Publications by authors named "Said M Sebti"

140 Publications

Global Phosphoproteomics Reveal CDK Suppression as a Vulnerability to KRas Addiction in Pancreatic Cancer.

Clin Cancer Res 2021 Apr 20. Epub 2021 Apr 20.

Department of Drug Discovery, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida.

Purpose: Among human cancers that harbor mutant (mt) KRas, some, but not all, are dependent on mt KRas. However, little is known about what drives KRas dependency.

Experimental Design: Global phosphoproteomics, screening of a chemical library of FDA drugs, and genome-wide CRISPR/Cas9 viability database analysis were used to identify vulnerabilities of KRas dependency.

Results: Global phosphoproteomics revealed that KRas dependency is driven by a cyclin-dependent kinase (CDK) network. CRISPR/Cas9 viability database analysis revealed that, in mt KRas-driven pancreatic cancer cells, knocking out the cell-cycle regulators CDK1 or CDK2 or the transcriptional regulators CDK7 or CDK9 was as effective as knocking out KRas. Furthermore, screening of a library of FDA drugs identified AT7519, a CDK1, 2, 7, and 9 inhibitor, as a potent inducer of apoptosis in mt KRas-dependent, but not in mt KRas-independent, human cancer cells. AT7519 inhibited the phosphorylation of CDK1, 2, 7, and 9 substrates and suppressed growth of xenografts from 5 patients with pancreatic cancer. AT7519 also abrogated mt KRas and mt p53 primary and metastatic pancreatic cancer in three-dimensional (3D) organoids from 2 patients, 3D cocultures from 8 patients, and mouse 3D organoids from pancreatic intraepithelial neoplasia, primary, and metastatic tumors.

Conclusions: A link between CDK hyperactivation and mt KRas dependency was uncovered and pharmacologically exploited to abrogate mt KRas-driven pancreatic cancer in highly relevant models, warranting clinical investigations of AT7519 in patients with pancreatic cancer.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-4781DOI Listing
April 2021

Pacritinib Combined with Sirolimus and Low-Dose Tacrolimus for GVHD Prevention after Allogeneic Hematopoietic Cell Transplantation: Preclinical and Phase I Trial Results.

Clin Cancer Res 2021 May 22;27(10):2712-2722. Epub 2021 Mar 22.

Division of Hematology, Oncology, and Transplantation, Department of Medicine, Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota.

Purpose: In this first-in-human, phase I, GVHD prevention trial (NCT02891603), we combine pacritinib (PAC), a JAK2 inhibitor, with sirolimus to concurrently reduce T-cell costimulation via mTOR and IL6 activity. We evaluate the safety of pacritinib when administered with sirolimus plus low-dose tacrolimus (PAC/SIR/TAC) after allogeneic hematopoietic cell transplantation.

Patients And Methods: The preclinical efficacy and immune modulation of PAC/SIR were investigated in xenogeneic GVHD. Our phase I trial followed a 3+3 dose-escalation design, including dose level 1 (pacritinib 100 mg daily), level 2 (pacritinib 100 mg twice daily), and level 3 (pacritinib 200 mg twice daily). The primary endpoint was to identify the lowest biologically active and safe dose of pacritinib with SIR/TAC ( = 12). Acute GVHD was scored through day +100. Allografts included 8/8 HLA-matched related or unrelated donor peripheral blood stem cells.

Results: In mice, we show that dual JAK2/mTOR inhibition significantly reduces xenogeneic GVHD and increases peripheral regulatory T cell (Treg) potency as well as Treg induction from conventional CD4 T cells. Pacritinib 100 mg twice a day was identified as the minimum biologically active and safe dose for further study. JAK2/mTOR inhibition suppresses pathogenic Th1 and Th17 cells, spares Tregs and antileukemia effector cells, and exhibits preliminary activity in preventing GVHD. PAC/SIR/TAC preserves donor cytomegalovirus (CMV) immunity and permits timely engraftment without cytopenias.

Conclusions: We demonstrate that PAC/SIR/TAC is safe and preliminarily limits acute GVHD, preserves donor CMV immunity, and permits timely engraftment. The efficacy of PAC/SIR/TAC will be tested in our ongoing phase II GVHD prevention trial.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-4725DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8127396PMC
May 2021

CD28 Costimulatory Domain-Targeted Mutations Enhance Chimeric Antigen Receptor T-cell Function.

Cancer Immunol Res 2021 01 13;9(1):62-74. Epub 2020 Nov 13.

Department of Blood and Marrow Transplant and Cellular Immunotherapy, Division of Clinical Science, H. Lee Moffitt Cancer Center, Tampa, Florida.

An obstacle to the development of chimeric antigen receptor (CAR) T cells is the limited understanding of CAR T-cell biology and the mechanisms behind their antitumor activity. We and others have shown that CARs with a CD28 costimulatory domain drive high T-cell activation, which leads to exhaustion and shortened persistence. This work led us to hypothesize that by incorporating null mutations of CD28 subdomains (YMNM, PRRP, or PYAP), we could optimize CAR T-cell costimulation and enhance function. , we found that mice given CAR T cells with only a PYAP CD28 endodomain had a significant survival advantage, with 100% of mice alive after 62 days compared with 50% for mice with an unmutated endodomain. We observed that mutant CAR T cells remained more sensitive to antigen after antigen and PD-L1 stimulation, as demonstrated by increased cytokine production. The mutant CAR T cells also had a reduction of exhaustion-related transcription factors and genes such as , and Our results demonstrated that CAR T cells with a mutant CD28 endodomain have better survival and function. This work allows for the development of enhanced CAR T-cell therapies by optimizing CAR T-cell costimulation.
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http://dx.doi.org/10.1158/2326-6066.CIR-20-0253DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7864379PMC
January 2021

Metabolic reprogramming augments potency of human pSTAT3-inhibited iTregs to suppress alloreactivity.

JCI Insight 2020 05 7;5(9). Epub 2020 May 7.

Division of Hematology, Oncology, and Transplantation, Department of Medicine, Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota, USA.

