The STAT3 transcription factor is hyperactivated in many cancers where it induces cancer progression by driving drug resistance, metastasis and pro-cancer inflammation. We therefore sought new strategies to target the activation of STAT3. Many kinases and signalling pathways can cause STAT3 activation, and it is not well understood how this is regulated in cancer cells. In some rare solid and hematological cancers, such as NK-/T-cell large granular lymphocytic leukemia and inflammatory hepatocellular adenomas, STAT3 has also been found to be mutated, resulting in a constitutively active STAT3 protein.

In testing mock transfected vs. STAT3(Y640F)-transfected cells, as a means to introduce activated STAT3 in already transformed cells, in a screen against 525 anticancer agents, we identified that activated STAT3 protects the cells from cytotoxic effects of several classes of anticancer compounds. Therefore, we performed RNAi screens to identify kinases and phosphatases that regulate STAT3 activity and therefore may act as drug-sensitizing targets. Using STAT3 transcription-driven luciferase reporter cell lines expressing active wild type (WT) or mutant (Y640F) STAT3, we identified several candidate genes (CDC7, CDK8, CSK, CSNK2A1, PI4KII, PTPRH, DDR2) whose downregulation led to differential STAT3 transcriptional activity. Strikingly, none of the hits caused highly selective effect on either mutant or wild-type STAT3 expressing cells, suggesting that the phosphoregulation of hyperactive mutant and IL6 induced wild-type STAT3 are mechanistically similar. Using small molecule inhibitors targeting CDK8, CSNK2A1, DDR2 and CDC7, we confirmed that STAT3 transcriptional activity was inhibited in a dose and time dependent fashion without severely affecting cell viability. Small molecules targeting of CSNK2A1, DDR2 and CDC7 kinase activity caused a slow inhibition of STAT3 transcriptional activity where the strongest effects were only seen after 72 h. CDK8 kinase inhibition, on the other hand, reduced STAT3 activity effectively already after 4 h. This suggests that CDK8 regulates STAT3 activity more directly than CSNK2A1, DDR2 and CDC7. Inhibition of CDK8 and CSNK2A1 resulted in decrease of STAT3 phosphorylation in the STAT3(Y640F)-transfected cells, and knockdown of CSNK2A1 inhibited the nuclear localization of STAT3.

In conclusion, we have found that hyperactive STAT3(Y640F) can protect cancer cells from several drug classes, mimicking the case when hyperactivated wild type STAT3 is acting as malignancy promoting protein in cancers. In a functional genetics screen, we found six kinases and a phosphatase that regulate transcriptional activity of STAT3. Using small molecule inhibitors, we could confirm that targeting the hit kinases regulate STAT3 transcriptional activity. Together, our data suggest that there are multiple kinases that may be targeted to counteract STAT3 mediated drug resistance in cancers.

Citation Format: Elina Parri, Heikki Kuusanmäki, Arjan van Adrichem, Meri Kaustio, Laura Turunen, Krister Wennerberg. Identifying kinases and phosphatases that regulate STAT3 activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3343.