4-Pyridone-3-carboxylic acid as a benzoic acid bioisostere: Design, synthesis, and evaluation of EP300/CBP histone acetyltransferase inhibitors.

Histone acetyltransferases (HATs) play a crucial role in post-translational modification. Among them, overexpression, mutation, or hyperfunction of EP300/CBP has been associated with various cancers. In this study, we identified the novel compound 2-chloro-5-[5-[(E)-[1-(3-chlorophenyl)-3-methyl-5-oxo-pyrazol-4-ylidene]methyl]-2-furyl]benzoic acid (1) as an EP300 HAT inhibitor via virtual screening. Further research has been focused on the design, synthesis, and in vitro biological evaluation of virtual hit derivatives. The studies revealed that 4-pyridone-3-carboxylic acid derivatives exhibited bioisosterism of benzoic acid. Replacement proved effective, providing compounds with similar EP300 HAT-inhibitory activity and improved cell growth-inhibitory activity compared to the benzoic acid analogs. Through these studies, we identified a potent and selective EP300/CBP HAT inhibitor.

Radiation-Induced Overexpression of TGFß and PODXL Contributes to Colorectal Cancer Cell Radioresistance through Enhanced Motility.

The primary cause of colorectal cancer (CRC) recurrence is increased distant metastasis after radiotherapy, so there is a need for targeted therapeutic approaches to reduce the metastatic-relapse risk. Dysregulation of the cell-surface glycoprotein podocalyxin-like protein (PODXL) plays an important role in promoting cancer-cell motility and is associated with poor prognoses for many malignancy types. We found that CRC cells exposed to radiation demonstrated increased TGFß and PODXL expressions, resulting in increased migration and invasiveness due to increased extracellular matrix deposition. In addition, both TGFß and PODXL were highly expressed in tissue samples from radiotherapy-treated CRC patients compared to those from patients without this treatment. However, it is unclear whether TGFß and PODXL interactions are involved in cancer-progression resistance after radiation exposure in CRC. Here, using CRC cells, we showed that silencing PODXL blocked radiation-induced cell migration and invasiveness. Cell treatment with galunisertib (a TGFß-pathway inhibitor) also led to reduced viability and migration, suggesting that its clinical use may enhance the cytotoxic effects of radiation and lead to the effective inhibition of CRC progression. Overall, the results demonstrate that downregulation of TGFß and its-mediated PODXL may provide potential therapeutic targets for patients with radiotherapy-resistant CRC.

Evidence for Delivery of Abraxane via a Denatured-Albumin Transport System.

Abraxane, an albumin-bound paclitaxel nanoparticle formulation, is superior to conventional paclitaxel preparations because it has better efficacy against unresectable pancreatic cancer. Previous reports suggest that this better efficacy of Abraxane than conventional paclitaxel preparation is probably due to its transport through Gp60, an albumin receptor on the surface of vascular endothelial cells. The increased tumor accumulation of Abraxane is also caused by the secreted protein acid and rich in cysteine in the tumor stroma. However, the uptake mechanism of Abraxane remains poorly understood. In this study, we demonstrated that the delivery of Abraxane occurred via different receptor pathways from that of endogenous albumin. Our results showed that the uptake of endogenous albumin was inhibited by a Gp60 pathway inhibitor in the process of endocytosis through endothelial cells or tumor cells. In contrast, the uptake of Abraxane-derived HSA was less affected by the Gp60 pathway inhibitor but significantly reduced by denatured albumin receptor inhibitors. In conclusion, these data indicate that Abraxane-derived HSA was taken up into endothelial cells or tumor cells by a mechanism different from normal endogenous albumin. These new data on distinct cellular transport pathways of denatured albumin via gp family proteins different from those of innate albumin shed light on the mechanisms of tumor delivery and antitumor activity of Abraxane and provide new scientific rationale for the development of a novel albumin drug delivery strategy via a denatured albumin receptor.

E7386, a Selective Inhibitor of the Interaction between ß-Catenin and CBP, Exerts Antitumor Activity in Tumor Models with Activated Canonical Wnt Signaling.

