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

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 14-3-3 zeta, which was confirmed by co-immunoprecipitation. The capacity of Wnt5a to induce ROR1 to complex with 14-3-3 zeta could be blocked in CLL cells by treatment with cirmtuzumab, a humanized mAb targeting ROR1. Silencing 14-3-3 zeta 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 14-3-3 zeta in ROR1-negative CLL cell-line MEC1, and in MEC1 cells transfected to express ROR1 (MEC1-ROR1), demonstrated that 14-3-3 zeta was necessary for the growth/engraftment advantage of MEC1-ROR1 over MEC1 cells. We identified a binding motif (RSPS(857)SAS) in ROR1 for 14-3-3 zeta. Site-directed mutagenesis of ROR1 demonstrated that serine-857 was required for the recruitment of 14-3-3. and ARHGEF2 to ROR1, and activation of RhoA and Rac1. Collectively, this study reveals that 14-3-3 zeta plays a critical role in Wnt5a/ROR1 signaling, leading to enhanced CLL migration and proliferation.