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1.  Sustained inhibition of receptor tyrosine kinases and macrophage depletion by PLX3397 and rapamycin as a potential new approach for the treatment of MPNSTs 
Purpose
Malignant peripheral nerve sheath tumor (MPNST) is a highly aggressive tumor type that is resistant to chemotherapy and there are no effective therapies. MPNSTs have been shown to have gene amplification for receptor tyrosine kinases (RTKs), PDGFR and c-Kit. We tested the c-Kit inhibitor, imatinib, and PLX3397, a selective c-Fms and c-Kit inhibitor, to evaluate their efficacy against MPNST cells in vitro and in vivo.
Experimental Design
We tested the efficacy of imatinib or PLX3397 either alone or in combination with TORC1 inhibitor rapamycin in a cell proliferation assay in vitro and by immunoblotting to determine target inhibition. Immunoblotting and immunohistochemical analysis was further carried out using xenograft samples in vivo.
Results
Our in vitro studies show that imatinib and PLX3397 similarly inhibit cell growth and this can be enhanced with rapamycin with comparable target specificity. However, in vivo studies clearly demonstrate that compared to imatinib, PLX3397 results in sustained blockade of c-Kit, c-Fms and PDGFRβ, resulting in significant suppression of tumor growth. Moreover, staining for Iba-1, a marker for macrophages, indicates that PLX3397 results in significant depletion of macrophages in the growing tumors. The combination of PLX3397 and rapamycin results in even greater macrophage depletion with continued growth suppression, even when the drug treatment is discontinued.
Conclusions
Taken together, our data strongly suggests that PLX3397 is superior to imatinib in the treatment of MPNST, and the combination of PLX3397 with a TORC1 inhibitor could provide a new therapeutic approach for the treatment of this disease.
doi:10.1158/1078-0432.CCR-13-2576
PMCID: PMC4060793  PMID: 24718867
PLX3397; rapamycin; mTOR; Fms-Kit
2.  Study design and rationale for a randomised, placebo-controlled, double-blind study to assess the efficacy of selumetinib (AZD6244; ARRY-142886) in combination with dacarbazine in patients with metastatic uveal melanoma (SUMIT) 
BMC Cancer  2015;15:467.
Background
Uveal melanoma is characterised by mutations in GNAQ and GNA11, resulting in Ras/Raf/MEK/ERK pathway activation. Treatment with selumetinib (AZD6244, ARRY-142886), a MEK1/2 inhibitor, results in antitumour effects in uveal melanoma pre-clinical models. A randomised phase II trial demonstrated improved progression-free survival (PFS) and response rate (RR) with selumetinib monotherapy versus chemotherapy with temozolomide or dacarbazine in patients with metastatic uveal melanoma. Pre-clinically, selumetinib in combination with alkylating agents enhanced antitumour activity compared with chemotherapy alone. We hypothesise that selumetinib in combination with dacarbazine will result in improved clinical outcomes in patients with metastatic uveal melanoma versus dacarbazine alone.
Methods/Design
SUMIT is a randomised, international, double-blind, placebo-controlled, phase III study assessing the efficacy and safety of selumetinib in combination with dacarbazine in patients with metastatic uveal melanoma who have not received prior systemic therapy. Primary endpoint is PFS. Secondary endpoints include objective RR, duration of response, change in tumour size at Week 6, overall survival, safety and tolerability. Exploratory endpoints include efficacy in tumours with GNAQ or GNA11 mutations. Eligible patients must have: ≥1 lesion that can be accurately measured at baseline, and is suitable for accurate repeated measurements; ECOG performance status 0–1; life expectancy >12 weeks. Mutation status for GNAQ/GNA11 will be assessed retrospectively.
An estimated 128 patients from approximately 50 sites globally will be randomised (3:1) to selumetinib 75 mg twice daily or placebo in combination with dacarbazine 1000 mg/m2 on Day 1 of every 21-day cycle until objective disease progression, intolerable toxicity or occurrence of another discontinuation criterion. Randomisation will be stratified by the presence/absence of liver metastases. Tumours will be evaluated by RECIST v1.1 every 6 weeks. All patients have the option of receiving selumetinib with or without dacarbazine at disease progression. Study enrolment began in April 2014 and is expected to complete in early 2015.
Discussion
Treatment of patients with metastatic uveal melanoma represents an area of high unmet medical need. This study evaluating selumetinib in combination with dacarbazine was designed with input from the US FDA, and is the first potential registration trial to be conducted in patients with metastatic uveal melanoma.
Trial registration
Clinicaltrials.gov (Date of registration, October 10, 2013)
Registration number: NCT01974752
Trial abbreviation: SUMIT
doi:10.1186/s12885-015-1470-z
PMCID: PMC4460965  PMID: 26059332
Dacarbazine; GNAQ; GNA11; Metastatic uveal melanoma; Phase III; Selumetinib
3.  Phase II Trial of Gemcitabine and Docetaxel with Bevacizumab in Soft Tissue Sarcoma 
Sarcoma  2015;2015:532478.
Gemcitabine (G) and docetaxel (D) are commonly used to treat recurrent/metastatic soft tissue sarcoma. This study tested the hypothesis that outcomes would be improved by addition of bevacizumab (B). The initial design was randomized double-blind trial of G + D + B versus G + D + placebo. Due to slow accrual this was modified to single-arm open-label G + D + B. Eligible patients had diagnosis of leiomyosarcoma, pleomorphic undifferentiated sarcoma, pleomorphic liposarcoma, or angiosarcoma. Treatment was B 15 mg/kg on d1, G 900 mg/m2 on d1 and d8, and D 75 mg/m2 on d8, q21d. Primary endpoint was progression-free survival (PFS) at 6 months and would be met if ≥17 patients were progression-free at 6 m. Secondary endpoints are response rate, PFS at 3 m, overall survival, and toxicity. Of 44 patients enrolled, 35 were treated with GDB and evaluable for safety and efficacy. Median age was 55, 50% male, most ECOG 0. Toxicity is mostly myelosuppression with one deep vein thrombosis and one small bowel perforation possibly related to B. There were 17 partial responses (49%) by RECIST 1.1. Among 35 patients, the number who remained on study and progression-free was 24 at 3 m and 15 at 6 m. 9 withdrew prior to 6 m for reasons other than toxicity or progression. PFS at 6 m was 65% (95% CI: 51–85%). The primary endpoint of 6 m PFS was not met due to censoring of patients who withdrew. However PFS at 3 m (76%) was promising and response rate was higher than expected from G + D.
doi:10.1155/2015/532478
PMCID: PMC4446476  PMID: 26074722
4.  The Phosphoinositide 3-Kinaseα Selective Inhibitor, BYL719, Enhances the Effect of the Protein Kinase C Inhibitor, AEB071, in GNAQ/GNA11 Mutant Uveal Melanoma Cells 
Molecular cancer therapeutics  2014;13(5):1044-1053.
