Characterization of CADD522, a small molecule that inhibits RUNX2-DNA binding and exhibits antitumor activity

Abstract

The RUNX2 transcription factor promotes breast cancer growth and metastasis through interactions with a variety of cofactors that activate or repress target genes. Using a direct drug discovery approach we identified CADD522 as a small molecule that inhibits the DNA binding of the runt box domain protein, RUNX2. The current study defines the effect of CADD522 on breast cancer growth and metastasis, and addresses the mechanisms by which it exerts its anti-tumor activity.CADD522 treatment resulted in significant growth inhibition, clonogenic survival, tumorsphere formation, and invasion of breast cancer cells. CADD522 negatively regulated transcription of RUNX2 target genes such as matrix metalloproteinase-13, vascular endothelial growth factor and glucose transporter-1, but upregulated RUNX2 expression by increasing RUNX2 stability. CADD522 reduced RUNX2-mediated increases in glucose uptake and decreased the level of CBF-β and RUNX2 phosphorylation at the S451 residue. These results suggest several potential mechanisms by which CADD522 exerts an inhibitory function on RUNX2-DNA binding; interference with RUNX2 for the DNA binding pocket, inhibition of glucose uptake leading to cell cycle arrest, down-regulation of CBF-β, and reduction of S451-RUNX2 phosphorylation.The administration of CADD522 into MMTV-PyMT mice resulted in significant delay in tumor incidence and reduction in tumor burden. A significant decrease of tumor volume was also observed in a CADD522-treated human triple-negative breast cancer-patient derived xenograft model. CADD522 impaired the lung retention and outgrowth of breast cancer cells in vivo with no apparent toxicity to the mice. Therefore, by inhibiting RUNX2-DNA binding, CADD522 may represent a potential antitumor drug.

Targeting breast cancer metabolism with a novel inhibitor of mitochondrial ATP synthesis

“…We previously reported that the transcription factor runt-related transcription factor 2 (RUNX2) promotes glycolytic switching in BC cells by increasing expression of genes regulating glycolytic pathways [52]. RUNX2 decreases pyruvate dehydrogenase (PDH) activity but RUNX2 KD increases mitochondrial oxygen consumption rate (OCR) by increasing the activity of PDH, a rate limiting step for entry into the TCA cycle at the branch point for pyruvate utilization.…”

“…These findings led us to hypothesize that targeting RUNX2 might inhibit BC growth and/or progression by reversing tumor cell dependence on glycolysis. In our effort to find small molecules that target RUNX2 by interfering with RUNX2 binding to the specific DNA sequences, we used Computer-Assisted Drug Design (CADD) [52,53]. Based on this approach, CADD522 ( Supplementary Figure 2, left) was identified as a potent inhibitor of RUNX2-DNA binding [52,53].…”

“…In our effort to find small molecules that target RUNX2 by interfering with RUNX2 binding to the specific DNA sequences, we used Computer-Assisted Drug Design (CADD) [52,53]. Based on this approach, CADD522 ( Supplementary Figure 2, left) was identified as a potent inhibitor of RUNX2-DNA binding [52,53]. www.oncotarget.com CADD522 inhibited the in vitro DNA binding activity of RUNX2 at nanomolar concentrations (IC50 ≅ 10 nM) [53] and inhibited BC growth and metastasis in both in vitro and in vivo models by specifically downregulating RUNX2-mediated transcription of downstream targets [52].…”

“…Based on this approach, CADD522 ( Supplementary Figure 2, left) was identified as a potent inhibitor of RUNX2-DNA binding [52,53]. www.oncotarget.com CADD522 inhibited the in vitro DNA binding activity of RUNX2 at nanomolar concentrations (IC50 ≅ 10 nM) [53] and inhibited BC growth and metastasis in both in vitro and in vivo models by specifically downregulating RUNX2-mediated transcription of downstream targets [52]. Since CADD522 suppressed RUNX2-mediated Glut-1 expression as well as glucose uptake and utilization [52], we initially hypothesized that CADD522 could restore the mitochondrial metabolic pathways that RUNX2 repressed.…”

Systems genetics identifies a macrophage cholesterol network associated with physiological wound healing

“…In order to validate Runx2 regulation of MMHN genes in vitro, we cultured BMDMs and incubated them for 48 hours with 20 μM CADD522, a drug previously shown to selectively block Runx2 DNA binding activity (37). Expression of genes positively and negatively correlated with Runx2 was then assessed by quantitative reverse-transcription PCR (qRT-PCR).…”