Publications by authors named "Samuel K Kulp"

100 Publications

Inhibition of androgen/AR signaling inhibits diethylnitrosamine (DEN) induced tumour initiation and remodels liver immune cell networks.

Sci Rep 2021 Feb 11;11(1):3646. Epub 2021 Feb 11.

The Comprehensive Cancer Center, The Ohio State University, Columbus, OH, 43210, USA.

A promotional role for androgen receptor (AR) signaling in hepatocellular carcinogenesis is emerging. In pre-clinical models, including diethylnitrosamine- (DEN-) induced hepatocellular carcinoma (HCC), anti-androgen therapies delay hepatocarcinogenesis. However, pharmacologic anti-androgen therapy in advanced HCC patients fails, suggesting that AR plays a role in HCC onset. This study aims to characterize AR expression and function throughout DEN-induced liver inflammation and carcinogenesis and evaluate the efficacy of prophylactic AR antagonism to prevent hepatocarcinogenesis. We demonstrate that pharmacologic AR antagonism with enzalutamide inhibits hepatocellular carcinogenesis. With enzalutamide treatment, we observe decreased CYP2E1 expression, reducing DEN-induced hepatocyte death and DNA ethyl-adducts. AR protein expression analyses show that DEN causes an initial upregulation of AR in portal fibroblasts and leukocytes, but not hepatocytes, suggesting that hepatocyte-autonomous AR signaling is not essential for DEN-induced carcinogenesis. Ablating androgen signaling by surgical castration reduced pre-carcinogen Kupffer cell populations but did not alter DEN-mediated immune cell recruitment nor AR expression. In this study, we identified that anti-androgen interventions modulate mutagenic DNA adducts, tumour initiation, and immune cell composition. Additionally, we find that AR expression in hepatocytes is not present during nor required for early DEN-mediated carcinogenesis.
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http://dx.doi.org/10.1038/s41598-021-82252-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7878907PMC
February 2021

Targeting DNA Damage Repair Functions of Two Histone Deacetylases, HDAC8 and SIRT6, Sensitizes Acute Myeloid Leukemia to NAMPT Inhibition.

Clin Cancer Res 2021 Apr 4;27(8):2352-2366. Epub 2021 Feb 4.

Division of Hematology, Department of Internal Medicine, The Ohio State University, Columbus, Ohio.

Purpose: Nicotinamide phosphoribosyltransferase (NAMPT) inhibitors (NAMPTi) are currently in development, but may be limited as single-agent therapy due to compound-specific toxicity and cancer metabolic plasticity allowing resistance development. To potentially lower the doses of NAMPTis required for therapeutic benefit against acute myeloid leukemia (AML), we performed a genome-wide CRISPRi screen to identify rational disease-specific partners for a novel NAMPTi, KPT-9274.

Experimental Design: Cell lines and primary cells were analyzed for cell viability, self-renewal, and responses at RNA and protein levels with loss-of-function approaches and pharmacologic treatments. efficacy of combination therapy was evaluated with a xenograft model.

Results: We identified two histone deacetylases (HDAC), and , whose knockout conferred synthetic lethality with KPT-9274 in AML. Furthermore, HDAC8-specific inhibitor, PCI-34051, or clinical class I HDAC inhibitor, AR-42, in combination with KPT-9274, synergistically decreased the survival of AML cells in a dose-dependent manner. AR-42/KPT-9274 cotreatment attenuated colony-forming potentials of patient cells while sparing healthy hematopoietic cells. Importantly, combined therapy demonstrated promising efficacy compared with KPT-9274 or AR-42 monotherapy. Mechanistically, genetic inhibition of SIRT6 potentiated the effect of KPT-9274 on PARP-1 suppression by abolishing mono-ADP ribosylation. AR-42/KPT-9274 cotreatment resulted in synergistic attenuation of homologous recombination and nonhomologous end joining pathways in cell lines and leukemia-initiating cells.

Conclusions: Our findings provide evidence that HDAC8 inhibition- or shSIRT6-induced DNA repair deficiencies are potently synergistic with NAMPT targeting, with minimal toxicity toward normal cells, providing a rationale for a novel-novel combination-based treatment for AML.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-3724DOI Listing
April 2021

Soy-tomato enriched diet reduces inflammation and disease severity in a pre-clinical model of chronic pancreatitis.

Sci Rep 2020 12 11;10(1):21824. Epub 2020 Dec 11.

James Comprehensive Cancer Center, The Ohio State University, Columbus, USA.

