Differential Kat3 Usage Orchestrates the Integration of Cellular Metabolism with Differentiation.

The integration of cellular status with metabolism is critically important and the coupling of energy production and cellular function is highly evolutionarily conserved. This has been demonstrated in stem cell biology, organismal, cellular and tissue differentiation and in immune cell biology. However, a molecular mechanism delineating how cells coordinate and couple metabolism with transcription as they navigate quiescence, growth, proliferation, differentiation and migration remains in its infancy. The extreme N-termini of the Kat3 coactivator family members, CBP and p300, by far the least homologous regions with only 66% identity, interact with members of the nuclear receptor family, interferon activated Stat1 and transcriptionally competent ß-catenin, a critical component of the Wnt signaling pathway. We now wish to report based on multiomic and functional investigations, utilizing p300 knockdown, N-terminal p300 edited and p300 S89A edited cell lines and p300 S89A knockin mice, that the N-termini of the Kat3 coactivators provide a highly evolutionarily conserved hub to integrate multiple signaling cascades to coordinate cellular metabolism with the regulation of cellular status and function.

Targeting the CBP/ß-Catenin Interaction to Suppress Activation of Cancer-Promoting Pancreatic Stellate Cells.

BACKGROUND: Although cyclic AMP-response element binding protein-binding protein (CBP)/ß-catenin signaling is known to promote proliferation and fibrosis in various organ systems, its role in the activation of pancreatic stellate cells (PSCs), the key effector cells of desmoplasia in pancreatic cancer and fibrosis in chronic pancreatitis, is largely unknown. METHODS: To investigate the role of the CBP/ß-catenin signaling pathway in the activation of PSCs, we have treated mouse and human PSCs with the small molecule specific CBP/ß-catenin antagonist ICG-001 and examined the effects of treatment on parameters of activation. RESULTS: We report for the first time that CBP/ß-catenin antagonism suppresses activation of PSCs as evidenced by their decreased proliferation, down-regulation of "activation" markers, e.g., α-smooth muscle actin (α-SMA/Acta2), collagen type I alpha 1 (Col1a1), Prolyl 4-hydroxylase, and Survivin, up-regulation of peroxisome proliferator activated receptor gamma (Ppar-γ) which is associated with quiescence, and reduced migration; additionally, CBP/ß-catenin antagonism also suppresses PSC-induced migration of cancer cells. CONCLUSION: CBP/ß-catenin antagonism represents a novel therapeutic strategy for suppressing PSC activation and may be effective at countering PSC promotion of pancreatic cancer.

The Mode of Stem Cell Division Is Dependent on the Differential Interaction of ß-Catenin with the Kat3 Coactivators CBP or p300.

Normal long-term repopulating somatic stem cells (SSCs) preferentially divide asymmetrically, with one daughter cell remaining in the niche and the other going on to be a transient amplifying cell required for generating new tissue in homeostatic maintenance and repair processes, whereas cancer stem cells (CSCs) favor symmetric divisions. We have previously proposed that differential ß-catenin modulation of transcriptional activity via selective interaction with either the Kat3 coactivator CBP or its closely related paralog p300, regulates symmetric versus asymmetric division in SSCs and CSCs. We have previously demonstrated that SSCs that divide asymmetrically per force retain one of the dividing daughter cells in the stem cell niche, even when treated with specific CBP/ß-catenin antagonists, whereas CSCs can be removed from their niche via forced stochastic symmetric differentiative divisions. We now demonstrate that loss of p73 in early corticogenesis biases ß-catenin Kat3 coactivator usage and enhances ß-catenin/CBP transcription at the expense of ß-catenin/p300 transcription. Biased ß-catenin coactivator usage has dramatic consequences on the mode of division of neural stem cells (NSCs), but not neurogenic progenitors. The observed increase in symmetric divisions due to enhanced ß-catenin/CBP interaction and transcription leads to an immediate increase in NSC symmetric differentiative divisions. Moreover, we demonstrate for the first time that the complex phenotype caused by the loss of p73 can be rescued in utero by treatment with the small-molecule-specific CBP/ß-catenin antagonist ICG-001. Taken together, our results demonstrate the causal relationship between the choice of ß-catenin Kat3 coactivator and the mode of stem cell division.

Convergence of Canonical and Non-Canonical Wnt Signal: Differential Kat3 Coactivator Usage.