The Wnt/ß-catenin signaling pathway plays crucial roles in embryonic development and the development of multiple types of cancer, and its aberrant activation provides cancer cells with escape mechanisms from immune checkpoint inhibitors. E7386, an orally active selective inhibitor of the interaction between ß-catenin and CREB binding protein, which is part of the Wnt/ß-catenin signaling pathway, disrupts the Wnt/ß-catenin signaling pathway in HEK293 and adenomatous polyposis coli (APC)-mutated human gastric cancer ECC10 cells. It also inhibited tumor growth in an ECC10 xenograft model and suppressed polyp formation in the intestinal tract of ApcMin /+ mice, in which mutation of Apc activates the Wnt/ß-catenin signaling pathway. E7386 demonstrated antitumor activity against mouse mammary tumors developed in mouse mammary tumor virus (MMTV)-Wnt1 transgenic mice. Gene expression profiling using RNA sequencing data of MMTV-Wnt1 tumor tissue from mice treated with E7386 showed that E7386 downregulated genes in the hypoxia signaling pathway and immune responses related to the CCL2, and IHC analysis showed that E7386 induced infiltration of CD8+ cells into tumor tissues. Furthermore, E7386 showed synergistic antitumor activity against MMTV-Wnt1 tumor in combination with anti-PD-1 antibody. In conclusion, E7386 demonstrates clear antitumor activity via modulation of the Wnt/ß-catenin signaling pathway and alteration of the tumor and immune microenvironments, and its antitumor activity can be enhanced in combination with anti-PD-1 antibody. SIGNIFICANCE: These findings demonstrate that the novel anticancer agent, E7386, modulates Wnt/ß-catenin signaling, altering the tumor immune microenvironment and exhibiting synergistic antitumor activity in combination with anti-PD-1 antibody.

PODXL might be a new prognostic biomarker in various cancers: a meta-analysis and sequential verification with TCGA datasets.

ABSRACT: BACKGROUND: Several studies have investigated the associations between the podocalyxin-like protein (PODXL) expression quantity or locations and cancers survival, but the results were far from conclusive. Therefore, we proceeded a meta-analysis on PODXL in various human cancers to find its prognostic value and followed confirmation using the TCGA datasets. METHODS: We performed a systematic search, and 18 citations, including 5705 patients were pooled in meta-analysis. The results were verified with TCGA datasets. RESULTS: Total eligible studies comprised 5705 patients with 10 types of cancer. And the result indicated that PODXL high-expression or membrane-expression were significantly related to poor overall survival (OS). However, subgroup analysis showed a significant association between high expressed PODXL and poor OS in the colorectal cancer, pancreatic cancer, urothelial bladder cancer, renal cell carcinoma and glioblastoma multiforme. Then, we validated the inference using TCGA datasets, and the consistent results were demonstrated in patients with pancreatic cancer, glioblastoma multiforme, gastric cancer, esophageal cancer and lung adenocarcinoma. CONCLUSION: The result of meta-analysis showed that high expressed PODXL was significantly linked with poor OS in pancreatic cancer and glioblastoma multiforme, but not in gastric cancer, esophageal cancer or lung adenocarcinoma. And the membrane expression of PODXL might also associate with poor OS. PODXL may act as tumor promotor and may serve as a potential target for antitumor therapy.

Therapeutic targeting of p300/CBP HAT domain for the treatment of NUT midline carcinoma.

Nuclear protein of the testis (NUT) midline carcinoma (NMC), is a rare and highly aggressive form of undifferentiated squamous cell carcinoma. NMC is molecularly characterized by chromosomal rearrangement of the NUT gene to another gene, most commonly the bromodomain and extraterminal domain (BET) gene BRD4, forming the BRD4-NUT fusion oncogene. Therefore, inhibiting BRD4-NUT oncogenic function directly by BET inhibitors represents an attractive therapeutic approach but toxicity may limit the use of pan-BET inhibitors treating this cancer. We thus performed a drug screening approach using a library consisting of epigenetic compounds and 'Donated Chemical Probes' collated by the Structural Genomics Consortium (SGC) and identified the p300/CBP HAT inhibitor A-485, in addition to the well-known BET inhibitor JQ1, to be the most active candidate for NMC treatment. In contrast to JQ1, A-485 was selectively potent in NMC compared to other cell lines tested. Mechanistically, A-485 inhibited p300-mediated histone acetylation, leading to disruption of BRD4-NUT binding to hyperacetylated megadomains. Consistently, BRD4-NUT megadomain-associated genes MYC, CCAT1 and TP63 were downregulated by A-485. A-485 strongly induced squamous differentiation, cell cycle arrest and apoptosis. Combined inhibition of p300/CBP and BET showed synergistic effects. In summary, we identified the p300/CBP HAT domain as a putative therapeutic target in highly therapy-resistant NMC.

