Xiangshu Xiao - US grants

DESCRIPTION (provided by applicant): The goal of the proposed research is to develop small molecule inhibitors of CREB (cAMP-response element binding protein)-mediated gene transcription as potential anticancer agents. Recently, accumulating evidence has revealed that CREB participates in the regulation of immortalization, transformation and metastasis of cancer cells. In some cancer patients (e.g. acute leukemia, prostate cancer and breast cancer), expression of CREB correlated with cancer phenotypes including cancer cell differentiation, metastasis, time to relapse and survival. This suggests that inhibiting the transcription activity of CREB could be a promising strategy for anticancer treatment. Consistent with this hypothesis, expression of a dominant-negative CREB mutant in melanoma and lung cancer cell lines or down-regulating the expression of CREB in leukemia cell lines reduced the transforming phenotypes of the cancer cells. Moreover, delivery of a CRE decoy oligonucleotide into human breast cancer or prostate cancer cells led to significant inhibition of tumor cell growth both in vitro and in vivo while similar experiments in normal cells showed no toxicity. These results indicate that pharmacologically inhibiting CREB activity could be an excellent strategy for cancer treatment. However, small molecule inhibitors of CREB-mediated have not been developed for evaluation of anticancer activity. We recently developed a novel assay to specifically look at CREB-CBP interaction and discovered naphthol AS-E as a small molecule inhibitor of CREB-CBP interaction both in vitro and in cells. Moreover, we found that this compound was able to inhibit cancer cell growth by inducing apoptosis in a number of different cancer cells irrespective of their p53 status. Based on these promising preliminary results, we propose to further evaluate its therapeutic potential and optimize its anticancer activity. The following aims will be addressed: 1) To define the structure-activity relationships (SAR) of naphthol AS-E as an anticancer agent and inhibitor of CREB-CBP interaction by designing and synthesizing analogs with different substitutions on the appendant phenyl ring;2) To develop a novel series of naphthol AS-E derivatives with enhanced potency and aqueous solubility as probes for structural studies;3) To investigate the hypothesis that CREB-mediated gene transcription is required for anticancer activity displayed by naphthol AS-E and its derivatives. Development of small molecule inhibitors CREB-mediated gene transcription will not only lead to potential therapeutics for cancer, but also provide a powerful tool to manipulate CREB's activity in vitro and in vivo to further dissect its biological functions. PUBLIC HEALTH RELEVANCE: The proposed research is to develop chemical inhibitors of CREB-mediated gene transcription. These inhibitors are expected to be potential candidates as anticancer agents for a variety of cancers.

Nuclear lamins are type V intermediate filament (IF) proteins known to be structural components of nuclear lamina that lie underneath the inner nuclear membrane. Recently, lamins have been implicated in nuclear metabolism, in particular DNA damage repair process. However, the underlying molecular mechanisms are largely unknown. One of the challenges to address these mechanisms is that we lack appropriate tools to manipulate this system other than genetic knockouts, which remove the proteins entirely. In this regard, small molecule modulators of lamins will provide invaluable tools to dissect the underlying mechanisms of DNA damage repair by lamins. Lamins' involvement in DNA repair pathways is consistent with the findings that expression of lamins is often misregulated in cancer cells. DNA replication stress and reactive oxygen species are prevalent in cancer cells due to activation of oncogenes. Thus cancer cells constantly generate DNA double-strand breaks (DSBs). These DSBs must be repaired in order for the cancer cells to survive. Accordingly, over the course of development of cancer, cancer cells have co-evolved efficient DSB repair mechanisms that protect them from endogenous DNA replication stress. By exploiting the unique feature of endogenous DSBs prevalent in cancer cells, such therapeutics can potentially offer selective toxicity in cancer cells without harming normal cells. Therefore, small molecule lamin modulators can also provide potential cancer therapeutics. We recently discovered a novel compound called LBL1 that was selectively toxic to cancer cells. We further found that LBL1 selectively binds to nuclear lamins. In this application, we propose the following three specific aims to further develop LBL1 and its derivatives as potential anti-breast cancer agents and understand their mechanism of action: 1) To characterize the binding between LBL1 and lamins; 2) To define the structure-activity relationships of LBL1 as a lamin-binding ligand and an anti-breast cancer agent; 3) To investigate the mechanism of dynamic interplay between LMNA and Rad51 and how LBL1 modulates this process.

