Recurrent SETBP1 mutations in atypical chronic myeloid leukemia

Abstract

Atypical chronic myeloid leukemia (aCML) shares clinical and laboratory features with CML, but it lacks the BCR-ABL1 fusion. We performed exome sequencing of eight aCMLs and identified somatic alterations of SETBP1 (encoding a p.Gly870Ser alteration) in two cases. Targeted resequencing of 70 aCMLs, 574 diverse hematological malignancies and 344 cancer cell lines identified SETBP1 mutations in 24 cases, including 17 of 70 aCMLs (24.3%; 95% confidence interval (CI) = 16-35%). Most mutations (92%) were located between codons 858 and 871 and were identical to changes seen in individuals with Schinzel-Giedion syndrome. Individuals with mutations had higher white blood cell counts (P = 0.008) and worse prognosis (P = 0.01). The p.Gly870Ser alteration abrogated a site for ubiquitination, and cells exogenously expressing this mutant exhibited higher amounts of SETBP1 and SET protein, lower PP2A activity and higher proliferation rates relative to those expressing the wild-type protein. In summary, mutated SETBP1 represents a newly discovered oncogene present in aCML and closely related diseases.

Figure 1

Distribution of alterations on the SETBP1 protein. Five exons (blue bars) encode isoform A of the protein (1,596 amino acids). The SETBP1 sequence contains three AT hook domains (amino acids 584–596, 1,016–1,028, 1,451–1,463), a SKI homologous region (amino acids 706–917), a SET-binding domain (amino acids 1,292–1,488) and a repeat domain (amino acids 1,520–1,543). Altered amino acids identified in our analysis are highlighted: black circles represent alterations found in aCML samples, and green circles represent alterations found in other diseases. Variants confirmed as somatic are indicated in bold. SETBP1 numbering refers to the NCBI reference sequence NM_015559.2.

Figure 2

Mutation profile of 61 aCML cases for a panel of 15 genes. (a) Total numbers of mutations for each case and each gene are reported. The different kinds of mutations are indicated by color. Total numbers of mutations are given on the right. (b) Distribution of mutations in aCML. For each gene, the percentage of mutations associated with either wild-type or mutated SETBP1 is reported. IDH1, RBBP4, NPM1, JAK2, FLT3 and DNMT3A were also analyzed, but no mutations were identified.

Figure 3

Clinical findings in cases with wild-type and mutated SETBP1. (a–d) Overall survival (P = 0.01) (a), white blood cell count (P = 0.008) (b), hemoglobin concentration (P = 0.44) (c) and platelet number (P = 0.16) (d) in 14 aCML cases with mutated SETBP1 and 24 cases with wild-type SETBP1 (WT). Values are shown as median (horizontal line), 25th and 75th percentiles (boxes), and maximum-minimum ranges (dotted lines). Error bars, s.e.m.

Figure 4

Interaction between β-TrCP1 and SETBP1. (a) The β-TrCP1 degron motif (amino acids 868–873) is highlighted in red on the SETBP1 protein schematic. The sequences of biotinylated phosphorylated peptides (amino acids 859–879) used in the experiments are given. Black circles represent alterations found in aCML samples; green circles represent alterations found in other diseases. (b) Peptide pulldown experiment performed using TF1 total cell lysate and phosphorylated peptides. Beads with no peptides were used to control for nonspecific binding. Immunoblotting for β-TrCP1 was performed on the bound fractions. Immunoblotting for actin on the unbound fractions was used as a loading control. (c) Peptide pulldown experiment using recombinant SCF–β-TrCP1 complex on phosphorylated (+ P) and dephosphorylated (− P) peptides representing either wild-type SETBP1 or SETBP1 Gly870Ser. Beads with no peptide were used to control for nonspecific binding. Immunoblotting for β-TrCP1 was performed on bound as well as unbound (control) fractions.

Figure 5

Effects of the SETBP1 Gly870Ser alteration on SETBP1 and SET protein expression, PP2A activity and cell growth. (a–d) TF1 cells were transfected with empty vector (pMIGR1, EV) or vector expressing wild-type SETBP1 or SETBP1 Gly870Ser. (a) Immunoblotting for SETBP1 on whole-cell lysates. Whole normal fetal stomach lysate was used as a control for SETBP1 protein. Immunoblotting for actin was used as a loading control. Lanes were derived from the same gel and were juxtaposed. (b) Immunoblotting for SET, phosphorylated PP2A (pPP2A), PP2A and actin on whole-cell lysates. Immunoblotting for actin was used as a loading control. Densitometric analysis of the amount of SET protein normalized over actin signal is shown in the bar graph. Mean and s.e.m. values from three independent experiments are plotted. ***P < 0.0001 compared to cells expressing wild-type SETBP1. (c) Lysates were used to assess the activity of PP2A. The activity relative to cells expressing wild-type SETBP1 is reported. Mean and s.e.m. values from three independent experiments are plotted. ***P < 0.0001. (d) Growth rate of the cells as measured by tritiated thymidine incorporation. Each curve was normalized to its value at time 0. Mean and s.e.m. values from two independent experiments are plotted. ***P < 0.0001 compared to cells expressing wild-type SETBP1.