Role of cereblon in angiogenesis and in mediating the antiangiogenic activity of immunomodulatory drugs.

Cereblon (CRBN) is a substrate recruiter element of the E3 cullin 4-RING ubiquitin ligase complex, and a binding target of immunomodulatory agents (IMiDs). CRBN is responsible for the pleiotropic effects of IMiDs, yet its function in angiogenesis and in mediating the antiangiogenic effects of IMiDs remains unclear. We investigated the role of CRBN in the angiogenic process and in propagating the antiangiogenic effects of IMiDs in vitro. siRNA-mediated CRBN knock down in human endothelial cells (HUVEC and HMVEC-L), did not affect endothelial cell proliferation, migration, or tube formation. Using CRBN-deficient mice, we further demonstrated that microvessal formation can occur independently of cereblon in the ex vivo mouse aortic ring model. The cereblon E3 ubiquitin ligase complex can recruit endothelial cell-specific factors, AGO2 (associated with angiogenesis), and SALL4 (associated with embryogenesis/angiogenesis), for ubiquitin-mediated degradation. Knockdown of CRBN caused a corresponding increase in AGO2 and SALL4 protein expression and IMiD treatment was able to rescue the siCRBN effect to increase the CRBN expression. These findings suggest one potential mechanism of action that likely involves a tightly coordinated regulation of CRBN with endothelial cell targets and highlight the need to further elucidate the mechanism(s), which could include cereblon-independent pathways, through which IMiDs exert their antiangiogenic effects.

Association between immunosuppressive cytokines and PSA progression in biochemically recurrent prostate cancer treated with intermittent hormonal therapy.

BACKGROUND: Immunosuppressive cytokines have the potential to promote prostate cancer progression. Assessing their longitudinal changes may implicate mechanisms of progression, treatment resistance, and suggest new therapeutic targets. METHODS: Thirty-seven men with biochemically recurrent (BCR) prostate cancer who received 6 months of androgen deprivation therapy (ADT) and were monitored until the time to prostate-specific antigen progression (TTPP) were identified from a completed phase III trial (NCT00020085). Serum samples were archived at baseline, 3 months after ADT, and at TTPP. Cytokine concentrations were quantified using a 36-parameter electrochemiluminescence assay. The Wilcoxon signed-rank sum test was used to compare observations between time points. Kaplan-Meier analysis was used to calculate TTPP dichotomized by cytokine values above or below the median. Pearson's rank correlation coefficient was used to compare continuous variables. RESULTS: Median TTPP was 399 days (range, 114-1641). Median prostate-specific antigen (PSA) at baseline and progression were 8.5 and 5.3 ng/mL, respectively. Twenty-three patients (62%) achieved undetectable PSA with ADT. Castrate levels of testosterone (<50 ng/dL) after 3 months of ADT occurred in 35 patients (95%). TNF-α (P = .002), IL-23 (P = .002), and CXCL10 (P = .001) significantly increased from baseline to post ADT. Certain cytokines correlated longitudinally: TNF-α correlated with IL-23 (r = .72; P < .001) and IL-8 (r = .59; P < .001) from baseline to post ADT and to PSA progression. Neutrophil-to-lymphocyte ratio correlated with IL-27 (r = .57; P < .001) and MIP-3α (r = .56; P < .001). Patients with a detectable PSA after ADT had elevated levels of IL-6 (P = .049) and IL-8 (P = .013) at PSA progression as compared with those with an undetectable PSA. There was a trend toward shorter TTPP in patients with TNF-α levels above the median (P = .042). CONCLUSIONS: Several innate cytokines were associated with biochemically recurrent prostate cancer.

Severe Hepatotoxicity of Mithramycin Therapy Caused by Altered Expression of Hepatocellular Bile Transporters.