Immunosuppressive donor Tregs can prevent graft-versus-host disease (GVHD) or solid-organ allograft rejection. We previously demonstrated that inhibiting STAT3 phosphorylation (pSTAT3) augments FOXP3 expression, stabilizing induced Tregs (iTregs). Here we report that human pSTAT3-inhibited iTregs prevent human skin graft rejection and xenogeneic GVHD yet spare donor antileukemia immunity. pSTAT3-inhibited iTregs express increased levels of skin-homing cutaneous lymphocyte-associated antigen, immunosuppressive GARP and PD-1, and IL-9 that supports tolerizing mast cells. Further, pSTAT3-inhibited iTregs significantly reduced alloreactive conventional T cells, Th1, and Th17 cells implicated in GVHD and tissue rejection and impaired infiltration by pathogenic Th2 cells. Mechanistically, pSTAT3 inhibition of iTregs provoked a shift in metabolism from oxidative phosphorylation (OxPhos) to glycolysis and reduced electron transport chain activity. Strikingly, cotreatment with coenzyme Q10 restored OxPhos in pSTAT3-inhibited iTregs and augmented their suppressive potency. These findings support the rationale for clinically testing the safety and efficacy of metabolically tuned, human pSTAT3-inhibited iTregs to control alloreactive T cells.
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http://dx.doi.org/10.1172/jci.insight.136437DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7253027PMC
May 2020

The GTPase KRAS suppresses the p53 tumor suppressor by activating the NRF2-regulated antioxidant defense system in cancer cells.

J Biol Chem 2020 03 30;295(10):3055-3063. Epub 2020 Jan 30.

Drug Discovery Department, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida 33612 Department of Oncologic Sciences, University of South Florida, Tampa, Florida 33612. Electronic address:

In human cancer cells that harbor mutant KRAS and WT p53 (p53), KRAS contributes to the maintenance of low p53 levels. Moreover, KRAS depletion stabilizes and reactivates p53 and thereby inhibits malignant transformation. However, the mechanism by which KRAS regulates p53 is largely unknown. Recently, we showed that KRAS depletion leads to p53 Ser-15 phosphorylation (P-p53) and increases the levels of p53 and its target p21/WT p53-activated fragment 1 (WAF1)/CIP1. Here, using several human lung cancer cell lines, siRNA-mediated gene silencing, immunoblotting, quantitative RT-PCR, promoter-reporter assays, and reactive oxygen species (ROS) assays, we demonstrate that KRAS maintains low p53 levels by activating the NRF2 (NFE2-related factor 2)-regulated antioxidant defense system. We found that KRAS depletion led to down-regulation of NRF2 and its targets NQO1 (NAD(P)H quinone dehydrogenase 1) and SLC7A11 (solute carrier family 7 member 11), decreased the GSH/GSSG ratio, and increased ROS levels. We noted that the increase in ROS is required for increased P-p53, p53, and p21 levels following KRAS depletion. Downstream of KRAS, depletion of RalB (RAS-like proto-oncogene B) and IκB kinase-related TANK-binding kinase 1 (TBK1) activated p53 in a ROS- and NRF2-dependent manner. Consistent with this, the IκB kinase inhibitor BAY11-7085 and dominant-negative mutant IκBαM inhibited NF-κB activity and increased P-p53, p53, and p21 levels in a ROS-dependent manner. In conclusion, our findings uncover an important role for the NRF2-regulated antioxidant system in KRAS-mediated p53 suppression.
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http://dx.doi.org/10.1074/jbc.RA119.011930DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7062149PMC
March 2020

A Phase I Study of GGTI-2418 (Geranylgeranyl Transferase I Inhibitor) in Patients with Advanced Solid Tumors.

Target Oncol 2019 10;14(5):613-618

University of Pennsylvania, Philadelphia, Pennsylvania, USA.

Background: Geranylgeranyltransferase I (GGTase I) catalyzes geranylgeranylation, a modification required for the function of many oncogenic RAS-related proteins. GGTI-2418 is a peptidomimetic small molecule inhibitor of GGTase I.

Objective: The aim of this study was to establish the maximum tolerated dose of GGTI-2418 in patients with advanced solid tumors.

Patients And Methods: This was a phase I, open-label, dose-escalation study conducted in two US centers (University of Pennsylvania and Indiana University) in adults with treatment-refractory advanced solid tumors. An accelerated dose-escalation schema was used across eight dose levels, from 120 to 2060 mg/m, administered on days 1-5 of each 21-day cycle.

Results: Fourteen patients were enrolled in the dose-escalation cohort. No dose-limiting toxicities were observed, and 2060 mg/m was determined to be the maximum tolerated dose. The only potential drug-related grade 3 or 4 toxicities were elevated bilirubin and alkaline phosphatase in a single patient with concurrent malignant biliary obstruction. No objective responses were observed. Four of thirteen evaluable patients had stable disease for up to 6.7 months. The study was terminated prior to dose expansion based on a sponsor decision. Pharmacokinetic analysis demonstrated a mean terminal half-life of 1.1 h.

Conclusions: GGTI2418 was safe and tolerable at all tested dose levels with some evidence of disease stability. Due to rapid elimination, dosing of GGTI2418 in this study may have been inadequate to achieve optimal inhibition of its target, GGTase I.
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http://dx.doi.org/10.1007/s11523-019-00661-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7771543PMC
October 2019

Vitamin E δ-tocotrienol sensitizes human pancreatic cancer cells to TRAIL-induced apoptosis through proteasome-mediated down-regulation of c-FLIP.

Cancer Cell Int 2019 22;19:189. Epub 2019 Jul 22.

1Gastrointestinal Oncology Program, Department of Gastrointestinal Oncology, H. Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, Tampa, FL 33612 USA.

Background: Vitamin E δ-tocotrienol (VEDT), a vitamin E compound isolated from sources such as palm fruit and annatto beans, has been reported to have cancer chemopreventive and therapeutic effects.

Methods: We report a novel function of VEDT in augmenting tumor necrosis factor-related apoptosis-inducing ligand- (TRAIL-) induced apoptosis in pancreatic cancer cells. The effects of VEDT were shown by its ability to trigger caspase-8-dependent apoptosis in pancreatic cancer cells.

