ABSTRACT
Bruton tyrosine kinase (BTK), a nonreceptor tyrosine kinase, is a major therapeutic target for B-cell-driven malignancies. However, approved covalent BTK inhibitors (cBTKis) are associated with treatment limitations because of off-target side effects, suboptimal oral pharmacology, and development of resistance mutations (eg, C481) that prevent inhibitor binding. Here, we describe the preclinical profile of pirtobrutinib, a potent, highly selective, noncovalent (reversible) BTK inhibitor. Pirtobrutinib binds BTK with an extensive network of interactions to BTK and water molecules in the adenosine triphosphate binding region and shows no direct interaction with C481. Consequently, pirtobrutinib inhibits both BTK and BTK C481 substitution mutants in enzymatic and cell-based assays with similar potencies. In differential scanning fluorimetry studies, BTK bound to pirtobrutinib exhibited a higher melting temperature than cBTKi-bound BTK. Pirtobrutinib, but not cBTKis, prevented Y551 phosphorylation in the activation loop. These data suggest that pirtobrutinib uniquely stabilizes BTK in a closed, inactive conformation. Pirtobrutinib inhibits BTK signaling and cell proliferation in multiple B-cell lymphoma cell lines, and significantly inhibits tumor growth in human lymphoma xenografts in vivo. Enzymatic profiling showed that pirtobrutinib was highly selective for BTK in >98% of the human kinome, and in follow-up cellular studies pirtobrutinib retained >100-fold selectivity over other tested kinases. Collectively, these findings suggest that pirtobrutinib represents a novel BTK inhibitor with improved selectivity and unique pharmacologic, biophysical, and structural attributes with the potential to treat B-cell-driven cancers with improved precision and tolerability. Pirtobrutinib is being tested in phase 3 clinical studies for a variety of B-cell malignancies.
Subject(s)
Agammaglobulinaemia Tyrosine Kinase , Lymphoma , Agammaglobulinaemia Tyrosine Kinase/antagonists & inhibitors , Humans , Animals , Xenograft Model Antitumor Assays , Lymphoma/drug therapy , Drug Evaluation, Preclinical , Cell Line, Tumor , Mice, Inbred NOD , Male , Mice, SCID , Molecular Conformation , MiceABSTRACT
The efficacy of the highly selective RET inhibitor selpercatinib is now established in RET-driven cancers, and we sought to characterize the molecular determinants of response and resistance. We find that the pre-treatment genomic landscape does not shape the variability of treatment response except for rare instances of RAS-mediated primary resistance. By contrast, acquired selpercatinib resistance is driven by MAPK pathway reactivation by one of two distinct routes. In some patients, on- and off-target pathway reactivation via secondary RET solvent front mutations or MET amplifications are evident. In other patients, rare RET-wildtype tumor cell populations driven by an alternative mitogenic driver are selected for by treatment. Multiple distinct mechanisms are often observed in the same patient, suggesting polyclonal resistance may be common. Consequently, sequential RET-directed therapy may require combination treatment with inhibitors targeting alternative MAPK effectors, emphasizing the need for prospective characterization of selpercatinib-treated tumors at the time of monotherapy progression.