Mithramycin demonstrates preclinical anticancer activity, but its therapeutic dose is limited by the development of hepatotoxicity that remains poorly characterized. A pharmacogenomics characterization of mithramycin-induced transaminitis revealed that hepatotoxicity is associated with germline variants in genes involved in bile disposition: ABCB4 (multidrug resistance 3) rs2302387 and ABCB11 [bile salt export pump (BSEP)] rs4668115 reduce transporter expression (P < 0.05) and were associated with ≥grade 3 transaminitis developing 24 hours after the third infusion of mithramycin (25 mcg/kg, 6 hours/infusion, every day ×7, every 28 days; P < 0.0040). A similar relationship was observed in a pediatric cohort. We therefore undertook to characterize the mechanism of mithramycin-induced acute transaminitis. As mithramycin affects cellular response to bile acid treatment by altering the expression of multiple bile transporters (e.g., ABCB4, ABCB11, sodium/taurocholate cotransporting polypeptide, organic solute transporter α/ß) in several cell lines [Huh7, HepaRG, HepaRG BSEP (-/-)] and primary human hepatocytes, we hypothesized that mithramycin inhibited bile-mediated activation of the farnesoid X receptor (FXR). FXR was downregulated in all hepatocyte cell lines and primary human hepatocytes (P < 0.0001), and mithramycin inhibited chenodeoxycholic acid- and GW4046-induced FXR-galactose-induced gene 4 luciferase reporter activity (P < 0.001). Mithramycin promoted glycochenodeoxycholic acid-induced cytotoxicity in ABCB11 (-/-) cells and increased the overall intracellular concentration of bile acids in primary human hepatocytes grown in sandwich culture (P < 0.01). Mithramycin is a FXR expression and FXR transactivation inhibitor that inhibits bile flow and potentiates bile-induced cellular toxicity, particularly in cells with low ABCB11 function. These results suggest that mithramycin causes hepatotoxicity through derangement of bile acid disposition; results also suggest that pharmacogenomic markers may be useful to identify patients who may tolerate higher mithramycin doses. SIGNIFICANCE STATEMENT: The present study characterizes a novel mechanism of drug-induced hepatotoxicity in which mithramycin not only alters farnesoid X receptor (FXR) and small heterodimer partner gene expression but also inhibits bile acid binding to FXR, resulting in deregulation of cellular bile homeostasis. Two novel single-nucleotide polymorphisms in bile flow transporters are associated with mithramycin-induced liver function test elevations, and the present results are the rationale for a genotype-directed clinical trial using mithramycin in patients with thoracic malignancies.

Preclinical Evaluation of Discorhabdins in Antiangiogenic and Antitumor Models.

Elements of the hypoxia inducible factor (HIF) transcriptional system, a key regulator of the cellular hypoxic response, are up-regulated in a range of cancer cells. HIF is fundamentally involved in tumor angiogenesis, invasion, and energy metabolism. Inhibition of the transcriptional activity of HIF may be of therapeutic benefit to cancer patients. We recently described the identification of two marine pyrroloiminoquinone alkaloids with potent activity in inhibiting the interaction between the oncogenic transcription factor HIF-1α and the coactivator protein p300. Herein, we present further characterization data for these two screening hits: discorhabdin H (1) and discorhabdin L (2), with a specific focus on their anti-angiogenic and anti-tumor effects. We demonstrated that only discorhabdin L (2) possesses excellent anti-angiogenic activity in inhibiting endothelial cell proliferation and tube formation, as well as decreasing microvessel outgrowth in the ex vivo rat aortic ring assay. We further showed that discorhabdin L (2) significantly inhibits in vivo prostate tumor growth in a LNCaP xenograft model. In conclusion, our findings suggest that discorhabdin L (2) represents a promising HIF-1α inhibitor worthy of further drug development.

Detection of Extracellular Vesicle-Derived RNA as Potential Prostate Cancer Biomarkers: Role of Cancer-type SLCO1B3 and ABCC3.