Results: When combined with TRAIL, VEDT significantly augmented TRAIL-induced apoptosis of pancreatic cancer cells. VEDT decreased cellular FLICE inhibitory protein (c-FLIP) levels without consistently modulating the expression of decoy death receptors 1, 2, 3 or death receptors 4 and 5. Enforced expression of c-FLIP substantially attenuated VEDT/TRAIL-induced apoptosis. Thus, c-FLIP reduction plays an important part in mediating VEDT/TRAIL-induced apoptosis. Moreover, VEDT increased c-FLIP ubiquitination and degradation but did not affect its transcription, suggesting that VEDT decreases c-FLIP levels through promoting its degradation. Of note, degradation of c-FLIP and enhanced TRAIL-induced apoptosis in pancreatic cancer cells were observed only with the anticancer bioactive vitamin E compounds δ-, γ-, and β-tocotrienol but not with the anticancer inactive vitamin E compounds α-tocotrienol and α-, β-, γ-, and δ-tocopherol.

Conclusions: c-FLIP degradation is a key event for death receptor-induced apoptosis by anticancer bioactive vitamin E compounds in pancreatic cancer cells. Moreover, VEDT augmented TRAIL inhibition of pancreatic tumor growth and induction of apoptosis in vivo. Combination therapy with TRAIL agonists and bioactive vitamin E compounds may offer a novel strategy for pancreatic cancer intervention.
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http://dx.doi.org/10.1186/s12935-019-0876-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6647259PMC
July 2019

Dual Farnesyl and Geranylgeranyl Transferase Inhibitor Thwarts Mutant KRAS-Driven Patient-Derived Pancreatic Tumors.

Clin Cancer Res 2019 10 21;25(19):5984-5996. Epub 2019 Jun 21.

Drug Discovery Department, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida.

Purpose: Mutant KRAS is a major driver of pancreatic oncogenesis and therapy resistance, yet KRAS inhibitors are lacking in the clinic. KRAS requires farnesylation for membrane localization and cancer-causing activity prompting the development of farnesyltransferase inhibitors (FTIs) as anticancer agents. However, KRAS becomes geranylgeranylated and active when cancer cells are treated with FTIs. To overcome this geranylgeranylation-dependent resistance to FTIs, we designed FGTI-2734, a RAS C-terminal mimetic dual FT and geranylgeranyltransferase-1 inhibitor (GGTI).

Experimental Design: Immunofluorescence, cellular fractionation, and gel shift assays were used to assess RAS membrane association, Western blotting to evaluate FGTI-2734 effects on signaling, and mouse models to demonstrate its antitumor activity.

Results: FGTI-2734, but not the selective FTI-2148 and GGTI-2418, inhibited membrane localization of KRAS in pancreatic, lung, and colon human cancer cells. FGTI-2734 induced apoptosis and inhibited the growth in mice of mutant KRAS-dependent but not mutant KRAS-independent human tumors. Importantly, FGTI-2734 inhibited the growth of xenografts derived from four patients with pancreatic cancer with mutant KRAS (2 G12D and 2 G12V) tumors. FGTI-2734 was also highly effective at inhibiting, in three-dimensional cocultures with resistance promoting pancreatic stellate cells, the viability of primary and metastatic mutant KRAS tumor cells derived from eight patients with pancreatic cancer. Finally, FGTI-2734 suppressed oncogenic pathways mediated by AKT, mTOR, and cMYC while upregulating p53 and inducing apoptosis in patient-derived xenografts .

Conclusions: The development of this novel dual FGTI overcomes a major hurdle in KRAS resistance, thwarting growth of patient-derived mutant KRAS-driven xenografts from patients with pancreatic cancer, and as such it warrants further preclinical and clinical studies.
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http://dx.doi.org/10.1158/1078-0432.CCR-18-3399DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6774803PMC
October 2019

GSK3 suppression upregulates β-catenin and c-Myc to abrogate KRas-dependent tumors.

Nat Commun 2018 12 4;9(1):5154. Epub 2018 Dec 4.

Drug Discovery Department, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, 33612, USA.

Mutant KRas is a significant driver of human oncogenesis and confers resistance to therapy, underscoring the need to develop approaches that disable mutant KRas-driven tumors. Because targeting KRas directly has proven difficult, identifying vulnerabilities specific for mutant KRas tumors is an important alternative approach. Here we show that glycogen synthase kinase 3 (GSK3) is required for the in vitro and in vivo growth and survival of human mutant KRas-dependent tumors but is dispensable for mutant KRas-independent tumors. Further, inhibiting phosphorylation of GSK3 substrates c-Myc on T58 and β-catenin on S33/S37/T41 and their subsequent upregulation contribute to the antitumor activity of GSK3 inhibition. Importantly, GSK3 blockade inhibits the in vivo growth of G12D, G12V, and G12C mutant KRas primary and metastatic patient-derived xenografts from pancreatic cancer patients who progressed on chemo- and radiation therapies. This discovery opens new avenues to target mutant KRas-dependent cancers.
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http://dx.doi.org/10.1038/s41467-018-07644-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6279809PMC
December 2018

Consensus report of the 8 and 9th Weinman Symposia on Gene x Environment Interaction in carcinogenesis: novel opportunities for precision medicine.

Cell Death Differ 2018 11 15;25(11):1885-1904. Epub 2018 Oct 15.

MRC Toxicology Unit, Leicester, UK.

The relative contribution of intrinsic genetic factors and extrinsic environmental ones to cancer aetiology and natural history is a lengthy and debated issue. Gene-environment interactions (G x E) arise when the combined presence of both a germline genetic variant and a known environmental factor modulates the risk of disease more than either one alone. A panel of experts discussed our current understanding of cancer aetiology, known examples of G × E interactions in cancer, and the expanded concept of G × E interactions to include somatic cancer mutations and iatrogenic environmental factors such as anti-cancer treatment. Specific genetic polymorphisms and genetic mutations increase susceptibility to certain carcinogens and may be targeted in the near future for prevention and treatment of cancer patients with novel molecularly based therapies. There was general consensus that a better understanding of the complexity and numerosity of G × E interactions, supported by adequate technological, epidemiological, modelling and statistical resources, will further promote our understanding of cancer and lead to novel preventive and therapeutic approaches.
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http://dx.doi.org/10.1038/s41418-018-0213-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6219489PMC
November 2018

PTEN counteracts FBXL2 to promote IP3R3- and Ca-mediated apoptosis limiting tumour growth.

Nature 2017 06 14;546(7659):554-558. Epub 2017 Jun 14.

Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, 522 First Avenue, SRB 1107, New York, New York 10016, USA.