Subject(s)
Lung Neoplasms , Thyroid Neoplasms , Humans , Lung , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Mutation , Prospective Studies , Proto-Oncogene Proteins c-ret/genetics , Thyroid Neoplasms/drug therapy , Thyroid Neoplasms/geneticsABSTRACT
Patients with melanoma receiving drugs targeting BRAFV600E and mitogen-activated protein (MAP) kinase kinases 1 and 2 (MEK1/2) invariably develop resistance and face continued progression. Based on preclinical studies, intermittent treatment involving alternating periods of drug withdrawal and rechallenge has been proposed as a method to delay the onset of resistance. The beneficial effect of intermittent treatment has been attributed to drug addiction, where drug withdrawal reduces the viability of resistant cells due to MAP kinase pathway hyperactivation. However, the mechanistic basis of the intermittent effect is incompletely understood. We show that intermittent treatment with the BRAFV600E inhibitor, LGX818/encorafenib, suppresses growth compared with continuous treatment in human melanoma cells engineered to express BRAFV600E, p61-BRAFV600E, or MEK2C125 oncogenes. Analysis of the BRAFV600E-overexpressing cells shows that, while drug addiction clearly occurs, it fails to account for the advantageous effect of intermittent treatment. Instead, growth suppression is best explained by resensitization during periods of drug removal, followed by cell death after drug readdition. Continuous treatment leads to transcriptional responses prominently associated with chemoresistance in melanoma. By contrast, cells treated intermittently reveal a subset of transcripts that reverse expression between successive cycles of drug removal and rechallenge and include mediators of cell invasiveness and the epithelial-to-mesenchymal transition. These transcripts change during periods of drug removal by adaptive switching, rather than selection pressure. Resensitization occurs against a background of sustained expression of melanoma resistance genes, producing a transcriptome distinct from that of the initial drug-naive cell state. We conclude that phenotypic plasticity leading to drug resensitization can underlie the beneficial effect of intermittent treatment.
Subject(s)
Melanoma , Proto-Oncogene Proteins B-raf , Cell Line, Tumor , Drug Resistance, Neoplasm/genetics , Humans , MAP Kinase Signaling System , Melanoma/drug therapy , Melanoma/genetics , Melanoma/pathology , Mitogen-Activated Protein Kinases/metabolism , Mutation , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins B-raf/metabolismABSTRACT
INTRODUCTION: Novel rearranged in transfection (RET)-specific tyrosine kinase inhibitors (TKIs) such as selpercatinib (LOXO-292) have shown unprecedented efficacy in tumors positive for RET fusions or mutations, notably RET fusion-positive NSCLC and RET-mutated medullary thyroid cancer (MTC). However, the mechanisms of resistance to these agents have not yet been described. METHODS: Analysis was performed of circulating tumor DNA and tissue in patients with RET fusion-positive NSCLC and RET-mutation positive MTC who developed disease progression after an initial response to selpercatinib. Acquired resistance was modeled preclinically using a CCDC6-RET fusion-positive NSCLC patient-derived xenograft. The inhibitory activity of anti-RET multikinase inhibitors and selective RET TKIs was evaluated in enzyme and cell-based assays. RESULTS: After a dramatic initial response to selpercatinib in a patient with KIF5B-RET NSCLC, analysis of circulating tumor DNA revealed emergence of RET G810R, G810S, and G810C mutations in the RET solvent front before the emergence of clinical resistance. Postmortem biopsy studies reported intratumor and intertumor heterogeneity with distinct disease subclones containing G810S, G810R, and G810C mutations in multiple disease sites indicative of convergent evolution on the G810 residue resulting in a common mechanism of resistance. Acquired mutations in RET G810 were identified in tumor tissue from a second patient with CCDC6-RET fusion-positive NSCLC and in plasma from patients with additional RET fusion-positive NSCLC and RET-mutant MTC progressing on an ongoing phase 1 and 2 trial of selpercatinib. Preclinical studies reported the presence of RET G810R mutations in a CCDC6-RET patient-derived xenograft (from a patient with NSCLC) model of acquired resistance to selpercatinib. Structural modeling predicted that these mutations sterically hinder the binding of selpercatinib, and in vitro assays confirmed loss of activity for both anti-RET multikinase inhibitors and selective RET TKIs. CONCLUSIONS: RET G810 solvent front mutations represent the first described recurrent mechanism of resistance to selective RET inhibition with selpercatinib. Development of potent inhibitor of these mutations and maintaining activity against RET gatekeeper mutations could be an effective strategy to target resistance to selective RET inhibitors.