The novel BET-CBP/p300 dual inhibitor NEO2734 is active in SPOP mutant and wild-type prostate cancer.

CULLIN3-based E3 ubiquitin ligase substrate-binding adaptor gene SPOP is frequently mutated in prostate cancer (PCa). PCa harboring SPOP hotspot mutants (e.g., F133V) are resistant to BET inhibitors because of aberrant elevation of BET proteins. Here, we identified a previously unrecognized mutation Q165P at the edge of SPOP MATH domain in primary and metastatic PCa of a patient. The Q165P mutation causes structural changes in the MATH domain and impairs SPOP dimerization and substrate degradation. Different from F133V hotspot mutant tumors, Q165P mutant patient-derived xenografts (PDXs) and organoids were modestly sensitive to the BET inhibitor JQ1. Accordingly, protein levels of AR, BRD4 and downstream effectors such as RAC1 and phosphorylated AKT were not robustly elevated in Q165P mutant cells as in F133V mutant cells. However, NEO2734, a novel dual inhibitor of BET and CBP/p300, is active in both hotspot mutant (F133V) and non-hotspot mutant (Q165P) PCa cells in vitro and in vivo. These data provide a strong rationale to clinically investigate the anti-cancer efficacy of NEO2734 in SPOP-mutated PCa patients.

Regulation of Human Airway Epithelial Tissue Stem Cell Differentiation by ß-Catenin, P300, and CBP.

The wingless/integrase-1 (WNT)/ß-catenin signaling pathway is active in several chronic lung diseases including idiopathic pulmonary fibrosis, asthma, and chronic obstructive pulmonary disease. Although this WNT/ß-catenin pathway activity is associated with an increase in mucus cell frequency and a decrease in ciliated cell frequency, a cause and consequence relationship between signaling and cell frequency has not been established. We previously demonstrated that genetic stabilization of ß-catenin inhibited differentiation of mouse bronchiolar tissue stem cells (TSC). This study determined the effect of ß-catenin and its co-factors P300 (E1A-binding protein, 300 kDa) and cAMP response element binding (CREB)-binding protein (CBP) on human bronchial epithelial TSC differentiation to mucus and ciliated cells. We developed a modified air-liquid interface (ALI) culture system in which mucus and ciliated cell frequency is similar. These cultures were treated with the ß-catenin agonist CHIR99021 (CHIR) and antagonists to ß-catenin (XAV939), P300 (IQ1), and CBP (ICG001). We report that human TSC differentiation to mucus and ciliated cells can be divided into two stages, specification and commitment. CHIR treatment inhibited mucus and ciliated cell commitment while XAV939 treatment demonstrated that ß-catenin was necessary for mucus and ciliated cell specification. Additional studies demonstrate that a ß-catenin/P300 complex promotes mucus cell specification and that ß-catenin interacts with either P300 or CBP to inhibit ciliated cell commitment. These data indicate that activation of ß-catenin-dependent signaling in chronic lung disease leads to changes in mucus and ciliated cell frequency and that P300 and CBP tune the ß-catenin signal to favor mucus cell differentiation. Stem Cells 2018;36:1905-12.

CBP/p300 inhibitor C646 prevents high glucose exposure induced neuroepithelial cell proliferation.