G-protein mutations are one of the most common mutations occurring in uveal melanoma activating the protein kinase C (PKC)/mitogen-activated protein kinase (MAPK) and phosphoinositide 3-Kinase (PI3K)/AKT pathways. In this study, we described the effect of dual pathway inhibition in uveal melanoma harboring GNAQ and GNA11 mutations via PKC inhibition with AEB071 (Sotrastaurin) and PI3k/AKT inhibition with BYL719, a selective PI3Kα inhibitor. Growth inhibition was observed in GNAQ/GNA11 mutant cells with AEB071 versus no activity in WT cells. In the GNAQ-mutant cells, AEB071 decreased phosphorylation of MARCKS, a substrate of PKC, along with ERK1/2 and ribosomal S6, but persistent AKT activation was present. BYL719 had minimal anti-proliferative activity in all uveal melanoma cell lines, and inhibited phosphorylation of AKT in most cell lines. In the GNA11 mutant cell line, similar effects were observed with ERK1/2 inhibition, mostly inhibited by BYL719. With the combination treatment, both GNAQ and GNA11 mutant cell lines showed synergistic inhibition of cell proliferation and apoptotic cell death. In vivo studies correlated with in vitro findings showing reduced xenograft tumor growth with the combination therapy in a GNAQ mutant model. These findings suggest a new therapy treatment option for G-protein mutant uveal melanoma with a focus on specific targeting of multiple downstream pathways as part of combination therapy.
doi:10.1158/1535-7163.MCT-13-0550
PMCID: PMC4146424  PMID: 24563540
Pi3Kα; PKC; BYL719; AEB071
5.  Cholinergic neurons in the dorsomedial hypothalamus regulate mouse brown adipose tissue metabolism 
Molecular Metabolism  2015;4(6):483-492.
Objective
Brown adipose tissue (BAT) thermogenesis is critical in maintaining body temperature. The dorsomedial hypothalamus (DMH) integrates cutaneous thermosensory signals and regulates adaptive thermogenesis. Here, we study the function and synaptic connectivity of input from DMH cholinergic neurons to sympathetic premotor neurons in the raphe pallidus (Rpa).
Methods
In order to selectively manipulate DMH cholinergic neuron activity, we generated transgenic mice expressing channelrhodopsin fused to yellow fluorescent protein (YFP) in cholinergic neurons (choline acetyltransferase (ChAT)-Cre::ChR2-YFP) with the Cre-LoxP technique. In addition, we used an adeno-associated virus carrying the Cre recombinase gene to delete the floxed Chat gene in the DMH. Physiological studies in response to optogenetic stimulation of DMH cholinergic neurons were combined with gene expression and immunocytochemical analyses.
Results
A subset of DMH neurons are ChAT-immunopositive neurons. The activity of these neurons is elevated by warm ambient temperature. A phenotype-specific neuronal tracing shows that DMH cholinergic neurons directly project to serotonergic neurons in the Rpa. Optical stimulation of DMH cholinergic neurons decreases BAT activity, which is associated with reduced body core temperature. Furthermore, elevated DMH cholinergic neuron activity decreases the expression of BAT uncoupling protein 1 (Ucp1) and peroxisome proliferator-activated receptor γ coactivator 1 α (Pgc1α) mRNAs, markers of BAT activity. Injection of M2-selective muscarinic receptor antagonists into the 4th ventricle abolishes the effect of optical stimulation. Single cell qRT-PCR analysis of retrogradely identified BAT-projecting neurons in the Rpa shows that all M2 receptor-expressing neurons contain tryptophan hydroxylase 2. In animals lacking the Chat gene in the DMH, exposure to warm temperature reduces neither BAT Ucp1 nor Pgc1α mRNA expression.
Conclusion
DMH cholinergic neurons directly send efferent signals to sympathetic premotor neurons in the Rpa. Elevated cholinergic input to this area reduces BAT activity through activation of M2 mAChRs on serotonergic neurons. Therefore, the direct DMHACh–Rpa5-HT pathway may mediate physiological heat-defense responses to elevated environmental temperature.
Graphical abstract
doi:10.1016/j.molmet.2015.03.006
PMCID: PMC4443291  PMID: 26042202
Acetylcholine; Muscarinic; Nicotinic; Neuronal tracing; Serotonin; Hypothalamus
6.  MDM2 turnover and expression of ATRX determine the choice between quiescence and senescence in response to CDK4 inhibition 
Oncotarget  2015;6(10):8226-8243.
CDK4 inhibitors (CDK4i) earned Breakthrough Therapy Designation from the FDA last year and are entering phase III clinical trials in several cancers. However, not all tumors respond favorably to these drugs. CDK4 activity is critical for progression through G1 phase and into the mitotic cell cycle. Inhibiting this kinase induces Rb-positive cells to exit the cell cycle into either a quiescent or senescent state. In this report, using well-differentiated and dedifferentiated liposarcoma (WD/DDLS) cell lines, we show that the proteolytic turnover of MDM2 is required for CDK4i-induced senescence. Failure to reduce MDM2 does not prevent CDK4i-induced withdrawal from the cell cycle but the cells remain in a reversible quiescent state. Reducing MDM2 in these cells drives them into the more stable senescent state. CDK4i-induced senescence associated with loss of MDM2 is also observed in some breast cancer, lung cancer and glioma cell lines indicating that this is not limited to WD/DDLS cells in which MDM2 is overexpressed or in cells that contain wild type p53. MDM2 turnover depends on its E3 ligase activity and expression of ATRX. Interestingly, in seven patients the changes in MDM2 expression were correlated with outcome. These insights identify MDM2 and ATRX as new regulators controlling geroconversion, the process by which quiescent cells become senescent, and this insight may be exploited to improve the activity of CDK4i in cancer therapy.
