Bromodomain and extra-terminal domain (BET) proteins regulate melanocyte differentiation.

BACKGROUND: Pharmacologic inhibition of bromodomain and extra-terminal (BET) proteins is currently being explored as a new therapeutic approach in cancer. Some studies have also implicated BET proteins as regulators of cell identity and differentiation through their interactions with lineage-specific factors. However, the role of BET proteins has not yet been investigated in melanocyte differentiation. Melanocyte inducing transcription factor (MITF) is the master regulator of melanocyte differentiation, essential for pigmentation and melanocyte survival. In this study, we tested the hypothesis that BET proteins regulate melanocyte differentiation through interactions with MITF. RESULTS: Here we show that chemical inhibition of BET proteins prevents differentiation of unpigmented melanoblasts into pigmented melanocytes and results in de-pigmentation of differentiated melanocytes. BET inhibition also slowed cell growth, without causing cell death, increasing the number of cells in G1. Transcriptional profiling revealed that BET inhibition resulted in decreased expression of pigment-specific genes, including many MITF targets. The expression of pigment-specific genes was also down-regulated in melanoma cells, but to a lesser extent. We found that RNAi depletion of the BET family members, bromodomain-containing protein 4 (BRD4) and bromodomain-containing protein 2 (BRD2) inhibited expression of two melanin synthesis enzymes, TYR and TYRP1. Both BRD4 and BRD2 were detected on melanocyte promoters surrounding MITF-binding sites, were associated with open chromatin structure, and promoted MITF binding to these sites. Furthermore, BRD4 and BRD2 physically interacted with MITF. CONCLUSION: These findings indicate a requirement for BET proteins in the regulation of pigmentation and melanocyte differentiation. We identified changes in pigmentation specific gene expression that occur upon BET inhibition in melanoblasts, melanocytes, and melanoma cells.

Interleukin 21 (IL-21) regulates chronic allograft vasculopathy (CAV) in murine heart allograft rejection.

IL-21 is the most recently discovered common gamma-chain cytokine that promotes persistent T-cell responses in chronic infections, autoimmunity and cancer. However, the therapeutic potential of inhibiting the IL-21-BATF signaling axis, particularly in transplant rejection, remains unclear. We used heart transplant models to examine the effects of IL-21 blockade in prevention of chronic cardiac allograft vasculopathy (CAV) using genetic knock-out and therapeutic approaches. Both wild-type C57BL/6 and IL-21-/- strains acutely rejected Balb/c skin grafts and once immunized with this skin graft, rejected Balb/c heart allografts in an accelerated fashion. However, when transplanted with heart grafts from the class-II major histocompatibility complex mutant, B6bm12 mice; wild-type recipients developed CAV, while IL-21-/- recipients were protected, even at day 100 post-transplant. Similarly, BATF-/- recipients, lacking the transcription factor BATF responsible for IL-21 production, did not develop CAV in B6-bm12 heart allografts. Strikingly, in a transient treatment protocol, the development of CAV in wild-type recipients of B6-bm12 hearts allografts was blocked by the administration of IL-21 receptor fusion protein (R-Fc). Thus, we demonstrate that CAV is regulated at least in part by IL-21 signaling and its blockade by genetic approaches or therapy with IL-21R-Fc prevents CAV in mice.

Exposure to the Harmful Algal Bloom (HAB) Toxin Microcystin-LR (MC-LR) Prolongs and Increases Severity of Dextran Sulfate Sodium (DSS)-Induced Colitis.

