Radiotherapy-induced Immune Response Enhanced by Selective HDAC6 Inhibition.

Radiotherapy is a curative cancer treatment modality that imparts damage to cellular DNA, induces immunogenic cell death, and activates antitumor immunity. Despite the radiotherapy-induced direct antitumor effect seen within the treated volume, accumulating evidence indicates activation of innate antitumor immunity. Acute proinflammatory responses mediated by anticancer M1 macrophages are observed in the immediate aftermath following radiotherapy. However, after a few days, these M1 macrophages are converted to anti-inflammatory and pro-cancer M2 phenotype, leading to cancer resistance and underlying potential tumor relapse. Histone deacetylase 6 (HDAC6) plays a crucial role in regulating macrophage polarization and innate immune responses. Here, we report targeting HDAC6 function with a novel selective inhibitor (SP-2-225) as a potential therapeutic candidate for combination therapy with radiotherapy. This resulted in decreased tumor growth and enhanced M1/M2 ratio of infiltrating macrophages within tumors. These observations support the use of selective HDAC6 inhibitors to improve antitumor immune responses and prevent tumor relapse after radiotherapy.

Radiotherapy Induces Innate Immune Responses in Patients Treated for Prostate Cancers.

PURPOSE: Radiotherapy is a curative therapeutic modality used to treat cancers as a single agent or in combination with surgery and chemotherapy. Advanced radiotherapy technologies enable treatment with large fractions and highly conformal radiation doses to effect free-radical damage to cellular DNA leading to cell-cycle arrest, cell death, and innate immune response (IIR) stimulation. EXPERIMENTAL DESIGN: To understand systemic clinical responses after radiation exposure, proteomic and metabolomic analyses were performed on plasma obtained from patients with cancer at intervals after prostate stereotactic body radiotherapy. Pathway and multivariate analyses were used to delineate molecular alterations following radiotherapy and its correlation with clinical outcomes. RESULTS: DNA damage response increased within the first hour after treatment and returned to baseline by 1 month. IIR signaling also increased within 1 hour of treatment but persisted for up to 3 months thereafter. Furthermore, robust IIR and metabolite elevations, consistent with an early proinflammatory M1-mediated innate immune activation, were observed in patients in remission, whereas patients experiencing prostate serum antigen-determined disease progression demonstrated less robust immune responses and M2-mediated metabolite elevations. CONCLUSIONS: To our knowledge, these data are the first report of longitudinal proteomic and metabolomic molecular responses in patients after radiotherapy for cancers. The data supports innate immune activation as a critical clinical response of patients receiving radiotherapy for prostate cancer. Furthermore, we propose that the observed IIR may be generalized to the treatment of other cancer types, potentially informing multidisciplinary therapeutic strategies for cancer treatment.

Novel Paired Normal Prostate and Prostate Cancer Model Cell Systems Derived from African American Patients.

Prostate cancer is the most frequently diagnosed solid malignancy in men. African American (AA) men are at greater risk for developing prostate cancer, and experience higher mortality rates, as compared with Caucasian American men. However, mechanistic studies to understand this health disparity have been limited by the lack of relevant in vitro and in vivo models. There is an urgent need for preclinical cellular models to investigate molecular mechanisms underlying prostate cancer in AA men. We collected clinical specimens from radical prostatectomies of AA patients and established 10 paired tumor-derived and normal epithelial cell cultures from the same donors, which were further cultivated to extend the growth under "conditional reprogramming." Clinical and cellular annotations characterized these model cells as intermediate risk and predominantly diploid. Immunocytochemical analyses demonstrated variable expression levels of luminal (CK8) and basal (CK5, p63) markers in both normal and tumor cells. However, expression levels of TOPK, c-MYC, and N-MYC were markedly increased only in tumor cells. To determine cell utility for drug testing, we examined viability of cells following exposure to the antiandrogen (bicalutamide) and two PARP inhibitors (olaparib and niraparib) and observed decreased viability of tumor-derived cells as compared with viability of normal prostate-derived cells. Significance: Cells derived from prostatectomies of AA patients conferred a bimodal cellular phenotype, recapitulating clinical prostate cellular complexity in this model cell system. Comparisons of viability responses of tumor derived to normal epithelial cells offer the potential for screening therapeutic drugs. Therefore, these paired prostate epithelial cell cultures provide an in vitro model system suitable for studies of molecular mechanisms in health disparities.

18Oxygen Substituted Nucleosides Combined with Proton Beam Therapy: Therapeutic Transmutation In Vitro.