In response to environmental cues that promote IP3 (inositol 1,4,5-trisphosphate) generation, IP3 receptors (IP3Rs) located on the endoplasmic reticulum allow the 'quasisynaptical' feeding of calcium to the mitochondria to promote oxidative phosphorylation. However, persistent Ca release results in mitochondrial Ca overload and consequent apoptosis. Among the three mammalian IP3Rs, IP3R3 appears to be the major player in Ca-dependent apoptosis. Here we show that the F-box protein FBXL2 (the receptor subunit of one of 69 human SCF (SKP1, CUL1, F-box protein) ubiquitin ligase complexes) binds IP3R3 and targets it for ubiquitin-, p97- and proteasome-mediated degradation to limit Ca influx into mitochondria. FBXL2-knockdown cells and FBXL2-insensitive IP3R3 mutant knock-in clones display increased cytosolic Ca release from the endoplasmic reticulum and sensitization to Ca-dependent apoptotic stimuli. The phosphatase and tensin homologue (PTEN) gene is frequently mutated or lost in human tumours and syndromes that predispose individuals to cancer. We found that PTEN competes with FBXL2 for IP3R3 binding, and the FBXL2-dependent degradation of IP3R3 is accelerated in Pten mouse embryonic fibroblasts and PTEN-null cancer cells. Reconstitution of PTEN-null cells with either wild-type PTEN or a catalytically dead mutant stabilizes IP3R3 and induces persistent Ca mobilization and apoptosis. IP3R3 and PTEN protein levels directly correlate in human prostate cancer. Both in cell culture and xenograft models, a non-degradable IP3R3 mutant sensitizes tumour cells with low or no PTEN expression to photodynamic therapy, which is based on the ability of photosensitizer drugs to cause Ca-dependent cytotoxicity after irradiation with visible light. Similarly, disruption of FBXL2 localization with GGTi-2418, a geranylgeranyl transferase inhibitor, sensitizes xenotransplanted tumours to photodynamic therapy. In summary, we identify a novel molecular mechanism that limits mitochondrial Ca overload to prevent cell death. Notably, we provide proof-of-principle that inhibiting IP3R3 degradation in PTEN-deregulated cancers represents a valid therapeutic strategy.
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http://dx.doi.org/10.1038/nature22965DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5627969PMC
June 2017

Combined HMG-COA reductase and prenylation inhibition in treatment of CCM.

Proc Natl Acad Sci U S A 2017 05 12;114(21):5503-5508. Epub 2017 May 12.

Department of Neurosurgery, Yale School of Medicine, New Haven, CT 06520;

Cerebral cavernous malformations (CCMs) are common vascular anomalies that develop in the central nervous system and, more rarely, the retina. The lesions can cause headache, seizures, focal neurological deficits, and hemorrhagic stroke. Symptomatic lesions are treated according to their presentation; however, targeted pharmacological therapies that improve the outcome of CCM disease are currently lacking. We performed a high-throughput screen to identify Food and Drug Administration-approved drugs or other bioactive compounds that could effectively suppress hyperproliferation of mouse brain primary astrocytes deficient for CCM3. We demonstrate that fluvastatin, an inhibitor of 3-hydroxy-3-methyl-glutaryl (HMG)-CoA reductase and the -bisphosphonate zoledronic acid monohydrate, an inhibitor of protein prenylation, act synergistically to reverse outcomes of CCM3 loss in cultured mouse primary astrocytes and in glial cells in vivo. Further, the two drugs effectively attenuate neural and vascular deficits in chronic and acute mouse models of CCM3 loss in vivo, significantly reducing lesion burden and extending longevity. Sustained inhibition of the mevalonate pathway represents a potential pharmacological treatment option and suggests advantages of combination therapy for CCM disease.
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http://dx.doi.org/10.1073/pnas.1702942114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5448170PMC
May 2017

δ-Tocotrienol, a natural form of vitamin E, inhibits pancreatic cancer stem-like cells and prevents pancreatic cancer metastasis.

Oncotarget 2017 May;8(19):31554-31567

Departments of Gastrointestinal Oncology, Tampa, FL, USA.

The growth, metastasis, and chemotherapy resistance of pancreatic ductal adenocarcinoma (PDAC) is characterized by the activation and growth of tumor-initiating cells in distant organs that have stem-like properties. Thus, inhibiting growth of these cells may prevent PDAC growth and metastases. We have demonstrated that δ-tocotrienol, a natural form of vitamin E (VEDT), is bioactive against cancer, delays progression, and prevents metastases in transgenic mouse models of PDAC. In this report, we provide the first evidence that VEDT selectively inhibits PDAC stem-like cells. VEDT inhibited the viability, survival, self-renewal, and expression of Oct4 and Sox2 transcription factors in 3 models of PDAC stem-like cells. In addition, VEDT inhibited the migration, invasion, and several biomarkers of epithelial-to-mesenchymal transition and angiogenesis in PDAC cells and tumors. These processes are critical for tumor metastases. Furthermore, in the L3.6pl orthotopic model of PDAC metastases, VEDT significantly inhibited growth and metastases of these cells. Finally, in an orthotopic xenograft model of human PDAC stem-like cells, we showed that VEDT significantly retarded the growth and metastases of gemcitabine-resistant PDAC human stem-like cells. Because VEDT has been shown to be safe and to reach bioactive levels in humans, this work supports investigating VEDT for chemoprevention of PDAC metastases.
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http://dx.doi.org/10.18632/oncotarget.15767DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5458229PMC
May 2017

Targeting Aurora kinase A and JAK2 prevents GVHD while maintaining Treg and antitumor CTL function.

Sci Transl Med 2017 01;9(372)

Department of Blood and Marrow Transplantation, Moffitt Cancer Center, Tampa, FL 33612, USA.

