Publications by authors named "Nicholas J Lawrence"

51 Publications

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

Structural Insights into JAK2 Inhibition by Ruxolitinib, Fedratinib, and Derivatives Thereof.

J Med Chem 2021 02 11;64(4):2228-2241. Epub 2021 Feb 11.

Drug Discovery DepartmentMoffitt Cancer Center, Tampa, Florida 33612, United States.

The discovery that aberrant activity of Janus kinase 2 (JAK2) is a driver of myeloproliferative neoplasms (MPNs) has led to significant efforts to develop small molecule inhibitors for this patient population. Ruxolitinib and fedratinib have been approved for use in MPN patients, while baricitinib, an achiral analogue of ruxolitinib, has been approved for rheumatoid arthritis. However, structural information on the interaction of these therapeutics with JAK2 remains unknown. Here, we describe a new methodology for the large-scale production of JAK2 from mammalian cells, which enabled us to determine the first crystal structures of JAK2 bound to these drugs and derivatives thereof. Along with biochemical and cellular data, the results provide a comprehensive view of the shape complementarity required for chiral and achiral inhibitors to achieve highest activity, which may facilitate the development of more effective JAK2 inhibitors as therapeutics.
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http://dx.doi.org/10.1021/acs.jmedchem.0c01952DOI Listing
February 2021

Mesenchymal stem cell-derived interleukin-28 drives the selection of apoptosis resistant bone metastatic prostate cancer.

Nat Commun 2021 02 1;12(1):723. Epub 2021 Feb 1.

Tumor Biology Department, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA.

Bone metastatic prostate cancer (PCa) promotes mesenchymal stem cell (MSC) recruitment and their differentiation into osteoblasts. However, the effects of bone-marrow derived MSCs on PCa cells are less explored. Here, we report MSC-derived interleukin-28 (IL-28) triggers prostate cancer cell apoptosis via IL-28 receptor alpha (IL-28Rα)-STAT1 signaling. However, chronic exposure to MSCs drives the selection of prostate cancer cells that are resistant to IL-28-induced apoptosis and therapeutics such as docetaxel. Further, MSC-selected/IL-28-resistant prostate cancer cells grow at accelerated rates in bone. Acquired resistance to apoptosis is PCa cell intrinsic, and is associated with a shift in IL-28Rα signaling via STAT1 to STAT3. Notably, STAT3 ablation or inhibition impairs MSC-selected prostate cancer cell growth and survival. Thus, bone marrow MSCs drive the emergence of therapy-resistant bone metastatic prostate cancer yet this can be disabled by targeting STAT3.
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http://dx.doi.org/10.1038/s41467-021-20962-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7851397PMC
February 2021

Cereblon harnesses Myc-dependent bioenergetics and activity of CD8+ T lymphocytes.

Blood 2020 08;136(7):857-870

Department of Tumor Biology, Moffitt Cancer Center and Research Institute, Tampa, FL.

Immunomodulatory drugs, such as thalidomide and related compounds, potentiate T-cell effector functions. Cereblon (CRBN), a substrate receptor of the DDB1-cullin-RING E3 ubiquitin ligase complex, is the only molecular target for this drug class, where drug-induced, ubiquitin-dependent degradation of known "neosubstrates," such as IKAROS, AIOLOS, and CK1α, accounts for their biological activity. Far less clear is whether these CRBN E3 ligase-modulating compounds disrupt the endogenous functions of CRBN. We report that CRBN functions in a feedback loop that harnesses antigen-specific CD8+ T-cell effector responses. Specifically, Crbn deficiency in murine CD8+ T cells augments their central metabolism manifested as elevated bioenergetics, with supraphysiological levels of polyamines, secondary to enhanced glucose and amino acid transport, and with increased expression of metabolic enzymes, including the polyamine biosynthetic enzyme ornithine decarboxylase. Treatment with CRBN-modulating compounds similarly augments central metabolism of human CD8+ T cells. Notably, the metabolic control of CD8+ T cells by modulating compounds or Crbn deficiency is linked to increased and sustained expression of the master metabolic regulator MYC. Finally, Crbn-deficient T cells have augmented antigen-specific cytolytic activity vs melanoma tumor cells, ex vivo and in vivo, and drive accelerated and highly aggressive graft-versus-host disease. Therefore, CRBN functions to harness the activation of CD8+ T cells, and this phenotype can be exploited by treatment with drugs.
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http://dx.doi.org/10.1182/blood.2019003257DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7426646PMC
August 2020

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

Targeting the BRD4-HOXB13 Coregulated Transcriptional Networks with Bromodomain-Kinase Inhibitors to Suppress Metastatic Castration-Resistant Prostate Cancer.