Extracellular vesicles (EVs) provide a minimally invasive liquid biopsy source of tumor-specific markers for patients who have already undergone prostatectomies. Our laboratory has previously demonstrated enrichment of the cancer-type solute carrier organic anion transporter family 1B3 (ct-SLCO1B3) and the ATP Binding Cassette Subfamily Member C (ABCC3) in castration-resistant cell lines (CRPC). However, their expression in EVs has yet to be explored. Our study demonstrated that ct-SLCO1B3 and ABCC3 are highly detectable in CRPC cell line-derived EVs. We also showed that ct-SLCO1B3 and ABCC3 were detectable in a CRPC xenograft mouse model, both intratumorally and in plasma-derived EVs. Our results provide evidence for EV-contained ct-SLCO1B3 and ABCC3 as novel, EV-based tumor markers for prostate cancer progression.

Expansion of neurofilament medium C terminus increases axonal diameter independent of increases in conduction velocity or myelin thickness.

Maturation of the peripheral nervous system requires specification of axonal diameter, which, in turn, has a significant influence on nerve conduction velocity. Radial axonal growth initiates with myelination, and is dependent upon the C terminus of neurofilament medium (NF-M). Molecular phylogenetic analysis in mammals suggested that expanded NF-M C termini correlated with larger-diameter axons. We used gene targeting and computational modeling to test this new hypothesis. Increasing the length of NF-M C terminus in mice increased diameter of motor axons without altering neurofilament subunit stoichiometry. Computational modeling predicted that an expanded NF-M C terminus extended farther from the neurofilament core independent of lysine-serine-proline (KSP) phosphorylation. However, expansion of NF-M C terminus did not affect the distance between adjacent neurofilaments. Increased axonal diameter did not increase conduction velocity, possibly due to a failure to increase myelin thickness by the same proportion. Failure of myelin to compensate for larger axonal diameters suggested a lack of plasticity during the processes of myelination and radial axonal growth.

Antitumor Activity of NLG207 (Formerly CRLX101) in Combination with Enzalutamide in Preclinical Prostate Cancer Models.

Effective treatments for patients with metastatic castration-resistant prostate cancer following disease progression on enzalutamide are currently an unmet clinical need. Simultaneous inhibition of the hypoxia-inducible factor (HIF)-1α and androgen receptor (AR) pathways has been previously shown to overcome enzalutamide resistance in vitro Combination treatment with NLG207, a nanoparticle-drug conjugate of camptothecin and inhibitor of HIF-1α, and enzalutamide was evaluated in preclinical prostate cancer models of enzalutamide resistance. The effect of NLG207 and enzalutamide on average tumor volume and tumor re-growth after 3 weeks of treatment was evaluated in vivo using the subcutaneous 22Rv1 xenograft and castrated subcutaneous VCaP xenograft models. Correlative assessments of antitumor activity were evaluated in vitro using cell proliferation and qPCR assays. NLG207 8 mg/kg alone and in combination with enzalutamide reduced average tumor volume by 93% after 3 weeks of treatment (P < 0.05) in comparison with vehicle control in the subcutaneous 22Rv1 xenograft model. Notably, the addition of NLG207 also enhanced the efficacy of enzalutamide alone in the castrated subcutaneous VCaP xenograft model, decreasing the median rate of tumor growth by 51% (P = 0.0001) in comparison with enzalutamide alone. In vitro assessments of cell proliferation and gene expression further demonstrated antitumor activity via AR-HIF-1α crosstalk inhibition. Combination treatment with NLG207 and enzalutamide was shown to be effective in preclinical prostate cancer models of enzalutamide resistance. Clinical investigation of this treatment combination is ongoing (NCT03531827).

Abiraterone induces SLCO1B3 expression in prostate cancer via microRNA-579-3p.