BACKGROUND: The ancient and highly evolutionarily conserved Wnt signaling pathway is critical in nearly all tissues and organs for an organism to develop normally from embryo through adult. Wnt signaling is generally parsed into "canonical" or Wnt-ß-catenin-dependent or "non-canonical" ß-catenin-independent signaling. Even though designating Wnt signaling as either canonical or noncanonical allows for easier conceptual discourse about this signaling pathway, in fact canonical and non-canonical Wnt crosstalk regulates complex nonlinear networks. OBJECTIVE: In this perspective, we discuss the integration of canonical and non-canonical Wnt signaling via differential Kat3 (CBP and p300) coactivator usage, thereby regulating and coordinating gene expression programs associated with both proliferation and cellular differentiation and morphogenesis. METHODS: Pharmacologic inhibitors, cell culture, real-time PCR, chromatin immunoprecipitation, protein immunoprecipitation, Western blotting, reporter-luciferase, protein purification, site-directed mutagenesis, in vitro phosphorylation and binding assays, and immunofluorescence were utilized. CONCLUSION: Coordinated integration between both canonical and non-canonical Wnt pathways appears to be crucial not only in the control of fundamental morphologic processes but also in the regulation of normal as well as pathologic events. Such integration between both canonical and non-canonical Wnt signaling is presumably effected via reversible phosphorylation mechanism (e.g., protein kinase C) to regulate differential ß -catenin/Kat3 coactivator usage in order to coordinate proliferation with differentiation and adhesion.

Enhanced Kat3A/Catenin transcription: a common mechanism of therapeutic resistance.

Cancers are heterogeneous at the cellular level. Cancer stem cells/tumor initiating cells (CSC/TIC) both initiate tumorigenesis and are responsible for therapeutic resistance and disease relapse. Elimination of CSC/TIC should therefore be able to reverse therapy resistance. In principle, this could be accomplished by either targeting cancer stem cell surface markers or "stemness" pathways. Although the successful therapeutic elimination of "cancer stemness" is a critical goal, it is complex in that it should be achieved without depletion of or increases in somatic mutations in normal tissue stem cell populations. In this perspective, we will discuss the prospects for this goal via pharmacologically targeting differential Kat3 coactivator/Catenin usage, a fundamental transcriptional control mechanism in stem cell biology.

Differentiation Therapy Targeting the ß-Catenin/CBP Interaction in Pancreatic Cancer.

BACKGROUND: Although canonical Wnt signaling is known to promote tumorigenesis in pancreatic ductal adenocarcinoma (PDAC), a cancer driven principally by mutant K-Ras, the detailed molecular mechanisms by which the Wnt effector ß-catenin regulates such tumorigenesis are largely unknown. We have previously demonstrated that ß-catenin's differential usage of the Kat3 transcriptional coactivator cyclic AMP-response element binding protein-binding protein (CBP) over its highly homologous coactivator p300 increases self-renewal and suppresses differentiation in other types of cancer. AIM/METHODS: To investigate Wnt-mediated carcinogenesis in PDAC, we have used the specific small molecule CBP/ß-catenin antagonist, ICG-001, which our lab identified and has extensively characterized, to examine its effects in human pancreatic cancer cells and in both an orthotopic mouse model and a human patient-derived xenograft (PDX) model of PDAC. RESULTS/CONCLUSION: We report for the first time that K-Ras activation increases the CBP/ß-catenin interaction in pancreatic cancer; and that ICG-001 specific antagonism of the CBP/ß-catenin interaction sensitizes pancreatic cancer cells and tumors to gemcitabine treatment. These effects were associated with increases in the expression of let-7a microRNA; suppression of K-Ras and survivin; and the elimination of drug-resistant cancer stem/tumor-initiating cells.

Wnt signaling orchestration with a small molecule DYRK inhibitor provides long-term xeno-free human pluripotent cell expansion.

An optimal culture system for human pluripotent stem cells should be fully defined and free of animal components. To date, most xeno-free culture systems require human feeder cells and/or highly complicated culture media that contain activators of the fibroblast growth factor (FGF) and transforming growth factor-ß (TGFß) signaling pathways, and none provide for replacement of FGF/TGFß ligands with chemical compounds. The Wnt/ß-catenin signaling pathway plays an important role in mouse embryonic stem cells in leukemia inhibitory factor-independent culture; however, the role of Wnt/ß-catenin signaling in human pluripotent stem cell is still poorly understood and controversial because of the dual role of Wnts in proliferation and differentiation. Building on our previous investigations of small molecules modulating Wnt/ß-catenin signaling in mouse embryonic stem cells, we identified a compound, ID-8, that could support Wnt-induced human embryonic stem cell proliferation and survival without differentiation. Dual-specificity tyrosine phosphorylation-regulated kinase (DYRK) is the target of the small molecule ID-8. Its role in human pluripotent cell renewal was confirmed by DYRK knockdown in human embryonic stem cells. Using Wnt and the DYRK inhibitor ID-8, we have developed a novel and simple chemically defined xeno-free culture system that allows for long-term expansion of human pluripotent stem cells without FGF or TGFß activation. These culture conditions do not include xenobiotic supplements, serum, serum replacement, or albumin. Using this culture system, we have shown that several human pluripotent cell lines maintained pluripotency (>20 passages) and a normal karyotype and still retained the ability to differentiate into derivatives of all three germ layers. This Wnt-dependent culture system should provide a platform for complete replacement of growth factors with chemical compounds.

