/STAT upregulation, corresponding with publicly available patient data. Bcl11b inactivation together with mutant JAK3 transformed pro-T cells to cytokine-independent growth. With this data, we characterized tumor suppressors and oncogenes in T-ALL, providing insight in the mechanisms of leukemia development.