Understanding mechanisms of resistance to abiraterone, one of the primary drugs approved for the treatment of castration resistant prostate cancer, remains a priority. The organic anion polypeptide 1B3 (OATP1B3, encoded by SLCO1B3) transporter has been shown to transport androgens into prostate cancer cells. In this study we observed and investigated the mechanism of induction of SLCO1B3 by abiraterone. Prostate cancer cells (22Rv1, LNCaP, and VCAP) were treated with anti-androgens and assessed for SLCO1B3 expression by qPCR analysis. Abiraterone treatment increased SLCO1B3 expression in 22Rv1 cells in vitro and in the 22Rv1 xenograft model in vivo. MicroRNA profiling of abiraterone-treated 22Rv1 cells was performed using a NanoString nCounter miRNA panel followed by miRNA target prediction. TargetScan and miRanda prediction tools identified hsa-miR-579-3p as binding to the 3'-untranslated region (3'UTR) of the SLCO1B3. Using dual luciferase reporter assays, we verified that hsa-miR-579-3p indeed binds to the SLCO1B3 3'UTR and significantly inhibited SLCO1B3 reporter activity. Treatment with abiraterone significantly downregulated hsa-miR-579-3p, indicating its potential role in upregulating SLCO1B3 expression. In this study, we demonstrated a novel miRNA-mediated mechanism of abiraterone-induced SLCO1B3 expression, a transporter that is also responsible for driving androgen deprivation therapy resistance. Understanding mechanisms of abiraterone resistance mediated via differential miRNA expression will assist in the identification of potential miRNA biomarkers of treatment resistance and the development of future therapeutics.

Pilot study of gadoxetate disodium-enhanced mri for localized and metastatic prostate cancers.

OATP1B3 is expressed de novo in primary prostate cancer tissue and to a greater degree in prostate cancer metastases. Gadoxetate disodium is a substrate of OATP1B3, and its uptake has been shown to correlate with OATP1B3 expression in other cancers. We aimed to evaluate use of gadoxetate disodium to image prostate cancer and to track its utility as a biomarker. A single center open-label non-randomized pilot study recruited men with (1) localized, and (2) metastatic castration resistant prostate cancer (mCRPC). Gadoxetate disodium-enhanced MRI was performed at four timepoints post-injection. The Wilcoxon signed rank test was used to compare MRI contrast enhancement ratio (CER) pre-injection and post-injection. OATP1B3 expression was evaluated via immunohistochemistry (IHC) and a pharmacogenomic analysis of OATP1B3, NCTP and OATP1B1 was conducted. The mCRPC subgroup (n = 9) demonstrated significant enhancement compared to pre-contrast images at 20-, 40- and 60-min timepoints (p < 0.0078). The localized cancer subgroup (n = 11) demonstrated earlier enhancement compared to the mCRPC group, but no retention over time (p > 0.05). OATP1B3 expression on IHC trended higher contrast enhancement between 20-40 min (p ≤ 0.064) and was associated with contrast enhancement at 60 min (p = 0.0422). OATP1B1 haplotype, with N130D and V174A substitutions, impacted enhancement at 40-60 min (p ≤ 0.038). mCRPC lesions demonstrate enhancement after injection of gadoxetate disodium on MRI and retention over 60 min. As inter-individual variability in OATP1B3 expression and function has both predictive and prognostic significance, gadoxetate disodium has potential as a biomarker in prostate cancer.

COVID-19 Clinical Diagnostics and Testing Technology.

Given the global nature of the coronavirus disease 2019 (COVID-19) pandemic, the need for disease detection and expanding testing capacity remains critical priorities. This review discusses the technological advances in testing capability and methodology that are currently used or in development for detecting the novel coronavirus. We describe the current clinical diagnostics and technology, including molecular and serological testing approaches, for severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) testing as well as address their advantages and limitations. Nucleic acid amplification technology for molecular diagnostics remains the gold standard for virus detection. We highlight alternative molecular detection techniques used for developing novel COVID-19 diagnostics on the horizon. Antibody response against SARS-CoV-2 remains poorly understood and proper validation of serology tests is necessary to demonstrate their accuracy and clinical utility. In order to bring the pandemic under control, we must speed up the development of rapid and widespread testing through improvements in clinical diagnostics and testing technology as well as access to these tools.

