Public Abstract The goal of this application is to develop synergistic inhibitors of CREB-mediated gene transcription. Cyclic-AMP-response element binding protein (CREB) is a 43 kD nuclear transcription factor. Its transcription activity is critically dependent on phosphorylation on Ser133 to be induced by extracellular signals including growth factors and hormones. CREB is overexpressed and/or overactivated in tumor tissues of different organs compared to normal tissue. CREB's transcription activity is activated upon phosphorylation at Ser133 by a variety of protein kinases including protein kinase A (PKA), mitogen-activated protein kinases (MAPKs), protein kinase B (PKB/Akt) and protein ribosomal S6 kinase (pp90RSK). Once phosphorylated, CREB can bind with CREB-binding protein (CBP) and its paralog p300 to recruit other components in the transcriptional machinery to the CREB promoter to initiate CREB-dependent gene transcription. CREB's phosphorylation in normal cells is tightly regulated and very dynamic. Until now, three protein phosphatases have been identified to be able to remove the phosphate from CREB to attenuate its transcription activity. They are protein phosphatase 1 (PP1), protein phosphatase 2A (PP2A) and phosphatase and tensin homolog (PTEN). Mechanistically, the kinases are often overactivated in cancer cells while the phosphatases are often inactivated in cancer cells. As a consequence, CREB is often overactivated in cancer cells compared to normal cells. Higher expression and/or activation of CREB is associated with poorer prognosis in cancer patients. Preclinical studies have validated CREB as an appealing target for various cancers. We recently identified various chemotypes as small molecule inhibitors of CREB or potentiators to exhibit synergistic activity in inhibiting CREB-mediated gene transcription. In this application, we will further develop these inhibitors and potentiators as synergistic inhibitors of CREB-mediated gene transcription. We will further study their mechanism of action and synergistic anti-breast cancer activity in vitro and in vivo. Accomplishing the proposed studies will provide novel insights into the mechanism of CREB regulation and provide potential cancer therapeutics.