The STAT3 Pathway in Uterine Leiomyoma: A Therapeutic Target - ABSTRACT/SUMMARY Uterine leiomyomas (LMs, fibroids) are the most important neoplastic threat to women worldwide. As no long- term non-invasive treatment option exists for LMs, deeper insight into tumor etiology is key to develop more effective medical therapies. Accordingly, this proposal is impactful as it suggests a novel etiological basis for the predominant MED12-mutated LM subtype and offers proof of concept for therapeutic intervention in this specific genetic setting. LMs arise from recurrent and mutually exclusive genetic alterations in a limited number of driver genes. Among these, mutations in the RNA Polymerase II Mediator subunit MED12 (mut-MED12) are by far the most prevalent, accounting for 77.4% of LMs. Recently, we showed that LM driver mutations in MED12 disrupt CDK8/19 kinase activity in Mediator, revealing the first and heretofore only known biochemical defect arising from these pathogenic mutations and further implying a new etiological role for CDK8/19 in LM pathogenesis. Herein, we identify signal transducer and activator of transcription 3 (STAT3), a transcription factor implicated in tumor growth and fibrosis, as a direct MED12-dependent CDK8/19 substrate. Physiologically, we show that CDK8/19, by direct phosphorylation of STAT3 serine 727, is a negative regulator of STAT3 transcriptional activity. However, the role of the STAT3 pathway in LM growth and whether this activity is altered by mut-MED12 remains unexplored. Our preliminary data indicate that STAT3 activation by CDK8/19 disruption is a key mediator of mut-MED12-driven LM growth. (i) Compared with wild-type (wt)-MED12 cells, mut-MED12 primary LM cells or a human myometrial smooth muscle cell line with CRISPR-engineered mut-MED12 responded to STAT3 inhibitors with greater sensitivity and decreased growth. (ii) CDK8/19 disruption by mut-MED12 reduced STAT3 serine 727 phosphorylation, leading to an increase in tyrosine 705 phosphorylation and strikingly enhancing the transcriptional activity and chromatin binding properties of STAT3 that drives expression of genes crucial for LM growth. (iii) STAT3-activating cytokines were specifically upregulated in myometrium adjacent to mut-MED12 LM, activated the JAK/STAT3 pathway, and promoted mut-MED12 LM cell growth. (iv) Progesterone receptor binds to the STAT3 gene promoter and stimulates its expression. We hypothesize that Mediator kinase disruption as a consequence of LM driver mutations in MED12 alters the phosphorylation pattern of STAT3 to favor a hyperactive form, one with enhanced chromatin binding properties leading to transcriptional activation of a unique set of genes responsible for LM growth. We propose that STAT3 inhibition using FDA-approved or in- pipeline agents targeting the JAK/STAT3 pathway will block steroid hormone-dependent LM growth, especially for the mut-MED12 subtype. Using a clinically relevant patient-derived xenograft LM mouse model and a cutting- edge single-nucleus multiomics approach, we will: (1) determine the functional role of the STAT3 pathway in LM growth in vivo; and (2) define the cell populations with unique epigenetic and transcriptomic processes responsible for STAT3-driven growth in mut-MED12 LM and verify the mechanistic findings in clinical samples.