Chronic pancreatitis (CP) is a fibro-inflammatory syndrome in individuals who develop persistent pathological responses to parenchymal injury or stress. Novel therapeutic or dietary interventions that could lessen inflammation in this disease could significantly improve quality of life in patients with CP. Complex dietary foods like soy and tomatoes are composed of active metabolites with anti-inflammatory effects. Data from our group reports that bioactive agents in soy and tomatoes can reduce pro-inflammatory cytokines and suppressive immune populations. Additionally, our team has developed a novel soy-tomato juice currently being studied in healthy individuals with no toxicities, and good compliance and bioavailability. Thus, we hypothesize that administration of a soy-tomato enriched diet can reduce inflammation and severity of CP. C57BL/6 mice were injected intraperitoneally with 50 μg/kg caeurlein (7 hourly injections, twice weekly) for 6 weeks to induce CP. After 4 weeks of caerulein injections, mice were administered a control or a soy-tomato enriched diet for 2 weeks. Disease severity was measured via immunohistochemical analysis of pancreata measuring loss of acini, fibrosis, inflammation, and necrosis. Serum lipase and amylase levels were analyzed at the end of the study. Inflammatory factors in the serum and pancreas, and immune populations in the spleen of mice were analyzed by cytokine multiplex detection, qRT-PCR, and flow cytometry respectively. Infra-red (IR) sensing of mice was used to monitor spontaneous activity and distress of mice. Mice fed a soy-tomato enriched diet had a significantly reduced level of inflammation and severity of CP (p = 0.032) compared to mice administered a control diet with restored serum lipase and amylase levels (p < 0.05). Mice with CP fed a soy-tomato diet had a reduction in inflammatory factors (TNF-α, IL-1β, IL-5) and suppressive immune populations (myeloid-derived suppressor cells; MDSC) compared to control diet fed mice (p < 0.05). Infra-red sensing to monitor spontaneous activity of mice showed that soy-tomato enriched diet improved total activity and overall health of mice with CP (p = 0.055) and CP mice on a control diet were determined to spend more time at rest (p = 0.053). These pre-clinical results indicate that a soy-tomato enriched diet may be a novel treatment approach to reduce inflammation and pain in patients with CP.
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http://dx.doi.org/10.1038/s41598-020-78762-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7733503PMC
December 2020

Pharmacokinetics and Tolerability of the Novel Non-immunosuppressive Fingolimod Derivative, OSU-2S, in Dogs and Comparisons with Data in Mice and Rats.

AAPS J 2020 07 16;22(4):92. Epub 2020 Jul 16.

Division of Pharmaceutics and Pharmacology, College of Pharmacy, The Ohio State University, 506 Riffe Building, 496 W. 12th Ave., Columbus, Ohio, 43210, USA.

In this study, we characterized the pharmacokinetics of OSU-2S, a fingolimod-derived, non-immunosuppressive phosphatase activator, in mice, rats, and dogs, as well as tolerability and food effects in dogs. Across all species tested, plasma protein binding for OSU-2S was > 99.5%, and metabolic stability and hepatic intrinsic clearance were in the moderate range. OSU-2S did not significantly modulate CYP enzyme activity up until 50 μM, and Caco-2 data suggested low permeability with active efflux at 2 μM. Apparent oral bioavailability in mice was 16% and 69% at 10 and 50 mg/kg, respectively. In rats, bioavailability was 24%, 35%, and 28% at 10, 30, and 100 mg/kg, respectively, while brain/plasma ratio was 36 at 6-h post-dose at 30 mg/kg. In dogs, OSU-2S was well tolerated with oral capsule bioavailability of 27.5%. Plasma OSU-2S exposures increased proportionally over a 2.5-20 mg/kg dose range. After 4 weeks of 3 times weekly, oral administration (20 mg/kg), plasma AUC (26.1 μM*h), and C (0.899 μM) were nearly 2-fold greater than those after 1 week of dosing, and no food effects were observed. The elimination half-life (29.7 h), clearance (22.9 mL/min/kg), and plasma concentrations of repeated oral doses support a 3-times weekly dosing schedule in dogs. No significant CBC, serum biochemical, or histopathological changes were observed. OSU-2S has favorable oral PK properties similar to fingolimod in rodents and dogs and is well tolerated in healthy animals. This work supports establishing trials of OSU-2S efficacy in dogs with spontaneous tumors to guide its clinical development as a cancer therapeutic for human patients.
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http://dx.doi.org/10.1208/s12248-020-00474-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7814188PMC
July 2020

Retraction: Non-epigenetic function of HDAC8 in regulating breast cancer stem cells by maintaining Notch1 protein stability.

Oncotarget 2020 03 24;11(12):1096. Epub 2020 Mar 24.

Division of Medicinal Chemistry, College of Pharmacy and Comprehensive Cancer Center, Columbus, Ohio, USA.

[This retracts the article DOI: 10.18632/oncotarget.6427.].
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http://dx.doi.org/10.18632/oncotarget.27533DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7105163PMC
March 2020

Overcoming resistance to anabolic SARM therapy in experimental cancer cachexia with an HDAC inhibitor.

EMBO Mol Med 2020 02 13;12(2):e9910. Epub 2020 Jan 13.

Division of Pharmaceutics and Pharmacology, College of Pharmacy, The Ohio State University, Columbus, OH, USA.