The Wnt signaling pathway in cellular proliferation and differentiation: A tale of two coactivators.

Wnt signaling pathways play divergent roles during development, normal homeostasis and disease. The responses that result from the activation of the pathway control both proliferation and differentiation. Tight regulation and controlled coordination of the Wnt signaling cascade is required to maintain the balance between proliferation and differentiation. The non-redundant roles of the coactivator proteins CBP and p300, within the context of Wnt signaling are discussed. We highlight their roles as integrators of the various inputs that a cell receives to elicit the correct and coordinated response. We propose that essentially all cellular information - i.e. from other signaling pathways, nutrient levels, etc. - is funneled down into a choice of coactivators usage, either CBP or p300, by their interacting partner beta-catenin (or catenin-like molecules in the absence of beta-catenin) to make the critical decision to either remain quiescent, or once entering cycle to proliferate without differentiation or to initiate the differentiation process.

Wnt/beta-catenin/CBP signaling maintains long-term murine embryonic stem cell pluripotency.

Embryonic stem cells (ESCs) represent an important research tool and a potential resource for regenerative medicine. Generally, ESCs are cocultured with a supportive feeder cell layer of murine embryonic fibroblasts, which maintain the ESCs' capacity for self-renewal and block spontaneous differentiation. These cumbersome conditions, as well as the risk of xenobiotic contamination of human ESCs grown on murine embryonic fibroblasts, make it a priority to develop chemically defined methods that can be safely used for the expansion of ESCs. Using a high-throughput, cell-based assay, we identified the small molecule IQ-1 that allows for the Wnt/beta-catenin-driven long-term expansion of mouse ESCs and prevents spontaneous differentiation. We demonstrate that IQ-1, by targeting the PR72/130 subunit of the serine/threonine phosphatase PP2A, prevents beta-catenin from switching coactivator usage from CBP to p300. The increase in beta-catenin/CBP-mediated transcription at the expense of beta-catenin/p300-mediated transcription is critical for the maintenance of murine stem cell pluripotency.

Specific inhibition of CBP/beta-catenin interaction rescues defects in neuronal differentiation caused by a presenilin-1 mutation.

Wnt/beta-catenin signaling has been shown to promote self-renewal in a variety of tissue stem cells, including neuronal stem cells and hematopoietic stem cells. However, activation of the Wnt/beta-catenin pathway promoted and inhibition of the pathway prevented differentiation of neuronal precursor cells. A clear explanation for the differential effects of Wnt/beta-catenin activation on neuronal precursors is not available at present. Presenilin-1 (PS-1) is a polytopic protein comprised of six to eight transmembrane domains. PS-1, as part of the gamma-secretase complex, is required for the intramembrane proteolysis of both amyloid precursor protein (APP) and Notch. Additionally, through interactions with beta-catenin, PS-1 is associated with modulation of Wnt/beta-catenin signaling. A familial Alzheimer's disease-associated PS-1 mutant, PS-1(L286V), causes a dramatic increase in T cell factor (TCF)/beta-catenin transcription in PC-12 cells, which prevents normal nerve growth factor (NGF)-induced neuronal differentiation and neurite outgrowth. Selective inhibition of TCF/beta-catenin/cAMP-response element-binding protein (CREB)-binding protein (CBP)-mediated transcription, but not TCF/beta-catenin/p300, with the recently described small molecule antagonist ICG-001 corrects these defects in neuronal differentiation, highlighting the importance of Wnt/beta-catenin signaling in this process. We propose that increased TCF/beta-catenin/CBP-mediated transcription, as well as a failure to switch to TCF/beta-catenin/p300-mediated transcription, play an important role in decreasing neuronal differentiation.

A small molecule inhibitor of beta-catenin/CREB-binding protein transcription [corrected].