BACKGROUND: Maternal diabetes related neural tube defects (NTDs) are a result of oxidative stress and apoptosis. However, the molecular mechanism behind the pathogenesis is not fully understood. Here, we report that high glucose exposure-induced epigenetic changes influence histone H4 acetylation and neuroepithelial cell proliferation. We also show that the acetyltransferase inhibitor C646 can prevent high glucose induced changes in histone H4 acetylation and neuroepithelial cell proliferation. METHODS: By using LC-MS/MS as an unbiased approach, we screened the histone acetylation profile in an E9 neuroepithelial cell line (NE-4C) under high glucose exposure. We further explored the mechanism in cells in vitro and in maternal diabetes-induced mouse embryos in vivo. RESULTS: We identified 35 core histone acetylation marks in normal E9 neuroepithelial cells, whereas high glucose exposure resulted in novel acetylation sites on H4K31 and H4K44. Acetylation levels of embryonic development associated H4K5/K8/K12/K16 increased in neuroepithelial cells exposed to high glucose in vitro and in brain tissue from maternal diabetes induced exencephalic embryos in vivo. Further, mRNA level of histone acetyltransferase CBP encoded gene Crebbp was significantly increased both in vitro and in vivo. The addition of C646, a selective inhibitor for CBP/p300, significantly rescued increase of H4K5/K8/K12/K16 acetylation levels and H3S10pi-labeled neuroepithelial cell proliferation induced by high glucose exposure. CONCLUSION: Our data provide complementary insights for potential mechanisms of maternal diabetes induced NTDs.

DSPP-MMP20 gene silencing downregulates cancer stem cell markers in human oral cancer cells.

BACKGROUND: Recent findings indicate that dentin sialophosphoprotein (DSPP) and matrix metalloproteinase (MMP) 20 interact in oral squamous cell carcinoma (OSCC). The objective of this study was to determine the effects of DSPP/MMP20 gene silencing on oral cancer stem cell (OCSC) markers. METHODS: The expression of well-established OCSC markers: ABCG2; ALDH1; CD133; CD44; BMI1; LGR4, and Podoplanin in DSPP/MMP20-silenced OSCC cell line, OSC2, and controls were assayed by western blot (WB), and flow cytometry techniques. The sensitivity of OSC2 cells to cisplatin following DSPP/MMP20 silencing was also determined. RESULTS: DSPP/MMP20 silencing resulted in downregulation of OCSC markers, more profoundly ABCG2 (84%) and CD44 (81%), following double silencing. Furthermore, while treatment of parent (pre-silenced) OSC2 cells with cisplatin resulted in upregulation of OCSC markers, DSPP/MMP20-silenced OSC2 cells similarly treated resulted in profound downregulation of OCSC markers (72 to 94% at 50 µM of cisplatin), and a marked reduction in the proportion of ABCG2 and ALDH1 positive cells (~ 1%). CONCLUSIONS: We conclude that the downregulation of OCSC markers may signal a reduction in OCSC population following MMP20/DSPP silencing in OSCC cells, while also increasing their sensitivity to cisplatin. Thus, our findings suggest a potential role for DSPP and MMP20 in sustaining OCSC population in OSCCs, possibly, through mechanism(s) that alter OCSC sensitivity to treatment with chemotherapeutic agents such as cisplatin.

Discovery and optimization of 1-(1H-indol-1-yl)ethanone derivatives as CBP/EP300 bromodomain inhibitors for the treatment of castration-resistant prostate cancer.

The CREB (cAMP responsive element binding protein) binding protein (CBP) and its homolog EP300 have emerged as new therapeutic targets for the treatment of cancer and inflammatory diseases. Here we report the identification, optimization and evaluation of 1-(1H-indol-1-yl)ethanone derivatives as CBP/EP300 inhibitors starting from fragment-based virtual screening (FBVS). A cocrystal structure of the inhibitor (22e) in complex with CBP provides a solid structural basis for further optimization. The most potent compound 32h binds to the CBP bromodomain and has an IC50 value of 0.037 µM in the AlphaScreen assay which was 2 times more potent than the reported CBP bromodomain inhibitor SGC-CBP30 in our hands. 32h also exhibit high selectivity for CBP/EP300 over other bromodomain-containing proteins. Notably, the ester derivative (29h) of compound 32h markedly inhibits cell growth in several prostate cancer cell lines including LNCaP, 22Rv1 and LNCaP derived C4-2B. Compound 29h suppresses the mRNA expression of full length AR (AR-FL), AR target genes and other oncogene in LNCaP cells. 29h also reduces the expression of PSA, the biomarker of prostate cancer. CBP/EP300 inhibitor 29h represents a promising lead compound for the development of new therapeutics for the treatment of castration-resistant prostate cancer.

Sam68 Allows Selective Targeting of Human Cancer Stem Cells.

