Wnt5a Induces ROR1 to Interact Grb2 to Enhance Ras Activation in Chronic Lymphocytic Leukemia

ROR1 (Receptor tyrosine kinase-like orphan receptor 1) is an evolutionarily conserved, oncoembryonic antigen found on chronic lymphocytic leukemia (CLL) cells, but not on healthy postpartum tissues. In prior studies we found that higher-level CLL-cell expression of ROR1 associated with accelerated disease progression and unfavorable clinical outcome (Cui B. et al., Blood, 128:2931, 2016). ROR1 is a receptor for Wnt5a, which is found at high levels in plasma of patients with CLL. We found Wnt5a could enhance ERK1/2 phosphorylation and leukemia-cell proliferation (Hasan MK. et al., Leukemia, 35:1621, 2021). Moreover, Wnt5a-enhanced ERK1/2 phosphorylation and leukemia-cell proliferation could be inhibited by UC-961, a first-in-class humanized mAb specific for a functional epitope of the ROR1 extracellular domain that is undergoing phase II clinical testing (Choi MY. et al., Cell Stem Cell, 22:951, 2018). However, it is not known how Wnt5a/ROR1-signaling induces ERK1/2 activation. We performed mass spectrometry-based proteomics to interrogate immune-precipitates of Wnt5a-activated ROR1 from CLL cells and identified Grb2 (Growth Factor Receptor Bound Protein 2), a cytoplasmic protein that may interact with SOS (son of sevenless) to induce activation of Ras, which can activate the mitogen-activated protein kinase (MAPK) pathway, leading to activation of ERK1/2. We found that treatment of serum-starved CLL cells with Wnt5a induced Grb2 to associate with ROR1 and activate Ras and ERK1/2; these effects could be blocked by siRNA silencing of Grb2 or by treatment with UC-961, indicating that Wnt5a induced activation of ERK1/2 is dependent upon expression of ROR1 and Grb2. To examine the structure-function relationship(s) in the interaction(s) between ROR1 and Grb2, we used the CLL-derived leukemia cell line MEC1, which we found expresses high-levels of Wnt5a, but not ROR1. Subclones of MEC1 cells transfected to express ROR1 (MEC1-ROR1) had significantly higher levels of ERK1/2 activation than MEC1 cells; immune-precipitation studies confirmed constitutive association of Grb2 and SOS1 with ROR1. This was not observed in MEC1-ROR1-Wnt5a ^-/- cells in which we disrupted both alleles of WNT5a via CRISPR-Cas9. Moreover, MEC1-ROR1 cells, or MEC1-ROR1-Wnt5a ^-/- cells treated with Wnt5a, had significantly higher levels of activated Ras and ERK1/2 than MEC1, MEC1-Wnt5a ^-/-, or MEC1-ROR1-Wnt5a ^-/-, demonstrating that activation of Ras, MAPK, and ERK1/2 in MEC1 was dependent upon on expression of ROR1 and Wnt5a. We next examined single amino acid substitutions of proline (P) to alanine (A) in the P-X-X-P- motifs of the proline-rich domain (PRD) of ROR1 at positions 784, 808, 826 or 841. Interestingly we found that P808A of ROR1 (MEC1-ROR1 ^P808A), a binding site of DOCK2 and critical for activation of ERK1/2, failed to complex with Grb2, SOS1, or enhance activation of Ras compared to MEC1-ROR1 cells. Overall, our results indicate that, upon Wnt5a stimulation, a novel complex of DOCK2/Grb2/SOS1 may form at position 808 of ROR1 that leads to activation of Ras and ERK1/2, which can promote proliferation of CLL cells and potentially contribute to the noted unfavorable prognostic implication of high-level expression of ROR1 in CLL.