Graft-versus-host disease (GVHD) is a leading cause of nonrelapse mortality after allogeneic hematopoietic cell transplantation. T cell costimulation by CD28 contributes to GVHD, but prevention is incomplete when targeting CD28, downstream mammalian target of rapamycin (mTOR), or Aurora A. Likewise, interleukin-6 (IL-6)-mediated Janus kinase 2 (JAK2) signaling promotes alloreactivity, yet JAK2 inhibition does not eliminate GVHD. We provide evidence that blocking Aurora A and JAK2 in human T cells is synergistic in vitro, prevents xenogeneic GVHD, and maintains antitumor responses by cytotoxic T lymphocytes (CTLs). Aurora A/JAK2 inhibition is immunosuppressive but permits the differentiation of inducible regulatory T cells (iT) that are hyperfunctional and CD39 bright and efficiently scavenge adenosine triphosphate (ATP). Increased iT potency is primarily a function of Aurora A blockade, whereas JAK2 inhibition suppresses T helper 17 (T17) differentiation. Inhibiting either Aurora A or JAK2 significantly suppresses T1 T cells. However, CTL generated in vivo retains tumor-specific killing despite Aurora A/JAK2 blockade. Thus, inhibiting CD28 and IL-6 signal transduction pathways in donor T cells can increase the T/T ratio, prevent GVHD, and preserve antitumor CTL.
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http://dx.doi.org/10.1126/scitranslmed.aai8269DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6368389PMC
January 2017

EGR-1/Bax pathway plays a role in vitamin E δ-tocotrienol-induced apoptosis in pancreatic cancer cells.

J Nutr Biochem 2015 Aug 1;26(8):797-807. Epub 2015 Apr 1.

Department of Gastrointestinal Oncology, H. Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, Tampa, FL; Department of Drug Discovery, H. Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, Tampa, FL. Electronic address:

The anticancer activity of δ-tocotrienol, a bioactive vitamin E present in whole grain cereals, annatto beans and palm fruit, is strongly dependent on its effect on the induction of apoptosis. δ-Tocotrienol-induced apoptosis is associated with consistent induction in the expression of the proapoptotic protein Bcl-2-associated X protein (Bax). The molecular mechanism by which δ-tocotrienol regulates Bax expression is unknown. We carried out a DNA microarray study that identified δ-tocotrienol induction of the zinc finger transcription factor EGR-1 in pancreatic cancer cells. Here, we provide evidence linking δ-tocotrienol-induced apoptosis in pancreatic cancer cells to EGR-1 regulation of Bax expression. Forced expression of EGR-1 induces Bax expression and apoptosis in pancreatic cancer cells. In contrast, knockdown of δ-tocotrienol-induced EGR-1 by small interfering RNA attenuated δ-tocotrienol-induced Bax expression and reduced δ-tocotrienol-induced apoptosis. Further analyses showed that de novo protein synthesis was not required for δ-tocotrienol-induced EGR-1 expression, suggesting a direct effect of δ-tocotrienol on EGR-1 expression. Furthermore, a chromatin immunoprecipitation assay demonstrated that EGR-1 binds to the Bax gene promoter. Finally, δ-tocotrienol treatment induced Bax expression and activated EGR-1 in the pancreatic neoplastic cells of the PDX-Cre Kras genetically engineered model of pancreatic cancer. Our study provides the first evidence for EGR-1 as a direct target of vitamin E δ-tocotrienol, suggesting that EGR-1 may act as a proapoptotic factor in pancreatic cancer cells via induction of Bax.
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http://dx.doi.org/10.1016/j.jnutbio.2015.02.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4576995PMC
August 2015

CD4+ T cell STAT3 phosphorylation precedes acute GVHD, and subsequent Th17 tissue invasion correlates with GVHD severity and therapeutic response.

J Leukoc Biol 2015 Apr 6;97(4):807-19. Epub 2015 Feb 6.

Departments of *Blood and Marrow Transplantation, Immunology, Hematopathology and Laboratory Medicine, Drug Discovery, and Biostatistics, Analytic Microscopy Core, H. Lee Moffitt Cancer Center, Tampa, Florida, USA.

Th17 cells contribute to severe GVHD in murine bone marrow transplantation. Targeted deletion of the RORγt transcription factor or blockade of the JAK2-STAT3 axis suppresses IL-17 production and alloreactivity by Th17 cells. Here, we show that pSTAT3 Y705 is increased significantly in CD4(+) T cells among human recipients of allogeneic HCT before the onset of Grade II-IV acute GVHD. Examination of target-organ tissues at the time of GVHD diagnosis indicates that the amount of RORγt + Th17 cells is significantly higher in severe GVHD. Greater accumulation of tissue-resident Th17 cells also correlates with the use of MTX- compared with Rapa-based GVHD prophylaxis, as well as a poor therapeutic response to glucocorticoids. RORγt is optimally suppressed by concurrent neutralization of TORC1 with Rapa and inhibition of STAT3 activation with S3I-201, supporting that mTOR- and STAT3-dependent pathways converge upon RORγt gene expression. Rapa-resistant T cell proliferation can be totally inhibited by STAT3 blockade during initial allosensitization. We conclude that STAT3 signaling and resultant Th17 tissue accumulation are closely associated with acute GVHD onset, severity, and treatment outcome. Future studies are needed to validate the association of STAT3 activity in acute GVHD. Novel GVHD prevention strategies that incorporate dual STAT3 and mTOR inhibition merit investigation.
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http://dx.doi.org/10.1189/jlb.5A1114-532RRDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4370048PMC
April 2015

Design, synthesis and evaluation of marinopyrrole derivatives as selective inhibitors of Mcl-1 binding to pro-apoptotic Bim and dual Mcl-1/Bcl-xL inhibitors.

Eur J Med Chem 2015 Jan 20;90:315-331. Epub 2014 Nov 20.

Department of Drug Discovery, Chemical Biology & Molecular Medicine Program, Moffitt Cancer Center, 12902 Magnolia Drive, Tampa, FL 33612, United States.

Inhibition of anti-apoptotic Mcl-1 is a promising anticancer strategy to overcome the survival and chemoresistance of a broad spectrum of human cancers. We previously reported on the identification of a natural product marinopyrrole A (1) that induces apoptosis in Mcl-1-dependent cells through Mcl-1 degradation. Here, we report the design and synthesis of novel marinopyrrole-based analogs and their evaluation as selective inhibitors of Mcl-1 as well as dual Mcl-1/Bcl-xL inhibitors. The most selective Mcl-1 antagonists were 34, 36 and 37 with 16-, 13- and 9-fold more selectivity for disrupting Mcl-1/Bim over Bcl-xL/Bim binding, respectively. Among the most potent dual inhibitors is 42 which inhibited Mcl-1/Bim and Bcl-xL/Bim binding 15-fold (IC50 = 600 nM) and 33-fold (500 nM) more potently than (±)-marinopyrrole A (1), respectively. Fluorescence quenching, NMR analysis and molecular docking indicated binding of marinopyrroles to the BH3 binding site of Mcl-1. Several marinopyrroles potently decreased Mcl-1 cellular levels and induced caspase 3 activation in human breast cancer cells. Our studies provide novel "lead" marinopyrroles for further optimization as selective Mcl-1 inhibitors and dual Mcl-1 and Bcl-xL inhibitors.
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http://dx.doi.org/10.1016/j.ejmech.2014.11.035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4445146PMC
January 2015

Ral GTPase down-regulation stabilizes and reactivates p53 to inhibit malignant transformation.