Mol Cancer Ther 2018 12 21;17(12):2796-2810. Epub 2018 Sep 21.

Tumor Biology Department, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida.

Resistance to androgen receptor (AR) antagonists is a significant problem in the treatment of castration-resistant prostate cancers (CRPC). Identification of the mechanisms by which CRPCs evade androgen deprivation therapies (ADT) is critical to develop novel therapeutics. We uncovered that CRPCs rely on BRD4-HOXB13 epigenetic reprogramming for androgen-independent cell proliferation. Mechanistically, BRD4, a member of the BET bromodomain family, epigenetically promotes HOXB13 expression. Consistently, genetic disruption of or pharmacological suppression of its mRNA and protein expression by the novel dual-activity BET bromodomain-kinase inhibitors directly correlates with rapid induction of apoptosis, potent inhibition of tumor cell proliferation and cell migration, and suppression of CRPC growth. Integrative analysis revealed that the BRD4-HOXB13 transcriptome comprises a proliferative gene network implicated in cell-cycle progression, nucleotide metabolism, and chromatin assembly. Notably, although the core HOXB13 target genes responsive to BET inhibitors (HOTBIN10) are overexpressed in metastatic cases, in ADT-treated CRPC cell lines and patient-derived circulating tumor cells (CTC) they are insensitive to AR depletion or blockade. Among the HOTBIN10 genes, AURKB and MELK expression correlates with HOXB13 expression in CTCs of mCRPC patients who did not respond to abiraterone (ABR), suggesting that AURKB inhibitors could be used additionally against high-risk HOXB13-positive metastatic prostate cancers. Combined, our study demonstrates that BRD4-HOXB13-HOTBIN10 regulatory circuit maintains the malignant state of CRPCs and identifies a core proproliferative network driving ADT resistance that is targetable with potent dual-activity bromodomain-kinase inhibitors.
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http://dx.doi.org/10.1158/1535-7163.MCT-18-0602DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6528782PMC
December 2018

Ligand-mediated protein degradation reveals functional conservation among sequence variants of the CUL4-type E3 ligase substrate receptor cereblon.

J Biol Chem 2018 04 15;293(16):6187-6200. Epub 2018 Feb 15.

From the Department of Immunology,

Upon binding to thalidomide and other immunomodulatory drugs, the E3 ligase substrate receptor cereblon (CRBN) promotes proteosomal destruction by engaging the DDB1-CUL4A-Roc1-RBX1 E3 ubiquitin ligase in human cells but not in mouse cells, suggesting that sequence variations in CRBN may cause its inactivation. Therapeutically, CRBN engagers have the potential for broad applications in cancer and immune therapy by specifically reducing protein expression through targeted ubiquitin-mediated degradation. To examine the effects of defined sequence changes on CRBN's activity, we performed a comprehensive study using complementary theoretical, biophysical, and biological assays aimed at understanding CRBN's nonprimate sequence variations. With a series of recombinant thalidomide-binding domain (TBD) proteins, we show that CRBN sequence variants retain their drug-binding properties to both classical immunomodulatory drugs and dBET1, a chemical compound and targeting ligand designed to degrade bromodomain-containing 4 (BRD4) via a CRBN-dependent mechanism. We further show that dBET1 stimulates CRBN's E3 ubiquitin-conjugating function and degrades BRD4 in both mouse and human cells. This insight paves the way for studies of CRBN-dependent proteasome-targeting molecules in nonprimate models and provides a new understanding of CRBN's substrate-recruiting function.
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http://dx.doi.org/10.1074/jbc.M117.816868DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5912449PMC
April 2018

Blockade of ACK1/TNK2 To Squelch the Survival of Prostate Cancer Stem-like Cells.

Sci Rep 2018 01 31;8(1):1954. Epub 2018 Jan 31.

Tumor Biology Department, Moffitt Cancer Center, 12902 Magnolia Drive, Tampa, FL, 33612, USA.