PURPOSE: Proton therapy precisely delivers radiation to cancers to cause damaging strand breaks to cellular DNA, kill malignant cells, and stop tumor growth. Therapeutic protons also generate short-lived activated nuclei of carbon, oxygen, and nitrogen atoms in patients as a result of atomic transmutations that are imaged by positron emission tomography (PET). We hypothesized that the transition of 18O to 18F in an 18O-substituted nucleoside irradiated with therapeutic protons may result in the potential for combined diagnosis and treatment for cancer with proton therapy. MATERIALS AND METHODS: Reported here is a feasibility study with a therapeutic proton beam used to irradiate H2 18O to a dose of 10 Gy produced by an 85 MeV pristine Bragg peak. PET imaging initiated >45 minutes later showed an 18F decay signal with T1/2 of ∼111 minutes. RESULTS: The 18O to 18F transmutation effect on cell survival was tested by exposing SQ20B squamous carcinoma cells to physiologic 18O-thymidine concentrations of 5 µM for 48 hours followed by 1- to 9-Gy graded doses of proton radiation given 24 hours later. Survival analyses show radiation sensitization with a dose modification factor (DMF) of 1.2. CONCLUSIONS: These data support the idea of therapeutic transmutation in vitro as a biochemical consequence of proton activation of 18O to 18F in substituted thymidine enabling proton radiation enhancement in a cancer cell. 18O-substituted molecules that incorporate into cancer targets may hold promise for improving the therapeutic window of protons and can be evaluated further for postproton therapy PET imaging.

Discovery of Metabolic Biomarkers Predicting Radiation Therapy Late Effects in Prostate Cancer Patients.

Patients presenting with prostate cancers undergo clinical staging evaluations to determine the extent of disease to guide therapeutic recommendations. Management options may include watchful waiting, surgery, or radiation therapy. Thus, initial risk stratification of prostate cancer patients is important for achieving optimal therapeutic results or cancer cure and preservation of quality of life. Predictive biomarkers for risks of complications or late effects of treatment are needed to inform clinical decisions for treatment selection. Here, we analyzed pre-treatment plasma metabolites in a cohort of prostate cancer patients (N = 99) treated with Stereotactic Body Radiation Therapy (SBRT) at Medstar-Georgetown University Hospital in a longitudinal, quality-of-life study to determine if individuals experiencing radiation toxicities can be identified by a molecular profile in plasma prior to treatment. We used a multiple reaction mass spectrometry-based molecular phenotyping of clinically annotated plasma samples in a retrospective outcome analysis to identify candidate biomarker panels correlating with adverse clinical outcomes following radiation therapy. We describe the discovery of candidate biomarkers, based on small molecule metabolite panels, showing high correlations (AUCs ≥ 95%) with radiation toxicities, suitable for validation studies in an expanded cohort of patients.

Aminopeptidase P Mediated Targeting for Breast Tissue Specific Conjugate Delivery.

Cytotoxic chemotherapies are used to treat breast cancer, but are limited by systemic toxicity. The key to addressing this important issue is the development of a nontoxic, tissue selective, and molecular specific delivery system. In order to potentially increase the therapeutic index of clinical reagents, we designed an Aminopeptidase P (APaseP) targeting tissue-specific construct conjugated to a homing peptide for selective binding to human breast-derived cancer cells. Homing peptides are short amino acid sequences derived from phage display libraries that have the unique property of localizing to specific organs. Our molecular construct allows for tissue-specific drug delivery, by binding to APaseP in the vascular endothelium. The breast homing peptide evaluated in our studies is a cyclic nine-amino-acid peptide with the sequence CPGPEGAGC, referred to as PEGA. We show by confocal microscopy that the PEGA peptide and similar peptide conjugates distribute to human breast tissue xenograft specifically and evaluate the interaction with the membrane-bound proline-specific APaseP (KD = 723 ± 3 nM) by binding studies. To achieve intracellular breast cancer cell delivery, the incorporation of the Tat sequence, a cell-penetrating motif derived from HIV, was conjugated with the fluorescently labeled PEGA peptide sequence. Ultimately, tissue specific peptides and their conjugates can enhance drug delivery and treatment by their ability to discriminate between tissue types. Tissue specific conjugates as we have designed may be valuable tools for drug delivery and visualization, including the potential to treat breast cancer, while simultaneously minimizing systemic toxicity.

Small-molecule "BRCA1-mimetics" are antagonists of estrogen receptor-α.

