Kifocitanib, A Novel Small Molecule Inhibitor Of STAT3, Displays Preclinical Activity For Multiple Myeloma

Background and Purpose: Signal transducer and activator of transcription3(STAT3) is a transcription factor that can be activated by various cytokines and growthfactors and mediates the expression of a variety of important genes responsible for cellproliferation, cell cycle progress, cell survival, angiogenesis and migration. STAT3hasbeen found to be overexpressed in many human cancers including solid tumor cancerssuch as lung cancer, breast cancer and prostate cancer. In hematological malignancies,STAT3has been reported in leukemia, lymphoma and multiple myeloma (MM). STAT3is closely related to the pathogenesis and development of malignant tumors. Tumor cellscan be induced apoptosis if STAT3activity is inhibited. Recent studies have beendemonstrated that blocking the STAT3signaling pathway can overcome drug resistancein retinoblastoma, lung cancer, leukemia and other tumors. In the present study, weidentified a novel small chemical compound Kifocitanib that inhibits the activity ofSTAT3in multiple myeloma. Therefore, the purpose of this study is to determinewhether Kifocitanib can induce MM cell death and to find out the underlyingmechanisms.Methods:(1) Trypan blue exclusion assay was used to analyse the effects of Kifocitanib oncell viability and proliferation in multiple myeloma cells.(2) Flow cytometry was used to measure the effects of Kifocitanib on cellapoptosis and cell cycle in myeloma cells.(3) Western blotting was performed to examine the expression of proteins thatinvolved in cell apoptosis, cell cycle and STAT3signaling pathway.(4) Immunofluorescence assay was used to determine the cell expression ofp-STAT3and cellular localization in RPMI-8226cells.(5) Cell scratch assay was used to evaluate the effect of Kifocitanib on cellmigration in human umbilical vein endothelial cells (HUVEC). (6)A myeloma xenograft experiment was used to detect the effect of Kifocitanib ontumor growth in vivo.Results:(1) Kifocitanib inhibited the growth of multiple myeloma cells. We found that KIFinhibited cell viability and proliferation of multiple myeloma cells in a dose dependentmanner.(2) Kifocitanib induced multiple myeloma cells apoptosis. We analysed cellapoptosis by Annexin V-FITC and PI staining and found that the percentage of apoptoticcells raised in a concentration-dependent manner after the drug treatment. The results ofWestern blotting showed that Kifocitanib activated caspase-3, caspase-9and PARP.(3) Kifocitanib arrested MM cells at the G0/G1phase of the cell cycle. We detectedthe expression of proteins involved in cell cycle, including Cyclins, CDK4/6, E2F-1andp-RB. The results showed a dose dependent inhibition by Kifocitanib. And the flowcytometry result demonstrated that the percentage of cells at G0/G1phase was raised ina dose dependent manner.(4) Kifocitanib suppressed the activation of STAT3. We found that Kifocitanib notonly inhibited the constitutive STAT3phosphorylation, but also suppressed the IL-6–activated STAT3signals. Immunofluorescence analysis also showed that it decreasedthe expression of STAT3phosphorylation in RPMI-8226cells. In the myeloma–stromalmicroenvironment, we found Kifocitanib inhibited STAT3activation.(5) Kifocitanib blocked the nuclear STAT3export. We isolated the nuclear andcytosol fractions for STAT3analysis. The results showed that the expression levels ofSTAT3in nuclei were increased after drug treatment under both complete medium orstarved conditions.(6) Kifocitanib inhibited the STAT3signaling pathway. We detected the upstreamand downstream events in the STAT3signal by Western blotting. And the resultsdemonstrated that Kifocitanib not only inhibited the JAK2and c-Src phosphorylation,but also decreased the expression of Mcl-1, Bcl-2, CCND2, and VEGF.(7) Kifocitanib inhibited cell migration. The cell scratching assay demonstratedthat Kifocitanib inhibited cell migration in human umbilical vein endothelial cells(HUVEC).(8) Kifocitanib delayed tumor growth in a MM xenograft model. We established ahuman myeloma xenograft model. By daily monitoring of tumor sizes and body weight, we found that Kifocitanib markedly decreased tumor growth without significant loseweight within14-day treatment.Conclusion: In the present study, we demonstrate that Kifocitanib induces cellapoptosis and arrests cell cycle by inhibiting STAT3activity. Our date suggest thatKifocitanib may be appromising candidate for the treatment of multiple myeloma.