Prostate cancer stem-like cells (PCSCs) are not only enriched in the CD44PSA subpopulation but also employ androgen-independent signaling mechanisms for survival. CD44 PCSCs defy androgen deprivation, resist chemo- and radiotherapy and are highly tumorigenic. Human prostate tissue microarray (TMA) staining revealed an increased membranous staining of CD44 in the luminal compartment in higher grade G7-G9 tumors versus staining of the basal layer in benign hyperplasia. To uncover tyrosine kinase/s critical for the survival of the CD44PSA subpopulation, we performed an unbiased screen targeting 87 tyrosine kinases with gene specific siRNAs. Among a subset of tyrosine kinases crucial for PCSC survival, was a non-receptor tyrosine kinase, ACK1/TNK2, a critical regulator of castration resistant prostate cancer (CRPC) growth. Consistently, activated ACK1 as measured by phosphorylation at Tyr284 was significant in the CD44PSA population. Conversely, pharmacological inhibition by ACK1 inhibitor, (R)-9bMS mitigated CD44PSA sphere formation, overcame resistance to radiation-induced cell death, induced significant apoptosis in PCSCs and inhibited CD44PSA xenograft tumor growth in castrated mice suggesting dependency of PCSCs on ACK1 for survival. Thus, blockade of ACK1/TNK2 could be a new therapeutic modality to target recalcitrant PCSCs.
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http://dx.doi.org/10.1038/s41598-018-20172-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5792546PMC
January 2018

ACK1/TNK2 Regulates Histone H4 Tyr88-phosphorylation and AR Gene Expression in Castration-Resistant Prostate Cancer.

Cancer Cell 2017 06;31(6):790-803.e8

Drug Discovery Department, Moffitt Cancer Center, 12902 Magnolia Drive, Tampa, FL 33612, USA; Department of Oncological Sciences, University of South Florida, Tampa, FL 33612, USA. Electronic address:

The androgen receptor (AR) is critical for the progression of prostate cancer to a castration-resistant (CRPC) state. AR antagonists are ineffective due to their inability to repress the expression of AR or its splice variant, AR-V7. Here, we report that the tyrosine kinase ACK1 (TNK2) phosphorylates histone H4 at tyrosine 88 upstream of the AR transcription start site. The WDR5/MLL2 complex reads the H4-Y88-phosphorylation marks and deposits the transcriptionally activating H3K4-trimethyl marks promoting AR transcription. Reversal of the pY88-H4 epigenetic marks by the ACK1 inhibitor (R)-9bMS-sensitized naive and enzalutamide-resistant prostate cancer cells and reduced AR and AR-V7 levels to mitigate CRPC tumor growth. Thus, a feedforward ACK1/pY88-H4/WDR5/MLL2/AR epigenetic circuit drives CRPC and is necessary for maintenance of the malignant state.
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http://dx.doi.org/10.1016/j.ccell.2017.05.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5512571PMC
June 2017

Potent Dual BET Bromodomain-Kinase Inhibitors as Value-Added Multitargeted Chemical Probes and Cancer Therapeutics.

Mol Cancer Ther 2017 06 23;16(6):1054-1067. Epub 2017 Mar 23.

Drug Discovery Department, Moffitt Cancer Center, Tampa, Florida.

Synergistic action of kinase and BET bromodomain inhibitors in cell killing has been reported for a variety of cancers. Using the chemical scaffold of the JAK2 inhibitor TG101348, we developed and characterized single agents which potently and simultaneously inhibit BRD4 and a specific set of oncogenic tyrosine kinases including JAK2, FLT3, RET, and ROS1. Lead compounds showed on-target inhibition in several blood cancer cell lines and were highly efficacious at inhibiting the growth of hematopoietic progenitor cells from patients with myeloproliferative neoplasm. Screening across 931 cancer cell lines revealed differential growth inhibitory potential with highest activity against bone and blood cancers and greatly enhanced activity over the single BET inhibitor JQ1. Gene drug sensitivity analyses and drug combination studies indicate synergism of BRD4 and kinase inhibition as a plausible reason for the superior potency in cell killing. Combined, our findings indicate promising potential of these agents as novel chemical probes and cancer therapeutics. .
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http://dx.doi.org/10.1158/1535-7163.MCT-16-0568-TDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5457702PMC
June 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

Inhibition of Shp2 suppresses mutant EGFR-induced lung tumors in transgenic mouse model of lung adenocarcinoma.

Oncotarget 2015 Mar;6(8):6191-202

Department of Molecular Oncology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida, USA.

