RUNX proteins desensitize multiple myeloma to lenalidomide via protecting IKZFs from degradation.

Ikaros family zinc finger protein 1 and 3 (IKZF1 and IKZF3) are transcription factors that promote multiple myeloma (MM) proliferation. The immunomodulatory imide drug (IMiD) lenalidomide promotes myeloma cell death via Cereblon (CRBN)-dependent ubiquitylation and proteasome-dependent degradation of IKZF1 and IKZF3. Although IMiDs have been used as first-line drugs for MM, the overall survival of refractory MM patients remains poor and demands the identification of novel agents to potentiate the therapeutic effect of IMiDs. Using an unbiased screen based on mass spectrometry, we identified the Runt-related transcription factor 1 and 3 (RUNX1 and RUNX3) as interactors of IKZF1 and IKZF3. Interaction with RUNX1 and RUNX3 inhibits CRBN-dependent binding, ubiquitylation, and degradation of IKZF1 and IKZF3 upon lenalidomide treatment. Inhibition of RUNXs, via genetic ablation or a small molecule (AI-10-104), results in sensitization of myeloma cell lines and primary tumors to lenalidomide. Thus, RUNX inhibition represents a valuable therapeutic opportunity to potentiate IMiDs therapy for the treatment of multiple myeloma.

Regulation and function of runt-related transcription factors (RUNX1 and RUNX2) in goat granulosa cells.

Transcription factors, runt-related transcription factor 1 (RUNX1) and 2 (RUNX2), belong to the runt-related (RUNX) gene family and play critical roles in mammalian reproduction processes. However, the regulatory mechanisms of RUNX1 and RUNX2 expression or their functions in goat follicles remain largely unknown. Herein, RUNX1 and RUNX2 proteins were detected in the oocytes and granulosa cells of preantral and antral follicles, as well as corpus luteum by immunohistochemistry. Treatments with human chorionic gonadotropin (hCG) or with the agonists and inhibitors of hCG-induced intracellular signaling pathways in granulosa cells in vitro, we found that hCG increased RUNX1 expression by activating PKC and PI3K signaling molecules, and increased RUNX2 expression by activating adenylate cyclase, PKC, and PI3K signaling molecules. We also demonstrated that miR-181b expression is dependent on the hCG-induced activation of PKC and PKA, and miR-222 expression is dependent on the hCG-induced activation of PI3K and PKC in cultured granulosa cells. Meanwhile, miR-181b and miR-222 suppressed RUNX1 and RUNX2 expression by targeting RUNX1 and RUNX2 3' untranslated regions (3'UTRs) with or without hCG, respectively. These results suggested that hCG-mediated miR-181b and miR-222 expression are important for the regulation of RUNX1 and RUNX2 expression levels in granulosa cells. To explore the specific functions of RUNX1 and RUNX2, we transfected RUNX1 and RUNX2 small interfering RNAs into primary cultured granulosa cells. Knockdown of RUNX1 and RUNX2 significantly decreased progesterone productions and the mRNA abundance of key steroidogenic enzymes (StAR, CYP11A1 and HSD3B) after hCG treatment. But only miR-222 increased estradiol secretion in goat granulosa cells. In addition, knockdown of RUNX1 and RUNX2 also promoted granulosa cell proliferation. The hormonally regulated expression of RUNX1 and RUNX2 in granulosa cells, their involvement in progesterone production, and promoted granulosa cell proliferation suggest important roles of RUNX1 and RUNX2 in follicular development and luteinization.

Small molecule inhibition of the CBFß/RUNX interaction decreases ovarian cancer growth and migration through alterations in genes related to epithelial-to-mesenchymal transition.

OBJECTIVE: Ovarian cancer survival and treatment have improved minimally in the past 20years. Novel treatment strategies are needed to combat this disease. This study investigates the effects of chemical inhibition of the CBFß/RUNX protein-protein interaction on ovarian cancer cell lines. METHODS: Ovarian cancer cell lines were treated with CBFß/RUNX inhibitors, and the effects on proliferation, DNA replication, wound healing, and anchorage-independent growth were measured. RNA-Seq was performed on compound-treated cells to identify differentially expressed genes. Genes altered by compound treatment were targeted with siRNAs, and effects on DNA replication and wound healing were measured. RESULTS: Chemical inhibition of the CBFß/RUNX interaction decreases ovarian cancer cell proliferation. Inhibitor treatment leads to an S-phase cell cycle delay, as indicated by an increased percentage of cells in S-phase, and a decreased DNA replication rate. Inhibitor treatment also reduces wound healing and anchorage-independent growth. RNA-Seq on compound-treated cells revealed changes in a small number of genes related to proliferation and epithelial-to-mesenchymal transition. siRNA-mediated knockdown of INHBA and MMP1 - two genes whose expression decreases with compound treatment - slowed DNA replication and impaired wound healing. CONCLUSIONS: Chemical inhibition of the CBFß/RUNX interaction is a viable strategy for the treatment of ovarian cancer.