PMCID: PMC4480747  PMID: 25803170
CDK4 inhibitors; MDM2; ATRX; senescence; geroconversion
7.  Night Blood Pressure Responses to Atenolol and Hydrochlorothiazide in Black and White Patients With Essential Hypertension 
American Journal of Hypertension  2013;27(4):546-554.
BACKGROUND
Night blood pressure (BP) predicts patient outcomes. Variables associated with night BP response to antihypertensive agents have not been fully evaluated in essential hypertension.
METHODS
We sought to measure night BP responses to hydrochlorothiazide (HCTZ), atenolol (ATEN), and combined therapy using ambulatory blood pressure (ABP) monitoring in 204 black and 281 white essential hypertensive patients. Initial therapy was randomized; HCTZ and ATEN once daily doses were doubled after 3 weeks and continued for 6 more weeks with the alternate medication added for combined therapy arms. ABP was measured at baseline and after completion of each drug. Night, day, and night/day BP ratio responses (treatment − baseline) were compared in race/sex subgroups.
RESULTS
Baseline night systolic BP and diastolic BP, and night/day ratios were greater in blacks than whites (P < 0.01, all comparisons). Night BP responses to ATEN were absent and night/day ratios increased significantly in blacks (P < 0.05). At the end of combined therapy, women, blacks, and those starting with HCTZ as opposed to ATEN had significantly greater night BP responses (P < 0.01). Variables that significantly associated with ATEN response differed from those that associated with HCTZ response and those that associated with night BP response differed from those that associated with day BP response.
CONCLUSIONS
In summary, after completion of HCTZ and ATEN therapy, women, blacks, and those who started with HCTZ had greater night BP responses. Reduced night BP response and increased night/day BP ratios occured with ATEN in blacks. Given the prognostic significance of night BP, strategies for optimizing night BP antihypertensive therapy should be considered.
CLINICAL TRIAL REGISTRATION
Clinicaltrials.gov identifier NCT00246519
doi:10.1093/ajh/hpt124
PMCID: PMC3958600  PMID: 23886594
ambulatory blood pressure; atenolol; blood pressure; hydrochlorothiazide; hypertension; night/day ratio.
8.  High-Fat Feeding Impairs Nutrient Sensing and Gut Brain Integration in the Caudomedial Nucleus of the Solitary Tract in Mice 
PLoS ONE  2015;10(3):e0118888.
Hyperphagic obesity is characterized in part by a specific increase in meal size that contributes to increased daily energy intake, but the mechanisms underlying impaired activity of meal size regulatory circuits, particularly those converging at the caudomedial nucleus of the solitary tract in the hindbrain (cmNTS), remain poorly understood. In this paper, we assessed the consequences of high-fat (HF) feeding and diet-induced obesity (DIO) on cmNTS nutrient sensing and metabolic integration in the control of meal size. Mice maintained on a standard chow diet, low-fat (LF) diet or HF diet for 2 weeks or 6 months were implanted with a bilateral brain cannula targeting the cmNTS. Feeding behavior was assessed using behavioral chambers and meal-pattern analysis following cmNTS L-leucine injections alone or together with ip CCK. Molecular mechanisms implicated in the feeding responses were assessed using western blot, immunofluorescence and pharmacological inhibition of the amino acid sensing mTORC1 pathway (mammalian target of rapamycin complex 1). We found that HF feeding blunts the anorectic consequences of cmNTS L-leucine administration. Increased baseline activity of the L-leucine sensor P70 S6 kinase 1 and impaired L-leucine-induced activation of this pathway in the cmNTS of HF-fed mice indicate that HF feeding is associated with an impairment in cmNTS mTOR nutritional and hormonal sensing. Interestingly, the acute orexigenic effect of the mTORC1 inhibitor rapamycin was preserved in HF-fed mice, supporting the assertion that HF-induced increase in baseline cmNTS mTORC1 activity underlies the defect in L-leucine sensing. Last, the synergistic feeding-suppressive effect of CCK and cmNTS L-leucine was abrogated in DIO mice. These results indicate that HF feeding leads to an impairment in cmNTS nutrient sensing and metabolic integration in the regulation of meal size.
doi:10.1371/journal.pone.0118888
PMCID: PMC4361711  PMID: 25774780
9.  Molecular Subtypes of Uterine Leiomyosarcoma and Correlation with Clinical Outcome12 
Neoplasia (New York, N.Y.)  2015;17(2):183-189.
The molecular etiology of uterine leiomyosarcoma (ULMS) is poorly understood, which accounts for the wide disparity in outcomes among women with this disease. We examined and compared the molecular profiles of ULMS and normal myometrium (NL) to identify clinically relevant molecular subtypes. Discovery cases included 29 NL and 23 ULMS specimens. RNA was hybridized to Affymetrix U133A 2.0 transcription microarrays. Differentially expressed genes and pathways were identified using standard methods. Fourteen NL and 44 ULMS independent archival samples were used for external validation. Molecular subgroups were correlated with clinical outcome. Pathway analyses of differentially expressed genes between ULMS and NL samples identified overrepresentation of cell cycle regulation, DNA repair, and genomic integrity. External validation confirmed differential expression in 31 genes (P < 4.4 × 10− 4, Bonferroni corrected), with 84% of the overexpressed genes, including CDC7, CDC20, GTSE1, CCNA2, CCNB1, and CCNB2, participating in cell cycle regulation. Unsupervised clustering of ULMS identified two clades that were reproducibly associated with progression-free (median, 4.0 vs 26.0 months; P = .02; HR, 0.33) and overall (median, 18.2 vs 77.2 months; P = .04; HR, 0.33) survival. Cell cycle genes play a key role in ULMS sarcomagenesis, providing opportunities for therapeutic targeting. Reproducible molecular subtypes associated with clinical outcome may permit individualized adjuvant treatment after clinical trial validation.
doi:10.1016/j.neo.2014.12.007
PMCID: PMC4351299  PMID: 25748237
FDR, false discovery rate; FFPE, formalin-fixed and paraffin-embedded; GSEA, gene set enrichment analysis; LMS, leiomyosarcoma; MSKCC, Memorial Sloan Kettering Cancer Center; NL, normal myometrium; OS, overall survival; PFS, progression-free survival; ULMS, uterine leiomyosarcoma
10.  Synthetic lethality in ATM-deficient RAD50-mutant tumors underlie outlier response to cancer therapy 
Cancer discovery  2014;4(9):1014-1021.
