Activation of the Wnt signaling pathway in chronic lymphocytic leukemia

Abstract The canonical Wnt signaling pathway is pathogenic in a variety of cancers. We previously identified aberrant expression of the Wnt pathway transcription factor and target gene lymphoid enhancer binding factor-1 (LEF1) in chronic lymphocytic leukemia (CLL). This suggested that the Wnt signaling pathway has a role in the biology of CLL. In this study, we performed a Wnt pathway analysis using gene expression profiling and identified aberrant regulation of Wnt pathway target genes, ligands, and signaling members in CLL cells. Furthermore, we identified aberrant protein expression of LEF-1 specifically in CLL but not in normal mature B-cell subsets or after B-cell activation. Using the T cell–specific transcription factor/LEF (TCF/LEF) dual luciferase reporter assay, we demonstrated constitutive Wnt pathway activation in CLL, although the pathway was inactive in normal peripheral B cells. Importantly, LEF-1 knockdown decreased CLL B-cell survival. We also identified LEF-1 expression in CD19+/CD5+ cells obtained from patients with monoclonal B-cell lymphocytosis, suggesting a role for LEF-1 early in CLL leukemogenesis. This study has identified the constitutive activation and prosurvival function of LEF-1 and the Wnt pathway in CLL and uncovered a possible role for these factors in the preleukemic state of monoclonal B-cell lymphocytosis.

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Cirmtuzumab Inhibits Non-Canonical Wnt Signaling without Enhancing Canonical Wnt/β-Catenin Signaling in Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood-2018-99-119606 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2652-2652

Author(s):

Han Zhang ◽

Suping Zhang ◽

Emanuela M. Ghia ◽

Michael Y. Choi ◽

Jieyu Zhang ◽

...

Keyword(s):

Stem Cell ◽

Chronic Lymphocytic Leukemia ◽

Wnt Signaling ◽

Signaling Pathway ◽

Wnt Signaling Pathway ◽

Lymphocytic Leukemia ◽

Canonical Wnt Signaling ◽

Gene Set Enrichment ◽

Canonical Wnt ◽

Cell Stem Cell

Abstract ROR1 is a receptor tyrosine kinase-like orphan receptor for Wnt5a that is expressed by cells during embryogenesis and by the neoplastic cells of a variety of cancers, including chronic lymphocytic leukemia (CLL). ROR1 can induce activation of β-catenin-independent non-canonical Wnt-signaling. Studies reveal a cross-talk between the non-canonical Wnt-signaling pathway and the β-catenin-dependent canonical Wnt-signaling pathway, which we previously found was also activated in CLL (Lu D, et al, PNAS 101:31118-3123, 2004). Prior studies indicated that silencing a related Wnt5a receptor, ROR2, could augment canonical Wnt-signaling induced by Wnt3a. In this study, we examined whether genetic silencing of ROR1 or inhibition of ROR1-signaling also could influence canonical Wnt signaling. To inhibit ROR1 signaling we used the humanized anti-ROR1 mAb cirmtuzumab, which is being evaluated in patients with CLL (Choi MY, et al, Cell Stem Cell, 22:951, 2018). Surprisingly, we found that CRISPR/Cas9 deletion of ROR1 in 293T cells also could enhance the capacity of Wnt3a to activate canonical Wnt-signaling, albeit to a lesser extent than CRISPR/Cas9 deletion of ROR2; conversely, re-introduction of ROR1 into ROR1-deleted 293T cells suppressed Wnt3a-induced activation of canonical Wnt-signaling. In contrast, treatment of wildtype 293T cells with cirmtuzumab did not enhance Wnt3a-induced activation of canonical Wnt-signaling, but nonetheless suppressed ROR1-dependent non-canonical signaling induced by Wnt5a. We examined the influence of ROR1 on canonical Wnt-signaling in CLL. First, we examined whether the relative expression of ROR1 influenced the relative levels of genes induced by activation of the canonical Wnt-signaling pathway. Gene set enrichment analysis (GSEA) of gene-expression data on CLL cells of different patients (n=448, GSE13204) revealed that CLL cells with low-level expression of ROR1 (ROR1Low) did not have increased levels of genes induced by activation of canonical Wnt-signaling relative to those noted in CLL cells with high-level expression of ROR1 (ROR1Hi). Nonetheless, ROR1Hi CLL cells did have increased levels of genes induced by activation of non-canonical Wnt signaling compared to ROR1Low CLL cells. As in 293T cells, siRNA-mediated knockdown of ROR1 in ROR1Hi CLL cells could enhance the capacity of Wnt3a to increase the levels of genes induced by canonical Wnt-signaling (e.g. MYC, CCND1). However, treatment of the same CLL cells with cirmtuzumab did not enhance the levels of such genes in response to Wnt3a, even at concentrations that exceeded those required to inhibit Wnt5a-induced ROR1-dependent non-canonical Wnt-signaling. We examined whether these findings also applied to CLL cells treated with cirmtuzumab in vivo. For this, we performed gene-set enrichment analyses on the transcriptomes of CLL cells collected from patients before and after treatment with cirmtuzumab in a recently completed phase I clinical trial (Choi MY, et al, Cell Stem Cell, 22:951, 2018). Although treatment with cirmtuzumab repressed expression of genes induced by activation of non-canonical Wnt signaling, we did not observe changes in the levels of genes induced by activation of the canonical Wnt signaling pathway. Collectively, this study demonstrates that cirmtuzumab can inhibit non-canonical Wnt signaling without enhancing canonical Wnt signaling in CLL, in contrast to what we observed in CLL cells silenced for ROR1. As such, treatment with cirmtuzumab may represent a more selective approach to suppressing non-canonical Wnt5a signaling than strategies aimed at genetic down-modulation or silencing of ROR1. Disclosures Choi: Rigel: Consultancy; Pharmacyclics: Consultancy, Research Funding, Speakers Bureau; Gilead: Speakers Bureau; AbbVie, Inc: Consultancy, Speakers Bureau; Genentech: Speakers Bureau.