No approved therapy exists for cancer-associated cachexia. The colon-26 mouse model of cancer cachexia mimics recent late-stage clinical failures of anabolic anti-cachexia therapy and was unresponsive to anabolic doses of diverse androgens, including the selective androgen receptor modulator (SARM) GTx-024. The histone deacetylase inhibitor (HDACi) AR-42 exhibited anti-cachectic activity in this model. We explored combined SARM/AR-42 therapy as an improved anti-cachectic treatment paradigm. A reduced dose of AR-42 provided limited anti-cachectic benefits, but, in combination with GTx-024, significantly improved body weight, hindlimb muscle mass, and grip strength versus controls. AR-42 suppressed the IL-6/GP130/STAT3 signaling axis in muscle without impacting circulating cytokines. GTx-024-mediated β-catenin target gene regulation was apparent in cachectic mice only when combined with AR-42. Our data suggest cachectic signaling in this model involves catabolic signaling insensitive to anabolic GTx-024 therapy and a blockade of GTx-024-mediated anabolic signaling. AR-42 mitigates catabolic gene activation and restores anabolic responsiveness to GTx-024. Combining GTx-024, a clinically established anabolic therapy, with AR-42, a clinically evaluated HDACi, represents a promising approach to improve anabolic response in cachectic patients.
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http://dx.doi.org/10.15252/emmm.201809910DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7005646PMC
February 2020

SUV39H1 Represses the Expression of Cytotoxic T-Lymphocyte Effector Genes to Promote Colon Tumor Immune Evasion.

Cancer Immunol Res 2019 03 4;7(3):414-427. Epub 2019 Jan 4.

Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, Georgia.

Despite the presence of CTLs in the tumor microenvironment, the majority of immunogenic human colon cancer does not respond to immune checkpoint inhibitor immunotherapy, and microsatellite instable (MSI) tumors are not naturally eliminated. The molecular mechanism underlying the inactivity of tumor-infiltrating CTLs is unknown. We report here that CTLs were present in both MSI and microsatellite stable colon tumors. The expression of the H3K9me3-specific histone methyltransferase SUV39H1 was significantly elevated in human colon carcinoma compared with normal colon tissues. Using a mouse colon carcinoma model, we further determined that tumor-infiltrating CTLs in the colon tumor microenvironment have high expression of SUV39H1. To target SUV39H1 in the tumor microenvironment, a virtual chemical library was screened on the basis of the SET (suppressor of variegation 3-9, enhancer of zeste and trithorax) domain structure of the human SUV39H1 protein. Functional enzymatic activity assays identified a small molecule that inhibits SUV39H1 enzymatic activity. On the basis of the structure of this small molecule, we modified it and chemically synthesized a small molecule, termed F5446, which has an EC of 0.496 μmol/L for SUV39H1 enzymatic activity. H3K9me3 was enriched in the promoters of , and in quiescent T cells. F5446 inhibited H3K9me3, thereby upregulating expression of these effectors in tumor-infiltrating CTLs and suppressing colon carcinoma growth in a CD8 CTL-dependent manner Our data indicate that SUV39H1 represses CTL effector gene expression and, in doing so, confers colon cancer immune escape.
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http://dx.doi.org/10.1158/2326-6066.CIR-18-0126DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6397681PMC
March 2019

Sensitivity of osteosarcoma cells to HDAC inhibitor AR-42 mediated apoptosis.

BMC Cancer 2017 01 21;17(1):67. Epub 2017 Jan 21.

Department of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH, 43210, USA.

Background: Osteosarcoma (OS) is the most common primary bone tumor in both humans and dogs and is the second leading cause of cancer related deaths in children and young adults. Limb sparing surgery along with chemotherapy has been the mainstay of treatment for OS. Many patients are not cured with current therapies, presenting a real need for developing new treatments. Histone deacetylase (HDAC) inhibitors are a promising new class of anticancer agents. In this study, we investigated the activity of the novel HDAC inhibitor AR-42 in a panel of human and canine OS cell lines.

Methods: The effect of AR-42 and suberoylanilide hydroxamic acid (SAHA) alone or in combination with doxorubicin on OS cell viability was assessed. Induction of histone acetylation after HDAC inhibitor treatment was confirmed by Western blotting. Drug-induced apoptosis was analyzed by FACS. Apoptosis was assessed further by measuring caspase 3/7 enzymatic activity, nucleosome fragmentation, and caspase cleavage. Effects on Akt signaling were demonstrated by assessing phosphorylation of Akt and downstream signaling molecules.

Results: AR-42 was a potent inhibitor of cell viability and induced a greater apoptotic response compared to SAHA when used at the same concentrations. Normal osteoblasts were much less sensitive. The combination of AR-42 with doxorubicin resulted in a potent inhibition of cell viability and apparent synergistic effect. Furthermore, we showed that AR-42 and SAHA induced cell death via the activation of the intrinsic mitochondrial pathway through activation of caspase 3/7. This potent apoptotic activity was associated with the greater ability of AR-42 to downregulate survival signaling through Akt.

Conclusions: These results confirm that AR-42 is a potent inhibitor of HDAC activity and demonstrates its ability to significantly inhibit cell survival through its pleiotropic effects in both canine and human OS cells and suggests that spontaneous OS in pet dogs may be a useful large animal model for preclinical evaluation of HDAC inhibitors. HDAC inhibition in combination with standard doxorubicin treatment offers promising potential for chemotherapeutic intervention in both canine and human OS.
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http://dx.doi.org/10.1186/s12885-017-3046-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5251323PMC
January 2017

Pharmacological strategies to target oncogenic KRAS signaling in pancreatic cancer.

Pharmacol Res 2017 03 8;117:370-376. Epub 2017 Jan 8.

Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, OH, USA; Institute of Biological Chemistry, Academia Sinica, 128 Sec. 2, Academia Road, Nangang, Taipei 115, Taiwan. Electronic address:

The clear importance of mutated KRAS as a therapeutic target has driven the investigation of multiple approaches to inhibit oncogenic KRAS signaling at different molecular levels. However, no KRAS-targeted therapy has reached the clinic to date, which underlies the intrinsic difficulty in developing effective, direct inhibitors of KRAS. Thus, this article provides an overview of the history and recent progress in the development of pharmacological strategies to target oncogenic KRAS with small molecule agents. Mechanistically, these KRAS-targeted agents can be classified into the following four categories. (1) Small-molecule RAS-binding ligands that prevent RAS activation by binding within or outside the nucleotide-binding motif. (2) Inhibitors of KRAS membrane anchorage. (3) Inhibitors that bind to RAS-binding domains of RAS-effector proteins. (4) Inhibitors of KRAS expression. The advantage and limitation of each type of these anti-KRAS agents are discussed.
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http://dx.doi.org/10.1016/j.phrs.2017.01.006DOI Listing
March 2017

Targeting integrin-linked kinase to suppress oncogenic KRAS signaling in pancreatic cancer.

Small GTPases 2018 11 9;9(6):452-456. Epub 2016 Dec 9.

a Institute of Biological Chemistry , Academia Sinica , Taipei , Taiwan.

Although oncogenic KRAS represents a therapeutically relevant target in pancreatic cancer, it is deemed "non-druggable" because of the intrinsic difficulty in designing direct inhibitors of KRAS. Our recent work demonstrated a KRAS-integrin-linked kinase (ILK) regulatory feedback loop that allows pancreatic cancer cells to regulate KRAS expression and to interact with the tumor microenvironment to promote aggressive phenotype. KRAS induces E2F1-mediated transcriptional activation of ILK expression, and ILK, in turn, controls KRAS expression via hnRNPA1, which binds and destabilizes the G-quadruplex in the KRAS promoter. Moreover, ILK inhibition blocked KRAS-driven EMT and growth factor-stimulated KRAS expression. This regulatory loop, however, was not noted in KRAS mutant colorectal and lung cancer cells examined as knockdown of KRAS or ILK did not affect each other's expression, suggesting that this KRAS-ILK feedback regulation is specific for pancreatic cancer. In sum, this regulatory loop provides a strong mechanistic rationale for suppressing oncogenic KRAS signaling through targeting ILK, and this creating a potential new therapeutic strategy for pancreatic cancer.
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http://dx.doi.org/10.1080/21541248.2016.1251383DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6204986PMC
November 2018

Suppression of Tumor Growth and Muscle Wasting in a Transgenic Mouse Model of Pancreatic Cancer by the Novel Histone Deacetylase Inhibitor AR-42.

Neoplasia 2016 Dec 25;18(12):765-774. Epub 2016 Nov 25.

Department of Biochemistry and Molecular Biology, College of Medicine, National Cheng Kung University, 1 University Rd., Tainan 701, Taiwan; Institute of Basic Medical Sciences, College of Medicine, National Cheng Kung University, 1 University Rd., Tainan 701, Taiwan. Electronic address:

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer death in the United States. This study was aimed at evaluating the efficacy of AR-42 (formerly OSU-HDAC42), a novel histone deacetylase (HDAC) inhibitor currently in clinical trials, in suppressing tumor growth and/or cancer-induced muscle wasting in murine models of PDAC.

Experimental Design: The in vitro antiproliferative activity of AR-42 was evaluated in six human pancreatic cancer cell lines (AsPC-1, COLO-357, PANC-1, MiaPaCa-2, BxPC-3, SW1990). AsPC-1 subcutaneous xenograft and transgenic KPC (LSL-Kras;Trp53;Pdx-1-Cre) mouse models of pancreatic cancer were used to evaluate the in vivo efficacy of AR-42 in suppressing tumor growth and/or muscle wasting.

Results: Growth suppression in AR-42-treated cells was observed in all six human pancreatic cancer cell lines with dose-dependent modulation of proliferation and apoptotic markers, which was associated with the hallmark features of HDAC inhibition, including p21 upregulation and histone H3 hyperacetylation. Oral administration of AR-42 at 50 mg/kg every other day resulted in suppression of tumor burden in the AsPC-1 xenograft and KPC models by 78% and 55%, respectively, at the end of treatment. Tumor suppression was associated with HDAC inhibition, increased apoptosis, and inhibition of proliferation. Additionally, AR-42 as a single agent preserved muscle size and increased grip strength in KPC mice. Finally, the combination of AR-42 and gemcitabine in transgenic mice demonstrated a significant increase in survival than either agent alone.

Conclusions: These results suggest that AR-42 represents a therapeutically promising strategy for the treatment of pancreatic cancer.
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http://dx.doi.org/10.1016/j.neo.2016.10.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5126135PMC
December 2016

Minocycline, a putative neuroprotectant, co-administered with doxorubicin-cyclophosphamide chemotherapy in a xenograft model of triple-negative breast cancer.

Exp Toxicol Pathol 2016 Oct 21;68(9):505-515. Epub 2016 Aug 21.

Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA. Electronic address:

Minocycline is purported to have neuroprotective properties in experimental models of some human neurologic diseases, and has therefore been identified as a putative neuroprotectant for chemotherapy-induced cognitive impairment (CICI) in breast cancer patients. However, because its mechanism of action is believed to be mediated through anti-inflammatory, anti-apoptotic, and anti-oxidant pathways, co-administration of minocycline with chemotherapeutic agents has the potential to reduce the efficacy of anticancer drugs. The objective of this study is to evaluate the effect of minocycline on the activity of the AC chemotherapeutic regimen (Adriamycin [doxorubicin], Cytoxan [cyclophosphamide]) in in vitro and in vivo models of triple-negative breast cancer (TNBC). Clonogenic and methylthiazol tetrazolium (MTT) assays were used to assess survival and viability in two TNBC cell lines treated with increasing concentrations of AC in the presence or absence of minocycline. Biomarkers of apoptosis, cell stress, and DNA damage were evaluated by western blot. The in vivo effects of AC and minocycline, each alone and in combination, were assessed in a xenograft model of TNBC in female athymic nude mice by weekly tumor volume measurement, body and organ weight measurement, and histopathology. Apoptosis and proliferation were characterized by immunohistochemistry in the xenografts tumors. Brains from tumor-bearing mice were evaluated for microglial activation, glial scars, and the proportion of neural progenitor cells. Data from these in vitro and in vivo studies demonstrate that minocycline does not diminish the cytotoxic and tumor-suppressive effects of this chemotherapeutic drug combination in TNBC cells. Moreover, minocycline appeared to prevent the reduction in doublecortin-positive neural progenitor cells observed in AC-treated mice. We posit that minocycline may be useful clinically for its reported neuroprotective activity in breast cancer patients receiving AC without loss of chemotherapeutic efficacy.
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http://dx.doi.org/10.1016/j.etp.2016.08.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5203928PMC
October 2016

Preclinical Pharmacokinetics Study of R- and S-Enantiomers of the Histone Deacetylase Inhibitor, AR-42 (NSC 731438), in Rodents.

AAPS J 2016 05 4;18(3):737-45. Epub 2016 Mar 4.

College of Pharmacy, The Ohio State University, 500 W. 12th Avenue, Columbus, Ohio, 43210, USA.

AR-42, a new orally bioavailable, potent, hydroxamate-tethered phenylbutyrate class I/IIB histone deacetylase inhibitor currently is under evaluation in phase 1 and 2 clinical trials and has demonstrated activity in both hematologic and solid tumor malignancies. This report focuses on the preclinical characterization of the pharmacokinetics of AR-42 in mice and rats. A high-performance liquid chromatography-tandem mass spectrometry assay has been developed and applied to the pharmacokinetic study of the more active stereoisomer, S-AR-42, when administered via intravenous and oral routes in rodents, including plasma, bone marrow, and spleen pharmacokinetics (PK) in CD2F1 mice and plasma PK in F344 rats. Oral bioavailability was estimated to be 26 and 100% in mice and rats, respectively. R-AR-42 was also evaluated intravenously in rats and was shown to display different pharmacokinetics with a much shorter terminal half-life compared to that of S-AR-42. Renal clearance was a minor elimination pathway for parental S-AR-42. Oral administration of S-AR-42 to tumor-bearing mice demonstrated high uptake and exposure of the parent drug in the lymphoid tissues, spleen, and bone marrow. This is the first report of the pharmacokinetics of this novel agent, which is now in early phase clinical trials.
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http://dx.doi.org/10.1208/s12248-016-9876-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5256597PMC
May 2016

Integrin-linked kinase as a novel molecular switch of the IL-6-NF-κB signaling loop in breast cancer.

Carcinogenesis 2016 Apr 19;37(4):430-442. Epub 2016 Feb 19.

Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA.

Substantial evidence has clearly demonstrated the role of the IL-6-NF-κB signaling loop in promoting aggressive phenotypes in breast cancer. However, the exact mechanism by which this inflammatory loop is regulated remains to be defined. Here, we report that integrin-linked kinase (ILK) acts as a molecular switch for this feedback loop. Specifically, we show that IL-6 induces ILK expression via E2F1 upregulation, which, in turn, activates NF-κB signaling to facilitate IL-6 production. shRNA-mediated knockdown or pharmacological inhibition of ILK disrupted this IL-6-NF-κB signaling loop, and blocked IL-6-induced cancer stem cells in vitro and estrogen-independent tumor growth in vivo Together, these findings establish ILK as an intermediary effector of the IL-6-NF-κB feedback loop and a promising therapeutic target for breast cancer.
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http://dx.doi.org/10.1093/carcin/bgw020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5006214PMC
April 2016

Non-epigenetic function of HDAC8 in regulating breast cancer stem cells by maintaining Notch1 protein stability.

Oncotarget 2016 Jan;7(2):1796-807

Division of Medicinal Chemistry, College of Pharmacy and Comprehensive Cancer Center, Columbus, Ohio, USA.