Inflammatory Bowel Disease (IBD) represents a collection of gastrointestinal disorders resulting from genetic and environmental factors. Microcystin-leucine arginine (MC-LR) is a toxin produced by cyanobacteria during algal blooms and demonstrates bioaccumulation in the intestinal tract following ingestion. Little is known about the impact of MC-LR ingestion in individuals with IBD. In this study, we sought to investigate MC-LR's effects in a dextran sulfate sodium (DSS)-induced colitis model. Mice were separated into four groups: (a) water only (control), (b) DSS followed by water (DSS), (c) water followed by MC-LR (MC-LR), and (d) DSS followed by MC-LR (DSS + MC-LR). DSS resulted in weight loss, splenomegaly, and severe colitis marked by transmural acute inflammation, ulceration, shortened colon length, and bloody stools. DSS + MC-LR mice experienced prolonged weight loss and bloody stools, increased ulceration of colonic mucosa, and shorter colon length as compared with DSS mice. DSS + MC-LR also resulted in greater increases in pro-inflammatory transcripts within colonic tissue (TNF-α, IL-1ß, CD40, MCP-1) and the pro-fibrotic marker, PAI-1, as compared to DSS-only ingestion. These findings demonstrate that MC-LR exposure not only prolongs, but also worsens the severity of pre-existing colitis, strengthening evidence of MC-LR as an under-recognized environmental toxin in vulnerable populations, such as those with IBD.

Estrogen receptor α and aromatase polymorphisms affect risk, prognosis, and therapeutic outcome in men with castration-resistant prostate cancer treated with docetaxel-based therapy.

CONTEXT: Reactive estrogen species cause genotoxicity and interfere with docetaxel-mediated tubulin polymerization resulting in shortened survival in men with castrate-resistant prostate cancer (CRPC). OBJECTIVE: We hypothesized that polymorphisms in estrogen synthesis and estrogen targets (i.e., CYP19 and ERα) would be linked to interindividual variation in CRPC risk, docetaxel response, and overall survival in men with CRPC. MATERIALS AND METHODS: Patients with CRPC (n=115) treated with docetaxel, single-agent thalidomide (n=42), or healthy controls (n=289) were genotyped for the CYP19 R264C (rs700519) and the ERα PvuII T>C (rs2234693) and XbaI A>G (rs9340799) polymorphisms. RESULTS: Patients carrying two copies of ERα polymorphisms had shorter progression-free survival on docetaxel than other patients (median survival difference ≥ 3.1 months; P ≤ 0.036). When the analysis was limited to nonobese patients, the relationship between the ERα XbaI A>G polymorphism and PFS improved (median survival difference = 3.5 months; P = 0.0078). The CYP19 R264C variant was related to the duration of survival after docetaxel in patients who were >70 years old (median survival difference =10.6 months; P=0.041). Both ERα polymorphisms were also associated with increases in CRPC risk [P ≤ 0.032; double variants vs. wild-type odds ratio ≥ 2.6], and the association with the ERα PvuII T>C also improved in those men who were <70 years old (P = 0.0073; odds ratio = 3.0). CONCLUSIONS: This study demonstrates that estrogen-related genetic variation affects docetaxel clinical response and that this relationship is dependent on age and body-type in men with CRPC. Moreover, this study suggests ERα polymorphisms confer risk of developing prostate cancer, especially in men under 70 years of age.

A SNP in CYP2C8 is not associated with the development of bisphosphonate-related osteonecrosis of the jaw in men with castrate-resistant prostate cancer.

A single nucleotide polymorphism (SNP) in CYP2C8 (rs1934951), was previously identified in a genome-wide association study as a risk factor for the development of osteonecrosis of the jaw (ONJ) in patients receiving bisphosphonates (BPs) for multiple myeloma. To determine if the same SNP is also associated with the development of ONJ in men receiving BPs for bone metastases from prostate cancer, we genotyped 100 men with castrate-resistant prostate cancer treated with bisphosphonates for bone metastases, 17 of whom developed ONJ. Important clinical characteristics, including type and duration of bisphosphonate therapy, were consistent among those who developed ONJ and those who did not. We found no significant correlation between the variant allele and the development of ONJ (OR = 0.63, 95% CI: 0.165-2.42, P > 0.47). This intronic SNP in CYP2C8 (rs1934951) does not seem to be a risk factor for the development of bisphosphonate-related ONJ in men with prostate cancer. It is important to note that this is only the second study to investigate the genetics associated with BP-related ONJ and the first to do so in men with prostate cancer. More studies are needed to identify genetic risk factors that may predict the development of this important clinical condition.

Androgen receptor CAG repeat length and association with prostate cancer risk: results from the prostate cancer prevention trial.