CONTEXT: Resistance to conventional antiestrogens is a major cause of treatment failure and, ultimately, death in breast cancer. OBJECTIVE: The objective of the study was to identify small-molecule estrogen receptor (ER)-α antagonists that work differently from tamoxifen and other selective estrogen receptor modulators. DESIGN: Based on in silico screening of a pharmacophore database using a computed model of the BRCA1-ER-α complex (with ER-α liganded to 17ß-estradiol), we identified a candidate group of small-molecule compounds predicted to bind to a BRCA1-binding interface separate from the ligand-binding pocket and the coactivator binding site of ER-α. Among 40 candidate compounds, six inhibited estradiol-stimulated ER-α activity by at least 50% in breast carcinoma cells, with IC50 values ranging between 3 and 50 µM. These ER-α inhibitory compounds were further studied by molecular and cell biological techniques. RESULTS: The compounds strongly inhibited ER-α activity at concentrations that yielded little or no nonspecific toxicity, but they produced only a modest inhibition of progesterone receptor activity. Importantly, the compounds blocked proliferation and inhibited ER-α activity about equally well in antiestrogen-sensitive and antiestrogen-resistant breast cancer cells. Representative compounds disrupted the interaction of BRCA1 and ER-α in the cultured cells and blocked the interaction of ER-α with the estrogen response element. However, the compounds had no effect on the total cellular ER-α levels. CONCLUSIONS: These findings suggest that we have identified a new class of ER-α antagonists that work differently from conventional antiestrogens (eg, tamoxifen and fulvestrant).

Novel carbazole inhibits phospho-STAT3 through induction of protein-tyrosine phosphatase PTPN6.

The aberrant activation of STAT3 occurs in many human cancers and promotes tumor progression. Phosphorylation of a tyrosine at amino acid Y705 is essential for the function of STAT3. Synthesized carbazole derived with fluorophore compound 12 was discovered to target STAT3 phosphorylation. Compound 12 was found to inhibit STAT3-mediated transcription as well as to reduce IL-6 induced STAT3 phosphorylation in cancer cell lines expressing both elevated and low levels of phospho-STAT3 (Y705). Compound 12 potently induced apoptosis in a broad number of TNBC cancer cell lines in vitro and was effective at inhibiting the in vivo growth of human TNBC xenograft tumors (SUM149) without any observed toxicity. Compound 12 also effectively inhibited the growth of human lung tumor xenografts (A549) harboring aberrantly active STAT3. In vitro and in vivo studies showed that the inhibitory effects of 12 on phospho-STAT3 were through up-regulation of the protein-tyrosine phosphatase PTPN6. Our present studies strongly support the continued preclinical evaluation of compound 12 as a potential chemotherapeutic agent for TNBC and cancers with constitutive STAT3 signaling.

Synthesis and biological evaluation of a fluorescent analog of phenytoin as a potential inhibitor of neuropathic pain and imaging agent.

Here we report on a novel fluorescent analog of phenytoin as a potential inhibitor of neuropathic pain with potential use as an imaging agent. Compound 2 incorporated a heptyl side chain and dansyl moiety onto the parent compound phenytoin and produced greater displacement of BTX from sodium channels and greater functional blockade with greatly reduced toxicity. Compound 2 reduced mechano-allodynia in a rat model of neuropathic pain and was visualized ex vivo in sensory neuron axons with two-photon microscopy. These results suggest a promising strategy for developing novel sodium channel inhibitors with imaging capabilities.

Histone deacetylase cytoplasmic trapping by a novel fluorescent HDAC inhibitor.

Inhibitors of histone deacetylases (HDAC) are an important emerging class of drugs for the treatment of cancers. HDAC inhibitors are currently under evaluation in clinical trials as single agents and as sensitizers in combinations with chemotherapies and radiation therapy. Although these drugs have important effects on cancer cell growth and functions, the mechanisms underlying HDAC inhibitor activities remain to be fully defined. By using rational drug design, compound 2, a fluorescent class II HDAC targeting inhibitor, was synthesized and observed to accumulate in the cytoplasmic compartments of treated cells, but not in the nuclei. Furthermore, immunostaining of inhibitor exposed cells for HDAC4 showed accumulation of this enzyme in the cytoplasmic compartment with concomitant increased acetylation of tubulin and nuclear histones. These observations support a mechanism by which nuclear histone acetylation is increased as a result of HDAC4 trapping and sequestration in the cytoplasm after binding to compound 2. The HDAC inhibitor offers potential as a novel theranostic agent, combining diagnostic and therapeutic properties in the same molecule.

Fluorescent cyclin-dependent kinase inhibitors block the proliferation of human breast cancer cells.

