Somatic mutation as a mechanism of Wnt/β-catenin pathway activation in CLL

Abstract

One major goal of cancer genome sequencing is to identify key genes and pathways that drive tumor pathogenesis. Although many studies have identified candidate driver genes based on recurrence of mutations in individual genes, subsets of genes with nonrecurrent mutations may also be defined as putative drivers if they affect a single biological pathway. In this fashion, we previously identified Wnt signaling as significantly mutated through large-scale massively parallel DNA sequencing of chronic lymphocytic leukemia (CLL). Here, we use a novel method of biomolecule delivery, vertical silicon nanowires, to efficiently introduce small interfering RNAs into CLL cells, and interrogate the effects of 8 of 15 mutated Wnt pathway members identified across 91 CLLs. In HEK293T cells, mutations in 2 genes did not generate functional changes, 3 led to dysregulated pathway activation, and 3 led to further activation or loss of repression of pathway activation. Silencing 4 of 8 mutated genes in CLL samples harboring the mutated alleles resulted in reduced viability compared with leukemia samples with wild-type alleles. We demonstrate that somatic mutations in CLL can generate dependence on this pathway for survival. These findings support the notion that nonrecurrent mutations at different nodes of the Wnt pathway can contribute to leukemogenesis.

Figure 1

The Wnt pathway is significantly mutated in CLL (P = .00067). (A) Cellular localization of mutations in Wnt pathway components in CLL (light red). *Pathway genes mutated in more than 1 CLL sample. (B) Clinical characteristics of CLL samples harboring Wnt pathway mutations, as well as the putative function of the mutated Wnt pathway genes and their genomic localization. A, pathway activator; R, pathway repressor.

Figure 2

Significantly mutated Wnt pathway genes. Type (missense, splice site, nonsense) and localization of mutations in the 12 unique Wnt pathway genes identified in CLL cases (top) compared with previously reported mutations in the literature or within the COSMIC database (version 64) (bottom).

Figure 3

Wnt pathway mutations do not contribute to the extent of dysregulated gene expression of Wnt pathway. (A) Expression profiles (from Affymetrix U133Plus2 arrays) of 60 Wnt pathway members that are significantly differentially expressed in 179 CLL-B cells compared with 24 normal CD19⁺ B cells (according to FDR corrected permutation test P values assessing significance of Student t test scores, FDR ≤0.05, with Student t test score ranging from −13.6-30.9). All tumor samples were comprised of >95% tumor purity. Genes were visualized in GENE-E. Wnt pathway genes downregulated in CLL are shown in the heatmap (top); Wnt pathway genes upregulated in CLL are shown (bottom). From these 60 genes, a “Wnt score” was calculated as a statistical measure of differential expression between the 37 known Wnt activators in the set (labeled in orange, in the column [right] of the heatmap) vs the 23 known repressors (labeled in black, separate column [right] of the heatmap). We generated a Student t test score for each CLL sample by comparing the differential expression of activators that are upregulated to repressors that are downregulated in each sample according to FDR-corrected permutation test P values assessing significance of Student t test scores, with FDR ≤0.05. Wnt scores ranged from −2.62 (blue) to 2.91 (red). Compared with normal B cells, overall, CLL cells demonstrate downregulation of Wnt pathway repressors and upregulation of Wnt pathway activators. (Bottom) CLL sample characteristics and whether samples also underwent whole-exome (WES) or whole-genome sequencing (WGS) (black-positive; white-negative). The rank of genes represented within the top 9% of genes differentially expressed array-wide is noted (in parentheses next to the gene names). Expression levels are log2 transformed and mean-centered for each gene for visualization. Induced levels are represented in red, repressed levels in blue, and no change is represented in white, in which levels are saturated at −0.5 and +0.5. (B) Unsupervised hierarchical clustering of Wnt pathway gene expression profiles from 12 mutated (with arrow) and 58 unmutated CLL-B cells (without arrow), performed using Pearson linear correlation with average linkage. (Right) Activators and repressors are shown (orange and black, respectively). (Upper panel) Wnt pathway genes and (lower panel) Wnt target genes, curated from the literature. A supervised analysis between the Wnt pathway mutated vs wild-type samples is presented in supplemental Figure 4. (C) The Wnt signaling pathway is chronically active in CLL-B cells. Normal and CLL-B cells were co-transfected with SuperTOPflash and pRL-TK constructs to measure endogenous TCF/LEF activity. CLL-B cells have fivefold greater normalized luciferase activity compared with normal B cells (n = 5; P < .01, Wilcoxon test, 2-tailed).