Subject(s)
Lung Neoplasms , Proto-Oncogene Proteins c-ret , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Mutation , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Proto-Oncogene Proteins c-ret/genetics , Pyrazoles , Pyridines , Solvents , TransfectionABSTRACT
KRAS is the most frequently mutated driver oncogene in human cancer, and KRAS mutations are commonly associated with poor prognosis and resistance to standard treatment. The ability to effectively target and block the function of mutated KRAS has remained elusive despite decades of research. Recent findings have demonstrated that directly targeting KRAS-G12C with electrophilic small molecules that covalently modify the mutated codon 12 cysteine is feasible. We have discovered a series of tetrahydropyridopyrimidines as irreversible covalent inhibitors of KRAS-G12C with in vivo activity. The PK/PD and efficacy of compound 13 will be highlighted.
ABSTRACT
Larotrectinib, a selective TRK tyrosine kinase inhibitor (TKI), has demonstrated histology-agnostic efficacy in patients with TRK fusion-positive cancers. Although responses to TRK inhibition can be dramatic and durable, duration of response may eventually be limited by acquired resistance. LOXO-195 is a selective TRK TKI designed to overcome acquired resistance mediated by recurrent kinase domain (solvent front and xDFG) mutations identified in multiple patients who have developed resistance to TRK TKIs. Activity against these acquired mutations was confirmed in enzyme and cell-based assays and in vivo tumor models. As clinical proof of concept, the first 2 patients with TRK fusion-positive cancers who developed acquired resistance mutations on larotrectinib were treated with LOXO-195 on a first-in-human basis, utilizing rapid dose titration guided by pharmacokinetic assessments. This approach led to rapid tumor responses and extended the overall duration of disease control achieved with TRK inhibition in both patients.Significance: LOXO-195 abrogated resistance in TRK fusion-positive cancers that acquired kinase domain mutations, a shared liability with all existing TRK TKIs. This establishes a role for sequential treatment by demonstrating continued TRK dependence and validates a paradigm for the accelerated development of next-generation inhibitors against validated oncogenic targets. Cancer Discov; 7(9); 963-72. ©2017 AACR.See related commentary by Parikh and Corcoran, p. 934This article is highlighted in the In This Issue feature, p. 920.
Subject(s)
Antineoplastic Agents/therapeutic use , Drug Resistance, Neoplasm/drug effects , Neoplasms/drug therapy , Protein Kinase Inhibitors/therapeutic use , Receptor, trkA/antagonists & inhibitors , Animals , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Female , Humans , Mice , Mice, Nude , NIH 3T3 Cells , Neoplasms/genetics , Neoplasms/metabolism , Protein Kinase Inhibitors/pharmacokinetics , Protein Kinase Inhibitors/pharmacology , Receptor, trkA/genetics , Receptor, trkA/metabolismABSTRACT
Numerous oncogenic mutations occur within the BRAF kinase domain (BRAF(KD)). Here we show that stable BRAF-MEK1 complexes are enriched in BRAF(WT) and KRAS mutant (MT) cells but not in BRAF(MT) cells. The crystal structure of the BRAF(KD) in a complex with MEK1 reveals a face-to-face dimer sensitive to MEK1 phosphorylation but insensitive to BRAF dimerization. Structure-guided studies reveal that oncogenic BRAF mutations function by bypassing the requirement for BRAF dimerization for activity or weakening the interaction with MEK1. Finally, we show that conformation-specific BRAF inhibitors can sequester a dormant BRAF-MEK1 complex resulting in pathway inhibition. Taken together, these findings reveal a regulatory role for BRAF in the MAPK pathway independent of its kinase activity but dependent on interaction with MEK.