Epidermal growth factor receptor (EGFR) mutants drive lung tumorigenesis and are targeted for therapy. However, resistance to EGFR inhibitors has been observed, in which the mutant EGFR remains active. Thus, it is important to uncover mediators of EGFR mutant-driven lung tumors to develop new treatment strategies. The protein tyrosine phosphatase (PTP) Shp2 mediates EGF signaling. Nevertheless, it is unclear if Shp2 is activated by oncogenic EGFR mutants in lung carcinoma or if inhibiting the Shp2 PTP activity can suppress EGFR mutant-induced lung adenocarcinoma. Here, we generated transgenic mice containing a doxycycline (Dox)-inducible PTP-defective Shp2 mutant (tetO-Shp2CSDA). Using the rat Clara cell secretory protein (CCSP)-rtTA-directed transgene expression in the type II lung pneumocytes of transgenic mice, we found that the Gab1-Shp2 pathway was activated by EGFRL858R in the lungs of transgenic mice. Consistently, the Gab1-Shp2 pathway was activated in human lung adenocarcinoma cells containing mutant EGFR. Importantly, Shp2CSDA inhibited EGFRL858R-induced lung adenocarcinoma in transgenic animals. Analysis of lung tissues showed that Shp2CSDA suppressed Gab1 tyrosine phosphorylation and Gab1-Shp2 association, suggesting that Shp2 modulates a positive feedback loop to regulate its own activity. These results show that inhibition of the Shp2 PTP activity impairs mutant EGFR signaling and suppresses EGFRL858R-driven lung adenocarcinoma.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4467431PMC
http://dx.doi.org/10.18632/oncotarget.3356DOI Listing
March 2015

Development of novel ACK1/TNK2 inhibitors using a fragment-based approach.

J Med Chem 2015 Mar 17;58(6):2746-63. Epub 2015 Mar 17.

§Department of Oncologic Sciences, University of South Florida, Tampa, Florida 33620, United States.

The tyrosine kinase ACK1, a critical signal transducer regulating survival of hormone-refractory cancers, is an important therapeutic target, for which there are no selective inhibitors in clinical trials to date. This work reports the discovery of novel and potent inhibitors for ACK1 tyrosine kinase (also known as TNK2) using an innovative fragment-based approach. Focused libraries were designed and synthesized by selecting fragments from reported ACK inhibitors to create hybrid structures in a mix and match process. The hybrid library was screened by enzyme-linked immunosorbent assay-based kinase inhibition and (33)P HotSpot assays. Systematic structure-activity relationship studies led to the identification of compound (R)-9b, which shows potent in vitro (IC50 = 56 nM, n = 3, (33)P HotSpot assay) and in vivo (IC50 < 2 μM, human cancer cell lines) ACK1 inhibition. Both (R)-9b and (S)-9b were stable in human plasma and displayed a long half-life (t(1/2) > 6 h).
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http://dx.doi.org/10.1021/jm501929nDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4605435PMC
March 2015

ACK1 tyrosine kinase interacts with histone demethylase KDM3A to regulate the mammary tumor oncogene HOXA1.

J Biol Chem 2014 Oct 22;289(41):28179-91. Epub 2014 Aug 22.

From the Drug Discovery Department, Moffitt Cancer Center, and the Department of Oncologic Sciences, College of Medicine, University of South Florida, Tampa, Florida 33612

Hormone therapy with the selective estrogen-receptor modulator tamoxifen provides a temporary relief for patients with estrogen receptor α (ER)-positive breast cancers. However, a subset of patients exhibiting overexpression of the HER2 receptor tyrosine kinase displays intrinsic resistance to tamoxifen therapy. Therefore, elucidating the mechanisms promoting the estrogen (E2)-independent ER-regulated gene transcription in tamoxifen-resistant breast tumors is essential to identify new therapeutic avenues to overcome drug resistance and ameliorate poor prognosis. The non-receptor tyrosine kinase, ACK1 (also known as TNK2), has emerged as a major integrator of signaling from various receptor tyrosine kinases including HER2. We have uncovered that heregulin-mediated ACK1 activation promoted ER activity in the presence of tamoxifen, which was significantly down-regulated upon ACK1 knockdown or inhibition of ACK1 by small molecule inhibitors, AIM-100 or Dasatinib. We report that ACK1 phosphorylates the ER co-activator, KDM3A, a H3K9 demethylase, at an evolutionary conserved tyrosine 1114 site in a heregulin-dependent manner, even in the presence of tamoxifen. Consistent with this finding, ACK1 activation resulted in a significant decrease in the deposition of dimethyl H3K9 epigenetic marks. Conversely, inhibition of ACK1 by AIM-100 or Dasatinib restored dimethyl H3K9 methylation marks and caused transcriptional suppression of the ER-regulated gene HOXA1. Thus, by its ability to regulate the epigenetic activity of an ER co-activator KDM3A, ACK1 modulates HOXA1 expression in the absence of E2, conferring tamoxifen resistance. These data reveal a novel therapeutic option, suppression of ACK1 signaling by AIM-100 or Dasatinib, to mitigate HOXA1 up-regulation in breast cancer patients displaying tamoxifen resistance.
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http://dx.doi.org/10.1074/jbc.M114.584425DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4192474PMC
October 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

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

A novel inhibitor of STAT3 homodimerization selectively suppresses STAT3 activity and malignant transformation.