J Biol Chem 2014 Nov 10;289(45):31296-309. Epub 2014 Sep 10.

From the Drug Discovery Department, H. Lee Moffitt Cancer Center and Research Institute and the Departments of Oncologic Sciences and Molecular Medicine, University of South Florida, Tampa, Florida 33612

Ral GTPases are critical effectors of Ras, yet the molecular mechanism by which they induce malignant transformation is not well understood. In this study, we found the expression of K-Ras, RalB, and sometimes RalA, but not AKT1/2 and c-Raf, to be required for maintaining low levels of p53 in human cancer cells that harbor mutant K-Ras and wild-type p53. Down-regulation of K-Ras, RalB, and sometimes RalA increases p53 protein levels and results in a p53-dependent up-regulation of the expression of p21(WAF). K-Ras, RalA, and RalB depletion increases p53 stability as demonstrated by ataxia telangiectasia-mutated kinase activation, increased Ser-15 phosphorylation, and a significant (up to 6-fold) increase in p53 half-life. Furthermore, depletion of K-Ras and RalB inhibits anchorage-independent growth and invasion and interferes with cell cycle progression in a p53-dependent manner. Depletion of RalA inhibits invasion in a p53-dependent manner. Thus, expression of K-Ras and RalB and possibly RalA proteins is critical for maintaining low levels of p53, and down-regulation of these GTPases reactivates p53 by significantly enhancing its stability, and this contributes to suppression of malignant transformation.
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http://dx.doi.org/10.1074/jbc.M114.565796DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4223330PMC
November 2014

Imidazo[1,2-a]pyridine-based peptidomimetics as inhibitors of Akt.

Bioorg Med Chem Lett 2014 Oct 28;24(19):4650-4653. Epub 2014 Aug 28.

Drug Discovery Department, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612, USA.

We report the design, synthesis, and biological evaluation of imidazopyridine-based peptidomimetics based on the substrate consensus sequence of Akt, an AGC family serine/threonine kinase hyperactivated in over 50% of human tumors. Our ligand-based approach led to the identification of novel substrate mimetic inhibitors of Akt1 featuring an unnatural extended dipeptide surrogate. Compound 11 inhibits Akt isoforms in the sub-micromolar range and exhibits improved proteolytic stability relative to a parent pentapeptide.
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http://dx.doi.org/10.1016/j.bmcl.2014.08.040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4176526PMC
October 2014

Marinopyrrole derivatives with sulfide spacers as selective disruptors of Mcl-1 binding to pro-apoptotic protein Bim.

Mar Drugs 2014 Jul 29;12(8):4311-25. Epub 2014 Jul 29.

Department of Drug Discovery and Chemical Biology and Molecular Medicine Program, H. Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, Tampa, FL 33612, USA.

A series of novel marinopyrroles with sulfide and sulphone spacers were designed and synthesized. Their activity to disrupt the binding of the pro-apoptotic protein, Bim, to the pro-survival proteins, Mcl-1 and Bcl-xL, was evaluated using ELISA assays. Fluorescence-quenching (FQ) assays confirmed the direct binding of marinopyrroles to Mcl-1. Benzyl- and benzyl methoxy-containing sulfide derivatives 4 and 5 were highly potent dual Mcl-1/Bim and Bcl-xL/Bim disruptors (IC50 values of 600 and 700 nM), whereas carboxylate-containing sulfide derivative 9 exhibited 16.4-fold more selectivity for disrupting Mcl-1/Bim over Bcl-xL/Bim binding. In addition, a nonsymmetrical marinopyrrole 12 is as equally potent as the parent marinopyrrole A (1) for disrupting both Mcl-1/Bim and Bcl-xL/Bim binding. Some of the derivatives were also active in intact human breast cancer cells where they reduced the levels of Mcl-1, induced programd cell death (apoptosis) and inhibited cell proliferation.
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http://dx.doi.org/10.3390/md12084311DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4145318PMC
July 2014

Identification of novel inhibitors that disrupt STAT3-DNA interaction from a γ-AApeptide OBOC combinatorial library.

Chem Commun (Camb) 2014 Aug 25;50(63):8739-42. Epub 2014 Jun 25.

Department of Chemistry, University of South Florida, 4202 E. Fowler Ave, Tampa, FL 33620, USA.

From a γ-AApeptide-based one-bead-one-compound (OBOC) combinatorial library, we identified γ-AApeptides that can selectively inhibit STAT3-DNA interaction and suppress the expression levels of STAT3 target genes in intact cells. Our results demonstrate that in addition to the SH2 domain, the DNA binding domain of STAT3 is targetable for the development of a new generation of anti-cancer therapeutics.
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http://dx.doi.org/10.1039/c4cc03909bDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4128407PMC
August 2014

Dual Aurora A and JAK2 kinase blockade effectively suppresses malignant transformation.

Oncotarget 2014 May;5(10):2947-61

Drug Discovery Department, Chemical Biology and Molecular Medicine Program, Chemical Biology Core Moffitt Cancer Center and Research Institute.

