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Mutations of the FMS-like tyrosine kinase 3 (FLT3) have been reported in about a third of patients with acute myeloid leukemia (AML). The presence of FLT3 mutations confers a poor prognosis. Thus, pharmacological inhibitors of FLT3 are of therapeutic interest for AML. Go6976 is an indolocarbazole with a similar structural backbone to staurosporine. In the present study, we demonstrated that Go6976 displays a potent inhibitory activity against recombinant FLT3 using an in vitro kinase assay, with an IC50 value of 0.7nM. Go6976 markedly inhibited the proliferation of human leukemia cells having FLT3-ITD such as MV4-11 and MOLM13. We also observed that Go6976 showed minimal toxicity for human normal CD34(+) cells. Go6976 suppressed the phosphorylation of FLT3 and downstream signaling molecules such as STAT3/5, Erk1/2, and Akt in MV4-11 and MOLM13 cells. Interestingly, induction of apoptosis by Go6976 was associated with rapid and pronounced down-regulation of the anti-apoptotic protein survivin and MCL-1. Suppression of survivin protein expression by Go6976 was due to the inhibition of transcription via the suppression of STAT3/5. On the other hand, Go6976 induced proteasome-mediated degradation of MCL-1. Previously described FLT3 inhibitors such as PKC412 are bound by the human plasma protein, α1-acid glycoprotein, resulting in diminished inhibitory activity against FLT3. In contrast, we found that Go6976 potently inhibited phosphorylation of FLT3 and exerted cytotoxicity in the presence of human serum. In conclusion, Go6976 is a potent FLT3 inhibitor that displays a significant antiproliferative activity against leukemia cells with FLT3-ITD through the profound down-regulation of survivin and MCL-1.

The FMS-like tyrosine kinase 3 (FLT3) is a class III receptor tyrosine kinase involved in hematopoietic progenitor cell development. Mutations of FLT3 have been reported in about a third of patients with acute myeloid leukemia (AML). The presence of FLT3 mutations confers a poor prognosis, and thus recent studies are directed at developing and testing novel FLT3 inhibitors for the treatment of AML. Go6976 is an indolocarbazole with a simillar structural backbone to staurosporine. In the present study, we demonstrated that Go6976 displays a potent inhibitory activity against recombinant FLT3 using in vitro kinase assay. Its IC50 value is 0.7nM. We also tested the effect of Go6976 on several kinds of other kinases. Go6976 inhibited the kinase activity of Aurora-A, Aurora-B and JAK2 with IC50 values of 118.2 nM, 77.7 nM and 92.7 nM, respectively. Go6976 did not show the inhibitory activity against the FGFR3 even at 1 microM. These data indicated Go6976 preferentially and potently inhibit the FLT3. Go6976 significantly inhibited the proliferation of human leukemia cells having FLT3-ITD. The IC50 values of Go6976 against MV4-11 and MOLM-13 were 0.044 and 0.008 microM, respectively. In contrast, human leukemia HL-60 and U937 cells which lack FLT3-ITD showed strong resistance to Go6976 treatment. Furthermore, we observed that Go6976 shows minimal toxicity for purified human normal CD34(+) cells. Go6976 suppressed the phosphorylation of FLT3 in MV4-11 and MOLM13 cells. Consistent with FLT3 inhibition, Go6976 potently inhibited phosphorylation of constitutively activated STAT3/5, Erk1/2, and Akt. Western blotting analysis revealed that MV4-11 and MOLM13 cells possess abundant survivin and Mcl-1 protein. We hypothesized that the expression of survivin and Mcl-1 may be regulated by constitutive activation of FLT3. In order to test this hypothesis, the effect of siRNA for FLT3-ITD was examined. Indeed, we observed that siRNA-induced down-regulation of FLT3 decreased survivin and Mcl-1 expression in MOLM13 cells, suggesting that up-regulated survivin and Mcl-1 may be closely associated with FLT3 signaling. Interestingly, we found that both survivin mRNA and protein were rapidly downregulated by Go6976 treatment in MOLM13 and MV4-11 cells. It was also observed that Go6976 significantly suppressed Mcl-1 mRNA and protein. It has been reported that STAT-3 and STAT-5 signaling play a pivotal role in the regulation of survivin and Mcl-1, respectively. Thus, inhibitory effects of Go6976 on the expression Survivin and Mcl-1 may be a consequence of the suppression of phosphorylation of STAT-3 and STAT-5 by Go6976 in FLT3-ITD cells. This inhibition of anti-apoptotic proteins by Go6976 may be critical for its antiproliferative effect in FLT3-ITD cells. It has been known that previous FLT3 inhibitors such as PKC412 and CEP-701 bind to the human plasma protein, alpha1-acid glycoprotein, resulting in significantly diminished inhibitory activity against FLT3. Indeed, inhibitory effect of PKC412 on FLT3 was significantly decreased, when MOLM13 cells were treated with PKC412 in the presence of human serum. In contrast, we found that Go6976 potently inhibits phosphorylation of FLT3 and exerts cytotoxicity even in the presence of human serum or human alpha1-acid glycoprotein. In conclusion, our data indicate that Go6976 may have a unique therapeutic potential for FLT3-driven acute myeloid leukemia. Disclosures: No relevant conflicts of interest to declare.

Abstract 3823 Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic stem cell disorders characterized by ineffctive hematopoiesis. Survivin belongs to the inhibitors of apoptosis (IAP) family and inhibits apoptosis. Besides its role as IAP, Survivin appears to function as a subunit of the chromosomal passenger complex (CPC) for regulating mitosis with other CPC proteins including Aurora-B. As the CPC, Survivin and Aurora-B play an important role in the maintenance of a stable genome. Until now, there has been no report to examine whether expression of Survivin and Aurora-B kinase may be increased in CD34(+) cells prepared from the patients with MDS during disease progression. To investigate the contribution of Survivin and Aurora-B to the progression and prognosis of MDS, we evaluated the expression levels of these two genes in CD34(+) cells prepared from 57 patients with MDS and 50 patients with de novo AML using real-time quantitative polymerase chain reaction. The degree of Survivin and Aurora-B expression was highly correlated with the type of MDS, was much higher in RAEB-1, RAEB-2 and secondary AML following MDS (s-AML) compared with normal control, and increased during disease progression. Expression levels of both Survivin and Aurora-B were significantly correlated with International Prognostic Scoring System (IPSS). Furthermore, the amount of Aurora-B kinase was significantly correlated with Survivin expression in MDS and s-AML, but not in de novo AML. Interestingly, the levels of Survivin and Aurora-B were remarkably higher in patients with s-AML when compared with de novo AML. We demonstrated for the first time that high levels of Survivin and Aurora-B kinase expression are distinctive molecular feature of high-risk MDS and s-AML. Marked up-regulations of Survivin and Aurora-B Kinase may contribute to genetic instability and disease progression of MDS. Disclosures: No relevant conflicts of interest to declare.