Targeting of human cancer stem cells (CSCs) requires the identification of vulnerabilities unique to CSCs versus healthy resident stem cells (SCs). Unfortunately, dysregulated pathways that support transformed CSCs, such as Wnt/ß-catenin signaling, are also critical regulators of healthy SCs. Using the ICG-001 and CWP family of small molecules, we reveal Sam68 as a previously unappreciated modulator of Wnt/ß-catenin signaling within CSCs. Disruption of CBP-ß-catenin interaction via ICG-001/CWP induces the formation of a Sam68-CBP complex in CSCs that alters Wnt signaling toward apoptosis and differentiation induction. Our study identifies Sam68 as a regulator of human CSC vulnerability.

Bromodomain inhibition of the transcriptional coactivators CBP/EP300 as a therapeutic strategy to target the IRF4 network in multiple myeloma.

Pharmacological inhibition of chromatin co-regulatory factors represents a clinically validated strategy to modulate oncogenic signaling through selective attenuation of gene expression. Here, we demonstrate that CBP/EP300 bromodomain inhibition preferentially abrogates the viability of multiple myeloma cell lines. Selective targeting of multiple myeloma cell lines through CBP/EP300 bromodomain inhibition is the result of direct transcriptional suppression of the lymphocyte-specific transcription factor IRF4, which is essential for the viability of myeloma cells, and the concomitant repression of the IRF4 target gene c-MYC. Ectopic expression of either IRF4 or MYC antagonizes the phenotypic and transcriptional effects of CBP/EP300 bromodomain inhibition, highlighting the IRF4/MYC axis as a key component of its mechanism of action. These findings suggest that CBP/EP300 bromodomain inhibition represents a viable therapeutic strategy for targeting multiple myeloma and other lymphoid malignancies dependent on the IRF4 network.

Kaposi's Sarcoma-Associated Herpesvirus Viral Interferon Regulatory Factor 4 (vIRF4) Perturbs the G1-S Cell Cycle Progression via Deregulation of the cyclin D1 Gene.

Kaposi's sarcoma-associated herpesvirus (KSHV) infection modulates the host cell cycle to create an environment optimal for its viral-DNA replication during the lytic life cycle. We report here that KSHV vIRF4 targets the ß-catenin/CBP cofactor and blocks its occupancy on the cyclin D1 promoter, suppressing the G1-S cell cycle progression and enhancing KSHV replication. This shows that KSHV vIRF4 suppresses host G1-S transition, possibly providing an intracellular milieu favorable for its replication.

Disruption of ß-catenin/CBP signaling inhibits human airway epithelial-mesenchymal transition and repair.

The epithelium of asthmatics is characterized by reduced expression of E-cadherin and increased expression of the basal cell markers ck-5 and p63 that is indicative of a relatively undifferentiated repairing epithelium. This phenotype correlates with increased proliferation, compromised wound healing and an enhanced capacity to undergo epithelial-mesenchymal transition (EMT). The transcription factor ß-catenin plays a vital role in epithelial cell differentiation and regeneration, depending on the co-factor recruited. Transcriptional programs driven by the ß-catenin/CBP axis are critical for maintaining an undifferentiated and proliferative state, whereas the ß-catenin/p300 axis is associated with cell differentiation. We hypothesized that disrupting the ß-catenin/CBP signaling axis would promote epithelial differentiation and inhibit EMT. We treated monolayer cultures of human airway epithelial cells with TGFß1 in the presence or absence of the selective small molecule ICG-001 to inhibit ß-catenin/CBP signaling. We used western blots to assess expression of an EMT signature, CBP, p300, ß-catenin, fibronectin and ITGß1 and scratch wound assays to assess epithelial cell migration. Snai-1 and -2 expressions were determined using q-PCR. Exposure to TGFß1 induced EMT, characterized by reduced E-cadherin expression with increased expression of α-smooth muscle actin and EDA-fibronectin. Either co-treatment or therapeutic administration of ICG-001 completely inhibited TGFß1-induced EMT. ICG-001 also reduced the expression of ck-5 and -19 independent of TGFß1. Exposure to ICG-001 significantly inhibited epithelial cell proliferation and migration, coincident with a down regulation of ITGß1 and fibronectin expression. These data support our hypothesis that modulating the ß-catenin/CBP signaling axis plays a key role in epithelial plasticity and function.