Aurora A and JAK2 kinases are involved in cell division and tumor cell survival, respectively. Here we demonstrate that ectopic expression of Aurora A and JAK2 together is more effective than each alone at inducing non-transformed cells to grow in an anchorage-independent manner and to invade. Furthermore, siRNA silencing or pharmacological inhibition of Aurora A and JAK2 with Alisertib and Ruxolitinib, respectively, is more effective than blocking each kinase alone at suppressing anchorage-dependent and -independent growth and invasion as well as at inducing apoptosis. Importantly, we have developed dual Aurora and JAK inhibitors, AJI-214 and AJI-100, which potently inhibit Aurora A, Aurora B and JAK2 in vitro. In human cancer cells, these dual inhibitors block the auto-phosphorylation of Aurora A (Thr-288) and the phosphorylation of the Aurora B substrate histone H3 (Ser-10) and the JAK2 substrate STAT3 (Tyr-705). Furthermore, AJI-214 and AJI-100 inhibit anchorage dependent and independent cell growth and invasion and induce G2/M cell cycle accumulation and apoptosis. Finally, AJI-100 caused regression of human tumor xenografts in mice. Taken together, our genetic and pharmacological studies indicate that targeting Aurora A and JAK2 together is a more effective approach than each kinase alone at inhibiting malignant transformation and warrant further advanced pre clinical investigations of dual Aurora A/JAK2 inhibitors as potential anti tumor agents.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4102782PMC
http://dx.doi.org/10.18632/oncotarget.1615DOI Listing
May 2014

Cyclic marinopyrrole derivatives as disruptors of Mcl-1 and Bcl-x(L) binding to Bim.

Mar Drugs 2014 Mar 7;12(3):1335-48. Epub 2014 Mar 7.

Chemical Biology & Molecular Medicine Program, Department of Drug Discovery, H. Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, Tampa, FL 33612, USA.

A series of novel cyclic marinopyrroles were designed and synthesized. Their activity to disrupt the binding of the pro-apoptotic protein, Bim, to the pro-survival proteins, Mcl-1 and Bcl-x(L), was evaluated using ELISA assays. Both atropisomers of marinopyrrole A (1) show similar potency. A tetrabromo congener 9 is two-fold more potent than 1. Two novel cyclic marinopyrroles (3 and 4) are two- to seven-fold more potent than 1.
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http://dx.doi.org/10.3390/md12031335DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3967213PMC
March 2014

Discovery of PI-1840, a novel noncovalent and rapidly reversible proteasome inhibitor with anti-tumor activity.

J Biol Chem 2014 Apr 25;289(17):11906-11915. Epub 2014 Feb 25.

Drug Discovery Department, H. Lee Moffit Cancer Center and Research Institute, Tampa, Florida 33612; Department of Oncologic Sciences, University of South Florida, Tampa, Florida 33620. Electronic address:

The proteasome inhibitor bortezomib is effective in hematologic malignancies such as multiple myeloma but has little activity against solid tumors, acts covalently, and is associated with undesired side effects. Therefore, noncovalent inhibitors that are less toxic and more effective against solid tumors are desirable. Structure activity relationship studies led to the discovery of PI-1840, a potent and selective inhibitor for chymotrypsin-like (CT-L) (IC50 value = 27 ± 0.14 nm) over trypsin-like and peptidylglutamyl peptide hydrolyzing (IC50 values >100 μm) activities of the proteasome. Furthermore, PI-1840 is over 100-fold more selective for the constitutive proteasome over the immunoproteasome. Mass spectrometry and dialysis studies demonstrate that PI-1840 is a noncovalent and rapidly reversible CT-L inhibitor. In intact cancer cells, PI-1840 inhibits CT-L activity, induces the accumulation of proteasome substrates p27, Bax, and IκB-α, inhibits survival pathways and viability, and induces apoptosis. Furthermore, PI-1840 sensitizes human cancer cells to the mdm2/p53 disruptor, nutlin, and to the pan-Bcl-2 antagonist BH3-M6. Finally, in vivo, PI-1840 but not bortezomib suppresses the growth in nude mice of human breast tumor xenografts. These results warrant further evaluation of a noncovalent and rapidly reversible proteasome inhibitor as potential anticancer agents against solid tumors.
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http://dx.doi.org/10.1074/jbc.M113.533950DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4002098PMC
April 2014

Lysophosphatidic acid acyltransferase beta regulates mTOR signaling.

PLoS One 2013 31;8(10):e78632. Epub 2013 Oct 31.

Department of Experimental Therapeutics, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida, United States of America.

Lysophosphatidic acid acyltransferase (LPAAT-β) is a phosphatidic acid (PA) generating enzyme that plays an essential role in triglyceride synthesis. However, LPAAT-β is now being studied as an important regulator of cell growth and differentiation and as a potential therapeutic target in cancer since PA is necessary for the activity of key proteins such as Raf, PKC-ζ and mTOR. In this report we determine the effect of LPAAT-β silencing with siRNA in pancreatic adenocarcinoma cell lines. We show for the first time that LPAAT-β knockdown inhibits proliferation and anchorage-independent growth of pancreatic cancer cells. This is associated with inhibition of signaling by mTOR as determined by levels of mTORC1- and mTORC2-specific phosphorylation sites on 4E-BP1, S6K and Akt. Since PA regulates the activity of mTOR by modulating its binding to FKBP38, we explored the possibility that LPAAT-β might regulate mTOR by affecting its association with FKBP38. Coimmunoprecipitation studies of FKBP38 with mTOR show increased levels of FKBP38 associated with mTOR when LPAAT-β protein levels are knocked down. Furthermore, depletion of LPAAT-β results in increased Lipin 1 nuclear localization which is associated with increased nuclear eccentricity, a nuclear shape change that is dependent on mTOR, further confirming the ability of LPAAT-β to regulate mTOR function. Our results provide support for the hypothesis that PA generated by LPAAT-β regulates mTOR signaling. We discuss the implications of these findings for using LPAAT-β as a therapeutic target.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0078632PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3814986PMC
August 2014

Selective disruption of rb-raf-1 kinase interaction inhibits pancreatic adenocarcinoma growth irrespective of gemcitabine sensitivity.

Mol Cancer Ther 2013 Dec 9;12(12):2722-34. Epub 2013 Oct 9.

Corresponding Author: S.P. Chellappan, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612.

