B-cell receptor (BCR) signaling contributes to the pathogenesis of chronic lymphocytic leukemia (CLL). Spleen tyrosine kinase (SYK) activated directly downstream of the BCR is essential for the induction of proliferation and survival pathways. The SYK inhibitor fostamatinib disrupts BCR signaling and was the first such inhibitor to show significant clinical activity in patients with mature B-cell malignancies. Fostamatinib has been shown to both induce apoptosis in unstimulated CLL cells as well as to inhibit BCR induced anti-apoptotic signals in vitro (Gobessi et al., 2009; Quiroga et al., 2009). Similarly, using the Eμ-TCL1 transgenic mouse model, fostamatinib has been shown to inhibit the growth of malignant B-cells without significant alteration of normal B-cells (Suljagic et al., 2010). In the first phase I/II clinical trial investigating fostamatinib in relapsed B-cell non-Hodgkin's lymphoma (NHL) and CLL, clinical efficacy was observed in a variety of histologies with the highest response rate in CLL/SLL patients (Friedberg et al., 2010). Eleven CLL/SLL patients enrolled in this trial donated cellular material for correlative studies. Using these primary tumor samples, we evaluated the effects of fostamatinib on CLL cells in vivo after one cycle of treatment.
We first validated the on-target effect of fostamatinib by using quantitative RT-PCR to measure expression of validated pathway specific gene signatures. Fostamatinib greatly down-regulated 12/12 evaluated BCR signature genes and significantly reduced the BCR gene signature score (computed as the average expression of the pathway specific genes; p=.002) Effective inhibition of BCR signaling was confirmed by a significant reduction in the phosphorylation of both BTK and ERK; two key BCR signaling molecules located downstream of SYK activation. Interestingly, BCR signaling was inhibited in CLL cells from all patients regardless of response to therapy. We next expanded our analysis to look at NF-κB and MYC gene signatures. We found that 11/11 representative NF-κB signature genes and 5/5 MYC signature genes were also down-regulated resulting in a significant reduction in both gene signature scores (p=.004 and p=.020, respectively). Confirming these results, we also observed a significant reduction in JUNB (p<.001) and MYC (p=.026) at the protein level. Interestingly, the reduction in NF-κB and MYC signature scores was highly correlated with the degree of reduction in BCR signaling suggesting that these pathways are linked. In addition to changes in the gene signatures we also observed a significant reduction in the cellular activation immunephenotype; CD69 and CD86 expression were significantly reduced by fostamatinib (p=.033 and p=.004, respectively). Further, we found that CD38 (an activation marker with prognostic significance) was also reduced on treatment although not to a significant extent. Finally, fostamatinib significantly reduced tumor proliferation as determined by the percentage of CLL cells expressing Ki67 (p=.005). Eight of the 11 patients in this study achieved a clinically significant response; interestingly however, the 3 non-responders demonstrated significantly brighter CD38 expression with an MFI up to 9-times the CD38 MFI in responders. A possible role of CD38 as a biomarker for response should be further explored in patients treated with BCR directed kinase inhibitors.
In conclusion, fostamatinib and other inhibitors of BCR-related kinases constitute a major advance in the treatment of CLL. In vitro data with these compounds suggests that interruption of BCR signaling and survival pathways activated in the tissue microenvironments are likely responsible for the observed clinical response as only a moderate direct induction of apoptosis is seen in vitro. Here we demonstrate that inhibition of BCR-mediated signaling by fostamatinib results in a reduction in CLL proliferation and activation in vivo. Together these data provide a blueprint to further study the mechanism of action and resistance mechanisms of not only fostamatinib but also other BCR targeted therapeutics.
This work was supported in part by the Intramural Research Program of the National, Heart, Lung and Blood Institute and by the University of Rochester SPORE in lymphoma P50 CA13080503, Rigel and the James P. Wilmot Foundation.
Friedberg:Rigel: Research Funding.
Asterisk with author names denotes non-ASH members.