PURPOSE: We investigated the association between the length of the polymorphic trinucleotide CAG microsatellite repeats in exon 1 of the AR gene and the risk of prostate cancer. MATERIALS AND METHODS: This is a nested case-control study of 1,159 cases and 1,353 controls from the Prostate Cancer Prevention Trial, a randomized, placebo controlled trial testing whether the 5α-reductase inhibitor finasteride could decrease the 7-year prevalence of prostate cancer. During the course of the trial men underwent annual digital rectal examination and prostate specific antigen measurement. Prostate biopsy was recommended in all men with abnormal digital rectal examination or finasteride adjusted prostate specific antigen greater than 4.0 ng/ml. Cases were drawn from men with biopsy determined prostate cancer identified by for cause or end of study biopsy. Controls were selected from men who completed the end of study biopsy. RESULTS: Mean CAG repeat length did not differ between cases and controls. The frequency distribution of cases and controls for the AR CAG repeat length was similar. There were no significant associations of CAG repeat length with prostate cancer risk when stratified by treatment arm (finasteride or placebo), or when combined. There was also no significant association between CAG repeat length and the risk of low or high grade prostate cancer. CONCLUSIONS: There is no association of AR CAG repeat length with prostate cancer risk. Knowledge of AR CAG repeat length provides no clinically useful information to predict prostate cancer risk.

Hypertension and hand-foot skin reactions related to VEGFR2 genotype and improved clinical outcome following bevacizumab and sorafenib.

BACKGROUND: Hypertension (HT) and hand-foot skin reactions (HFSR) may be related to the activity of bevacizumab and sorafenib. We hypothesized that these toxicities would correspond to favorable outcome in these drugs, that HT and HFSR would coincide, and that VEGFR2 genotypic variation would be related to toxicity and clinical outcomes. METHODS: Toxicities (> or = grade 2 HT or HFSR), progression-free survival (PFS), and overall survival (OS) following treatment initiation were evaluated. Toxicity incidence and VEGFR2 H472Q and V297I status were compared to clinical outcomes. RESULTS: Individuals experiencing HT had longer PFS following bevacizumab therapy than those without this toxicity in trials utilizing bevacizumab in patients with prostate cancer (31.5 vs 14.9 months, n = 60, P = 0.0009), and bevacizumab and sorafenib in patients with solid tumors (11.9 vs. 3.7 months, n = 27, P = 0.052). HT was also linked to a > 5-fold OS benefit after sorafenib and bevacizumab cotherapy (5.7 versus 29.0 months, P = 0.0068). HFSR was a marker for prolonged PFS during sorafenib therapy (6.1 versus 3.7 months respectively, n = 113, P = 0.0003). HT was a risk factor for HFSR in patients treated with bevacizumab and/or sorafenib (OR(95%CI) = 3.2(1.5-6.8), P = 0.0024). Carriers of variant alleles at VEGFR2 H472Q experienced greater risk of developing HT (OR(95%CI) = 2.3(1.2 - 4.6), n = 170, P = 0.0154) and HFSR (OR(95%CI) = 2.7(1.3 - 5.6), n = 170, P = 0.0136). CONCLUSIONS: This study suggests that HT and HFSR may be markers for favorable clinical outcome, HT development may be a marker for HFSR, and VEGFR2 alleles may be related to the development of toxicities during therapy with bevacizumab and/or sorafenib.

Androgen receptor sequence and variations in several common prostate cancer cell lines.

The androgen receptor gene (AR) plays an important role in molecular signaling and regulation and the subsequent cellular growth of prostate cancer. In addition, it is a highly variable region of the genome. We used direct nucleotide sequencing to genotype the entire exogenous coding region of the androgen receptor in ten commonly used prostate cancer cell lines. Our analysis confirmed the presence or absence of several known SNPs in the cell lines studied. We also assayed the number of CAG-repeat and GGC-repeat sequences for each for the ten cell lines. Our analysis identified three new mutations, one each in the DU145, LnCAP and RWPE-2 cell lines. In DU145, the DNA isolated in our lab was heterozygous at G527G (T>C transition), a polymorphism not previously reported. The LnCAP cells cultured in our lab were found to have a T>C transition (heterozygous), resulting in a S641P change that was not present in the ATCC cell line DNA. Lastly, a homozygous G>T transversion was found in RWPE-2 cells, resulting in the S187I change. This is potentially significant for use in cell culture and future cell model development.

