13q12.2 deletions in acute lymphoblastic leukemia lead to upregulation of FLT3 through enhancer hijacking

Abstract

Mutations in the FMS-like tyrosine kinase 3 (FLT3) gene in 13q12.2 are among the most common driver events in acute leukemia, leading to increased cell proliferation and survival through activation of the phosphatidylinositol 3-kinase/AKT-, RAS/MAPK-, and STAT5-signaling pathways. In this study, we examine the pathogenetic impact of somatic hemizygous 13q12.2 microdeletions in B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) using 5 different patient cohorts (in total including 1418 cases). The 13q12.2 deletions occur immediately 5' of FLT3 and involve the PAN3 locus. By detailed analysis of the 13q12.2 segment, we show that the deletions lead to loss of a topologically associating domain border and an enhancer of FLT3. This results in increased cis interactions between the FLT3 promoter and another enhancer located distally to the deletion breakpoints, with subsequent allele-specific upregulation of FLT3 expression, expected to lead to ligand-independent activation of the receptor and downstream signaling. The 13q12.2 deletions are highly enriched in the high-hyperdiploid BCP ALL subtype (frequency 3.9% vs 0.5% in other BCP ALL) and in cases that subsequently relapsed. Taken together, our study describes a novel mechanism of FLT3 involvement in leukemogenesis by upregulation via chromatin remodeling and enhancer hijacking. These data further emphasize the role of FLT3 as a driver gene in BCP ALL.

Graphical abstract

Figure 1.

Genetic mapping and epigenetic landscape of the 13q12.2 locus. Map of the FLT3 and PAN3 loci in 13q12.2. Predicted topologically associating domains based on data from the lymphoblastic GM12878 cell line are indicated by diagonal dashed lines. (A) Hemizygous deletions identified in 27 BCP ALL samples by WGS (black) or SNP array or WES (gray). The violet square corresponds to the minimally deleted region. (B) DNase-seq signal based on the GM12878 cell line. (C) ATAC-seq signal based on the NALM-6 cell line. (D) ChIP-seq of histone modifications (H3K27ac and H3K4me3) based on the GM12878 (E) and the NALM-6 cell lines. (F) H3K27ac- and H3K4me3-enriched PLAC-seq interaction map based on the NALM-6 cell line. (G) Relative interaction frequencies in 13q12.2 in 6 primary ALL cases and the GM12878 cell line based on Hi-C, where higher values on the y-axis correspond to stronger interaction. Increased interactions with the enhancer element DS3 are seen in the case with 13q12.2 deletion.

Figure 2.

Gene-expression analysis of high-hyperdiploid cases with 13q12.2 deletions. RNA expression of the FLT3 and PAN3 genes in cases with and without 13q12.2 deletions.

Figure 3.

Allele-specific expression analysis of FLT3 in BCP ALL. (A) Scatterplot of allelic ratio (x-axis) and log-scale binomial P values (y-axis) of FLT3 in BCP ALL (n = 77) with informative SNVs. The horizontal line represents the binomial P value of .05 (log₁₀ scale) and the vertical lines represent the ratio for the expressed reference/nonreference allele (log₂ scale). All 7 cases with clonal 13q12.2 deletions displayed allele-specific expression of FLT3 (top right quadrant). (B) FLT3 mutant allele frequencies observed by RNA-seq (y-axis) and by genomic-sequencing data (x-axis) of 9 BCP ALL cases. The line y=x is shown in black. The 3 cases with concurrent FLT3 mutation and 13q12.2 deletion showed increased expression of the mutated allele.

Figure 4.

Changes in TADs and enhancer hijacking in ALL with 13q12.2 deletion. (A) Juicer KR-normalized Hi‐C interaction heatmaps at 13q12.2 (chr13:28.0 to 29.5 Mb) of the GM12878 cell line, the NALM-6 cell line, and 2 primary high-hyperdiploid ALL cases (25‐kb resolution). The black arrows indicate the TAD boundary between DS1 and DS3 and the red arrow indicates the disruption of this TAD boundary in the case with 13q12.2 deletion (case 3). (B) Insulation score profile at 13q12.2 derived from chromatin interaction sequencing of 6 primary ALL cases. The case with 13q12.2 deletion (case 3) displays a deviant insulation score in this region (blue rectangle), corresponding to aberrant chromatin organization likely due to loss of a TAD boundary. (C) Schematic figure of chromatin remodeling and enhancer hijacking in ALL with 13q12.2 deletion. In normal hematopoietic cells, FLT3 expression is primarily controlled by interactions between the FLT3 promoter element DS1 with the DS2 enhancer located in the 5′ region of PAN3 (top panel). In cells with 13q12.2 deletion, a TAD boundary is lost together with DS2, resulting in increased interactions between DS1 and the upstream enhancer element DS3, thereby upregulating FLT3 messenger RNA expression (bottom panel).