Subject(s)
MAP Kinase Kinase 1/chemistry , Proto-Oncogene Proteins B-raf/chemistry , Catalytic Domain , Crystallography, X-Ray , HCT116 Cells , HEK293 Cells , Humans , MAP Kinase Kinase 1/genetics , MAP Kinase Kinase 1/metabolism , Models, Molecular , Mutation, Missense , Point Mutation , Protein Structure, Quaternary , Protein Structure, Secondary , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins B-raf/metabolism , Proto-Oncogene Proteins p21(ras) , Signal Transduction , ras Proteins/geneticsABSTRACT
The protein kinase v-akt murine thymoma viral oncogene homolog (AKT), a key regulator of cell survival and proliferation, is frequently hyperactivated in human cancers. Intramolecular pleckstrin homology (PH) domain-kinase domain (KD) interactions are important in maintaining AKT in an inactive state. AKT activation proceeds after a conformational change that dislodges the PH from the KD. To understand these autoinhibitory interactions, we generated mutations at the PH-KD interface and found that most of them lead to constitutive activation of AKT. Such mutations are likely another mechanism by which activation may occur in human cancers and other diseases. In support of this likelihood, we found somatic mutations in AKT1 at the PH-KD interface that have not been previously described in human cancers. Furthermore, we show that the AKT1 somatic mutants are constitutively active, leading to oncogenic signaling. Additionally, our studies show that the AKT1 mutants are not effectively inhibited by allosteric AKT inhibitors, consistent with the requirement for an intact PH-KD interface for allosteric inhibition. These results have important implications for therapeutic intervention in patients with AKT mutations at the PH-KD interface.
Subject(s)
Neoplasms/enzymology , Neoplasms/genetics , Oncogenes/genetics , Proto-Oncogene Proteins c-akt/chemistry , Proto-Oncogene Proteins c-akt/genetics , Allosteric Regulation/drug effects , Allosteric Regulation/genetics , Animals , Cell Line, Tumor , Cell Membrane/drug effects , Cell Membrane/enzymology , Cell Transformation, Neoplastic/drug effects , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/pathology , Enzyme Activation/drug effects , Humans , Mice , Models, Molecular , Mutant Proteins/metabolism , Mutation/genetics , NIH 3T3 Cells , Protein Binding/drug effects , Protein Binding/genetics , Protein Kinase Inhibitors/pharmacology , Protein Transport/drug effects , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction/drug effects , Signal Transduction/geneticsABSTRACT
The discovery and optimization of a series of 6,7-dihydro-5H-cyclopenta[d]pyrimidine compounds that are ATP-competitive, selective inhibitors of protein kinase B/Akt is reported. The initial design and optimization was guided by the use of X-ray structures of inhibitors in complex with Akt1 and the closely related protein kinase A. The resulting compounds demonstrate potent inhibition of all three Akt isoforms in biochemical assays and poor inhibition of other members of the cAMP-dependent protein kinase/protein kinase G/protein kinase C extended family and block the phosphorylation of multiple downstream targets of Akt in human cancer cell lines. Biological studies with one such compound, 28 (GDC-0068), demonstrate good oral exposure resulting in dose-dependent pharmacodynamic effects on downstream biomarkers and a robust antitumor response in xenograft models in which the phosphatidylinositol 3-kinase-Akt-mammalian target of rapamycin pathway is activated. 28 is currently being evaluated in human clinical trials for the treatment of cancer.