Inhibitors of cyclin-dependent kinases (CDKs) are an emerging class of drugs for the treatment of cancers. CDK inhibitors are currently under evaluation in clinical trials as single agents and as sensitizers in combination with radiation therapy and chemotherapies. Drugs that target CDKs could have important inhibitory effects on cancer cell cycle progression, an extremely important mechanism in the control of cancer cell growth. Using rational drug design, we designed and synthesized fluorescent CDK inhibitors (VMY-1-101 and VMY-1-103) based on a purvalanol B scaffold. The new agents demonstrated more potent CDK inhibitory activity, enhanced induction of G2/M arrest and modest apoptosis as compared to purvalanol B. Intracellular imaging of the CDK inhibitor distribution was performed to reveal drug retention in the cytoplasm of treated breast cancer cells. In human breast cancer tissue, the compounds demonstrated increased binding as compared to the fluorophore. The new fluorescent CDK inhibitors showed undiminished activity in multidrug resistance (MDR) positive breast cancer cells, indicating that they are not a substrate for p-glycoprotein. Fluorescent CDK inhibitors offer potential as novel theranostic agents, combining therapeutic and diagnostic properties in the same molecule.

Effects of a fluorescent Myosin light chain phosphatase inhibitor on prostate cancer cells.

Myosin light chain phosphatase (MLCP) is an enzyme important to regulation of cell cycle and motility that is shown to be upregulated in aggressive prostate cancer cells and tissue. We developed a fluorescent small molecule inhibitor of MLCP using structure based design in recombinant protein phosphatase 1C. Several best fit compounds were synthesized and evaluated by their inhibition of MLCP/(32)P-MLC dephosphorylation, which resulted in the identification of novel MLCP inhibitors. Androgen dependent (AD) and castration resistant prostate cancer cell (CRPC) lines were treated with the lead inhibitor resulting in decreased growth rate, reduced DNA synthesis, and G2/M cell cycle arrest. Moreover, CRPC cell lines showed an increased sensitivity to drug treatment having GI(50) values four times lower than the AD prostate cancer cell line. This was reinforced by reduced BrdU DNA incorporation into CRPC cells compared to AD cells. ß-actin disruption was also seen at much lower drug concentrations in CR cells which caused a dose dependent reduction in cellular chemotaxis of PC-3 cells. Since there are currently few clinical therapeutics targeting CR prostate cancer, MLCP represents a new target for preclinical and clinical development of new potential therapeutics which inhibit this disease phenotype.

VMY-1-103, a dansylated analog of purvalanol B, induces caspase-3-dependent apoptosis in LNCaP prostate cancer cells.

The 2,6,9-trisubstituted purine group of cyclin dependent kinase inhibitors have the potential to be clinically relevant inhibitors of cancer cell proliferation. We have recently designed and synthesized a novel dansylated analog of purvalanol B, termed VMY-1-103, that inhibited cell cycle progression in breast cancer cell lines more effectively than did purvalanol B and allowed for uptake analyses by fluorescence microscopy. ErbB-2 plays an important role in the regulation of signal transduction cascades in a number of epithelial tumors, including prostate cancer (PCa). Our previous studies demonstrated that transgenic expression of activated ErbB-2 in the mouse prostate initiated PCa and either the overexpression of ErbB-2 or the addition of the ErbB-2/ErbB-3 ligand, heregulin (HRG), induced cell cycle progression in the androgen-responsive prostate cancer cell line, LNCaP. In the present study, we tested the efficacy of VMY-1-103 in inhibiting HRG-induced cell proliferation in LNCaP prostate cancer cells. At concentrations as low as 1 µM, VMY-1-103 increased both the proportion of cells in G(1) and p21(CIP1) protein levels. At higher concentrations (5 µM or 10 µM), VMY-1-103 induced apoptosis via decreased mitochondrial membrane polarity and induction of p53 phosphorylation, caspase-3 activity and PARP cleavage. Treatment with 10 µM Purvalanol B failed to either influence proliferation or induce apoptosis. Our results demonstrate that VMY-1-103 was more effective in inducing apoptosis in PCa cells than its parent compound, purvalanol B, and support the testing of VMY-1-103 as a potential small molecule inhibitor of prostate cancer in vivo.

Fluorescent epigenetic small molecule induces expression of the tumor suppressor ras-association domain family 1A and inhibits human prostate xenograft.