Antagonistic interplay between ThPOK and Runx in lineage choice of thymocytes.

Differentiation of CD4(+)CD8(+) double-positive (DP) thymocytes into either CD4(+)-helper or CD8(+)-cytotoxic lineages involves several phases. It has been suggested that, following initial specification to one of the lineages by a set of lineage-specific genes during positive selection, stable cell identity is established during the commitment process by eliminating differentiation potential toward the other lineage. While the Runx3 transcription factor fixes the Cd4 gene into a silenced state during cytotoxic-lineage cell differentiation, the ThPOK transcription factor is both necessary and sufficient to generate a CD4(+)CD8(-) phenotype in post-selection thymocytes, regardless of the MHC specificity of the TCRs. Recent studies have revealed that a reciprocal antagonistic interplay between Runx3 and ThPOK is a central component in the transcription factor network governing the helper versus cytotoxic-lineage decision.

The B cell translocation gene (BTG) family in the rat ovary: hormonal induction, regulation, and impact on cell cycle kinetics.

The B cell translocation gene (BTG) family regulates gene transcription and cellular differentiation and inhibits proliferation. The present study investigated the spatiotemporal expression pattern of BTG members and their potential role in the rat ovary during the periovulatory period. Immature female rats (22-23 d old) were injected with pregnant mare serum gonadotropin to stimulate follicular development. Ovaries or granulosa cells were collected at various times after hCG administration (n = 3 per time point). Real-time PCR analysis revealed that mRNA for Btg1, Btg2, and Btg3 were highly induced both in intact ovaries and granulosa cells by 4-8 h after hCG treatment, although their temporal expression patterns differed. In situ hybridization analysis demonstrated that Btg1 mRNA expression was highly induced in theca cells at 4 h after hCG, primarily localized to granulosa cells at 8 h, and decreased at 24 h. Btg2 and Btg3 mRNA was also induced in granulosa cells; however, Btg2 mRNA was observed in newly forming corpora lutea. Inhibition of progesterone action and the epidermal growth factor pathway did not change Btg1 and Btg2 mRNA expression, whereas inhibition of prostaglandin synthesis or RUNX activity diminished Btg2 mRNA levels. Overexpression of BTG1 or BTG2 arrested granulosa cells at the G0/G1 phase of the cell cycle and decreased cell apoptosis. In summary, hCG induced Btg1, Btg2, and Btg3 mRNA expression predominantly in the granulosa cell compartment. Our findings suggest that the induction of the BTG family may be important for theca and granulosa cell differentiation into luteal cells by arresting cell cycle progression.

Physical and functional interactions between STAT5 and Runx transcription factors.

The signal transducers and activators of transcription (STAT) and the Runt-related (Runx) are two of major transcription factor families that play essential roles in lymphocyte development. Although the interaction of Runx2 with STAT1 and STAT3 has been reported before, the interaction between STAT5 and Runx family proteins has not been characterized. In this study, we first showed that STAT5 physically interacts with Runx1, Runx2 and Runx3 by co-immunoprecipitation experiments. The Runt domain of Runx proteins and the DNA-binding domain and alpha-helix loop structure of STAT5 are responsible for the interaction. When expressed in CHO cells, STAT5 inhibits the nuclear localization of Runx proteins and retains them in the cytoplasm. In addition, we showed by reporter assay that the interaction between STAT5 and Runx proteins mutually inhibits their transcriptional activity. Furthermore, Runx proteins inhibit the DNA-binding activity of STAT5. Finally, we found that Runx proteins suppress the transcription of an endogenous STAT5 target gene, cytokine-inducible SH2 protein-1, in an interleukin-3-dependent pro-B cell line, Ba/F3. These results collectively suggested that STAT5 and Runx proteins physically and functionally interact to mutually inhibit their transcriptional activity. Thus, this study implies a potential role of the STAT5-Runx interaction in lymphocyte development.