Somatic Mutation As a Mechanism of Wnt/β-Catenin Pathway Activation in CLL

Abstract 559

One goal of cancer genome sequencing is to identify key genes and pathways that drive tumor pathogenesis. While many studies identify driver genes based on recurrence of mutations in individual genes, subsets of genes with non-recurrent mutations may also be defined as drivers if they affect components of a single biological pathway. By large-scale DNA sequencing, we recently identified the Wnt pathway as a significantly mutated pathway in chronic lymphocytic leukemia (CLL) (Wang et al NEJM 2011). This pathway is known to be critical for hematopoietic stem cell development and differentiation, and is highly dysregulated in CLL. However, the functional alterations generated by specific mutations in members of this pathway and their contributions to CLL pathogenesis have not been previously characterized.

In our previously reported DNA sequencing of 91 CLL samples, we identified a broad spectrum of mutated Wnt pathway members. Fifteen non-synonymous coding mutations were identified in 13 CLL samples (1–2 mutations per sample) within core Wnt pathway members. These nonsilent coding mutations involved components of the entire Wnt pathway–ranging from extracellular factors (DKK2, WNT1, WNT10A, RSPO4, CSNK1E), to cell surface receptors (FZD5, RYK), cytoplasmic factors (CSNK1E, PRICKLE1) and nuclear transcriptional factors (CHD8, BRD7, CREBBP, BCL9). All of the identified Wnt pathway mutations localized to evolutionarily conserved sites, supporting a role for these mutations in perturbing Wnt pathway function.

To assess the contribution of the Wnt pathway to CLL-B cell survival, we optimized a novel miniaturized transfection platform, silicon nanowires (SiNWs), to deliver siRNAs targeting core Wnt pathway members into normal and CLL-B cells. By exploring various nanowire sizes, lengths and densities, we achieved conditions to consistently deliver siRNAs into B cells (90%) without impacting cellular viability (>95%). Using SiNW-mediated delivery of gene-specific siRNAs, we established that silencing of the Wnt pathway members LEF1, CTTNB1, and DVL1 in CLL samples (n=3) resulted in greater loss of cell viability (by∼20–35%) compared to delivery of non-targeting controls (P=0.06, 0.01, and 0.005, respectively). These results demonstrate that perturbation of key nodes in the Wnt pathway reduce CLL-B cell survival, and support the importance of this pathway in CLL.

We then explored whether mutated Wnt pathway members could functionally alter Wnt pathway signaling. We focused our studies on two mutated Wnt pathway members, DKK2-R197H and BCL9-G548S. We generated plasmids expressing either wildtype or mutant alleles and measured Wnt pathway activation in 293T cells using a TCF/LEF-dependent luciferase reporter system following coexpression of WNT1 with the wildtype or mutant gene or both. For both DKK2 and BCL9, mutated forms abolished their normal repressive effects on Wnt activation. Elimination of the repressive effects of the wildtype protein was also observed when the wildtype and mutant alleles were expressed together, suggesting a dominant activating effect of the mutated allele. We hypothesized that chronic pathway activation by these mutations could lead to dependence of patient CLL cells on Wnt signaling for their survival. We directly addressed this question by examining the survival of normal B cells (n=4) and patient CLL-B cells with or without (n=7) mutated DKK2 or BCL9 following SiNW-mediated delivery of gene-specific siRNAs. This revealed that a BCL9-mutated CLL sample was more dependent on BCL9 expression for survival than normal B cells (p=0.02) or CLL samples lacking Wnt pathway mutations (p=0.07). Similarly, silencing of DKK2 in a DKK2- mutated CLL sample led to greater cell death than gene silencing in normal B cells, or in CLL samples without Wnt pathway mutations (p=0.003). These results demonstrate that CLL samples harboring mutations in DKK2 or BCL9 exhibit greater dependency on Wnt pathway signaling than samples without Wnt pathway mutations, and support a driving role for these mutations in CLL.

Taken together, our results demonstrate that somatic mutations in Wnt pathway components in CLL-B cells alter the activity of this pathway and increase its sensitivity to pathway inhibition. Our findings further support the notion that non-recurrent mutations at different nodes of the Wnt pathway contribute to CLL pathogenesis.