Here, we report a novel non-epigenetic function of histone deacetylase (HDAC) 8 in activating cancer stem cell (CSC)-like properties in breast cancer cells by enhancing the stability of Notch1 protein. The pan-HDAC inhibitors AR-42 and SAHA, and the class I HDAC inhibitor depsipeptide, suppressed mammosphere formation and other CSC markers by reducing Notch1 expression in MDA-MB-231 and SUM-159 cells. Interrogation of individual class I isoforms (HDAC1-3 and 8) using si/shRNA-mediated knockdown, ectopic expression and/or pharmacological inhibition revealed HDAC8 to be the primary mediator of this drug effect. This suppression of Notch1 in response to HDAC8 inhibition was abrogated by the proteasome inhibitor MG132 and siRNA-induced silencing of Fbwx7, indicating Notch1 suppression occurred through proteasomal degradation. However, co-immunoprecipitation analysis indicated that HDAC8 did not form complexes with Notch1 and HDAC inhibition had no effect on Notch1 acetylation. In a xenograft tumor model, the tumorigenicity of breast cancer cells was decreased by HDAC8 knockdown. These findings suggest the therapeutic potential of HDAC8 inhibition to suppress Notch1 signaling in breast cancer.
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http://dx.doi.org/10.18632/oncotarget.6427DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4811498PMC
January 2016

Preclinical Investigation of the Novel Histone Deacetylase Inhibitor AR-42 in the Treatment of Cancer-Induced Cachexia.

J Natl Cancer Inst 2015 Dec 12;107(12):djv274. Epub 2015 Oct 12.

Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy (YCT, SKK, ILL, ECH, CSC), Department of Molecular Virology, Immunology, and Medical Genetics (WAH, DCG), Department of Surgery (MB), Department of Internal Medicine (GBL, GM, TBS), and Center for Biostatistics (XM), College of Medicine, and Genomics Shared Resource (DEF, PSY), The Comprehensive Cancer Center, The Ohio State University, Columbus, OH; Institute of Basic Medical Sciences, National Cheng-Kung University, Tainan, Taiwan (CSC); Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan (CSC).

Background: Cancer cachexia is a debilitating condition that impacts patient morbidity, mortality, and quality of life and for which effective therapies are lacking. The anticachectic activity of the novel HDAC inhibitor AR-42 was investigated in murine models of cancer cachexia.

Methods: The effects of AR-42 on classic features of cachexia were evaluated in the C-26 colon adenocarcinoma and Lewis lung carcinoma (LLC) models. Effects on survival in comparison with approved HDAC inhibitors (vorinostat, romidepsin) were determined. The muscle metabolome and transcriptome (by RNA-seq), as well as serum cytokine profile, were evaluated. Data were analyzed using mixed effects models, analysis of variance, or log-rank tests. All statistical tests were two-sided.

Results: In the C-26 model, orally administered AR-42 preserved body weight (23.9±2.6 grams, AR-42-treated; 20.8±1.3 grams, vehicle-treated; P = .005), prolonged survival (P < .001), prevented reductions in muscle and adipose tissue mass, muscle fiber size, and muscle strength and restored intramuscular mRNA expression of the E3 ligases MuRF1 and Atrogin-1 to basal levels (n = 8). This anticachectic effect, confirmed in the LLC model, was not observed after treatment with vorinostat and romidepsin. AR-42 suppressed tumor-induced changes in inflammatory cytokine production and multiple procachexia drivers (IL-6, IL-6Rα, leukemia inhibitory factor, Foxo1, Atrogin-1, MuRF1, adipose triglyceride lipase, uncoupling protein 3, and myocyte enhancer factor 2c). Metabolomic analysis revealed cachexia-associated changes in glycolysis, glycogen synthesis, and protein degradation in muscle, which were restored by AR-42 to a state characteristic of tumor-free mice.

Conclusions: These findings support further investigation of AR-42 as part of a comprehensive therapeutic strategy for cancer cachexia.
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http://dx.doi.org/10.1093/jnci/djv274DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6280990PMC
December 2015

Development of Potent Adenosine Monophosphate Activated Protein Kinase (AMPK) Activators.

ChemMedChem 2015 Nov 9;10(11):1915-23. Epub 2015 Sep 9.

Division of Medicinal Chemistry & Pharmacognosy, College of Pharmacy, The Ohio State University, Room 336, Parks Hall, 500 West 12th Ave., Columbus, OH, 43210, USA.

Previously, we reported the identification of a thiazolidinedione-based adenosine monophosphate activated protein kinase (AMPK) activator, compound 1 (N-[4-({3-[(1-methylcyclohexyl)methyl]-2,4-dioxothiazolidin-5-ylidene}methyl)phenyl]-4-nitro-3-(trifluoromethyl)benzenesulfonamide), which provided a proof of concept to delineate the intricate role of AMPK in regulating oncogenic signaling pathways associated with cell proliferation and epithelial-mesenchymal transition (EMT) in cancer cells. In this study, we used 1 as a scaffold to conduct lead optimization, which generated a series of derivatives. Analysis of the antiproliferative and AMPK-activating activities of individual derivatives revealed a distinct structure-activity relationship and identified 59 (N-(3-nitrophenyl)-N'-{4-[(3-{[3,5-bis(trifluoromethyl)phenyl]methyl}-2,4-dioxothiazolidin-5-ylidene)methyl]phenyl}urea) as the optimal agent. Relative to 1, compound 59 exhibits multifold higher potency in upregulating AMPK phosphorylation in various cell lines irrespective of their liver kinase B1 (LKB1) functional status, accompanied by parallel changes in the phosphorylation/expression levels of p70S6K, Akt, Foxo3a, and EMT-associated markers. Consistent with its predicted activity against tumors with activated Akt status, orally administered 59 was efficacious in suppressing the growth of phosphatase and tensin homologue (PTEN)-null PC-3 xenograft tumors in nude mice. Together, these findings suggest that 59 has clinical value in therapeutic strategies for PTEN-negative cancer and warrants continued investigation in this regard.
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http://dx.doi.org/10.1002/cmdc.201500371DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4852482PMC
November 2015