Subject(s)
Adenosine Triphosphate/metabolism , Binding, Competitive , Drug Discovery , Piperazines/metabolism , Piperazines/pharmacology , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/metabolism , Pyrimidines/metabolism , Pyrimidines/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/metabolism , Antineoplastic Agents/pharmacology , Humans , Inhibitory Concentration 50 , Models, Molecular , Piperazines/chemistry , Protein Conformation , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/metabolism , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-akt/chemistry , Pyrimidines/chemistry , Substrate SpecificityABSTRACT
The protein serine-threonine kinase Akt undergoes a substantial conformational change upon activation, which is induced by the phosphorylation of two critical regulatory residues, threonine 308 and serine 473. Paradoxically, treating cells with adenosine 5'-triphosphate (ATP)-competitive inhibitors of Akt results in increased phosphorylation of both residues. We show that binding of ATP-competitive inhibitors stabilized a conformation in which both phosphorylated sites were inaccessible to phosphatases. ATP binding also produced this protection of the phosphorylated sites, whereas interaction with its hydrolysis product adenosine 5'-diphosphate (ADP) or allosteric Akt inhibitors resulted in increased accessibility of these phosphorylated residues. ATP-competitive inhibitors mimicked ATP by targeting active Akt. Forms of Akt activated by an oncogenic mutation or myristoylation were more potently inhibited by the ATP-competitive inhibitors than was wild-type Akt. These data support a new model of kinase regulation, wherein nucleotides modulate an on-off switch in Akt through conformational changes, which is disrupted by ATP-competitive inhibitors.
Subject(s)
Adenosine Triphosphate/metabolism , Phosphoric Monoester Hydrolases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Adenosine Diphosphate/metabolism , Allosteric Regulation , Binding Sites , Models, Molecular , PhosphorylationABSTRACT
The synthesis and biological evaluation of non-oxime pyrazole based B-Raf inhibitors is reported. Several oxime replacements have been prepared and have shown excellent enzyme activity. Further optimization of fused pyrazole 2a led to compound 38, a selective and potent B-Raf inhibitor.
Subject(s)
Enzyme Inhibitors/pharmacology , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Pyrazoles/chemical synthesis , Pyrazoles/pharmacology , Animals , Crystallography, X-Ray , Dose-Response Relationship, Drug , Enzyme Activation/drug effects , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Inhibitory Concentration 50 , Mice , Molecular Structure , Oximes/chemistry , Pyrazoles/chemistryABSTRACT
A novel series of spirochromane pan-Akt inhibitors is reported. SAR optimization furnished compounds with improved enzyme potencies and excellent selectivity over the related AGC kinase PKA. Attempted replacement of the phenol hinge binder provided compounds with excellent Akt enzyme and cell activities but greatly diminished selectivity over PKA.
Subject(s)
Protein Kinase Inhibitors/chemistry , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Binding Sites , Crystallography, X-Ray , Drug Evaluation, Preclinical , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-akt/metabolism , Structure-Activity RelationshipABSTRACT
We describe the design and synthesis of novel bicyclic spiro sulfonamides as potent Akt inhibitors. Through structure-based rational design, we have successfully improved PKA selectivity of previously disclosed spirochromanes. Representative compounds showed favorable Akt potency while exhibiting up to 1000-fold selectivity against PKA.
Subject(s)
Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Protein Kinase Inhibitors/chemistry , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Spiro Compounds/chemistry , Sulfonamides/chemistry , Binding Sites , Computer Simulation , Crystallography, X-Ray , Cyclic AMP-Dependent Protein Kinases/metabolism , Drug Evaluation, Preclinical , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-akt/metabolism , Structure-Activity Relationship , Sulfonamides/chemical synthesis , Sulfonamides/pharmacologyABSTRACT
The development of inhibitors of B-Raf(V600E) serine-threonine kinase is described. Various head-groups were examined to optimize inhibitor activity and ADME properties. Several of the head-groups explored, including naphthol, phenol and hydroxyamidine, possessed good activity but had poor pharmacokinetic exposure in mice. Exposure was improved by incorporating more metabolically stable groups such as indazole and tricyclic pyrazole, while indazole could also be optimized for good cellular activity.
Subject(s)
Protein Kinase Inhibitors/chemistry , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Amino Acid Substitution , Animals , Binding Sites , Cell Line, Tumor , Crystallography, X-Ray , Humans , Indazoles/chemistry , Mice , Microsomes, Liver/metabolism , Mutation , Oximes/chemistry , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacokinetics , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins B-raf/metabolism , Pyrazoles/chemistry , Structure-Activity RelationshipABSTRACT
Virtual and high-throughput screening identified imidazo[1,2-a]pyrazines as inhibitors of B-Raf. We describe the rationale, SAR, and evolution of the initial hits to a series of furo[2,3-c]pyridine indanone oximes as highly potent and selective inhibitors of B-Raf.
