Wnt5a Can Induce Activation of Rac1 and Proliferation in Mantle Cell Lymphoma Via a ROR1-Dependent, but BTK-Independent, Signaling Pathway

Signaling via the B cell receptor (BCR) plays an important role in the pathogenesis and progression of chronic lymphocytic leukemia (CLL) or mantle cell lymphoma (MCL). This is underscored by the clinical effectiveness of an inhibitor of Bruton's tyrosine kinase (BTK), ibrutinib, which can block BCR-signaling. However, ibrutinib cannot induce complete responses (CR) or durable remissions without continued therapy, particularly in patients with MCL. This suggests that ancillary pathways contribute to the growth/survival of MCL/CLL that are independent of BCR-signaling and/or inhibition of BTK. Studies have found that Wnt5a can promote activation of Rho GTPase Rac1 to enhance CLL-cell proliferation and survival via a ROR1-dependent,but BTK-independent pathway (Yu J, et al., Leukemia 31:1333, 2017). Furthermore, Wnt5a-induced Rac1 activation could be blocked by cirmtuzumab, a humanized anti-ROR1 mAb, which significantly could enhance the capacity of ibrutinib to clear CLL cells in vivo . MCL cells also express ROR1. Moreover, we find that the median level of ROR1 on primary MCL cells is higher than that on CLL cells; the median absolute mean fluorescence intensity of ROR1 (ROR1 DMFI) on MCL (87.5 ± 18.5 (S.D.), N = 8) was significantly higher than the median ROR1 DMFI on CLL cells (38.5 ± 18.5 (S.D.), N = 1567; (Cui B, et al., Blood 128:2931, 2016)). We also found that the plasma of patients with MCL had high levels of Wnt5a that were comparable to those found in patients with CLL; in contrast Wnt5a was virtually undetectable in the plasma of age-matched healthy adults. We cultured primary MCL cells of different patients with ibrutinib, cirmtuzumab, or both ibrutinib and cirmtuzumab for 2 h, and then stimulated the cells with exogenous Wnt5a for 30 min. In parallel, the same MCL cells were cultured without exogenous Wnt5a. We found that Wnt5a induced Rac1 activation in the primary MCL cells. Cirmtuzumab, but not ibrutinib, could inhibit the capacity of Wnt5a to induce primary MCL cells to activate Rac1. We induced proliferation of MCL cells by co-culturing MCL cells with HeLa cells expressing CD154 (HeLaCD154) and recombinant interleukin (IL)-4 and IL-10. Addition of exogenous Wnt5a to co-cultures of MCL cells with HeLaCD154 cells and IL-4/10 significantly enhanced the proportion of dividing MCL entering S/G2 and the numbers of cell divisions over time. Treatment of the MCL cells with cirmtuzumab, but not ibrutinib, blocked Wnt5a-enhanced survival and proliferation of MCL cells from each of 3 different patients. This study demonstrates that Wnt5a can induce Rac1 activation, which leads to enhanced proliferation and survival of primary MCL cells via a ROR1-dependent signaling pathway that can be blocked by cirmtuzumab. On the other hand, this signaling pathway cannot be blocked by ibrutinib, even at concentrations that completely inhibit BTK or BCR-signaling. This implies that cirmtuzumab and ibrutinib may have additive, if not synergistic, activity in the treatment of patients with MCL.