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Upregulation of Genes Involved in the Beta-Catenin Signaling Pathway in a Mouse Model of Chronic Lymphocytic Leukemia.

Blood ◽

10.1182/blood.v104.11.1121.1121 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1121-1121

Author(s):

Qingli Wu ◽

Erik A. Ranheim

Keyword(s):

Bone Marrow ◽

B Cells ◽

Chronic Lymphocytic Leukemia ◽

Mouse Model ◽

Signaling Pathway ◽

Wnt Signaling Pathway ◽

Lymphocytic Leukemia ◽

Beta Catenin ◽

Rt Pcr ◽

Canonical Wnt

Abstract Binding of Wnt ligands to Frizzled (Fzd) family and LRP5/6 receptors results in stabilization of beta-catenin protein, its translocation to the nucleus, and in association with LEF/TCF family transcription factors, expression of target genes. This canonical Wnt signaling pathway plays a critical role in development and in the maintenance of tissue specific stem cell populations in the skin, gut, and bone marrow. Dysregulation of the beta-catenin signaling pathway has been described in numerous human malignancies, including chronic lymphocytic leukemia (CLL). Using the Eμ-TCL1 transgenic mouse model of CLL developed by C. Croce et al., we have examined expression of various components of the canonical Wnt pathway during lymphomagenesis by RT-PCR. The Eμ-TCL1 mouse spontaneously develops a hyperplasia of CD5+ B cells in the peripheral blood and peritoneum which progresses towards a monoclonal B cell leukemia/lymphoma with infiltration of spleen, bone marrow, and other organs. This provides the ability, largely lacking in human subjects, to analyze changes in gene expression during development of a lymphoid malignancy. We have FACS sorted normal CD5- B cells as well as CD5+ B cells from the hyperplastic, oligoclonal and malignant, monoclonal phases of leukemia development in Eμ-TCL1 mice and assessed their expression of various members of the Wnt, Fzd, LRP coreceptor, and LEF/TCF transcription factor families. Coreceptor LRP6 is expressed in all populations in approximately equal amounts as determined by quantitative RT-PCR. We find no expression of Fzd receptors 1, 2, 3, 4, or 9, nor Wnt4 or 5a in any population. We find mRNA for Fzd6 and LEF-1 are increased in hyperplastic and malignant CD5+ B cells in Eμ-TCL1 mice as compared to CD5- polyclonal B cells from the same animals. Of interest, both LEF-1 and Fzd6 expression also have been shown to be upregulated in human CLL B cells. To further assess Fzd6 expression during malignant transformation, we took advantage of the fact that only 3–5% of mouse B cells express lambda light chain. Therefore, large expansions of lambda expressing CD5+ B cells are most likely to be monoclonal in Eμ-TCL1 mice. In three separate mice, oligoclonal expansions of CD5+lambda- B cells coexisted with a clonal expansion of CD5+lambda+ cells, as confirmed by PCR analysis of germline immunoglobulin DNA. These two populations, as well as polyclonal CD5- B cells, were purified by FACS; and Fzd6 expression was quantified by real time RT-PCR. Fzd6 mRNA was increased in the oligoclonal CD5+ B cells by an average of 15.9 fold over the normal CD5- B cells (range 4.1-36.9). In the monoclonal lambda expressing subset, Fzd6 expression was increased 41.7 fold (range 23.7–68.3) over CD5- B cells, and increased over that seen in the oligoclonal CD5+ B cells of the same animal by 4.1 fold (range 1.9–5.7). These findings show that progressively increased expression of Fzd6 correlates with malignant transformation in B cell leukemogenesis in the Eμ-TCL1 mouse model and parallels the findings in fully transformed CLL B cells in humans. The functional consequences of enhanced Fzd6 and other components of the canonical Wnt signaling pathway in neoplastic B cells are currently being investigated.

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