Molecular basis of ETV6-mediated predisposition to childhood acute lymphoblastic leukemia

Abstract

There is growing evidence supporting an inherited basis for susceptibility to acute lymphoblastic leukemia (ALL) in children. In particular, we and others reported recurrent germline ETV6 variants linked to ALL risk, which collectively represent a novel leukemia predisposition syndrome. To understand the influence of ETV6 variation on ALL pathogenesis, we comprehensively characterized a cohort of 32 childhood leukemia cases arising from this rare syndrome. Of 34 nonsynonymous germline ETV6 variants in ALL, we identified 22 variants with impaired transcription repressor activity, loss of DNA binding, and altered nuclear localization. Missense variants retained dimerization with wild-type ETV6 with potentially dominant-negative effects. Whole-transcriptome and whole-genome sequencing of this cohort of leukemia cases revealed a profound influence of germline ETV6 variants on leukemia transcriptional landscape, with distinct ALL subsets invoking unique patterns of somatic cooperating mutations. 70% of ALL cases with damaging germline ETV6 variants exhibited hyperdiploid karyotype with characteristic recurrent mutations in NRAS, KRAS, and PTPN11. In contrast, the remaining 30% cases had a diploid leukemia genome and an exceedingly high frequency of somatic copy-number loss of PAX5 and ETV6, with a gene expression pattern that strikingly mirrored that of ALL with somatic ETV6-RUNX1 fusion. Two ETV6 germline variants gave rise to both acute myeloid leukemia and ALL, with lineage-specific genetic lesions in the leukemia genomes. ETV6 variants compromise its tumor suppressor activity in vitro with specific molecular targets identified by assay for transposase-accessible chromatin sequencing profiling. ETV6-mediated ALL predisposition exemplifies the intricate interactions between inherited and acquired genomic variations in leukemia pathogenesis.

Graphical abstract

Figure 1.

Functional characterization of germline ETV6 variants identified in pediatric ALL. (A) ETV6 transcription repressor activity was determined using a dual-luciferase reporter assay by cotransfecting an ETV6 expression construct, PF4 luciferase reporter construct, and Renilla luciferase reporter construct in HEK293T cells. Bars show the mean of triplicate experiments and repeated at least 3 times; error bars represent standard deviation (SD). (B) Electrophoretic mobility shift assays were performed using nuclear extracts from HEK293T cells ectopically expressing WT or variant ETV6. Bars show the mean of triplicate experiments; error bars represent SD. (C) Western blotting was performed after subcellular fractionation of HEK293T cells ectopically expressing WT or variant ETV6. Bars show the mean of nuclear to cytoplasmic ratio from 3 individual experiments; error bars represent SD. In panels A-C, a 1-way analysis of variance (ANOVA) with multiple comparison was performed to compare each variant with WT (*P < .01). (D) Germline ETV6 variants were classified as damaging or WT-like on the basis of functional characterization as shown in panels A-C.

Figure 2.

Dominant-negative effects of variant ETV6 on WT ETV6. (A) ETV6 transcription repressor activity was measured by cotransfecting HEK293T with constructs encoding WT ETV6, increasing amounts of variant ETV6, and a luciferase reporter. The top panel denotes missense variants, while the bottom panel indicates nonsense or frameshift variants. Bars show the mean of triplicate experiments and repeated at least 3 times; error bars represent SD. **P < .01 in ≥1 condition. (B) HEK293T cells were cotransfected with MYC-tagged WT and FLAG-tagged variant ETV6 constructs followed by pull-down with anti-FLAG beads for western blotting to examine dimerization. Seven representative variants were chosen for this assay. IP, immunoprecipitation. (C) HEK293T cells were cotransfected with MYC-tagged WT and the same 7 FLAG-tagged variant ETV6 constructs as in panel B followed by subcellular fractionation and western blotting of nuclear and cytoplasmic fractions using an anti-MYC antibody. Red bars indicate frameshift variants, orange bar indicates a nonsense variant, and lime green bars indicate missense variants. Bars represent the nuclear-to-cytoplasmic ratio from triplicate experiments; error bars represent SD. *P < .05. In panels A and C, a 1-way ANOVA with multiple comparison was performed to compare each variant with WT.

Figure 3.

Comprehensive genomic profiling of ALL harboring germline ETV6 variants. (A) Heatmap showing major somatic coding mutations and structural variations identified in cases with a germline ETV6 variant or ETV6-RUNX1 fusion. (B-C) Unsupervised hierarchical clustering of global gene expression profile from cases with germline ETV6 variants (B) or with addition of the 231 pediatric ALL cases of diverse subtypes (C). (D) Somatic genomic features in AML and ALL arising from the same ETV6 germline variant.

Figure 4.

Effects of ETV6 variants on oncogenic transformation in vitro. Ba/F3 cells were transduced with constructs encoding WT ETV6, damaging ETV6 (R359X or R399C), WT-like ETV6 (R353Q), and NRAS^G12D, followed by sorting for cells expressing mCherry and ZsGreen double-positive cells and IL-3 withdrawal 48 hours after transduction. (A) Cytokine-independent cell growth was monitored daily as an indicator of transformation in vitro. Data represent the mean from 3 individual experiments; error bars represent SD. Two-way ANOVA with multiple comparison was performed to compare each variant with WT (*P < .05). (B) RNA-seq and (C) ATAC-seq were performed using Ba/F3 cells at 48 hours after IL-3 withdrawal. Cells expressing a damaging ETV6 variant (R359X) exhibited similar RNA-seq and ATAC-seq patterns as cells transduced with empty vector (B). Differential ATAC-seq peaks in Ba/F3 cells expressing a damaging ETV6 variant (R359X) versus WT ETV6 correspond to upregulated genes identified by RNA-seq (C). (D) t-Distributed stochastic neighbor embedding clustering analysis was performed on the 253 ALL cases as shown in Figure 3C using 94 possible target genes identified by RNA-seq and ATAC-seq of Ba/F3 cells.

Figure 5.

Summary of in vitro functional characterization of ETV6 variants. A variety of in vitro assays were performed to comprehensively characterize the impacts of germline ETV6 variants. Dot colors represent different assays; blanks denote that the variant was not tested in the assay. The top panel includes “damaging” variants as determined by impairment of transcriptional repression in luciferase reporter assays. The bottom panel includes WT-like variants, which showed no impairment of transcriptional repression.