Inactivation of the retinoblastoma (Rb) tumor suppressor protein is widespread in human cancers. Inactivation of Rb is thought to be initiated by association with Raf-1 (C-Raf) kinase, and here we determined how RRD-251, a disruptor of the Rb-Raf-1 interaction, affects pancreatic tumor progression. Assessment of phospho-Rb levels in resected human pancreatic tumor specimens by immunohistochemistry (n = 95) showed that increased Rb phosphorylation correlated with increasing grade of resected human pancreatic adenocarcinomas (P = 0.0272), which correlated with reduced overall patient survival (P = 0.0186). To define the antitumor effects of RRD-251 (50 μmol/L), cell-cycle analyses, senescence, cell viability, cell migration, anchorage-independent growth, angiogenic tubule formation and invasion assays were conducted on gemcitabine-sensitive and -resistant pancreatic cancer cells. RRD-251 prevented S-phase entry, induced senescence and apoptosis, and inhibited anchorage-independent growth and invasion (P < 0.01). Drug efficacy on subcutaneous and orthotopic xenograft models was tested by intraperitoneal injections of RRD-251 (50 mg/kg) alone or in combination with gemcitabine (250 mg/kg). RRD-251 significantly reduced tumor growth in vivo accompanied by reduced Rb phosphorylation and lymph node and liver metastasis (P < 0.01). Combination of RRD-251 with gemcitabine showed cooperative effect on tumor growth (P < 0.01). In conclusion, disruption of the Rb-Raf-1 interaction significantly reduces the malignant properties of pancreatic cancer cells irrespective of their gemcitabine sensitivity. Selective targeting of Rb-Raf-1 interaction might be a promising strategy targeting pancreatic cancer.
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http://dx.doi.org/10.1158/1535-7163.MCT-12-0719DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3858536PMC
December 2013

Development of new -Arylbenzamides as STAT3 Dimerization Inhibitors.

Medchemcomm 2013 Jun;4(6):932-941

Departments of Drug Discovery, Moffitt Cancer Center, 12902 Magnolia Drive, Tampa, FL 33612, USA.

The -tosylsalicylamide () was used as a starting point for design and synthesis of novel STAT-3 dimerization inhibitors with improved drug-like qualities. The phosphonic acid and salicylic acids , with a shorter amide linker lacking the -tosyl group had improved STAT-3 inhibitory activity. The equivalent potencies observed by the replacement of phosphonic acid moiety of with 5-amino-2-hydroxybenzoic acid group as in further validates 5-amino-2-hydroxybenzoic acid as a phosphotyrosine mimic. The salicylic acid displayed improved whole cell activity. The focused library of salicylic acids with benzamide linker indicated that hydrophobic heptyl and cyclohexyl are the best tolerated R groups and a biphenyl ether (as the Ar group) significantly contributes to STAT3 inhibitory activity. Our docking studies indicated that the acidic groups of , and interact in the p-Tyr-705 binding site in a broadly similar manner, while the phenoxybenzoyl group and the cyclohexylbenzyl group occupying pY+1 and pY-X hydrophobic pockets respectively. The and cell based potency of warrants further development of this scaffold as STAT3 inhibitors.
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http://dx.doi.org/10.1039/C3MD20323ADOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3780559PMC
June 2013

Vitamin E δ-tocotrienol prolongs survival in the LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre (KPC) transgenic mouse model of pancreatic cancer.

Cancer Prev Res (Phila) 2013 Oct 20;6(10):1074-83. Epub 2013 Aug 20.

Department of Gastrointestinal Oncology, Moffitt Cancer Center, 12902 Magnolia Drive, Tampa, FL 33612.

Previous work has shown that vitamin E δ-tocotrienol (VEDT) prolongs survival and delays progression of pancreatic cancer in the LSL-Kras(G12D)(/+);Pdx-1-Cre mouse model of pancreatic cancer. However, the effect of VEDT alone or in combination with gemcitabine in the more aggressive LSL-Kras(G12D)(/+);LSL-Trp53(R172H)(/+);Pdx-1-Cre (KPC) mouse model is unknown. Here, we studied the effects of VEDT and the combination of VEDT and gemcitabine in the KPC mice. KPC mice were randomized into four groups: (i) vehicle [olive oil, 1.0 mL/kg per os twice a day and PBS 1.0 mL/kg intrapertoneally (i.p.) twice a week], (ii) gemcitabine (100 mg/kg i.p. twice a week), (iii) VEDT (200 mg/kg per os twice a day), and (iv) gemcitabine + VEDT. Mice received treatment until they displayed symptoms of impending death from pancreatic cancer, at which point animals were euthanized. At 16 weeks, survival was 10% in the vehicle group, 30% in the gemcitabine group, 70% in the VEDT group (P < 0.01), and 90% in the VEDT combined with gemcitabine group (P < 0.05). VEDT alone and combined with gemcitabine resulted in reversal of epithelial-to-mesenchymal transition in tumors. Biomarkers of apoptosis (plasma CK18), PARP1 cleavage, and Bax expression were more greatly induced in tumors subjected to combined treatment versus individual treatment. Combined treatment induced cell-cycle inhibitors (p27(Kip1) and p21(Cip1)) and inhibited VEGF, vascularity (CD31), and oncogenic signaling (pAKT, pMEK, and pERK) greater than individual drugs. No significant differences in body weight gain between drug treatment and control mice were observed. These results strongly support further investigation of VEDT alone and in combination with gemcitabine for pancreatic cancer prevention and treatment.
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http://dx.doi.org/10.1158/1940-6207.CAPR-13-0157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4165552PMC
October 2013

Akt2 and acid ceramidase cooperate to induce cell invasion and resistance to apoptosis.

Cell Cycle 2013 Jul 6;12(13):2024-32. Epub 2013 Jun 6.

Drug Discovery Department and Chemical Biology and Molecular Medicine Program, Moffitt Cancer Center, Tampa, FL, USA.

Both Akt 2 and acid ceramidase (ASAH1) are found aberrantly overexpressed in cancer cells, but whether these two enzymes cooperate to induce malignant transformation is not known. We found that in immortalized, non-transformed cells, ectopic co-expression of Akt2 and ASAH1 is significantly more effective than expression of each gene alone at inducing cell invasion and at conferring resistance to apoptosis. Consistent with these observations, siRNA-mediated depletion of both Akt2 and ASAH1 is much more potent than depleting each alone at inhibiting cell viability/proliferation and cell invasion. Furthermore, pharmacological inhibitors of Akt (TCN or MK-2206) and ASAH1 (B13) synergize to inhibit cell viability/proliferation, and combinations of these drugs are more effective than single-agent treatments at inhibiting cell invasion. Taken together, the results suggest that these two enzymes cooperate to induce malignant transformation and warrant further preclinical studies to evaluate the potential of combining inhibitors of Akt and ASAH1 to treat cancer.
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http://dx.doi.org/10.4161/cc.25043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3737305PMC
July 2013