Metastatic solid tumors are almost invariably fatal. Patients with disseminated small-cell cancers have a particularly unfavorable prognosis with most succumbing to their disease within two years. Here, we report on the genetic and functional analysis of an outlier curative response of a patient with metastatic small cell cancer to combined checkpoint kinase 1 (Chk1) inhibition and DNA damaging chemotherapy. Whole-genome sequencing revealed a clonal hemizygous mutation in the Mre11 complex gene RAD50 that attenuated ATM signaling which in the context of Chk1 inhibition contributed, via synthetic lethality, to extreme sensitivity to irinotecan. As Mre11 mutations occur in a diversity of human tumors, the results suggest a tumor-specific combination therapy strategy whereby checkpoint inhibition in combination with DNA damaging chemotherapy is synthetically lethal in tumor but not normal cells with somatic mutations that impair Mre11 complex function.
doi:10.1158/2159-8290.CD-14-0380
PMCID: PMC4155059  PMID: 24934408
DNA damage and repair; cancer genomics; exceptional responders; targeted and systemic therapy; RAD50
11.  Spexin is a Novel Human Peptide that Reduces Adipocyte Uptake of Long Chain Fatty Acids and Causes Weight Loss in Rodents with Diet-induced Obesity* 
Obesity (Silver Spring, Md.)  2014;22(7):1643-1652.
Objective
Microarray studies identified Ch12:orf39 (Spexin) as the most dysregulated gene in obese human fat. Therefore we examined its role in obesity pathogenesis.
Design and Methods
Spexin effects on food intake, meal patterns, body weight, Respiratory Exchange Ratio (RER), and locomotor activity were monitored electronically in C57BL/6J mice or Wistar rats with dietary-induced obesity (DIO). Its effects on adipocyte [3H]-oleate uptake were determined.
Results
In humans, Spexin gene expression was down-regulated 14.9-fold in obese omental and subcutaneous fat. Circulating Spexin changed in parallel, correlating (r = −0.797) with Leptin. In rats, Spexin (35 μg/kg/day s.c) reduced caloric intake ~32% with corresponding weight loss. Meal patterns were unaffected. In mice, Spexin (25 μg/kg/day i.p.) significantly reduced the RER at night, and increased locomotion. Spexin incubation in vitro significantly inhibited facilitated fatty acid (FA) uptake into DIO mouse adipocytes. Conditioned taste aversion testing (70μg/kg/day i.p.) demonstrated no aversive Spexin effects.
Conclusions
Spexin gene expression is markedly down-regulated in obese human fat. The peptide produces weight loss in DIO rodents. Its effects on appetite and energy regulation are presumably central; those on adipocyte FA uptake appear direct and peripheral. Spexin is a novel hormone involved in weight regulation, with potential for obesity therapy.
doi:10.1002/oby.20725
PMCID: PMC4077920  PMID: 24550067
12.  Calcium and Vitamin D Supplementation and Loss of Bone Mineral Density in Women Undergoing Breast Cancer Therapy 
Critical reviews in oncology/hematology  2013;88(3):10.1016/j.critrevonc.2013.07.002.
An unintended consequence of breast cancer therapies is an increased risk of osteoporosis due to accelerated bone loss. We conducted a systematic review of calcium and/or vitamin D (Ca±D) supplementation trials for maintaining bone mineral density (BMD) in women with breast cancer using the “before-after” data from the Ca±D supplemented comparison group of trials evaluating the effect of drugs such as bisphosphonates on BMD. Whether Ca±D supplements increase BMD in women undergoing breast cancer therapy has never been tested against an unsupplemented control group. However, results from 16 trials indicate that the Ca±D doses tested (500-1500 mg calcium; 200-1000 IU vitamin D) were inadequate to prevent BMD loss in these women. Cardiovascular disease is the main cause of mortality in women with breast cancer. Because calcium supplements may increase cardiovascular disease risk, future trials should evaluate the safety and efficacy of Ca±D supplementation in women undergoing breast cancer therapy.
doi:10.1016/j.critrevonc.2013.07.002
PMCID: PMC3844003  PMID: 23932583
breast cancer; calcium; vitamin D; osteoporosis; bone mineral density
13.  Effect of Selumetinib versus Chemotherapy on Progression-Free Survival in Uveal Melanoma: A Randomized Clinical Trial 
JAMA  2014;311(23):2397-2405.
Importance
Uveal melanoma is characterized by mutations in GNAQ and GNA11, resulting in MAPK pathway activation.
Objective
To assess the efficacy of selumetinib, a selective, non-ATP competitive inhibitor of MEK1 and MEK2, in uveal melanoma.
Design
Randomized open-label phase II clinical trial comparing selumetinib versus chemotherapy. Those receiving chemotherapy could receive selumetinib at the time of radiographic progression.
Setting
Fifteen academic oncology centers.
Participants
120 patients with metastatic uveal melanoma.
Interventions
101 patients were randomized on a 1:1 ratio to selumetinib 75 mg orally twice daily on a continual basis (n=50) or chemotherapy (temozolomide 150 mg/m2 orally daily for 5 of every 28 days or DTIC 1000 mg/m2 intravenously every 21 days; investigator choice; n=51) until disease progression, death, intolerable toxicity, or withdrawal of consent. Following primary outcome analysis, enrollment continued in a non-randomized fashion to the superior therapy.
