Syk Inhibition By TAK-659 Overcomes Proliferative, Survival and Migratory Signals from the Microenvironment in Chronic Lymphocytic Leukemia Cells

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation and proliferation of monoclonal CD5+ mature B cells in peripheral blood, lymph nodes (LN), and bone marrow (BM). The microenvironment found in BM and LN induces proliferation of CLL cells and protects them from spontaneous and chemotherapy-induced apoptosis. Syk protein is a tyrosine kinase essential for the BCR signaling pathway that also participates in signaling from chemokine receptors and has been shown to be deregulated in CLL. Therefore Syk has been hypothesized to be a rational candidate for targeted therapy in CLL and its inhibition has been tested with the non-specific Syk inhibitor fostamatinib (R406). Against this background we tested the effectiveness of the highly specific Syk inhibitor TAK-659 in suppressing the induction of survival, proliferation and migration of CLL cells by the microenvironment.

To mimic the microenvironment of the proliferative centers ex vivo, we co-cultured primary CLL cells with the BM stromal cells (BMSC), CD40L, CpG ODN and anti-IgM (BCR stimulation). This co-culture system protected CLL cells from apoptosis (mean % of viable cells relative to suspension: 137.52±26.17, P<0.05); proliferative responses were significantly observed after 72 hours (mean % of Ki-67-positive cells: 0.91±0.22 in suspension vs. 7.00±1.49 in co-culture, P<0.001); CLL cells activation according to CD69, CD38, and CD86 expression was markedly induced (mean MFI of CD69: 137.2±26.3 in suspension vs. 339.1±41.4 in co-culture, P<0.01; mean MFI of CD38: 14.1±1.67 in suspension vs. 29.8±6.26 in co-culture, P<0.01; mean MFI of CD86: 27.3±2.99 in suspension vs. 80.8±15.1 in co-culture, P<0.01). Moreover, in this system CLL cells became chemoresistant to fludarabine and bendamustine.

TAK-659 inhibited Syk^Tyr525, Btk, Akt and ERK1/2 phosphorylation after BCR cross-linking with anti-IgM in the B cell line Ramos and in primary CLL cells, as assessed by western blot. Syk inhibition by TAK-659 translated into an induction of apoptosis in primary CLL cells, obtaining a LD₅₀ for CLL cells in suspension of 40.39μM (95%CI 21.7-75.2μM) vs. 16.99μΜ (95%CI 7.67-37.67μM) for CLL cells in co-culture. Interestingly, TAK-659 displayed stronger capacity to induce apoptosis than R406 especially in co-cultured CLL cells (LD₅₀ TAK-659 16.99μΜ vs. LD₅₀ R406 not achieved). In addition, combination of TAK-659 with fludarabine, ibrutinib or idelalisib showed a synergistic effect in inducing apoptosis especially in co-cultured CLL cells (Cooperative index for TAK-659 0.1μΜ combined with fludarabine 1μM: 0.62, with ibrutinib 0.1μM: 0.68, and with idelalisib 0.1μM: 0.18). Treatment with TAK-659 also resulted in almost complete abrogation of co-culture-induced proliferation in a dose-dependent manner (mean % of Ki-67-positive cells: 7.00±1.49 in untreated controls vs. 3.39±0.76 after 0.1μM TAK-659 vs. 1.72±0.20 after 1μM TAK-659 vs. 1.27±0.18 after 10μM TAK-659, P<0.01), and CLL cells activation (mean MFI of CD38: 29.7±6.26 in untreated controls vs. 23.9±4.39 after 0.1μM TAK-659, P<0.01; mean MFI of CD86: 80.8±15.1 in untreated controls vs. 58.7±8.99 after 0.1Μm TAK-659, P<0.05). Since BCR signaling also promotes CLL cell chemotaxis toward BMSCs and the chemokines CXCL12 and CXCL13, we next evaluated the effect of treatment with TAK-659 on the migratory capacity of primary CLL cells and we observed that TAK-659 markedly decreased chemotaxis of CLL cells toward CXCL12, CXCL13 and BMSCs. Given the significant homology between Syk and ZAP-70 protein and the critical role of the latter in T cell signaling we aimed to assess the effects of TAK-659 on Jurkat T cells. Surprisingly, we observed that although TAK-659 inhibited ZAP-70^Tyr493 phosphorylation, this inhibition did not translate into inhibition of downstream signaling elements, such as Itk, Akt or ERK. We next analyzed the effects of TAK-659 in viability and activation of primary T cells and we observed that TAK-659 did not induce significant apoptosis neither inhibition of activation in terms of CD69 and CD38 expression.

In conclusion, these findings demonstrate that, in this ex vivo system, the specific inhibition of Syk by TAK-659 effectively overcomes the microenvironment signals that promote proliferation, activation, survival and chemoresistance of primary CLL cells. Altogether, this study provides a rationale for the clinical development of TAK-659 in CLL.