Pharmacogenetics of membrane transporters: an update on current approaches.

This review provides an overview of the pharmacogenetics of membrane transporters including selected ABC transporters (ABCB1, ABCC1, ABCC2, and ABCG2) and OATPs (OATP1B1 and OATP1B3). Membrane transporters are heavily involved in drug clearance and alters drug disposition by actively transporting substrate drugs between organs and tissues. As such, polymorphisms in the genes encoding these proteins may have significant effects on the absorption, distribution, metabolism and excretion of compounds, and may alter pharmacodynamics of many agents. This review discusses the techniques used to identify substrates and inhibitors of these proteins and subsequently to assess the effect of genetic mutation on transport, both in vitro and in vivo. A comprehensive list of substrates for the major drug transporters is included. Finally, studies linking transporter genotype with clinical outcomes are discussed.

Population pharmacokinetics of romidepsin in patients with cutaneous T-cell lymphoma and relapsed peripheral T-cell lymphoma.

PURPOSE: Romidepsin is a potent histone deacetylase inhibitor under clinical development. The objective of this study was to evaluate the effect of demographic, clinical, and pharmacogenetic covariates on the pharmacokinetics of romidepsin in patients with T-cell lymphoma. EXPERIMENTAL DESIGN: Pharmacokinetic assessment was done in 98 patients enrolled in a phase II study who received 14 or 18 mg/m2 of romidepsin as a 4-hour infusion on day 1 during their first treatment cycle. Population modeling was done using a nonlinear mixed effects modeling approach to explore the effects of polymorphic variations in CYP3A4, CYP3A5, SLCO1B3, and ABCB1, all of which encode genes thought to be involved in romidepsin disposition. RESULTS: A two-compartment model with linear kinetics adequately described the romidepsin disposition. Population clearance was 15.9 L/h with between-patient variability of 37%. ABCB1 2677G>T/A variant alleles tended toward a reduced clearance and lower volume of tissue distribution, but this was not supported by a statistical significance. Genetic variations in CYP3A4/5 and SCLO1B3 had no effect on the systemic exposure. CONCLUSION: The population pharmacokinetic analysis indicates moderate interindividual variability in romidepsin pharmacokinetics and no clinically relevant covariates associated with the unexplained pharmacokinetic variability of romidepsin in this population.

ABCB1 genetic variation influences the toxicity and clinical outcome of patients with androgen-independent prostate cancer treated with docetaxel.

PURPOSE: Polymorphisms that are associated with ABCB1 expression and function may be linked to treatment efficacy and the development of neutropenia and neurotoxicity in patients with androgen-independent prostate cancer receiving docetaxel. EXPERIMENTAL DESIGN: Patients with androgen-independent prostate cancer treated with docetaxel alone (n = 23) or docetaxel and thalidomide (n = 50) were genotyped for the ABCB1 1236C>T, 2677 G>T/A, and 3435 C>T alleles by direct sequencing, and diplotypes were constructed using an EM algorithm. The data were then compared with duration to onset of peripheral neuropathy, neutropenia grade, and survival after docetaxel. RESULTS: For patients receiving docetaxel alone, individuals carrying a diplotype consisting of the 1236C-2677G-3435C linked alleles had improved overall survival after treatment (P = 0.0017). Additionally, patients treated with docetaxel and thalidomide carrying a diplotype consisting of the 2677T-3435T haplotype had a shorter median survival (P = 0.045). After adjusting for a particular set of polymorphisms and diplotype groupings, a hazard ratio of 10.87 was found for patients carrying the 2677GG genotype versus patients carrying other genotypes (P = 0.0048) in the docetaxel and thalidomide cohort. Among both treatment arms together, individuals carrying the 2677GG genotype also had a significantly longer time to neuropathy (P = 0.035). Finally, there was a strong trend toward patients carrying the 2677TT-3435TT diplotype having higher grades of neutropenia (P = 0.053). CONCLUSION: The data suggest that docetaxel-induced neuropathy, neutropenia grade, and overall survival could be linked to ABCB1 allelic variants with ensuing negative implications for docetaxel treatment in patients carrying ABCB1 variant genotypes.