This application is to develop a novel targeted therapy for clear cell sarcoma of soft tissue (CCSST). CCSST is a rare but aggressive soft tissue sarcoma that typically develops in the lower extremity close to tendons and aponeuroses of adolescents and young adults. The 5-year survival is only 20% for metastatic disease. The current treatment option is to perform wide local surgical resection or amputation attempting to remove all the cancer cells. However, in metastatic cases, complete removal of cancer cells becomes impossible and systemic adjuvant therapy is the key to control this disease. Unfortunately, this disease is notorious for its insensitivity to current chemotherapies, underscoring an urgent need for developing novel therapies for CCSST. The hallmark of CCSST is characterized by a balanced t (12;22) (q13;q12) chromosomal translocation, which results in a fusion of the Ewing's sarcoma gene EWS with activating transcription factor 1 (ATF1) to give an oncogene EWS-ATF1. ATF1 is a member of the cAMP-responsive element binding protein (CREB) family transcription factor. EWS-ATF1 is constitutively active to drive the expression of target genes that are normally controlled by CREB/ATF1. Preclinical studies have shown that CCSST cancer cells are dependent on EWS-ATF1 for survival, suggesting that EWS-ATF1 is a powerful target to develop novel therapies for CCSST. We recently developed a small molecule called 666-15 as the first potent inhibitor CREB/ATF1-mediated gene transcription. 666-15 is well-tolerated in vivo. These preliminary results suggest that 666-15 is perfectly suited for a potential targeted therapy for CCSST. To test this hypothesis, we propose two specific aims: 1) To evaluate if 666-15 inhibits EWS-ATF1 and EWS- CREB-mediated gene transcription; 2) To investigate if 666-15 possesses anti-CCSST activity. Accomplishing these aims can potentially provide a novel targeted therapy for CCSST.

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 cues including growth factors and hormones. CREB is overexpressed and/or overactivated in tumor tissues of different cancer types compared to adjacent normal tissue. The goals of this application are to identify small molecule inhibitors of CREB (cyclic-AMP-response element binding protein)-mediated gene transcription and understand their mechanism of action as potential anti-cancer agents. Upon stimulation by extracellular cues, CREB is phosphorylated at Ser133 that promotes its association 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. Many oncogenic kinases including protein kinase A (PKA), mitogen-activated protein kinases (MAPKs), protein kinase B (PKB/Akt) and protein ribosomal S6 kinase (pp90RSK) can phosphorylate Ser133 in CREB. These kinases are often overactivated in cancer cells. On the other hand, three protein phosphatases, protein phosphatase 1 (PP1), protein phosphatase 2A (PP2A) and phosphatase and tensin homolog (PTEN), can dephosphorylate CREB to attenuate CREB-mediated gene transcription. These phosphatases are often inactivated or deleted in cancer cells. Because of this dual regulation of CREB's transcription activity, CREB has been frequently observed to be overactivated in cancer cells. Furthermore, overactivation of CREB inversely correlates with cancer patients survival. Therefore, CREB is intimately implicated in tumorigenesis and has been proposed as a valuable target for developing novel cancer therapeutics. Potentially, targeting CREB can simultaneously shut down multiple oncogenic pathways, providing a novel type of cancer therapeutics with delayed or no resistance. We recently designed and synthesized a potent CREB inhibitor, 666-15, with efficacious in vitro and in vivo anti-cancer activity. In this application, we will further study 666-15 to understand its mechanism of action and structure-activity relationship (SAR). To accomplish these goals, the following three specific aims will be addressed: 1) To identify the direct molecular target(s) of 666-15; 2) To investigate the mechanisms by which Hsp60 regulates CREB's transcription; 3) To identify derivatives of 666-15 with improved physicochemical properties.