A Novel Small-Molecule Inhibitor Targeting CREB-CBP Complex Possesses Anti-Cancer Effects along with Cell Cycle Regulation, Autophagy Suppression and Endoplasmic Reticulum Stress.

Lung adenocarcinoma, the most common subtype of lung cancer, is the leading cause of cancer death worldwide. Despite attempts for the treatment of lung cancer which have been accumulating, promising new therapies are still needed. Here, we found that cyclic-AMP response element-binding protein (CREB)-CREB binding protein (CBP) transcription factors complex inhibitor, Naphthol AS-TR phosphate (NASTRp), is a potential therapeutic agent for lung cancer. We show that NASTRp inhibited oncogenic cell properties through cell cycle arrest with concomitant suppression of tumor-promoting autophagy with down-regulations of Atg5-12 and Atg7, and accumulation of p62 in human lung cancer cell lines. In addition, NASTRp induced expression of endoplasmic reticulum stress markers such as DDIT3/CHOP, and led to apoptosis along with Bim induction. These findings suggest that transcription factor/co-activator complex, CREB-CBP, can be a potential therapeutic target and its inhibition could be a novel therapeutic strategy for lung cancer.

Podocalyxin enhances breast tumor growth and metastasis and is a target for monoclonal antibody therapy.

INTRODUCTION: Podocalyxin (gene name PODXL) is a CD34-related sialomucin implicated in the regulation of cell adhesion, migration and polarity. Upregulated expression of podocalyxin is linked to poor patient survival in epithelial cancers. However, it is not known if podocalyxin has a functional role in tumor progression. METHODS: We silenced podocalyxin expression in the aggressive basal-like human (MDA-MB-231) and mouse (4T1) breast cancer cell lines and also overexpressed podocalyxin in the more benign human breast cancer cell line, MCF7. We evaluated how podocalyxin affects tumorsphere formation in vitro and compared the ability of podocalyxin-deficient and podocalyxin-replete cell lines to form tumors and metastasize using xenogenic or syngeneic transplant models in mice. Finally, in an effort to develop therapeutic treatments for systemic cancers, we generated a series of antihuman podocalyxin antibodies and screened these for their ability to inhibit tumor progression in xenografted mice. RESULTS: Although deletion of podocalyxin does not alter gross cell morphology and growth under standard (adherent) culture conditions, expression of PODXL is required for efficient formation of tumorspheres in vitro. Correspondingly, silencing podocalyxin resulted in attenuated primary tumor growth and invasiveness in mice and severely impaired the formation of distant metastases. Likewise, in competitive tumor engraftment assays where we injected a 50:50 mixture of control and shPODXL (short-hairpin RNA targeting PODXL)-expressing cells, we found that podocalyxin-deficient cells exhibited a striking decrease in the ability to form clonal tumors in the lung, liver and bone marrow. Finally, to validate podocalyxin as a viable target for immunotherapy, we screened a series of novel antihuman podocalyxin antibodies for their ability to inhibit tumor progression in vivo. One of these antibodies, PODOC1, potently blocked tumor growth and metastasis. CONCLUSIONS: We show that podocalyxin plays a key role in the formation of primary tumors and distant tumor metastasis. In addition, we validate podocalyxin as potential target for monoclonal antibody therapy to inhibit primary tumor growth and systemic dissemination.

Safely targeting cancer stem cells via selective catenin coactivator antagonism.

Throughout our life, long-lived somatic stem cells (SSC) regenerate adult tissues both during homeostatic processes and repair after injury. The role of aberrant regulation of SSC has also recently gained prominence in the field of cancer research. Following malignant transformation, so termed cancer stem cells (CSC), endowed with the same properties as SSC (i.e. the ability to both self-renew and generate differentiated progenitors), play a major part in tumor initiation, therapy resistance and ultimately relapse. The same signaling pathways involved in regulating SSC maintenance are involved in the regulation of CSC. CSC exist in a wide array of tumor types, including leukemias, and brain, breast, prostate and colon tumors. Consequently, one of the key goals in cancer research over the past decade has been to develop therapeutic strategies to safely eliminate the CSC population without damaging the endogenous SSC population. A major hurdle to this goal lies in the identification of the key mechanisms that distinguish CSC from the normal endogenous tissue stem cells. This review will discuss the discovery of the specific CBP/catenin antagonist ICG-001 and the ongoing clinical development of the second generation CBP/catenin antagonist PRI-724. Importantly, specific CBP/catenin antagonists appear to have the ability to safely eliminate CSC by taking advantage of an intrinsic differential preference in the way SSC and CSC divide.

