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Wnt5a induces ROR1 to associate with 14-3-3ζ for enhanced chemotaxis and proliferation of chronic lymphocytic leukemia cells.

ABSTRACT: Wnt5a can activate Rho GTPases in chronic lymphocytic leukemia (CLL) cells by inducing the recruitment of ARHGEF2 to ROR1. Mass spectrometry on immune precipitates of Wnt5a-activated ROR1 identified 14-3-3ζ, which was confirmed by co-immunoprecipitation. The capacity of Wnt5a to induce ROR1 to complex with 14-3-3ζ could be blocked in CLL cells by treatment with cirmtuzumab, a humanized mAb targeting ROR1. Silencing 14-3-3ζ via small interfering RNA impaired the capacity of Wnt5a to: (1) induce recruitment of ARHGEF2 to ROR1, (2) enhance in vitro exchange activity of ARHGEF2 and (3) induce activation of RhoA and Rac1 in CLL cells. Furthermore, CRISPR/Cas9 deletion of 14-3-3ζ in ROR1-negative CLL cell-line MEC1, and in MEC1 cells transfected to express ROR1 (MEC1-ROR1), demonstrated that 14-3-3ζ was necessary for the growth/engraftment advantage of MEC1-ROR1 over MEC1 cells. We identified a binding motif (RSPS⁸⁵⁷SAS) in ROR1 for 14-3-3ζ. Site-directed mutagenesis of ROR1 demonstrated that serine-857 was required for the recruitment of 14-3-3ζ and ARHGEF2 to ROR1, and activation of RhoA and Rac1. Collectively, this study reveals that 14-3-3ζ plays a critical role in Wnt5a/ROR1 signaling, leading to enhanced CLL migration and proliferation.

SUBMITTER: Yu J

PROVIDER: S-EPMC5670032 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Wnt5a induces ROR1/ROR2 heterooligomerization to enhance leukemia chemotaxis and proliferation.

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Wnt5a enhances proliferation of chronic lymphocytic leukemia and ERK1/2 phosphorylation via a ROR1/DOCK2-dependent mechanism.

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High expression level of ROR1 and ROR1-signaling associates with venetoclax resistance in chronic lymphocytic leukemia.

Project description:Although the BH3-mimetic venetoclax is highly cytotoxic for chronic lymphocytic leukemia (CLL) cells, some patients with CLL fail to clear minimal residual disease (MRD). We examined the CLL cells of seven such patients (CLL1-7) and found each had high-level expression of ROR1. By examining the CLL cells from such patients prior to therapy at SC1 and then more than 1 year later (Sample Collection 2 (SC2)), when they had progressive increases in MRD despite continued venetoclax therapy, we found the levels of ROR1 expressed on CLL cells at SC2 were significantly higher than that on CLL cells collected at SC1. At SC2, we also observed upregulation of genes induced by Wnt5a-induced ROR1 signaling, including BCL2L1. Transduction of the CLL-cell-line MEC1 to express ROR1 enhanced expression of target genes induced by ROR1-signaling, increased expression of BCL-XL, and enhanced resistance to venetoclax, even in MEC1 made to express mutant forms of BCL2, which are associated with venetoclax resistance. Treatment of primary CLL cells with Wnt5a also increased their resistance to venetoclax, an effect that could be inhibited by the anti-ROR1 mAb (UC-961, zilovertamab). Collectively, these studies indicate that Wnt5a-induced ROR1-signaling can enhance resistance to venetoclax therapy.

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STAT3-Induced Wnt5a Provides Chronic Lymphocytic Leukemia Cells with Survival Advantage.