Main Outcomes
Final analysis of progression-free survival, the primary endpoint, was assessed as of April 22, 2013. Additional endpoints, including overall survival, response rate, and safety/toxicity, were assessed as of December 31, 2013.
Results
Median progression-free survival for those randomized to chemotherapy and selumetinib was 7 (95% CI, 4.3 – 8.4; median treatment duration of 8 weeks (IQR, 4.3–16)) and 15.9 weeks (95% CI, 8.4 – 21.1; median treatment duration of 16.1 weeks (IQR, 8.1–25.3)), respectively (hazard ratio 0.46; 95% CI, 0.30 – 0.71; p < 0.001). Median overall survival was 9.1 (95% CI, 6.1 – 11.1) and 11.8 months (95% CI, 9.8 – 15.7) for those randomized to chemotherapy and selumetinib, respectively (hazard ratio 0.66; 95% CI, 0.41–1.06; p=0.09). No objective responses were observed with chemotherapy. 49% of patients treated with selumetinib achieved tumor regression, with 14% achieving an objective radiographic response to therapy. Treatment-related adverse events were observed in 97% patients treated with selumetinib, with 37% requiring at least one dose reduction.
Conclusions and Relevance
In this hypothesis-generating study of patients with advanced uveal melanoma, selumetinib compared with chemotherapy resulted in a modestly improved progression-free survival and response rate; however, no improvement in overall survival was observed. Improvement in clinical outcomes was accompanied by a high adverse event rate.
doi:10.1001/jama.2014.6096
PMCID: PMC4249701  PMID: 24938562
Uveal; melanoma; MEK; GNAQ; GNA11
14.  Immunologic Response to Xenogeneic gp100 DNA in Melanoma Patients: Comparison of Particle Mediated Epidermal Delivery with Intramuscular Injection 
Purpose
Prior studies show that intramuscular (IM) injection of xenogeneic orthologues of melanosomal antigens (tyrosinase, gp100) induces CD8+ T cell responses to the syngeneic protein. To further define the optimal vaccination strategy, we conducted a pilot clinical trial comparing IM injection with particle-mediated epidermal delivery (PMED).
Experimental Design
Human leukocyte antigen (HLA)-A*0201+ disease-free melanoma patients were randomized to the PMED or IM arm, receiving 8 vaccinations over 4 months. Patients received 4 μg or 2000 μg per injection, respectively, of mouse gp100 DNA. Peripheral blood mononuclear cells (PBMCs) were collected, cultured with gp100 peptides and analyzed by tetramer and intracellular cytokine staining (ICS) for responses to HLA-A*0201-restricted gp100 epitopes [gp100209-217 (ITDQVPFSV) and gp100280-288 (YLEPGPVTA)].
Results
Twenty seven patients with stage IIB-IV melanoma were analyzable for immune response. The only common toxicity was grade I injection site reaction in 9 patients with no intergroup difference, while one dose-limiting toxicity of acute hypersensitivity occurred in a PMED patient with undiagnosed gold allergy. Four of 27 patients produced gp100 tetramer+CD8+ T cells, all carrying the CCR7loCD45RAlo effector-memory phenotype. Five of 27 patients generated interferon-γ+ (IFN-γ) CD8+ T cells, one who was also tetramer-positive. Overall, vaccination induced a response in 30% of patients, which was not significantly associated with study arm or clinical outcome. However, the PMED group showed a trend toward increased IFN-γ+CD8+ T cell generation (p=0.07).
Conclusion
A comparable efficacy and safety profile was demonstrated between the IM and PMED arms, despite a significantly decreased dose of DNA used for PMED injection.
doi:10.1158/1078-0432.CCR-10-1093
PMCID: PMC4241567  PMID: 20647477
gp100; DNA; vaccine; PMED; melanoma
15.  The Treatment of Hypertension During Pregnancy: When Should Blood Pressure Medications Be Started? 
Current cardiology reports  2013;15(11):10.1007/s11886-013-0412-0.
Hypertensive pregnancy disorders (HPD) are important causes of maternal and fetal morbidity and mortality worldwide. In addition, a history of HPD has been associated with an increased risk for maternal cardiovascular disease later in life, possibly due to irreversible vascular and metabolic changes that persist beyond the affected pregnancies. Therefore, treatment of HPD may not only improve immediate pregnancy outcomes, but also the maternal long-term cardiovascular health. Unlike the recommendations for hypertension treatment in the general population, treatment recommendations of HPD have not changed substantially for more than two decades. This is particularly true for mild to moderate hypertension in pregnancy, defined as a blood pressure of 140–159/90–109 mm Hg.
This review focuses on the goals of therapy, treatment strategies, and new developments in the field of HPD that should be taken into account when considering blood pressure targets and pharmacological options for treatment of hypertension in pregnant women.
doi:10.1007/s11886-013-0412-0
PMCID: PMC3859314  PMID: 24057769
Hypertension; Treatment; Pregnancy induced; Antihypertensive agents; Cardiovascular diseases in women; Blood pressure medications
16.  Prenatal Polycyclic Aromatic Hydrocarbon, Adiposity, Peroxisome Proliferator-Activated Receptor (PPAR) γ Methylation in Offspring, Grand-Offspring Mice 
PLoS ONE  2014;9(10):e110706.
Rationale
Greater levels of prenatal exposure to polycyclic aromatic hydrocarbon (PAH) have been associated with childhood obesity in epidemiological studies. However, the underlying mechanisms are unclear.
Objectives
We hypothesized that prenatal PAH over-exposure during gestation would lead to weight gain and increased fat mass in offspring and grand-offspring mice. Further, we hypothesized that altered adipose gene expression and DNA methylation in genes important to adipocyte differentiation would be affected.
Materials and Methods
Pregnant dams were exposed to a nebulized PAH mixture versus negative control aerosol 5 days a week, for 3 weeks. Body weight was recorded from postnatal day (PND) 21 through PND60. Body composition, adipose cell size, gene expression of peroxisome proliferator-activated receptor (PPAR) γ, CCAAT/enhancer-binding proteins (C/EBP) α, cyclooxygenase (Cox)-2, fatty acid synthase (FAS) and adiponectin, and DNA methylation of PPAR γ, were assayed in both the offspring and grand-offspring adipose tissue.