Cancer Res 2013 Mar 15;73(6):1922-33. Epub 2013 Jan 15.

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

STAT3-STAT3 dimerization, which involves reciprocal binding of the STAT3-SH2 domain to phosphorylated tyrosine-705 (Y-705), is required for STAT3 nuclear translocation, DNA binding, and transcriptional regulation of downstream target genes. Here, we describe a small molecule S3I-1757 capable of disrupting STAT3-STAT3 dimerization, activation, and malignant transforming activity. Fluorescence polarization assay and molecular modeling suggest that S3I-1757 interacts with the phospho-Y-705-binding site in the SH2 domain and displaces fluorescein-labeled GpYLPQTV phosphotyrosine peptide from binding to STAT3. We generated hemagglutinin (HA)-tagged STAT3 and FLAG-tagged STAT3 and showed using coimmunoprecipitation and colocalization studies that S3I-1757 inhibits STAT3 dimerization and STAT3-EGF receptor (EGFR) binding in intact cells. Treatment of human cancer cells with S3I-1757 (but not a closely related analog, S3I-1756, which does not inhibit STAT3 dimerization), inhibits selectively the phosphorylation of STAT3 over AKT1 and ERK1/2 (MAPK3/1), nuclear accumulation of P-Y705-STAT3, STAT3-DNA binding, and transcriptional activation and suppresses the expression levels of STAT3 target genes, such as Bcl-xL (BCL2L1), survivin (BIRC5), cyclin D1 (CCND1), and matrix metalloproteinase (MMP)-9. Furthermore, S3I-1757, but not S3I-1756, inhibits anchorage-dependent and -independent growth, migration, and invasion of human cancer cells, which depend on STAT3. Finally, STAT3-C, a genetically engineered mutant of STAT3 that forms a constitutively dimerized STAT3, rescues cells from the effects of S3I-1757 inhibition. Thus, we have developed S3I-1757 as a STAT3-STAT3 dimerization inhibitor capable of blocking hyperactivated STAT3 and suppressing malignant transformation in human cancer cells that depend on STAT3.
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http://dx.doi.org/10.1158/0008-5472.CAN-12-3175DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4030628PMC
March 2013

Pyridylthiazole-based ureas as inhibitors of Rho associated protein kinases (ROCK1 and 2).

Medchemcomm 2012 Jun 27;3(6):699-709. Epub 2012 Jan 27.

The Department of Drug Discovery, Moffitt Cancer Center, 12902 Magnolia Drive, Tampa, Florida, 33612, USA.

Potent ROCK inhibitors of a new class of 1-benzyl-3-(4-pyridylthiazol-2-yl)ureas have been identified. Remarkable differences in activity were observed for ureas bearing a benzylic stereogenic center. Derivatives with hydroxy, methoxy and amino groups at the meta position of the phenyl ring give rise to the most potent inhibitors (low nM). Substitutions at the para position result in substantial loss of potency. Changes at the benzylic position are tolerated resulting in significant potency in the case of methyl and methylenehydroxy groups. X-Ray crystallography was used to establish the binding mode of this class of inhibitors and provides an explanation for the observed differences of the enantiomer series. Potent inhibition of ROCK in human lung cancer cells was shown by suppression of the levels of phosphorylation of the ROCK substrate MYPT-1.
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http://dx.doi.org/10.1039/C2MD00320ADOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3531244PMC
June 2012

Inhibiting the interaction of cMET and IGF-1R with FAK effectively reduces growth of pancreatic cancer cells in vitro and in vivo.

Anticancer Agents Med Chem 2013 May;13(4):595-602

Department of Surgery, University of Florida College of Medicine, Gainesville, FL, USA.

Pancreatic cancer is one of the most lethal diseases with no effective treatment. Previously, we have shown that FAK is overexpressed in pancreatic cancer and plays a key role in cancer cell survival and proliferation. FAK has been shown to interact with growth factor receptors including cMET and IGF-1R. As a novel therapeutic approach, we targeted the protein interaction of FAK with growth factor receptors to block tumor growth, alter signaling pathways and sensitize cells to chemotherapy. We have selected a small molecule compound (INT2-31) that decreases phosphorylation of AKT via disrupting interaction of FAK with cMET and IGF-1R. Our results demonstrate that interaction of a small molecule compound with FAK decreases phosphorylation of FAK Y397 while increasing FAK Y407 phosphorylation, without inhibiting the kinase activity of FAK and dramatically reduces downstream signaling to AKT. Our lead compound, INT2-31, demonstrates significant inhibition of tumor cell growth in two orthotopic models of pancreatic cancer. In addition, INT2-31 increases sensitivity to gemcitabine chemotherapy in a direct fresh biopsy xenograft model of pancreatic cancer growth.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4052463PMC
http://dx.doi.org/10.2174/1871520611313040009DOI Listing
May 2013

Regulation of interlocking gene regulatory network subcircuits by a small molecule inhibitor of retinoblastoma protein (RB) phosphorylation: cancer cell expression of HLA-DR.