Figure 4

The expression of core Wnt pathway components is required for CLL survival. (A) Scanning electron micrographs (SEMs) of normal CD19⁺ B (left panels) and CLL-B cells (right panels) atop NWs taken 24 hours after plating. (B) Normal CD19⁺ B cells can grow and divide on NWs. Normal CD19⁺ B cells isolated from peripheral blood of healthy adult volunteers were stimulated with IL4 (2 ng/mL) and CD40L (0.1 mg/mLl) either in vitro (“culture”) or atop NWs (“SiNWs”) for 48 hours. Cell proliferation was measured using an adenosine triphosphate (ATP)-dependent Cell-Titer Glo assay. Cell growth rate was calculated based on the measurement of ATP amount after 48 hours of stimulation, normalized relative to the day 0 value. (Inset) Scanning electron microscope image showing that proliferating B cells on NWs grow in clusters. (C) Confocal scanning images of Alexa Fluor 546-labeled human anti-vimentin siRNA delivered into CLL-B cells. (Upper panel) siRNA delivery is calculated by manually counting the number of cells that have higher levels of fluorescent siRNAs compared with untransfected controls (not shown here). Alexa Fluor 546-labeled siRNA is shown (orange), whereas cell membranes are shown outlined (gray). (Lower panel) Viability was calculated as a percentage of the number of live cells in the total cells using a live–dead cell staining method. Intact cells (stained with Calcein-AM) are shown (green), whereas the nuclei of dead cells are shown in magenta (stained with EthD-1). (D) Core Wnt pathway components can be silenced in HEK293T using siRNA delivery. Gene expression of Wnt pathway members DVL1, CTNNB1, and LEF1 (relative to glyceraldehyde-3-phosphate dehydrogenase expression) were analyzed by quantitative Taqman RT-PCR using complementary DNA derived from HEK293T cells that were either untransfected (“untreated,” white bars) or transfected for 48 hours with control nontargeting siRNA (“control,” pink bars) or siRNA specific for DVL1, CTNNB1, or LEF1 (dark red bars). (E) Efficient knockdown of protein expression of Wnt pathway members in normal CD19⁺ and CLL-B cells via NW-mediated siRNA delivery. Representative images of target protein expression, detected by immunofluorescence 48 hours after siRNA delivery using gene-specific antibodies against the target proteins are shown. (F) Median decrease in cell survival (measured by CellTiter Glo) 48 hours after NW-mediated delivery of siRNAs against LEF1, DVL, and CTNNB1 in normal CD19⁺ (n = 3) and CLL-B (n = 3) compared with silencing using nontargeting siRNA controls (2 different siRNAs per target gene). Percentage of cell survival was normalized to ATP amount at day 0.

Figure 5

Heterozygous mutations alter Wnt pathway activities in HEK293T cells. (A,C,E) HEK293T cells were cotransfected with Wnt1 expression plasmid (amounts indicated), wild-type (WT), or mutant (MT), or equal amounts of WT and MT plasmids, along with the reporter plasmids SuperTOPflash and pRL-TK. Forty-eight hours after transfection, luciferase activity was measured from 3 independent experiments. All WT, MT, or WT/MT plasmids were introduced at 0.1 ng, 0.1 ng, 50 ng, and 5 ng for BCL9, DKK2, CSNK1E, and FZD5, respectively. LRP6 plasmid (10 ng) was also included in the FZD5 mutation characterization. MT1: Y46*; MT2: V290I. Downstream Wnt pathway targets were also assessed for mutated DKK2 by gene expression (see supplemental Figure 7 and supplemental Methods). (B) HEK293T cells were cotransfected with 20 or 80 ng of WT or MT RYK along with reporter plasmids. At 24 hours after the transfection, recombinant Wnt3a was added (25 ng/mL final concentration) and incubated for 24 hours before luciferase activity was measured. Detection of phosphorylation of downstream target DVL2 was assessed by western blot analysis (see supplemental Figure 7 and supplemental Methods). (D) HEK293T cells were cotransfected with either WT or MT WNT1 along with the reporter plasmids. Luciferase activity was measured from three independent experiments 48 hours after transfection. For more details on the conditions of Wnt activation, please see supplemental Methods.

Figure 6

Increased dependence on Wnt signaling by CLL samples harboring Wnt pathway mutations. Silencing of BCL9, RYK, and CSNK1E protein in normal CD19⁺ B cells 48 hours after NW-mediated delivery of gene-specific siRNAs compared with nontargeting control siRNAs (“control”) was confirmed by gene-specific immunofluorescence staining (red) and visualized by confocal microscopy (A,C,D) (top panels). Nuclei were probed with DAPI (blue). Protein level silencing efficiency was estimated using Image J software. Per gene, 2 different targeting siRNAs were tested and the representative results are shown. (B) (Top) Silencing of DKK2 in HEK293T cells using gene-specific siRNAs detected by quantitative Taqman RT-PCR of complementary DNA derived from HEK293T cells that were either untreated (white bar) or treated with control nontargeting siRNA (“control”, gray bars) or with siRNA specific for DKK2 (black bars). (A-D) (Lower panels) Cell survival rate was normalized to nontargeting control in normal B cells (n = 4 for BCL9 and DKK2; n = 2 for RYK and CSNK1E), the CLL B cells with either mutated BCL9 (P48), DKK2 (P46), RYK (P35), or CSNK1E (P42) (all n = 1) or CLL-B samples without Wnt pathway mutations (n = 7 for BCL9 and DKK2; n = 5 for RYK and CSNK1E), using the Cell-Titer Glo assay 48 hours after NW-mediated siRNA delivery. Three replicates per independent sample were performed.