Abstract PO-001: Heterogeneous overexpression of HES6 promotes the growth of uterine leiomyomas

Abstract

Abstract Introduction: Uterine leiomyomas are a common cause of infertility, pelvic pain and irregular vaginal bleeding. Robust evidence suggests that leiomyomas arise from a distinct population of myometrial-specific stem cells (mSC). However, the mechanisms responsible for driving the mal-differentiation of mSC and subsequent leiomyoma growth remain poorly understood. Here, we explore the role of HES6, a basic helix-loop-helix (bHLH) co-factor critical for promoting myogenic differentiation, in uterine leiomyomas. Methods: After obtaining IRB approval, real time PCR (qPCR) and Western blot were used to evaluate patterns of HES6 expression using RNA and protein prepared from matched specimens of myometrium, leiomyoma and uterine leiomyosarcoma (uLMS). Primary cultures were generated as previously described. HES6-specific siRNAs were used to target gene expression in vitro. Standardized assays were used to measure proliferation (CellTiter-Glo, Promega) and apoptosis (Caspase 3/7 assay). A commercially available antibody specific for HES6 was used to localize HES6 expression in formalin-fixed, paraffin-embedded (FFPE) tissue specimens by immunohistochemistry (IHC). Patterns of gene expression and splicing were profiled by Next Generation Sequencing. Student’s t-test was used to assess statistical significance. Results: We found that, on average, levels of HES6 transcript were >15-fold higher when specimens of leiomyomas were compared to adjacent normal myometrium (p<0.01). No relationship between expression of HES6 in myometrium or leiomyoma, menstrual phase (n=58), menopausal status (n=12) or clinical treatment of pre- or postmenopausal women with medroxyprogesterone (Pre: n=12; Post: n=6) was observed. When matched FFPE specimens were evaluated by IHC, we found that HES6 expression was selectively limited to organized subsets of cells within large leiomyomas as well as the blood vessels surrounding these tumors. Levels of HES6 were >4-fold lower in specimens of uLMS (n=6) than proliferative or secretory phase myometrium (p<0.01). In vitro, knockdown of HES6 expression significantly reduced rates of proliferation and enhanced rates of apoptosis across multiple primary cultures derived from matched specimens of myometrium and leiomyomas (n=10, p<0.05). Of the 123 gene products who expression was significantly altered (p<0.05) in response to targeting HES6 expression, levels of the mitochrondrial solute transporter SLC25A22 was most dramatically disrupted. Conclusions: Overexpression of HES6 is a reliable feature important for promoting the growth of benign but not malignant uterine smooth muscle tumors within metabolically defined subsets of uterine smooth muscle cells. We are currently working to determine how this highly novel pattern of intratumoral heterogeneity promotes leiomyoma growth and its hierarchical relationship to early events in the mal-differentiation of mSC. Citation Format: Joie Z. Guner, Venkata A. Jonnalagadda, Ninad A. Patel, Ramya P. Masand, Cecilia Valdes, Williams E. Gibbons, Matthew L. Anderson. Heterogeneous overexpression of HES6 promotes the growth of uterine leiomyomas [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-001.