Inhibition of SYK More Effectively Overcomes MCL-1 Mediated ABT-199 Resistance of BCR-Stimulated CLL Cells Than Inhibition of BTK or PI3Kdelta

The selective Bcl-2 antagonist ABT-199 has demonstrated promising clinical activity in patients with CLL. This drug is strongly cytotoxic against unstimulated peripheral blood CLL cells in vitro, but is much less effective against CLL cells that have received signals mimicking interactions with the lymph node microenvironment. In particular, stimulation of peripheral blood CLL cells with CD40L and IL-4 results in substantial resistance, which is mediated by induction of the antiapoptotic Bcl-2 family proteins Bcl-XL and Bfl-1. In the present study, we investigated whether resistance to ABT-199 can be conferred by engagement of the BCR, which is another stimulus that CLL cells receive primarily in the lymph nodes. Sustained engagement of the BCR with immobilized anti-IgM antibody, which is commonly used as a surrogate for a membrane-bound antigen, significantly protected CLL cells from ABT-199-mediated apoptosis (n=28; % viable CLL cells after 24 hours in culture without ABT-199: 52.2±14.8; with 2nM ABT-199: 28.3±15.1, with 2nM ABT-199 and immobilized anti-IgM: 43.0±19.5,  P<0.001).  Resistance of BCR-stimulated CLL cells from ABT-199 mediated apoptosis directly correlated with induction of the antiapoptotic Bcl-2 family protein Mcl-1 (n=14, Pearson correlation coefficient 0.675, P=0.008), whereas no significant changes were observed in the levels of Bcl-XL, Bcl-2 and Bim. Downregulation of Mcl-1 by RNA interference completely reversed resistance of anti-IgM stimulated CLL cells to ABT-199, suggesting that induction of this antiapoptotic protein is primarily responsible for BCR-mediated protection (n=5; % viable after nucleofection with control siRNA: 39.0±15.9; control siRNA and ABT-199: 14.2±4.1; control siRNA, ABT-199 and anti-IgM: 27.4±13.9; Mcl-1 siRNA, ABT-199 and anti-IgM: 14.6 ±6.0; P=0.022 for the comparison between the last two conditions). In line with this possibility, nucleofection of primary CLL cells with in vitro-transcribed Mcl-1 mRNA resulted in almost complete protection from ABT-199 induced apoptosis (n=3, % viable cells after nucleofection with control mRNA: 47.0±12.7; control mRNA and ABT-199: 27.5±16.2; Mcl-1 mRNA and ABT-199: 46.5±6.0). To investigate whether clinically tested BCR pathway inhibitors can prevent anti-IgM-mediated Mcl-1 induction and consequent ABT-199 resistance, we pretreated CLL cells with the SYK inhibitors R406 and GS-9973, the BTK inhibitor ibrutinib or the PI3Kδ inhibitor idelalisib (all used at 1μM concentration) and evaluated changes in Mcl-1 expression. Although all four inhibitors reduced Mcl-1 expression in anti-IgM stimulated CLL cells, the effect of SYK inhibitors was considerably more profound. In addition, only SYK inhibitors were capable of reducing Mcl-1 expression below basal levels. To understand the reasons for the greater activity of SYK inhibitors, we compared the capacity of R406, GS-9973, ibrutinib and idelalisib to inhibit the kinases AKT and GSK3, which are both important regulators of Mcl-1. Activated AKT increases Mcl-1 expression through post-transcriptional mechanisms, whereas activated GSK3 downregulates Mcl-1 by targeting it for proteasomal degradation. All four BCR pathway inhibitors efficiently blocked anti-IgM induced activation of AKT, but BCR-induced phosphorylation and inactivation of GSK3 was blocked only by R406 and GS-9973. These data suggest that all four drugs can prevent de novo Mcl-1 synthesis, but only SYK inhibitors can increase the rate of Mcl-1 turnover. The capacity of SYK inhibitors to overcome BCR-mediated ABT-199 resistance was further confirmed by investigating the viability of anti-IgM stimulated CLL cells (n=22) cultured in the presence or absence of ABT-199, R406 and GS-9973. Both R406 and GS-9973 significantly reduced protection of anti-IgM-stimulated CLL cells from ABT-199-induced apoptosis (ABT-199 and anti-IgM: 52.0±14.0; ABT-199, anti-IgM and R406: 41.7±12.0;  ABT-199, anti-IgM and GS-9973: 43.5±13.5, P<0.001). In conclusion, these data demonstrate that BCR signals induce resistance of CLL cells to ABT-199 by upregulating Mcl-1. Resistance to ABT-199 can be effectively overcome by SYK inhibitors, which target Mcl-1 by preventing AKT activation and GSK3 inactivation. Altogether, these data suggest that SYK inhibitors should be the most appropriate BCR pathway inhibitors to test in combination with ABT-199 in the clinic.