Acute lymphoblastic leukemia (ALL) is characterized by the presence of chromosomal changes, including numerical changes, translocations, and deletions, which are often associated with additional single-nucleotide mutations. In this study, we used single cell-targeted DNA sequencing to evaluate the clonal heterogeneity of B-ALL at diagnosis and during chemotherapy treatment. We designed a custom DNA amplicon library targeting mutational hotspot regions (in 110 genes) present in ALL, and we measured the presence of mutations and small insertions/deletions (indels) in bone marrow or blood samples from 12 B-ALL patients, with a median of 7973 cells per sample. Nine of the 12 cases showed at least 1 subclonal mutation, of which cases with PAX5 alterations or high hyperdiploidy (with intermediate to good prognosis) showed a high number of subclones (1 to 7) at diagnosis, defined by a variety of mutations in the JAK/STAT, RAS, or FLT3 signaling pathways. Cases with RAS pathway mutations had multiple mutations in FLT3, NRAS, KRAS, or BRAF in various clones. For those cases where we detected multiple mutational clones at diagnosis, we also studied blood samples during the first weeks of chemotherapy treatment. The leukemia clones disappeared during treatment with various kinetics, and few cells with mutations were easily detectable, even at low frequency (<0.1%). Our data illustrate that about half of the B-ALL cases show >2 subclones at diagnosis and that even very rare mutant cells can be detected at diagnosis or during treatment by single cell-targeted DNA sequencing.
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.
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