Findings
Offspring of dams exposed to greater PAH during gestation had increased weight, fat mass, as well as higher gene expression of PPAR γ, C/EBP α, Cox2, FAS and adiponectin and lower DNA methylation of PPAR γ. Similar differences in phenotype and DNA methylation extended through the grand-offspring mice.
Conclusions
Greater prenatal PAH exposure was associated with increased weight, fat mass, adipose gene expression and epigenetic changes in progeny.
doi:10.1371/journal.pone.0110706
PMCID: PMC4210202  PMID: 25347678
17.  Lysosomal drug sequestration as a mechanism of drug resistance in vascular sarcoma cells marked by high CSF-1R expression 
Vascular Cell  2014;6:20.
Background
Human angiosarcoma and canine hemangiosarcoma are thought to arise from vascular tissue or vascular forming cells based upon their histological appearance. However, recent evidence indicates a hematopoietic or angioblastic cell of origin for these tumors. In support of this idea, we previously identified an endothelial-myeloid progenitor cell population with high expression of endothelial cell markers and the myeloid cell marker, colony stimulating factor 1 receptor (CSF-1R). Here, we further characterized these cells to better understand how their cellular characteristics may impact current therapeutic applications.
Methods
We performed cell enrichment studies from canine hemangiosarcoma and human angiosarcoma cell lines to generate cell populations with high or low CSF-1R expression. We then utilized flow cytometry, side population and cell viability assays, and fluorescence based approaches to elucidate drug resistance mechanisms and to determine the expression of hematopoietic and endothelial progenitor cell markers.
Results
We demonstrated that cells with high CSF-1R expression enriched from hemangiosarcoma and angiosarcoma cell lines are more drug resistant than cells with little or no CSF-1R expression. We determined that the increased drug resistance may be due to increased ABC transporter expression in hemangiosarcoma and increased drug sequestration within cellular lysosomes in both hemangiosarcoma and angiosarcoma.
Conclusions
We identified drug sequestration within cellular lysosomes as a shared drug resistance mechanism in human and canine vascular sarcomas marked by high CSF-1R expression. Taken together, our results demonstrate that studies in highly prevalent canine hemangiosarcoma may be especially relevant to understanding and addressing drug resistance mechanisms in both the canine and human forms of this disease.
doi:10.1186/2045-824X-6-20
PMCID: PMC4188569  PMID: 25295160
Angiosarcoma; Chemoresistance; CSF-1R; Endothelial; Hemangiosarcoma; Lysosome; Myeloid
18.  Identification of a Loss-of-Function Mutation in Ube2l6 Associated With Obesity Resistance 
Diabetes  2013;62(8):2784-2795.
We previously mapped a locus on BALB/c chromosome 2 associated with protection from leptin-deficiency–induced obesity. Here, we generated the corresponding congenic mouse strain by introgression of a segment of C57BL/6J chromosome 2 to the BALB/c background to confirm the genotype–phenotype associations. We found that the BALB/c alleles decreased fat mass expansion by limiting adipocyte hyperplasia and adipocyte hypertrophy. This was concomitant to an increase in adipocyte triglyceride lipase (ATGL)-mediated triglyceride breakdown and prolongation of ATGL half-life in adipose tissue. In addition, BALB/c alleles on chromosome 2 exerted a cell-autonomous role in restraining the adipogenic potential of preadipocytes. Within a 9.8-Mb critical interval, we identified a nonsynonymous coding single nucleotide polymorphism in the gene coding for the ubiquitin-conjugating enzyme E2L6 (Ube2l6, also known as Ubch8) and showed that the BALB/c allele of Ube2l6 is a hypomorph leading to the lack of UBE2L6 protein expression. Ube2l6 knockdown in 3T3-L1 adipocytes repressed adipogenesis. Thus, altered adipogenic potential caused by Ube2l6 knockdown is likely critically involved in BALB/c obesity resistance by inhibiting adipogenesis and reducing adipocyte numbers. Overall, we have identified a loss-of-function mutation in Ube2l6 that contributes to the chromosome 2 obesity quantitative trait locus.
doi:10.2337/db12-1054
PMCID: PMC3717837  PMID: 23557705
19.  The Role of Plasma Renin Activity, Age, and Race in Selecting Effective Initial Drug Therapy for Hypertension 
American Journal of Hypertension  2013;26(8):957-964.
BACKGROUND
Strategies for initial drug therapy of hypertension are a thiazide diuretic for all or drug selection based on age/race criteria or on plasma renin activity (PRA). It is uncertain which of these strategies will achieve the highest control rate among patients with stage 1 essential hypertension. We sought to compare control rates among 3 drug selection strategies: (i) thiazide diuretic for all, (ii) thiazide diuretic for all black subjects and white subjects aged ≥50 years and a renin-angiotensin system blocker for white subjects aged <50 years, or (iii) thiazide diuretic for PRA < 0.6ng/ml/h (suppressed PRA) and a renin-angiotensin system blocker for PRA ≥ 0.6ng/ml/h (nonsuppressed PRA).
METHODS
Blood pressure responses from the Genetic Epidemiology of Responses to Antihypertensives (GERA) study were used to determine control rates for each of the 3 strategies. In GERA, hypertensive adults were treated with hydrochlorothiazide (n = 286 black subjects and 284 white subjects) or with candesartan (n = 248 black subjects and 278 white subjects).
RESULTS
In the overall sample, the PRA strategy was associated with the highest control rate of 69.4% vs. 61.3% with the age/race strategy (P < 0.001) and 53.8% with the thiazide for all strategy (P < 0.001). This was also true in each racial subgroup (in black subjects: 62.1% vs. 55.2% for the other 2 strategies, P = 0.02; in white subjects: 76.3% vs. 67.1% with the age/race strategy (P < 0.001) and 52.4% with the thiazide for all strategy (P < 0.001)).
CONCLUSIONS
This exploratory analysis suggests that choice of initial therapy for hypertension using a PRA strategy may be associated with higher control rates than alternative strategies recommended in current guidelines.
doi:10.1093/ajh/hpt047
PMCID: PMC3816320  PMID: 23591988
blood pressure; control rate; hypertension; pharmacotherapy.