Sperm­associated antigen 9 promotes astrocytoma cell invasion through the upregulation of podocalyxin.

Podocalyxin (PODXL) has been found to increase the aggressive phenotype of a number of cancers, including astrocytoma. In addition, the progression of astrocytoma has been associated with sperm­associated antigen 9 (SPAG9), a recently characterized oncoprotein. In the present study, the association between SPAG9 and PODXL in human astrocytoma invasion and the underlying mechanisms were investigated for the first time, to the best of our knowledge. Overexpression and knockdown of SPAG9 were performed in SW1783 (grade III astrocytoma) and U87 (grade IV astrocytoma; glioblastoma) cells, respectively. PODXL expression at both the mRNA and the protein level, as well as the PODXL gene promoter activity, were significantly increased and decreased in parallel with the overexpression and knockdown of SPAG9 in astrocytoma cells; these effects were blocked by the selective c­Jun N­terminal kinase (JNK) inhibitor SP600125 (5 µM) and restored by the JNK agonist anisomycin (25 ng/ml), respectively. SPAG9 overexpression significantly increased cell invasion and matrix metalloproteinase­9 (MMP­9) expression in SW1783 cells, and this effect was reversed by knockdown of PODXL. In U87 cells, knockdown of SPAG9 markedly decreased cell invasion and MMP­9 expression, which was completely restored by overexpression of PODXL. In conclusion, it was demonstrated in the present study that SPAG9 upregulates PODXL expression in human astrocytoma cells at the PODXL gene promoter/transcriptional level through a JNK­dependent mechanism and that PODXL is a critical mediator of the promoting effect of SPAG9 on astrocytoma cell invasion, possibly through upregulation of MMP­9 expression. This study provides novel insights into the molecular mechanisms involved in astrocytoma invasion.

Small-molecule inhibition of CBP/catenin interactions eliminates drug-resistant clones in acute lymphoblastic leukemia.

Drug resistance in acute lymphoblastic leukemia (ALL) remains a major problem warranting new treatment strategies. Wnt/catenin signaling is critical for the self-renewal of normal hematopoietic progenitor cells. Deregulated Wnt signaling is evident in chronic and acute myeloid leukemia; however, little is known about ALL. Differential interaction of catenin with either the Kat3 coactivator CREBBP (CREB-binding protein (CBP)) or the highly homologous EP300 (p300) is critical to determine divergent cellular responses and provides a rationale for the regulation of both proliferation and differentiation by the Wnt signaling pathway. Usage of the coactivator CBP by catenin leads to transcriptional activation of cassettes of genes that are involved in maintenance of progenitor cell self-renewal. However, the use of the coactivator p300 leads to activation of genes involved in the initiation of differentiation. ICG-001 is a novel small-molecule modulator of Wnt/catenin signaling, which specifically binds to the N-terminus of CBP and not p300, within amino acids 1-110, thereby disrupting the interaction between CBP and catenin. Here, we report that selective disruption of the CBP/ß- and γ-catenin interactions using ICG-001 leads to differentiation of pre-B ALL cells and loss of self-renewal capacity. Survivin, an inhibitor-of-apoptosis protein, was also downregulated in primary ALL after treatment with ICG-001. Using chromatin immunoprecipitation assay, we demonstrate occupancy of the survivin promoter by CBP that is decreased by ICG-001 in primary ALL. CBP mutations have been recently identified in a significant percentage of ALL patients, however, almost all of the identified mutations reported occur C-terminal to the binding site for ICG-001. Importantly, ICG-001, regardless of CBP mutational status and chromosomal aberration, leads to eradication of drug-resistant primary leukemia in combination with conventional therapy in vitro and significantly prolongs the survival of NOD/SCID mice engrafted with primary ALL. Therefore, specifically inhibiting CBP/catenin transcription represents a novel approach to overcome relapse in ALL.