Wnt5a Induces Interleukin-6 Via a ROR1-Dependent Pathway in Chronic Lymphocytic Leukemia, Leading to Leukemia-Cell Activation of STAT3

Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncoembryonic cell surface protein, which is expressed on the neoplastic B cells of patients with chronic lymphocytic leukemia (CLL), but not on virtually all normal post-partem tissues. ROR1 functions as a receptor for Wnt5a, which induces activation of Rho-GTPases, AKT, and HS-1 and promotes migration, proliferation, and survival of CLL B cells. We observed that freshly isolated CLL cells also had high levels of pSTAT3 (Y705), which attenuated over time in culture in serum-free media unless the CLL cells were stimulated with exogenous Wnt5a. This effect of Wnt5a could be blocked by cirmtuzumab, a humanized anti-ROR1 mAb that currently is undergoing clinical evaluation in patients with CLL. We examined the capacity of recombinant Wnt5a (rWnt5a) to induce activation of STAT3 in isolated CLL cells by performing a time-course study, evaluating for pSTAT3 in serum-starved CLL cells at time 0, 5', 15', 30' and 1, 2, 3, 4, and 24 hours after stimulation with exogenous Wnt5a. Surprisingly, activation of STAT3 in CLL cells was delayed, appearing first at 3 hours after addition of rWnt5a, and peaking at 24 hours, the last time point examined. In contrast, activation of CLL cells via treatment of CLL cells with interleukin (IL)-6, induced activation of STAT3 within 15' of the addition of this cytokine to the cultures. We hypothesized that the activation of STAT3 in CLL cells by Wnt5a was indirect, being caused by a factor(s) made by isolated CLL cells in response to stimulation with Wnt5a. To test this hypothesis, we examined CLL cells, and harvested supernatants of cultured CLL cells, at various times after stimulation with rWnt5a. RNA isolated from CLL cells was examined via real-time PCR for cytokine transcripts. This revealed a prominent, time-dependent increase in transcripts encoding IL-6 following stimulation of CLL cells with rWnt5a. Consistent with this observation we found Wnt5a induced CLL cells to secrete IL-6, which was detected via ELISA at increasing concentrations over time in culture-supernatants of CLL cells treated with rWnt5a, but not in the culture supernatants of unstimulated CLL cells. Culture supernatants collected from CLL cells stimulated for 24 hours with rWnt5a could induce activation of STAT3 within 15' in serum-starved CLL cells; this effect could be inhibited significantly by the anti-IL-6-receptor mAb, tocilizumab, but not by cirmtuzumab, even when used at concentrations that could block the capacity of concomitantly-added Wnt5a to induce latent STAT3 activation at 24 hours, suggesting that cirmtuzumab could inhibit Wnt5a-induced CLL-cell production of cytokines, such as IL-6. We examined for IL-6 in cultures of CLL cells treated for 24 hours with rWnt5a and cirmtuzumab, or rWnt5a and an antibody of irrelevant specificity. We found the concentration of IL-6 in the supernatants of CLL cells treated with rWnt5a and cirmtuzumab (110.4 ± 18.9 pg/ml) was significantly lower than that found in the supernatants of CLL cells treated with rWnt5a and control antibody (416.2 ± 53.7 pg/ml, p = 0.0007, Students t test). This study reveals that Wnt5a can induce CLL to secrete cytokines, such as IL-6, via a ROR1-dependent pathway, leading to latent, autocrine activation of STAT3.