20.  Genomic Association Analysis of Common Variants Influencing Antihypertensive Response to Hydrochlorothiazide 
Hypertension  2013;62(2):391-397.
To identify novel genes influencing blood pressure response to thiazide diuretic therapy for hypertension, we conducted genome-wide association meta-analyses of ≈1.1 million single nucleotide polymorphisms in a combined sample of 424 European Americans with primary hypertension treated with hydrochlorothiazide from the Pharmacogenomic Evaluation of Antihypertensive Responses Study (N=228) and the Genetic Epidemiology of Responses to Antihypertensive Study (N=196). Polymorphisms associated with blood pressure response at p<10-5 were tested for replication of the associations in independent samples of hydrochlorothiazide-treated European hypertensives. The rs16960228 polymorphism in protein kinase C, alpha replicated for same-direction association with diastolic blood pressure response in the Nordic Diltiazem Study (N=420) and the Genetics of Drug Responsiveness in Essential Hypertension Study (N=206), and the combined four-study meta-analysis p-value achieved genome-wide significance (p=3.3 × 10-8). Systolic/diastolic blood pressure responses were consistently greater in carriers of the rs16960228 A allele than in GG homozygotes (4/4 mmHg greater) across study samples. The rs2273359 polymorphism in the GNAS-EDN3 region also replicated for same-direction association with systolic blood pressure response in the Nordic Diltiazem Study, and the combined three-study meta-analysis p-value approached genome-wide significance (p=5.5 × 10-8). The findings document clinically-important effects of genetic variation at novel loci on blood pressure response to a thiazide diuretic, which may be a basis for individualization of antihypertensive drug therapy and identification of new drug targets.
doi:10.1161/HYPERTENSIONAHA.111.00436
PMCID: PMC3780966  PMID: 23753411
Hypertension; high blood pressure; antihypertensive therapy/diuretics; hydrochlorothiazide; genomics; pharmacogenomics; protein kinase C
21.  A Phase 2 Study of Flavopiridol (Alvocidib) in Combination with Docetaxel in Refractory, Metastatic Pancreatic Cancer (NCI#6366) 
Background/Aims
Pancreatic adenocarcinoma (PC) harbors frequent alterations of p16, resulting in cell cycle dysregulation. A phase I study of docetaxel and flavopiridol, a pan-cyclin dependent kinase inhibitor, demonstrated encouraging clinical activity in PC. This phase II study was designed to further define the efficacy and toxicity of this regimen in patients with previously treated PC.
Methods
Patients with gemcitabine-refractory, metastatic PC were treated with docetaxel 35 mg/m2 followed by flavopiridol 80 mg/m2 on days 1, 8, and 15 of a 28 day cycle. Tumor measurements were performed every two cycles. A Simon two-stage design was used to evaluate the primary endpoint of response.
Results
Ten patients were enrolled; nine were evaluable for response. No objective responses were observed; however, three patients (33%) achieved transient stable disease, with one of these patients achieving a 20% reduction in tumor size. Median survival was 4.2 months, with no patients alive at the time of analysis. Adverse events were significant, with seven patients (78%) requiring ≥1 dose reduction for transaminitis (11%), grade 4 neutropenia (33%), grade 3 fatigue (44%), and grade 3 diarrhea (22%)
Conclusions
The combination of flavopiridol and docetaxel has minimal activity and significant toxicity in this patient population. These results reflect the challenges of treating patients with PC in a second-line setting where the risk/benefit equation is tightly balanced.
doi:10.1159/000187135
PMCID: PMC4053191  PMID: 19451750
Docetaxel; Flavopiridol; Pancreatic adenocarcinoma
22.  Phase II Trial of the CDK4 Inhibitor PD0332991 in Patients With Advanced CDK4-Amplified Well-Differentiated or Dedifferentiated Liposarcoma 
Journal of Clinical Oncology  2013;31(16):2024-2028.
Purpose
CDK4 is amplified in > 90% of well-differentiated (WDLS) and dedifferentiated liposarcomas (DDLS). The selective cyclin-dependent kinase 4 (CDK4)/CDK6 inhibitor PD0332991 inhibits growth and induces senescence in cell lines and xenografts. In a phase I trial of PD0332991, several patients with WDLS or DDLS experienced prolonged stable disease. We performed an open-label phase II study to determine the safety and efficacy of PD0332991 in patients with advanced WDLS/DDLS.
Patients and Methods
Patients age ≥ 18 years experiencing disease progression while receiving systemic therapy before enrollment received PD0332991 200 mg orally once per day for 14 consecutive days in 21-day cycles. All were required to have CDK4 amplification by fluorescence in situ hybridization and retinoblastoma protein (RB) expression by immunohistochemistry (≥ 1+). The primary end point was progression-free survival (PFS) at 12 weeks, with 12-week PFS of ≥ 40% considered promising and ≤ 20% not promising. If ≥ nine of 28 patients were progression free at 12 weeks, PD0332991 would be considered active.
Results
We screened 48 patients (44 of 48 had CDK4 amplification; 41 of 44 were RB positive). Of those, 30 were enrolled, and 29 were evaluable for the primary end point. Grade 3 to 4 events included anemia (17%), thrombocytopenia (30%), neutropenia (50%), and febrile neutropenia (3%). At 12 weeks, PFS was 66% (90% CI, 51% to 100%), significantly exceeding the primary end point. The median PFS was 18 weeks. There was one partial response.
Conclusion
Treatment with the CDK4 inhibitor PD0332991 was associated with a favorable progression-free rate in patients with CDK4-amplified and RB-expressing WDLS/DDLS who had progressive disease despite systemic therapy.
doi:10.1200/JCO.2012.46.5476
PMCID: PMC3661937  PMID: 23569312
23.  Oral paricalcitol (19-nor-1,25-dihydroxyvitamin D2) in women receiving chemotherapy for metastatic breast cancer 
Cancer Biology & Therapy  2013;14(6):476-480.