Subject(s)
Indans/chemistry , Oximes/chemistry , Protein Kinase Inhibitors/chemistry , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Pyridines/chemistry , Animals , Binding Sites , Cell Line, Tumor , Computer Simulation , Drug Evaluation, Preclinical , Humans , Indans/chemical synthesis , Indans/pharmacokinetics , Microsomes, Liver/metabolism , Models, Chemical , Models, Molecular , Oximes/chemical synthesis , Oximes/pharmacokinetics , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacokinetics , Proto-Oncogene Proteins B-raf/metabolism , Rats , Structure-Activity RelationshipABSTRACT
AKT1 (NP_005154.2) is a member of the serine/threonine AGC protein kinase family involved in cellular metabolism, growth, proliferation and survival. The three human AKT isozymes are highly homologous multi-domain proteins with both overlapping and distinct cellular functions. Dysregulation of the AKT pathway has been identified in multiple human cancers. Several clinical trials are in progress to test the efficacy of AKT pathway inhibitors in treating cancer. Recently, a series of AKT isozyme-selective allosteric inhibitors have been reported. They require the presence of both the pleckstrin-homology (PH) and kinase domains of AKT, but their binding mode has not yet been elucidated. We present here a 2.7 Å resolution co-crystal structure of human AKT1 containing both the PH and kinase domains with a selective allosteric inhibitor bound in the interface. The structure reveals the interactions between the PH and kinase domains, as well as the critical amino residues that mediate binding of the inhibitor to AKT1. Our work also reveals an intricate balance in the enzymatic regulation of AKT, where the PH domain appears to lock the kinase in an inactive conformation and the kinase domain disrupts the phospholipid binding site of the PH domain. This information advances our knowledge in AKT1 structure and regulation, thereby providing a structural foundation for interpreting the effects of different classes of AKT inhibitors and designing selective ones.
Subject(s)
Down-Regulation , Enzyme Inhibitors/chemistry , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/chemistry , Allosteric Regulation , Amino Acid Sequence , Binding Sites , Crystallography, X-Ray , Enzyme Inhibitors/metabolism , Humans , Molecular Conformation , Molecular Sequence Data , Protein Binding , Protein Structure, Tertiary , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolismABSTRACT
Herein we report the discovery and synthesis of a novel series of dihydrothieno- and dihydrofuropyrimidines (2 and 3) as potent pan Akt inhibitors. Utilizing previous SAR and analysis of the amino acid sequences in the binding site we have designed inhibitors displaying increased PKA and general kinase selectivity with improved tolerability compared to the progenitor pyrrolopyrimidine (1). A representative dihydrothieno compound (34) was advanced into a PC3-NCI prostate mouse tumor model in which it demonstrated a dose-dependent reduction in tumor growth and stasis when dosed orally daily at 200 mg/kg.
Subject(s)
Prostatic Neoplasms/drug therapy , Protein Kinase Inhibitors/chemistry , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Pyrimidines/chemistry , Animals , Binding Sites , Disease Models, Animal , Dose-Response Relationship, Drug , Male , Mice , Protein Kinase Inhibitors/pharmacology , Pyrimidines/pharmacology , Structure-Activity Relationship , Tumor Burden/drug effectsABSTRACT
The discovery and optimization of a series of pyrrolopyrimidine based protein kinase B (Pkb/Akt) inhibitors discovered via HTS and structure based drug design is reported. The compounds demonstrate potent inhibition of all three Akt isoforms and knockdown of phospho-PRAS40 levels in LNCaP cells and tumor xenografts.