Inherited and somatic mutations in the adenomatous polyposis coli occur in most colon cancers, leading to activation of beta-catenin-responsive genes. To identify small molecule antagonists of this pathway, we challenged transformed colorectal cells with a secondary structure-templated chemical library, looking for compounds that inhibit a beta-catenin-responsive reporter. We identified ICG-001, a small molecule that down-regulates beta-catenin/T cell factor signaling by specifically binding to cyclic AMP response element-binding protein. ICG-001 selectively induces apoptosis in transformed cells but not in normal colon cells, reduces in vitro growth of colon carcinoma cells, and is efficacious in the Min mouse and nude mouse xenograft models of colon cancer.

Chemogenomics with peptide secondary structure mimetics.

There is increasing evidence that redox regulation of transcription, particularly activator protein-1 (AP-1) and nuclear factor kappa B (NF-kappaB), is important in inflammatory diseases. Human thioredoxin (TRX), a member of the oxidoreductase superfamily, was initially identified, as a factor which augments the production of interleukin-2 receptor alpha (IL-2R alpha) in human T-cell lymphotropic virus type 1 (HTLV-1) infected patient T-cells. Substrates for the redox activity of TRX bind the active site cleft in extended strand structure. The rapid generation of high numbers of peptide secondary structure mimetics through solid-phase synthesis is a key technology for the identification of pharmaceutical leads based on such protein-peptide interactions. In this manuscript, we describe a chemogenomic approach utilizing an extended strand templated library to develop small molecule inhibitors to validate oxidoreductase molecular targets in a murine asthma model.

Chemogenomic identification of Ref-1/AP-1 as a therapeutic target for asthma.

Asthma is characterized by an oxidantantioxidant imbalance in the lungs leading to activation of redox-sensitive transcription factors, nuclear factor kappaB (NF-kappaB), and activator protein-1 (AP-1). To develop therapeutic strategies for asthma, we used a chemogenomics approach to screen for small molecule inhibitor(s) of AP-1 transcription. We developed a beta-strand mimetic template that acts as a reversible inhibitor (pseudosubstrate) of redox proteins. This template incorporates an enedione moiety to trap reactive cysteine nucleophiles in the active sites of redox proteins. Specificity for individual redox factors was achieved through variations in X and Y functionality by using a combinatorial library approach. A limited array (2 x 6) was constructed where X was either NHCH(3) or NHCH(2) Ph and Y was methyl, phenyl, m-cyanophenyl, m-nitrophenyl, m-acetylaniline, or m-methylbenzoate. These analogs were evaluated for their ability to inhibit transcription in transiently transfected human lung epithelial A549 cells from either an AP-1 or NF-kappaB reporter. A small-molecule inhibitor, PNRI-299, was identified that selectively inhibited AP-1 transcription (IC(50) of 20 microM) without affecting NF-kappaB transcription (up to 200 microM) or thioredoxin (up to 200 microM). The molecular target of PNRI-299 was determined to be the oxidoreductase, redox effector factor-1 by an affinity chromatography approach. The selective redox effector factor-1 inhibitor, PNRI-299, significantly reduced airway eosinophil infiltration, mucus hypersecretion, edema, and IL-4 levels in a mouse asthma model. These data validate AP-1 as an important therapeutic target in allergic airway inflammation.

A small molecule inhibitor of redox-regulated NF-kappa B and activator protein-1 transcription blocks allergic airway inflammation in a mouse asthma model.

An oxidant/antioxidant imbalance is seen in the lungs of patients with asthma. This oxidative stress in asthmatic airways may lead to activation of redox-sensitive transcription factors, NF-kappaB and AP-1. We examined the effect of the small molecule inhibitor of redox-regulated NF-kappaB and AP-1 transcription, MOL 294 on airway inflammation and airway hyperreactivity (AHR) in a mouse model of asthma. MOL 294 is a potent nonpeptide inhibitor of NF-kappaB and AP-1 based upon a beta-strand template that binds to and inhibits the cellular redox protein thioredoxin. BALB/c mice after i.p. OVA sensitization (day 0) were challenged with intranasal OVA on days 14, 25, 26, and 27. MOL 294, administered intranasal on days 25-27, blocked the airway inflammatory response to OVA assessed 24 h after the last OVA challenge on day 28. MOL 294 reduced eosinophil, IL-13, and eotaxin levels in bronchoalveolar lavage fluid and airway tissue eosinophilia and mucus hypersecretion. MOL 294 also decreased AHR in vivo to methacholine. These results support redox-regulated transcription as a therapeutic target in asthma and demonstrate that selective inhibitors can reduce allergic airway inflammation and AHR.