SYK-Inhibitor Bay 61-3606 Induces Cell Cycle Arrest and Apoptosis in Multiple Myeloma Cells Independent of SYK Inhibitory Effects but Via Degradation of IKZF1/3

Introduction: Bay 61-3606 is a cell-permeable imidazopyrimidine compound that acts as a potent, ATP-competitive, reversible, and highly selective inhibitor of Syk tyrosine kinase activity with no inhibitory effect against Btk, Fyn, Itk, Lyn, and Src. BAY 61-3606 has been also shown to inhibit Syk-mediated cellular functions such as glucose-tyrosine phosphorylation of I κ B α and p65 nuclear translocation. It further exhibits a good oral bioavailability and in vivo efficacy in rat models. Recently, Bay 61-3606 was found to inhibit cell proliferation and SDF-1a-induced migration of MM cells ⁽¹⁾. Based on these promising preliminary data we further investigated the potential of Bay 61-3606 as new anti-MM agent.

Methods and Results: Bay 61-3606, at concentrations as low as 10 nM, induced significant (p<0.01) inhibition of cell growth in MM cells (MM.1S, H929 and RPMI-8266) as shown by WST-1 cell proliferation assay. The inhibition of proliferation was accompanied by increased cell cycle arrest at G0/G1 (from 60% to 75%). More importantly, Bay 61-3606 dose dependently induced MM cells apoptosis suggesting that Bay 61-3606 has potent anti-MM properties. Since Bay 61-3606 was originally reported as a SYK inhibitor, we analyzed whether the effects of Bay 61-3606 were due to SYK inhibition. For this purpose, we first checked the expression of SYK in all the MM cell lines (MM.1S, H929, U266, OPM2 and RPMI-8226). And to our surprise, although all these cells were sensitive to Bay 61-3606 treatment, several of these cell lines (H929, U266 and RPMI-8226) had no detectable SYK expression by Western Blot assay. This suggests that the anti-MM effects of Bay 61-3606 are not mediated by SYK inhibition, which was further confirmed by using another specific SYK inhibitor GS-9973. Further mechanistic studies showed that Bay 61-3606 significantly downregulated protein levels of IKZF1, IKZF3, c-MYC and IRF-4 in IMiD®-sensitive and resistant MM cell lines (MM.1S, H929 and RPMI-8266) by Western Blot assay. In contrast, real-time PCR assay indicated IKZF1/3-mRNA level was not altered, suggesting that Bay 61-3606 regulates IKZF1/3 at post-translational level. The proteinase inhibitor MG132 and cullin-dependent ubiquitin ligase inhibitor MLN4924 blocked the downregulation IKZF1/3 by Bay 61-3606 confirming that Bay 61-3606 induced IKZF1/3 protein degradation. Consistent with its proapoptotic effects, Bay 61-3606 downregulated MDM2 phosphorylation, upregulated P53 level and induced PARP cleavage as confirmed by Western Blot assy.

Conclusion: In summary, our results demonstrate that Bay 61-3606 has anti-MM effects by inducing cell cycle arrest and apoptosis, and this effect is independent of SYK inhibition. Similar to IMiD® compounds, Bay 61-3606 induces IKZF1 and IKZF3 protein degradation in MM cells. More importantly, Bay 61-3606 has effects on both IMiD®-sensitive and resistant myeloma cells and may represent a novel potent anti-MM agent.

References:

(1). Koerber RM, et al. Exp Hematol Oncol. 2015.