Project description:The wingless and integration site growth factor-5a (Wnt5a) is a ligand of the receptor tyrosine kinase-like orphan receptor-1 (ROR1). Because both Wnt5a and ROR1 are expressed in circulating chronic lymphocytic leukemia (CLL) cells, and because in other cell types, STAT3, which is constitutively activated in CLL, induces Wnt5a signaling, we wondered whether STAT3 induces the expression of Wnt5a in CLL cells. Sequence analysis detected four putative STAT3 binding sites in close proximity to the Wnt5a gene promoter's start codon. Chromatin immunoprecipitation and EMSA revealed that STAT3 binds to the Wnt5a gene promoter, and a luciferase assay showed that STAT3 activates the Wnt5a gene. Additionally, transfection of peripheral blood CLL cells with STAT3 short hairpin RNA downregulated Wnt5a mRNA and protein levels, suggesting that STAT3 binds to the Wnt5a gene promoter and induces the expression of Wnt5a in CLL cells. Flow cytometry and confocal microscopy determined that both Wnt5a and its receptor ROR1 are coexpressed on the surface of CLL cells, and Western immunoblotting showed an inverse correlation between Wnt5a and ROR1 protein levels, implying that, regardless of CLL cells' ROR1 levels, blocking the interaction between Wnt5a and ROR1 might be beneficial to patients with CLL. Indeed, transfection of CLL cells with Wnt5a small interfering RNA reduced Wnt5a mRNA and protein levels and significantly increased the spontaneous apoptotic rate of CLL cells. Taken together, our data unravel an autonomous STAT3-driven prosurvival circuit that provides circulating CLL cells with a microenvironment-independent survival advantage.

| S-EPMC6864283 | biostudies-literature

The tyrosine kinase receptor ROR1 is constitutively phosphorylated in chronic lymphocytic leukemia (CLL) cells.

Project description:Phosphorylation of receptor tyrosine kinases (RTKs) has a key role in cellular functions contributing to the malignant phenotype of tumor cells. We and others have previously demonstrated that RTK ROR1 is overexpressed in chronic lymphocytic leukemia (CLL). Silencing siRNA downregulated ROR1 and induced apoptosis of CLL cells. In the present study we analysed ROR1 isoforms and the phosphorylation pattern in CLL cells (n=38) applying western blot and flow-cytometry using anti-ROR1 antibodies and an anti-phospho-ROR1 antibody against the TK domain. Two major ROR1 bands with the size of 105 and 130 kDa respectively were identified, presumably representing unglycosylated (immature) and glycosylated (mature) ROR1 respectively as well as a 260 kDa band which may represent dimerized ROR1. A ROR1 band of 64 kDa that may correspond to a C-terminal fragment was also noted, present only in the nucleus. The 105 kDa ROR1 isoform was more frequently expressed in non-progressive as compared to progressive CLL patients (p=0.03). The 64, 105, 130 and 260 kDa bands were constitutively phosphorylated both at tyrosine and serine residues. Phosphorylation intensity of the mature (130 kDa) isoform was significantly higher in progressive than in non-progressive disease (p<0.001). Incubation of CLL cells with a mouse anti-ROR1 KNG or an anti-ROR1 CRD mAb respectively induced dephosphorylation of ROR1 before entering apoptosis. In conclusion CLL cells expressed different isoforms of ROR1 which were constitutively phosphorylated. The mature, phosphorylated ROR1 isoform was associated with a progressive disease stage. Targeting ROR1 by mAbs induced specific dephosphorylation and leukemic cell death. ROR1 might be an interesting therapeutic target.

| S-EPMC3813472 | biostudies-literature

High Expression Level of ROR1 and ROR1-signaling Associates With Venetoclax Resistance In Chronic Lymphocytic Leukemia

Project description:We examined the CLL cells transcriptome data of six patients who fail to clear their leukemia cells and develop progressive disease on venetoclax therapy. Methods: CLL cells were collected prior to venetoclax therapy (Sample Collection 1 (SC1)) and then more than 1 year later (Sample Collection 2 (SC2)). Negative isolation of CLL cells to ≥95% purity was performed prior to RNA isolation.

2022-06-23 | GSE192685 | GEO

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