Function of Integrin-Linked Kinase in Modulating the Stemness of IL-6-Abundant Breast Cancer Cells by Regulating γ-Secretase-Mediated Notch1 Activation in Caveolae.

Neoplasia 2015 Jun;17(6):497-508

Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University Comprehensive Cancer Center, Columbus, OH, USA; Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan. Electronic address:

Interleukin-6 (IL-6) and Notch signaling are important regulators of breast cancer stem cells (CSCs), which drive the malignant phenotype through self-renewal, differentiation, and development of therapeutic resistance. We investigated the role of integrin-linked kinase (ILK) in regulating IL-6-driven Notch1 activation and the ability to target breast CSCs through ILK inhibition. Ectopic expression/short hairpin RNA-mediated knockdown of ILK, pharmacological inhibition of ILK with the small molecule T315, Western blot analysis, immunofluorescence, and luciferase reporter assays were used to evaluate the regulation of IL-6-driven Notch1 activation by ILK in IL-6-producing triple-negative breast cancer cell lines (MDA-MB-231, SUM-159) and in MCF-7 and MCF-7(IL-6) cells. The effects of ILK on γ-secretase complex assembly and cellular localization were determined by immunofluorescence, Western blots of membrane fractions, and immunoprecipitation. In vivo effects of T315-induced ILK inhibition on CSCs in SUM-159 xenograft models were assessed by mammosphere assays, flow cytometry, and tumorigenicity assays. Results show that the genetic knockdown or pharmacological inhibition of ILK suppressed Notch1 activation and the abundance of the γ-secretase components presenilin-1, nicastrin, and presenilin enhancer 2 at the posttranscriptional level via inhibition of caveolin-1-dependent membrane assembly of the γ-secretase complex. Accordingly, knockdown of ILK inhibited breast CSC-like properties in vitro and the breast CSC subpopulation in vivo in xenograft tumor models. Based on these findings, we propose a novel function of ILK in regulating γ-secretase-mediated Notch1 activation, which suggests the targeting of ILK as a therapeutic approach to suppress IL-6-induced breast CSCs.
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http://dx.doi.org/10.1016/j.neo.2015.06.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4719004PMC
June 2015

A novel HIF-1α-integrin-linked kinase regulatory loop that facilitates hypoxia-induced HIF-1α expression and epithelial-mesenchymal transition in cancer cells.

Oncotarget 2015 Apr;6(10):8271-85

Division of Medicinal Chemistry, College of Pharmacy and Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio, USA.

Here, we described a novel regulatory feedback loop in which hypoxia induces integrin-linked kinase (ILK) expression through a HIF-1α-dependent mechanism and ILK, in turn, stimulates HIF-1α expression through cell type- and cell context-dependent pathways. HIF-1α increased ILK via transcriptional activation. ILK increased HIF-1α levels by promoting mTOR-mediated translation in PC-3 and MCF-7 cells, and by blocking GSK3β-mediated degradation in LNCaP cells, consistent with the cell line-/cellular context-specific functions of ILK as a Ser473-Akt kinase. We show that ILK can account for the effects of hypoxia on Akt, mTOR, and GSK3β phosphorylation. Also, ILK can de-repress HIF-1α signaling through the YB-1-mediated inhibition of Foxo3a expression. In concert with HIF-1α, these downstream effectors promote epithelial-mesenchymal transition (EMT) through modulation of Snail and Zeb1. Thus, the ILK-HIF-1α regulatory loop could underlie the maintenance of high HIF-1α expression levels and the promotion of EMT under hypoxic conditions. Finally, we show that the small-molecule ILK inhibitor T315 can disrupt this regulatory loop in vivo and suppress xenograft tumor growth, thereby providing proof-of-concept that targeting ILK represents an effective strategy to block HIF-1α expression and aggressive phenotype in cancer cells.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4480751PMC
http://dx.doi.org/10.18632/oncotarget.3186DOI Listing
April 2015

Exploitation of the ability of γ-tocopherol to facilitate membrane co-localization of Akt and PHLPP1 to develop PHLPP1-targeted Akt inhibitors.

J Med Chem 2015 Mar 27;58(5):2290-8. Epub 2015 Feb 27.

Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy and Comprehensive Cancer Center, The Ohio State University , Columbus, Ohio 43210, United States.

