Whole Genome Sequencing Identifies Functional Mutations In the Wnt Pathway In CLL

The molecular pathogenesis of chronic lymphocytic leukemia (CLL) has not been well-characterized. Next generation sequencing technologies provide an unprecedented opportunity to screen entire genomes for genetic changes associated with tumorigenesis. To examine the mutational spectrum in CLL, we completed Illumina sequencing of 2 genomes and 4 exomes from patients with advanced CLL in parallel with germline DNA from autologous skin fibroblasts (average coverage 66x/59x for CLL/normal). While the somatic mutation rates varied widely between individuals, ranging from 1–10 per megabase, we discovered that 2 of 6 tumors harbored novel nonsynonymous mutations in Wnt pathway members (DKK2 [R197H] from Patient 1 and BCL9 [G458S] from Patient 3), suggesting that perturbation of this pathway may be critical in CLL. By targeted quantitative RNA sequencing, we detected both mutated DKK2 and BCL9 as heterozygous mutations in tumor RNA from Patients 1 and 3, respectively.

We confirmed that the Wnt pathway is in fact highly dysregulated in CLL based on gene expression analysis of 141 Wnt pathway members, in a dataset comprised of 155 CLL and 20 normal CD19⁺ B cell samples. In CLL cells, Wnt family members (Wnt1, Wnt2, Wnt2B, Wnt3, Wnt4, Wnt5B, Wnt9A, Wnt10A, and Wnt16) and Wnt receptors (FZD3, FZD8, FZD9, LRP6) were overexpressed. Additionally, we observed downregulation of Wnt pathway repressors (TLE1, SOX4 and NKD1) and upregulation of activators (SMAD2, DVL2, DVL3 and PYGO1). Expression of the downstream transcription factor LEF1 was strikingly elevated in CLL, and we measured consistent 3–4 fold elevation of LEF1 protein in CLL-B cells (n=10) compared to normal B cells (n=5) by intracellular staining (p= 0.002). Patient CLL cells demonstrated 5-fold greater beta-catenin mediated activation of the Wnt pathway in CLL compared to normal B cells (p=0.01), as measured by direct introduction of the LEF1/TCF1 luciferase reporter genes into primary cells. Silencing of LEF1 in CLL cells (n=5) led to increased cell death in vitro by 70–90%. Collectively, these results demonstrate that Wnt pathway activation and dysregulation contributes to CLL survival.

To examine the possible effects of mutated DKK2 and BCL9 on Wnt pathway activity, we introduced plasmids encoding wild-type or mutated genes, together with those encoding Wnt1 protein and Wnt reporter genes, into 293T cells. DKK2 is a secreted factor that normally represses Wnt activation by competing with Wnt proteins for binding to its surface receptors. We observed that DKK2 [R197H] is mutated in its second cysteine-rich domain, which is necessary for its interaction with the Wnt receptor. BCL9 interacts with the LEF1/TCF transcriptional factor complex and has been described to have both repressive and activating effects. In 3 of 3 experiments, we observed that the mutated forms of both genes lost their repressive activity. The loss of repression generated for both mutated genes was most marked in the presence of low concentrations of Wnt protein. Moreover, both mutated genes act as dominant alleles, especially in the presence of low concentrations of Wnt protein.

Since DKK2 is a secreted factor, we examined the effects of plasma of Patient 1 on Wnt pathway activity. In both normal donors (n=4) and CLL patients (n=4), DKK2 protein could be consistently detected at equivalent amounts in blood plasma following immunoprecipitation and immunoblotting with DKK2-specific antibodies Co-culture of 293T cells expressing the Wnt pathway luciferase reporters with Patient 1 plasma (2-10%) resulted in dramatic elevation of Wnt activation, which was abolished by prior depletion of DKK2. We observed similar Wnt activation when testing plasma from 22 of 93 CLL patients, but not from 5 normal volunteers. Further sequencing of 100 Wnt pathway members from 60 additional CLL patients is in progress to define the extent to which somatic mutation is a common mechanism by which this pathway is altered.

Our studies suggest that Wnt pathway inhibitors represent a promising therapeutic approach for CLL.

No relevant conflicts of interest to declare.