The genetic basis of early T-cell precursor acute lymphoblastic leukaemia

Abstract

Early T-cell precursor acute lymphoblastic leukaemia (ETP ALL) is an aggressive malignancy of unknown genetic basis. We performed whole-genome sequencing of 12 ETP ALL cases and assessed the frequency of the identified somatic mutations in 94 T-cell acute lymphoblastic leukaemia cases. ETP ALL was characterized by activating mutations in genes regulating cytokine receptor and RAS signalling (67% of cases; NRAS, KRAS, FLT3, IL7R, JAK3, JAK1, SH2B3 and BRAF), inactivating lesions disrupting haematopoietic development (58%; GATA3, ETV6, RUNX1, IKZF1 and EP300) and histone-modifying genes (48%; EZH2, EED, SUZ12, SETD2 and EP300). We also identified new targets of recurrent mutation including DNM2, ECT2L and RELN. The mutational spectrum is similar to myeloid tumours, and moreover, the global transcriptional profile of ETP ALL was similar to that of normal and myeloid leukaemia haematopoietic stem cells. These findings suggest that addition of myeloid-directed therapies might improve the poor outcome of ETP ALL.

Figure 1. Circos plots of genetic alterations in four representative ETP ALL cases depicting structural genetic variants, including DNA copy number alterations, intra- and inter-chromosomal translocations, and sequence alterations

Loss-of-heterozygosity, orange; amplification, red; deletion, blue. Sequence mutations in RefSeq genes: silent single-nucleotide variants (SNVs), green; non-silent SNVs, brown; indels, red. Genes at structural variant breakpoints: genes involved in in-frame fusions, pink; others, blue. Circos plots for all cases are provided in Supplementary Fig. 6.

Figure 2. Recurring sequence mutations in T-ALL

Recurring mutations in ETP-ALL. The figures show mutations for the 12 WGS cases, and the recurrence cohort of 94 cases sequenced by Sanger sequencing. The majority of cases had matched remission DNA to distinguish somatic from inherited variants. Where remission DNA was not available, but variants are known or predicted to be deleterious, mutations are shown as ‘variants’. The results of recurrence screening for additional genes sequenced are shown in Supplementary Table 17 and Supplementary Figs 9 and 14–16). The schematics are based on the following NCBI protein reference sequences: GATA3 NP_001002295, DNM2 NP_001005360, ECT2L NP_001071174, EZH2 NP_001190176, PHF6 NP_001015877.1 and RUNX1 NP_001745.

Figure 3. Recurring mutations in T-lineage ALL

a, Data are shown for 106 T-ALL cases, including the 12 cases that were subjected to whole-genome sequencing (arrowed), and 94 recurrence cases (52 ETP ALL and 42 non-ETP T-ALL). Cases have been grouped by ETP status, and cases lacking any mutations are shown to the left, followed by cases with NRAS and FLT3 mutations. Genes identified as novel targets of mutation in T-ALL are labelled green. Four cases died while in remission and are excluded from outcome analysis. b, Frequency of somatic alterations targeting haematopoietic and lymphoid development in ETP and non-ETP T-ALL, showing an increased frequency of lesions in these pathways in ETP ALL. ***P < 0.0001.

Figure 4. IL7R mutations in T-ALL

a, Domain structure of IL7R, showing two hotspots of missense and in-frame insertion-deletion mutations (IL241–242 and VA253–254). The single case with a mutation in the amino-terminal region (V78M) is accompanied by a transmembrane domain mutation. b, c, Murine Il7r mutant alleles homologous to the human IL7R mutations were expressed in the murine haematopoietic IL-3-dependent Ba/F3 cell lines (b) or the murine IL-7-dependent MOHITO T-ALL cell line (c). WT, wild type. Expression of these mutant alleles resulted in transformation to cytokine-independent proliferation. MIG, empty MSCV-IRES-GFP vector. In b, growth curves have been offset to permit visualization of each allele. Error bars represent mean ± s.d. for three replicates. In c, transformation to cytokine-independent growth is shown as an increasing proportion of Il7r-mutant-expressing cells (or as a positive control, BCR– ABL1), as measured by the percentage of GFP-positive cells. d, Western blotting for Il7r in MOHITO cells transduced with wild-type or mutant Il7r alleles, showing the formation of Il7r dimers in cells expressing mutant alleles with an unpaired cysteine residue. e, Phosphosignalling analysis of MOHITO cells transduced with MIG, WT Il7r or four different Il7r mutant alleles, showing increased Stat5 phosphorylation in cells stimulated with IL7, or cells expressing mutant Il7r alleles in the absence of cytokine. Stat5 phosphorylation was reduced following exposure to Jak inhibitor I (inh) at 3μM for 1 hour. f, g, Clonogenic assays of lineage-negative WT or Arf^−/− murine bone marrow cells expressing mutant Il7r alleles show enhanced replating compared to cells transduced with empty vector (f), and enhanced replating compared to cells expressing WT Il7r cultured in the absence of IL7 (g). Columns show mean of two replicates ± s.e.m.