Gene 2013 Jan 3;512(2):403-7. Epub 2012 Oct 3.

Drug Discovery Program, Department of Oncologic Sciences, Morsani College of Medicine, University of South Florida, Tampa, FL 33612, USA.

The induction of the major histocompatibility (MHC), antigen-presenting class II molecules by interferon-gamma, in solid tumor cells, requires the retinoblastoma tumor suppressor protein (Rb). In the absence of Rb, a repressosome blocks the access of positive-acting, promoter binding proteins to the MHC class II promoter. However, a complete molecular linkage between Rb expression and the disassembly of the MHC class II repressosome has been lacking. By treating A549 lung carcinoma cells with a novel small molecule that prevents phosphorylation-mediated, Rb inactivation, we demonstrate that Rb represses the synthesis of an MHC class II repressosome component, YY1. The reduction in YY1 synthesis correlates with the advent of MHC class II inducibility; with loss of YY1 binding to the promoter of the HLA-DRA gene, the canonical human MHC class II gene; and with increased Rb binding to the YY1 promoter. These results support the concept that the Rb gene regulatory network (GRN) subcircuit that regulates cell proliferation is linked to a GRN subcircuit regulating a tumor cell immune function.
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http://dx.doi.org/10.1016/j.gene.2012.09.092DOI Listing
January 2013

RKI-1447 is a potent inhibitor of the Rho-associated ROCK kinases with anti-invasive and antitumor activities in breast cancer.

Cancer Res 2012 Oct 30;72(19):5025-34. Epub 2012 Jul 30.

Drug Discovery Department, H Lee Moffitt Cancer Center and Research Institute, University of South Florida, Tampa, Florida 33612, USA.

The Rho-associated kinases ROCK1 and ROCK2 are critical for cancer cell migration and invasion, suggesting they may be useful therapeutic targets. In this study, we describe the discovery and development of RKI-1447, a potent small molecule inhibitor of ROCK1 and ROCK2. Crystal structures of the RKI-1447/ROCK1 complex revealed that RKI-1447 is a Type I kinase inhibitor that binds the ATP binding site through interactions with the hinge region and the DFG motif. RKI-1447 suppressed phosphorylation of the ROCK substrates MLC-2 and MYPT-1 in human cancer cells, but had no effect on the phosphorylation levels of the AKT, MEK, and S6 kinase at concentrations as high as 10 μmol/L. RKI-1447 was also highly selective at inhibiting ROCK-mediated cytoskeleton re-organization (actin stress fiber formation) following LPA stimulation, but does not affect PAK-meditated lamellipodia and filopodia formation following PDGF and Bradykinin stimulation, respectively. RKI-1447 inhibited migration, invasion and anchorage-independent tumor growth of breast cancer cells. In contrast, RKI-1313, a much weaker analog in vitro, had little effect on the phosphorylation levels of ROCK substrates, migration, invasion or anchorage-independent growth. Finally, RKI-1447 was highly effective at inhibiting the outgrowth of mammary tumors in a transgenic mouse model. In summary, our findings establish RKI-1447 as a potent and selective ROCK inhibitor with significant anti-invasive and antitumor activities and offer a preclinical proof-of-concept that justify further examination of RKI-1447 suitability as a potential clinical candidate.
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http://dx.doi.org/10.1158/0008-5472.CAN-12-0954DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3463757PMC
October 2012

Development of o-chlorophenyl substituted pyrimidines as exceptionally potent aurora kinase inhibitors.

J Med Chem 2012 Sep 30;55(17):7392-7416. Epub 2012 Aug 30.

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

The o-carboxylic acid substituted bisanilinopyrimidine 1 was identified as a potent hit (Aurora A IC(50) = 6.1 ± 1.0 nM) from in-house screening. Detailed structure-activity relationship (SAR) studies indicated that polar substituents at the para position of the B-ring are critical for potent activity. X-ray crystallography studies revealed that compound 1 is a type I inhibitor that binds the Aurora kinase active site in a DFG-in conformation. Structure-activity guided replacement of the A-ring carboxylic acid with halogens and incorporation of fluorine at the pyrimidine 5-position led to highly potent inhibitors of Aurora A that bind in a DFG-out conformation. B-Ring modifications were undertaken to improve the solubility and cell permeability. Compounds such as 9m with water-solubilizing moieties at the para position of the B-ring inhibited the autophosphorylation of Aurora A in MDA-MB-468 breast cancer cells.
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http://dx.doi.org/10.1021/jm300334dDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4429609PMC
September 2012

HG-829 is a potent noncompetitive inhibitor of the ATP-binding cassette multidrug resistance transporter ABCB1.