The vitamin D hormone, [1,25(OH)2D, calcitriol], inhibits proliferation and angiogenesis in breast cancer but its therapeutic use is limited by hypercalcemia. Synthetic analogs of 1,25(OH)2D that are less calcemic, such as paricalcitol (19-nor-1,25-Dihydroxyvitamin D2), are used to treat hyperparathyroidism associated with chronic kidney disease. We sought to determine the safety and feasibility of taking oral paricalcitol with taxane-based chemotherapy in women with metastatic breast cancer (MBC). Oral paricalcitol was considered safe if it did not result in excessive toxicity, defined as grade 3 or higher serum calcium levels. It was considered feasible if the majority of women could take eight weeks of continuous therapy in the first three months. Serum calcium was monitored weekly and the paricalcitol dose was adjusted based on its calcemic effect. Intact parathyroid hormone (iPTH) was monitored as a marker of paricalcitol activity. Twenty-four women with MBC were enrolled. Twenty women (83%) received eight weeks of continuous therapy. Paricalcitol was well-tolerated with no instances of hypercalcemia grade 2 or greater. Fourteen women (54%) were able to escalate the dose. The dose range of paricalcitol in the first 3 mo was 2–7 ug/day. Serum iPTH levels at baseline were significantly higher in women with serum 25-Hydroxyvitamin D (25-OHD) levels less than 30 ng/ml (96.4 ± 40.9 pg/ml) vs. 46.2 ± 20.3 pg/ml (p = 0 0.001) (iPTH reference 12–72 pg/ml). We conclude that paricalcitol is safe and feasible in women with MBC who are receiving chemotherapy.
doi:10.4161/cbt.24350
PMCID: PMC3813563  PMID: 23760489
vitamin D; metastatic breast cancer; paricalcitol; cancer treatment
24.  Ipilimumab for Patients With Advanced Mucosal Melanoma 
The Oncologist  2013;18(6):726-732.
This multicenter, retrospective analysis assessed the efficacy and safety of ipilimumab in 33 patients with unresectable or metastatic mucosal melanoma. The study provides evidence that ipilimumab can result in durable antitumor effects in a subset of patients with mucosal melanoma, although the response rate was low.
The outcome of patients with mucosal melanoma treated with ipilimumab is not defined. To assess the efficacy and safety of ipilimumab in this melanoma subset, we performed a multicenter, retrospective analysis of 33 patients with unresectable or metastatic mucosal melanoma treated with ipilimumab. The clinical characteristics, treatments, toxicities, radiographic assessment of disease burden by central radiology review at each site, and mutational profiles of the patients' tumors were recorded. Available peripheral blood samples were used to assess humoral immunity against a panel of cancer-testis antigens and other antigens. By the immune-related response criteria of the 30 patients who underwent radiographic assessment after ipilimumab at approximately week 12, there were 1 immune-related complete response, 1 immune-related partial response, 6 immune-related stable disease, and 22 immune-related progressive disease. By the modified World Health Organization criteria, there were 1 immune-related complete response, 1 immune-related partial response, 5 immune-related stable disease, and 23 immune-related progressive disease. Immune-related adverse events (as graded by Common Terminology Criteria for Adverse Events version 4.0) consisted of six patients with rash (four grade 1, two grade 2), three patients with diarrhea (one grade 1, two grade 3), one patient with grade 1 thyroiditis, one patient with grade 3 hepatitis, and 1 patient with grade 2 hypophysitis. The median overall survival from the time of the first dose of ipilimumab was 6.4 months (range: 1.8–26.7 months). Several patients demonstrated serologic responses to cancer-testis antigens and other antigens. Durable responses to ipilimumab were observed, but the overall response rate was low. Additional investigation is necessary to clarify the role of ipilimumab in patients with mucosal melanoma.
doi:10.1634/theoncologist.2012-0464
PMCID: PMC4063400  PMID: 23716015
Mucosal melanoma; Ipilimumab; CTLA-4; Immunotherapy; Cancer-testis antigens
25.  Post-transcriptional activation of PPAR alpha by KLF6 in hepatic steatosis 
Journal of hepatology  2013;58(5):1000-1006.
Background & Aims
Dysregulated glucose homeostasis and lipid accumulation characterize non-alcoholic fatty liver disease (NAFLD), but underlying mechanisms are obscure. We report here that Krüppel-like factor 6 (KLF6), a ubiquitous transcription factor that promotes adipocyte differentiation, also provokes the metabolic abnormalities of NAFLD by post-transcriptionally activating PPARα-signaling.
Methods
Mice with either hepatocyte-specific depletion of KLF6 (‘DeltaHepKlf6’) or global KLF6 heterozygosity (Klf6 +/−) were fed a high fat diet (HFD) or chow for 8 or 16 weeks. Glucose and insulin tolerance tests were performed to assess insulin sensitivity. Overexpression and knockdown of KLF6 in cultured cells enabled the elucidation of underlying mechanisms. In liver samples from a cohort of 28 NAFLD patients, the expression of KLF6-related target genes was quantified.
Results
Mice with global- or hepatocyte-depletion of KLF6 have reduced body fat content and improved glucose and insulin tolerance, and are protected from HFD-induced steatosis. In hepatocytes, KLF6 deficiency reduces PPARα-regulated genes (Trb3, Pepck) with diminished PPARα-protein but no change in Pparα-mRNA which is explained by the discovery that KLF6 represses miRNA 10b, which leads to induction of PPARα. In NAFLD-patients with advanced disease and inflammation, the expression of miRNA 10b is significantly down-regulated, while PEPCK mRNA is up-regulated; KLF6 mRNA expression also correlates with TRB3 as well as PEPCK gene expression.
Conclusions
KLF6 increases PPAR -activity, whereas KLF6 loss leads to PPARα repression and attenuation of lipid and glucose abnormalities associated with a high fat diet. The findings establish KLF6 as a novel regulator of hepatic glucose and lipid metabolism in fatty liver.
doi:10.1016/j.jhep.2013.01.020
PMCID: PMC3631429  PMID: 23353867
KLF6; PPARα; TRB3; PEPCK; CD36; miRNA-10b; NAFLD

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