Previously, we reported that Akt inactivation by γ-tocopherol (2) in PTEN-negative prostate cancer cells resulted from its unique ability to facilitate membrane co-localization of Akt and PHLPP1 (PH domain leucine-rich repeat protein phosphatase isoform 1), a Ser473-specific Akt phosphatase, through pleckstrin homology (PH) domain binding. This finding provided a basis for exploiting 2 to develop a novel class of PHLPP1-targeted Akt inhibitors. Here, we used 3 (γ-VE5), a side chain-truncated 2 derivative, as a scaffold for lead optimization. The proof-of-concept of this structural optimization was obtained by 20, which exhibited higher antitumor efficacy than 3 in PTEN-negative cancer cells through PHLPP1-facilitated Akt inactivation. Like 3, 20 preferentially recognized the PH domains of Akt and PHLPP1, as its binding affinities for other PH domains, including those of ILK and PDK1, were an order-of-magnitude lower. Moreover, 20 was orally active in suppressing xenograft tumor growth in nude mice, which underlines the translational potential of this new class of Akt inhibitor in PTEN-deficient cancers.
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http://dx.doi.org/10.1021/jm501751bDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4720140PMC
March 2015

Sensitization of intracellular Salmonella enterica serovar Typhimurium to aminoglycosides in vitro and in vivo by a host-targeted antimicrobial agent.

Antimicrob Agents Chemother 2014 Dec 29;58(12):7375-82. Epub 2014 Sep 29.

Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, Taipei, Taiwan

Aminoglycosides exhibit relatively poor activity against intracellular Salmonella enterica serovar Typhimurium due to their low permeativity across eukaryotic cell membranes. Previously, we identified the unique ability of AR-12, a celecoxib-derived small-molecule agent, to eradicate intracellular Salmonella Typhimurium in macrophages by facilitating autophagosome formation and suppressing Akt kinase signaling. In light of this unique mode of antibacterial action, we investigated the ability of AR-12 to sensitize intracellular Salmonella to aminoglycosides in macrophages and in an animal model. The antibacterial activities of AR-12 combined with various aminoglycosides, including streptomycin, kanamycin, gentamicin, and amikacin, against intracellular S. Typhimurium in murine RAW264.7 macrophages were assessed. Cells were infected with S. Typhimurium followed by treatment with AR-12 or individual aminoglycosides or with combinations for 24 h. The in vivo efficacies of AR-12, alone or in combination with gentamicin or amikacin, were also assessed by treating S. Typhimurium-infected BALB/c mice daily for 14 consecutive days. Exposure of S. Typhimurium-infected RAW264.7 cells to a combination of AR-12 with individual aminoglycosides led to a reduction in bacterial survival (P < 0.05), both intracellular and extracellular, that was greater than that seen with the aminoglycosides alone. This sensitizing effect, however, was not associated with increased aminoglycoside penetration into bacteria or macrophages. Moreover, daily intraperitoneal injection of AR-12 at 0.1 mg/kg of body weight significantly increased the in vivo efficacy of gentamicin and amikacin in prolonging the survival of S. Typhimurium-infected mice. These findings indicate that the unique ability of AR-12 to enhance the in vivo efficacy of aminoglycosides might have translational potential for efforts to develop novel strategies for the treatment of salmonellosis.
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http://dx.doi.org/10.1128/AAC.03778-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4249550PMC
December 2014

AMPK reverses the mesenchymal phenotype of cancer cells by targeting the Akt-MDM2-Foxo3a signaling axis.

Cancer Res 2014 09 3;74(17):4783-95. Epub 2014 Jul 3.

Division of Medicinal Chemistry, College of Pharmacy and Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio. Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan.

In cancer cells, the epithelial-mesenchymal transition (EMT) confers the ability to invade basement membranes and metastasize to distant sites, establishing it as an appealing target for therapeutic intervention. Here, we report a novel function of the master metabolic kinase AMPK in suppressing EMT by modulating the Akt-MDM2-Foxo3 signaling axis. This mechanistic link was supported by the effects of siRNA-mediated knockdown and pharmacologic activation of AMPK on epithelial and mesenchymal markers in established breast and prostate cancer cells. Exposure of cells to OSU-53, a novel allosteric AMPK activator, as well as metformin and AICAR, was sufficient to reverse their mesenchymal phenotype. These effects were abrogated by AMPK silencing. Phenotypic changes were mediated by Foxo3a activation, insofar as silencing or overexpressing Foxo3a mimicked the effects of AMPK silencing or OSU-53 treatment on EMT, respectively. Mechanistically, Foxo3a activation led to the transactivation of the E-cadherin gene and repression of genes encoding EMT-inducing transcription factors. OSU-53 activated Foxo3a through two Akt-dependent pathways, one at the level of nuclear localization by blocking Akt- and IKKβ-mediated phosphorylation, and a second at the level of protein stabilization via cytoplasmic sequestration of MDM2, an E3 ligase responsible for Foxo3a degradation. The suppressive effects of OSU-53 on EMT had therapeutic implications illustrated by its ability to block invasive phenotypes in vitro and metastatic properties in vivo. Overall, our work illuminates a mechanism of EMT regulation in cancer cells mediated by AMPK, along with preclinical evidence supporting a tractable therapeutic strategy to reverse mesenchymal phenotypes associated with invasion and metastasis.
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http://dx.doi.org/10.1158/0008-5472.CAN-14-0135DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4155002PMC
September 2014