Cancer Res 2012 Aug 3;72(16):4204-13. Epub 2012 Jul 3.

Experimental Therapeutics, Moffitt Cancer Center, University of South Florida, Tampa, Florida 33612, USA.

Transmembrane drug export mediated by the ATP-binding cassette (ABC) transporter P-glycoprotein contributes to clinical resistance to antineoplastics. In this study, we identified the substituted quinoline HG-829 as a novel, noncompetitive, and potent P-glycoprotein inhibitor that overcomes in vitro and in vivo drug resistance. We found that nontoxic concentrations of HG-829 restored sensitivity to P-glycoprotein oncolytic substrates. In ABCB1-overexpressing cell lines, HG-829 significantly enhanced cytotoxicity to daunorubicin, paclitaxel, vinblastine, vincristine, and etoposide. Coadministration of HG-829 fully restored in vivo antitumor activity of daunorubicin in mice without added toxicity. Functional assays showed that HG-829 is not a Pgp substrate or competitive inhibitor of Pgp-mediated drug efflux but rather acts as a noncompetitive modulator of P-glycoprotein transport function. Taken together, our findings indicate that HG-829 is a potent, long-acting, and noncompetitive modulator of P-glycoprotein export function that may offer therapeutic promise for multidrug-resistant malignancies.
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http://dx.doi.org/10.1158/0008-5472.CAN-12-0743DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4557794PMC
August 2012

Ack1-mediated androgen receptor phosphorylation modulates radiation resistance in castration-resistant prostate cancer.

J Biol Chem 2012 Jun 7;287(26):22112-22. Epub 2012 May 7.

Department of Drug Discovery, Moffitt Cancer Center, Tampa, Florida 33612, USA.

Androgen deprivation therapy has been the standard of care in prostate cancer due to its effectiveness in initial stages. However, the disease recurs, and this recurrent cancer is referred to as castration-resistant prostate cancer (CRPC). Radiotherapy is the treatment of choice; however, in addition to androgen independence, CRPC is often resistant to radiotherapy, making radioresistant CRPC an incurable disease. The molecular mechanisms by which CRPC cells acquire radioresistance are unclear. Androgen receptor (AR)-tyrosine 267 phosphorylation by Ack1 tyrosine kinase (also known as TNK2) has emerged as an important mechanism of CRPC growth. Here, we demonstrate that pTyr(267)-AR is recruited to the ATM (ataxia telangiectasia mutated) enhancer in an Ack1-dependent manner to up-regulate ATM expression. Mice engineered to express activated Ack1 exhibited a significant increase in pTyr(267)-AR and ATM levels. Furthermore, primary human CRPCs with up-regulated activated Ack1 and pTyr(267)-AR also exhibited significant increase in ATM expression. The Ack1 inhibitor AIM-100 not only inhibited Ack1 activity but also was able to suppress AR Tyr(267) phosphorylation and its recruitment to the ATM enhancer. Notably, AIM-100 suppressed Ack1 mediated ATM expression and mitigated the growth of radioresistant CRPC tumors. Thus, our study uncovers a previously unknown mechanism of radioresistance in CRPC, which can be therapeutically reversed by a new synergistic approach that includes radiotherapy along with the suppression of Ack1/AR/ATM signaling by the Ack1 inhibitor, AIM-100.
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http://dx.doi.org/10.1074/jbc.M112.357384DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3381169PMC
June 2012

Ack1 tyrosine kinase activation correlates with pancreatic cancer progression.

Am J Pathol 2012 Apr 7;180(4):1386-93. Epub 2012 Feb 7.

Department of Drug Discovery, Moffitt Cancer Center, Tampa, Florida 33612, USA.

