Integrated Genomic, Functional, and Prognostic Characterization of Atypical Chronic Myeloid Leukemia

Abstract

Atypical chronic myeloid leukemia (aCML) is a BCR-ABL1-negative clonal disorder, which belongs to the myelodysplastic/myeloproliferative group. This disease is characterized by recurrent somatic mutations in SETBP1, ASXL1 and ETNK1 genes, as well as high genetic heterogeneity, thus posing a great therapeutic challenge. To provide a comprehensive genomic characterization of aCML we applied a high-throughput sequencing strategy to 43 aCML samples, including both whole-exome and RNA-sequencing data. Our dataset identifies ASXL1, SETBP1, and ETNK1 as the most frequently mutated genes with a total of 43.2%, 29.7 and 16.2%, respectively. We characterized the clonal architecture of 7 aCML patients by means of colony assays and targeted resequencing. The results indicate that ETNK1 variants occur early in the clonal evolution history of aCML, while SETBP1 mutations often represent a late event. The presence of actionable mutations conferred both ex vivo and in vivo sensitivity to specific inhibitors with evidence of strong in vitro synergism in case of multiple targeting. In one patient, a clinical response was obtained. Stratification based on RNA-sequencing identified two different populations in terms of overall survival, and differential gene expression analysis identified 38 significantly overexpressed genes in the worse outcome group. Three genes correctly classified patients for overall survival.

Figure 1

Oncoprint. Oncoprint showing somatic mutations for a panel of 12 genes in 37 patients.

Figure 2

Colony formation assay. X axis represents different treatments and Y axis represents total number of colonies formed, normalized to 100 (colony counts in control conditions). Results are shown as the mean± s.d. (n = 2). (A) Patient CMLPh-003 carrying NRAS G12R mutation. (B) Patient CMLPh-006 carrying KRAS A146 V mutation. (C) Patient CMLPh-039 carrying NRAS G12R mutation. (D) Patient CMLPh-010 carrying KIT D816 V mutation. (E) Patient CMLPh-040 carrying CSF3R T618I mutation.

Figure 3

Clonal architecture of aCML patients. Schematic representation of the clonal architecture of 7 aCML patients whose bone marrow mononuclear cells were grown in semisolid medium and underwent targeted resequencing based on previously identified somatic mutations. CBLhet indicates a heterozygous somatic mutation; CBLhom indicates a homozygous somatic CBL mutation.

Figure 4

Effects of trametinib and phosphoethanolamine in patient CMLPh-042. (A) Colony-forming assay at the onset: bone marrow derived cells were left untreated or treated with phosphoethanolamine 1 mm, trametinib 10 nM, trametinib 100 nM, or combination of the two drugs. Colonies were counted after 15 days. (B) Colony-forming assay performed at relapse after treatment with trametinib 1 mg/day.(C) Number of colonies at the onset. (D) Number of colonies at relapse.

Figure 5

In-vivo experiments. (A) Overall survival (OS) of mice treated with 1 mg/kg trametinib by oral gavage once a day (red line) as compared with controls (black line). OS were analyzed using Kaplan-Meier plot and the log-rank test. (B) Analysis of human CD45 cells in peripheral blood assessed by flow cytometry in PDX models treated with 1 mg/kg trametinib compared to controls. Representative plots are shown. (C) Immunohistochemistry (human CD45 expression) of bone and spleen PDX models treated with 1 mg/kg trametinib by oral gavage once a day compared to controls. Representative images are shown. Scale bar: 200 μm.

Figure 6

Overall survival curve (Kaplan-Meier curve). Overall survival curve censored at 24 months shows significantly different outcomes (low-rank p = 0.004).

Figure 7

Gene ontology and pathway heatmaps. (A) Heatmap showing expression for a set of selected cancer-related GO terms is presented. (B) Heatmap showing expression for a set of selected cancer-related pathways is presented.

Figure 8

Heatmap showing fold change for the threetop differentially expressed genes used to classify good vs bad prognosis subtypes is presented.