Abstract
Recent studies indicate that dual inhibition of the spleen tyrosine kinase (Syk) and Janus kinases (JAK) by agents such as R788 (tamatinib fosdium) are clinically efficacious in patients with non-Hodgkin’s lymphoma. Since Syk has been implicated in B cell activation, proliferation, and survival, the present study was designed to determine whether the specific inhibition of Syk alone would suffice to disrupt proliferation and survival of lymphoma cells. To test this, a series of compounds were synthesized and screened to identify those that specifically inhibited Syk in a panel of in vitro multi-kinase assays at 300nM. Of these, three were selected for this study; compounds P459-72, P505-15, and P420-89. All three compounds inhibited purified Syk with IC50’s in the 6-43nM range. P459-72 was highly Syk specific. P505-15 also inhibited purified Lyn with an IC50 of 199nM. P420-89 inhibited multiple kinases in the BCR signaling pathway, in addition to JAK1, 2, and 3; IC50’s of 0.63-6.2nM. Assays using the non-Hodgkin’s lymphoma B cell lines Ramos, SUDHL-4 and -6 showed that each compound also inhibited BCR-induced Syk auto-phosphorylation and BLNK phosphorylation (IC50’s in the 100–400nM range) as well as subsequent Ca2+ flux and ERK phosphorylation (IC50’s in the 50–156nM range). In contrast, the activity of the Src family member Lyn, upstream of Syk in the BCR signaling pathway and responsible for Syk tyrosine phosphorylation at amino acid position 352, was not affected. Cellular proliferation was also attenuated in these lymphomas (IC50’s in the 1–5μM range), and all three compounds induced apoptosis to various extents between 1 and 3μM. Interestingly, while P505-15 did inhibit purified Lyn with an IC50 of 199nM, this did not translate into inhibition of Lyn activity upon BCR cross-linking in cells at concentrations where Syk activity was inhibited. A structurally similar compound with a greater than one hundred fold lower Syk inhibitory activity (P528-85) had no effect on the proliferation and survival of these B cell lines. Finally, the cellular effects of the Syk inhibitors required an active BCR signaling pathway, as the B cell line Toledo which lacks BCR expression was insensitive to these compounds; proliferation was inhibited with IC50’s in the 9-38μM range with no induction of apoptosis below 10μM, the highest concentration tested. Taken together, these data suggest that the specific inhibition of Syk, without concomitant inhibition of JAK, may be sufficient for the treatment of non-Hodgkin’s lymphoma.
Expression and Function of the Chemokine, CXCL13, and Its Receptor, CXCR5, in Aids-Associated Non-Hodgkin's Lymphoma