Epigenetic silencing of Ras-association domain family 1A (RASSF1A) protein in cancer cells results in a disruption of cell cycle control, genetic instability, enhanced cell motility, and apoptotic resistance. Ectopic expression of RASSF1A reverses this tumorigenic phenotype. Thus, small molecules with the ability to restore RASSF1A expression may represent a new class of therapeutic agents. Recently, we designed and synthesized a fluorescent carbazole analogue of mahanine (alkaloid from Murraya koenigii) that restored RASSF1A mRNA expression. Our fluorescent lead compound up-regulated RASSF1A in vitro, potently inhibited human prostate cancer cell proliferation, and fluoresced at a visible wavelength, allowing for the observation of intracellular distribution. The small molecule lead was not acutely toxic up to 550 mg/kg, and dosing at 10 mg/kg reduced human xenograft tumor volume by about 40%.

Synthesis of C-glycoside analogues of beta-galactosamine-(1-->4)-3-O-methyl-D-chiro-inositol and assay as activator of protein phosphatases PDHP and PP2Calpha.

The glycan beta-galactosamine-(1-4)-3-O-methyl-D-chiro-inositol, called INS-2, was previously isolated from liver as a putative second messenger-modulator for insulin. Synthetic INS-2 injected intravenously in rats is both insulin-mimetic and insulin-sensitizing. This bioactivity is attributed to allosteric activation of pyruvate dehydrogenase phosphatase (PDHP) and protein phosphatase 2Calpha (PP2Calpha). Towards identification of potentially metabolically stable analogues of INS-2 and illumination of the mechanism of enzymatic activation, C-INS-2, the exact C-glycoside of INS-2, and C-INS-2-OH the deaminated analog of C-INS-2, were synthesized and their activity against these two enzymes evaluated. C-INS-2 activates PDHP comparable to INS-2, but failed to activate PP2Calpha. C-INS-2-OH was inactive against both phosphatases. These results and modeling of INS-2, C-INS-2 and C-INS-2-OH into the 3D structure of PDHP and PP2Calpha, suggest that INS-2 binds to distinctive sites on the two different phosphatases to activate insulin signaling. Thus the carbon analog could selectively favor glucose disposal via oxidative pathways.

Asymmetric synthesis of 2,3-dihydro-2-arylquinazolin-4-ones: methodology and application to a potent fluorescent tubulin inhibitor with anticancer activity.

For several decades the 2,3-dihydroquinazolinone (DHQZ) heterocycle has been known to possess a variety of important biological and medicinal properties. Despite the many interesting facets of these molecules, synthetic access to nonracemic DHQZ analogues has remained elusive. Herein, we disclose a synthetic route that allows access to either enantiomer of a variety of DHQZ derivatives. We illustrate the utility of this chemistry with the asymmetric preparation and biological evaluation of a new chiral fluorescent tubulin binding agent with extremely potent antiproliferative properties against human cancer cells. A computational rationale for the increased potency of the (S)-enantiomer over the (R)-enantiomer is given, based on the crystal structure of alpha,beta-tubulin complexed with colchicine. Taking advantage of the inherent fluorescence of these molecules, confocal images of GMC-5-193 (compound 7) in the cytoplasm of human melanoma cells (MDA-MB-435) cells are presented.

Meso-substituted [34]octaphyrin(1.1.1.0.1.1.1.0) and corrole formation in reactions of a dipyrromethanedicarbinol with 2,2'-bipyrrole.

The reaction of a dipyrromethanedicarbinol with 2,2'-bipyrrole leading to meso-substituted [34]octaphyrin(1.1.1.0.1.1.1.0) and/or corrole was investigated to determine the effect of key reaction parameters on the distribution of the two macrocycles. Solvent, acid catalyst, acid quantity, oxidant, oxidant quantity, and reaction time were surveyed for a model reaction affording 5,10,19,24,29,38-hexaphenyl[34]octaphyrin(1.1.1.0.1.1.1.0) (HPO) and/or meso-triphenylcorrole (TPC). HPO was found to be a fairly ubiquitous product, produced in yields as high as 23% (UV-vis), while TPC was observed infrequently, in yields up to 10% (UV-vis). A preparative-scale reaction provided HPO in an isolated yield of 25%. The methodology was extended to the synthesis of an octaphyrin bearing two different substituents in defined locations and to an octaphyrin possessing electron-withdrawing pentafluorophenyl substituents. Preferential formation of octaphyrin instead of corrole suggests that the anti conformation of 2,2'-bipyrrole is the relevant form under the reaction conditions surveyed. The spectral properties of the novel meso-substituted [34]octaphyrin(1.1.1.0.1.1.1.0) species are similar to those of the known beta-substituted analogue, including spectra consistent with the absence of macrocycle aromaticity despite a main conjugation path of 34 pi-electrons. Key to the overall study was the development of a refined synthesis of 2,2'-bipyrrole.