A Novel STAT3 Inhibitor K9 Displays Potent Preclinical Activity Against Multiple Myeloma

Objective: The signal transducer and activator of transcription 3(STAT3) is dysregulated in multiple myeloma, and is closely associated with poor clinical outcomes in such patients, therefore, targeting STAT3 signaling has been proposed as a novel strategy for multiple myeloma therapy. This study is aimed to find novel anti-myeloma STAT3 inhibitors by a high throughput screen and to thread light on its anti-myeloma mechanism.Methods:(1) A high throughput screen was performed to search for STAT3 inhibitors from 56,000 compounds purchased from Maybridge chemicals using luciferase as a reporter.(2) Immunoblotting and flow cytometer were performed to examine whether K9 affected cell apoptosis in multiple myeloma(MM) cells.(3) A human myeloma xenograft model was established in nude mice with RPMI-8226 cells to examine the effects of K9 on MM tumor growth in vivo. At the same time, body weight was measured and blood samples were analyzed to evaluate the toxicity of K9 in vivo.(4) Immunoblotting was performed to detect whether K9 regulated the constitutive and IL-6-induced STAT3 phosphorylation. In addition, we evaluated whether K9 regulated the phosphorylation of STAT3 when MM cells were co-cultured with bone marrow stromal cells.(5) Immunoblotting, molecular docking and cell-free assay were carried out to evaluate whether K9 affected the activation of STAT3 up-stream kinases.(6) Co-immunoprecipitation and luciferase assay were performed to examine whether K9 affected the dimerization and transcriptional activity of STAT3.(7) RT-PCR and immunoblotting were used to detect whether K9 regulated the expression of STAT3-targeted genes.(8) Immunoblotting and trypan blue assays were performed to detect whether hyper-activation or over-expression of STAT3 affected the drug sensitivity of K9 in MM cells.(9) Immunoblotting and trypan blue assays were performed to evaluate whether K9 enhanced doxorubicin toxicity in MM cells.Results:(1) A high throughput screen identified K9 as a candidate of STAT3 inhibitors.(2) K9 activated apoptotic protein PARP in five different MM cells(NCI-H929, KMS12, OCI-My5, RPMI-8226 and U266), and induced PARP and Caspase 3 activation in a concentration-dependent manner in NCI-H929 and RPMI-8226 cells. And flow cytometry confirmed that K9 induced MM cell apoptosis.(3) K9 delayed MM tumor growth in xenograft mice but had no effect on mice body weight. In addition, hematological analysis showed that there were no significant changes in blood cell and platelet counts, and hemoglobin measurement. These results indicated that K9 displayed potent anti-myeloma activity without overt toxicity.(4) STAT3 was hyper-activated in five MM cell lines. Meanwhile, K9 effectively suppressed both the constitutive and IL6-induced STAT3 phosphorylation, but had no effect on other STAT family proteins activation, including STAT1 and STAT5. In addition, K9 could inhibit STAT3 activation as well when MM cells were co-cultured with bone marrow stromal cells.(5) K9 displayed potent inhibitory effects on STAT3 activation via inhibiting JAK2 but not other kinases, such as mTOR, P38, ERK and c-Src. In addition, molecular docking showed that K9 was well docked into the ATP-binding pocket of JAK2, and strongly inhibited JAK2 activation.(6) K9 effectively inhibited STAT3-driving luciferase activity which contained several STAT3 response elements. And K9 also inhibited the promoter activity of STAT3-targeted gene CCND2 which contained STAT3 response elements, but had no effect on the luciferase activity of CCND2 which contained no STAT3 response elements. In addition, Co-IP showed that K9 effectively suppressed the dimerization of STAT3.(7) K9 obviously inhibited the expression of STAT3-targeted genes, including MCL1 and XIAP.(8) IL6-induced STAT3 activation partly suppressed K9-induced MM cell apoptosis. Moreover, ectopic STAT3 decreaseed MM sensitivity to K9.(9) The anti-myeloma efficacy of doxorubicin(DOX) was enhanced by K9.Conclusion:K9 displays a potent anti-myeloma activity in vitro and in vivo with low toxicity. Mechanistically, K9 suppresses MM cell survival via inhibiting the JAK2-STAT3 signaling pathway. K9 is thus a novel STAT3 inhibitor, and this study may be helpful for developing novel anti-MM drugs in clinical.