Pancreatic cancer is a significant cause of cancer mortality worldwide as the disease has advanced significantly in patients before symptoms are evident. The signal transduction pathways that promote this rapid progression are not well understood. Ack1 or TNK2, an ubiquitously expressed oncogenic non-receptor tyrosine kinase, integrates signals from ligand-activated receptor tyrosine kinases to modulate intracellular signaling cascades. In the present study, we investigated the Ack1 activation profile in a pancreatic cancer tumor microarray, and observed that expression levels of activated Ack1 and pTyr284-Ack1 positively correlated with the severity of disease progression and inversely correlated with the survival of patients with pancreatic cancer. To explore the mechanisms by which Ack1 promotes tumor progression, we investigated the role of AKT/PKB, an oncogene and Ack1-interacting protein. Ack1 activates AKT directly in pancreatic and other cancer cell lines by phosphorylating AKT at Tyr176 to promote cell survival. In addition, the Ack1 inhibitor AIM-100 not only inhibited Ack1 activation but also suppressed AKT tyrosine phosphorylation, leading to cell cycle arrest in the G1 phase. This effect resulted in a significant decrease in the proliferation of pancreatic cancer cells and induction of apoptosis. Collectively, our data indicate that activated Ack1 could be a prognostic marker for ascertaining early or advanced pancreatic cancer. Thus, Ack1 inhibitors hold promise for therapeutic intervention to inhibit pancreatic tumor growth.
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http://dx.doi.org/10.1016/j.ajpath.2011.12.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3349895PMC
April 2012

Enhanced anti-melanoma efficacy of interferon alfa-2b via inhibition of Shp2.

Cancer Lett 2012 Jul 1;320(1):81-5. Epub 2012 Feb 1.

Department of Molecular Oncology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL 33612, United States.

Interferon-α2b (IFN-α2b) is used to treat melanoma but there is a need to improve its efficacy. IFN-α2b signaling requires STAT1/STAT2 tyrosine phosphorylation and is subject to negative regulation by phosphatases. In this study, we determined whether inhibition of the protein tyrosine phosphatase Shp2 could enhance IFN-α2b responses in human melanoma cells. Shp2 knockdown increased IFN-α2b-stimulated STAT1 Tyr-701 phosphorylation and ISRE-luciferase activity even though it did not affect STAT2 Tyr-690 phosphorylation in A375 cells. In A375 tumor xenografts, Shp2 knockdown enhanced the anti-melanoma effect of IFN-α2b. Furthermore, the Shp2 inhibitor SPI-112Me increased the IFN-α2b-induced STAT1 activation and anti-proliferative response in A375 and SK-MEL-2 cells. These results demonstrate that inhibition of Shp2 can enhance the anti-melanoma activity of IFN-α2b.
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http://dx.doi.org/10.1016/j.canlet.2012.01.034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3319810PMC
July 2012

Fragment-based and structure-guided discovery and optimization of Rho kinase inhibitors.

J Med Chem 2012 Mar 15;55(5):2474-8. Epub 2012 Feb 15.

Department of Drug Discovery, H. Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, Tampa, Florida 33612, USA.

Using high concentration biochemical assays and fragment-based screening assisted by structure-guided design, we discovered a novel class of Rho-kinase inhibitors. Compound 18 was equipotent for ROCK1 (IC(50) = 650 nM) and ROCK2 (IC(50) = 670 nM), whereas compound 24 was more selective for ROCK2 (IC(50) = 100 nM) over ROCK1 (IC(50) = 1690 nM). The crystal structure of the compound 18-ROCK1 complex revealed that 18 is a type 1 inhibitor that binds the hinge region in the ATP binding site. Compounds 18 and 24 inhibited potently the phosphorylation of the ROCK substrate MLC2 in intact human breast cancer cells.
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http://dx.doi.org/10.1021/jm201289rDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4516226PMC
March 2012

A novel mechanism by which small molecule inhibitors induce the DFG flip in Aurora A.

ACS Chem Biol 2012 Apr 27;7(4):698-706. Epub 2012 Jan 27.

Drug Discovery Department, Moffitt Cancer Center and Research Institute, Tampa, Florida 33612, United States.

Most protein kinases share a DFG (Asp-Phe-Gly) motif in the ATP site that can assume two distinct conformations, the active DFG-in and the inactive DFG-out states. Small molecule inhibitors able to induce the DFG-out state have received considerable attention in kinase drug discovery. Using a typical DFG-in inhibitor scaffold of Aurora A, a kinase involved in the regulation of cell division, we found that halogen and nitrile substituents directed at the N-terminally flanking residue Ala273 induced global conformational changes in the enzyme, leading to DFG-out inhibitors that are among the most potent Aurora A inhibitors reported to date. The data suggest an unprecedented mechanism of action, in which induced-dipole forces along the Ala273 side chain alter the charge distribution of the DFG backbone, allowing the DFG to unwind. As the ADFG sequence and three-dimensional structure is highly conserved, DFG-out inhibitors of other kinases may be designed by specifically targeting the flanking alanine residue with electric dipoles.
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http://dx.doi.org/10.1021/cb200508bDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4429595PMC
April 2012