Disrupting B cell receptor- (BCR-) induced signaling by inhibiting the spleen tyrosine kinase (SYK) represents a novel therapeutic approach in CLL. R406 is an ATP-competitive SYK inhibitor, and its prodrug, R788 (Fostamatinib disodium), displays clinical activity in patients with B cell lymphoma and CLL (Friedberg J. et al. Ann Oncol. 2008 Jun; 19 Suppl 4). In normal lymphocytes BCR engagement and SYK activation modulate cell adhesion and migration, suggesting that this pathway affects chemokine receptor and adhesion molecule function/signaling. The effects of SYK on CLL cell migration, activation and survival are largely unknown. Therefore, we examined the effects of BCR stimulation and its inhibition with R406 on CLL cell chemotaxis, expression of adhesion- and costimulatory- molecules, and survival. Activation of BCR with polyclonal goat F(ab’)2 fragments to human IgM significantly increased chemotaxis towards CXCL12 to 135.4% ±5.2% of controls and towards CXCL13 to 170.5% ± 31.5% of controls (Mean ± SEM, n=15). Engagement of BCR also significantly increased expression of CD40, CD44, CD54, and CD62L and decreased the expression of CXCR4. Pre-treatment with R406, which was kindly provided by Rigel Pharmaceuticals, Inc., abrogated the increased chemotaxis, adhesion- and co-stimulatory molecule expression, and pro-survival signals seen in response to BCR engagement. As displayed in Fig. 1, CLL cell viability at 24 hours was significantly increased to 60±5.2% with anti-IgM, compared to controls (41.8±6.7%) or CLL cells treated with anti-IgM plus R406 5 μM (39±6.6%, mean± SEM, n=9). Moreover, we found that R406 induced apoptosis in CLL cells in suspension culture and co-cultures with nurselike cells (NLC) and marrow stromal cells (MSC). When compared to CLL cells cultured with NLC (100%), the mean relative viability of CLL cells at 48h was reduced to 71.2±6.4% with 5 μM R406 and to 75.4±5.6% with 2.5 μM R406, whereas the same CLL samples cultured without NLC displayed a mean relative viability of 87±2.9% in controls, 58.9±6.8% with 5 μM R406, and 59.9±7.3%% with 2.5 μM R406 (mean±SEM, n=10). Similar levels of apoptosis were observed when CLL cells were incubated with R406 in MSC co-cultures.

Collectively, this study demonstrates that BCR engagement increases CLL cell migration, adhesion- and co-stimulatory-molecule expression, and survival. R406 effectively antagonizes these responses and induces CLL cells apoptosis, providing an explanation for the clinical activity of R406 in CLL patients.

Figure 1
Figure 1.
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Topics:
adhesions, b-cell lymphomas, cd40 antigens, cd44 antigens, cell adhesion molecules, chemokine cxcl13, chemokine receptors, coculture techniques, cxcr4 receptors, fostamatinib

Disclosures: No relevant conflicts of interest to declare.

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2008, The American Society of Hematology
2008
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