Are sex discordant outcomes in COVID-19 related to sex hormones?

COVID-19 has a clear sex disparity in clinical outcome. Globally, infection rates between men and women are similar; however, men are more likely to have more severe disease and are more likely to die. The causes for this disparity are currently under investigation and are most likely multifactorial. Sex hormones play an important role in the immune response with estrogen seen as immune boosting and testosterone as immunosuppressing. Additionally, an important protease involved in viral entry, TMPRSS2, is regulated by androgens. Many observational and prospective studies are ongoing or initiating to further examine the role of sex hormones in SARS-CoV-2 infection and if modulation of them is a realistic treatment option.

Drug-drug Interactions in Patients with HIV and Cancer in Sub-Saharan Africa.

In Sub-Saharan Africa, the cancer burden is predicted to increase by > 85% by 2030, the largest increase worldwide. This region has a large HIV-positive population. Drug-drug interactions (DDIs) from concomitant use of multiple drugs increase the risk of drug toxicities, sub-optimal therapy, and drug resistance. With the increase in polypharmacy, involving antiretroviral (ARV), and anticancer drugs, there is a greater need for an appreciation of clinically relevant DDIs. Anticancer and ARV drugs studied in this review were from The World Health Organization's Model List of Essential Medicines 2017. We reviewed; drug package inserts, www.drugbank.ca and www.UpToDate.com, to evaluate pharmacokinetic interactions with cytochrome P450 (CYP450) and ABCB1. The DDIs between drugs were assessed using the University Of Liverpool, UK HIV Drug Interactions Checker, and the LexiComp Drug Interaction tool of www.UpToDate.com. About 70% of ARVs studied interact with CYP450, all involve CYP3A4, and 55% interact with ABCB1. About 65% of anticancer drugs interact with CYP450, 44% of which do so through CYP3A4. About 75% of anticancer drugs interact with ARV drugs, with nine absolute contraindications to concomitant therapy. There exist a substantial number of DDIs between ARV and anticancer drugs, primarily mediated through CYP450 enzymes. Dolutegravir based regimens offer the safest DDI profile for concurrent use with anticancer drugs. However, there are substantial gaps in our knowledge, and this study serves to highlight the need for additional research to better define these interactions and their effect on drug exposure, as attention to these DDIs is a relatively simple intervention that could lead to optimizing disease treatment.

Botryllamide G is an ABCG2 inhibitor that improves lapatinib delivery in mouse brain.

Introduction: Transporters comprising the blood-brain barrier complicate delivery of many therapeutics to the central nervous system. The present study ascertained whether the natural product botryllamide G is viable for in vivo inhibition of ABCG2 using lapatinib as a probe for ABCB1 and ABCG2-mediated efflux from the brain. Methods: Wild-type and Mdr1a/Mdr1b (-/-) mice were treated with botryllamide G and lapatinib ("doublet therapy"), and while a separate cohort of wild-type mice was treated with botryllamide, tariquidar and lapatinib ("triplet therapy"). Results: Botryllamide G demonstrates biphasic elimination with a rapid distribution, decreasing below the in vitro IC50 of 6.9 µM within minutes, yet with a relatively slower terminal half-life (4.6 h). In Mdr1a/Mdr1b (-/-) mice, doublet therapy resulted in a significant increase in brain lapatinib AUC at 8 h (2058 h*ng/mL vs 4007 h*ng/mL; P = .031), but not plasma exposure (P = .15). No significant differences were observed after 24 h. Lapatinib brain exposure was greater through 1 h when wild-type mice were administered triplet therapy (298 h*pg/mg vs 120 h*pg/mg; P < .001), but the triplet decreased brain AUC through 24 h vs. mice administered lapatinib alone (2878 h*pg/mg vs 4461hr*ng/mL; P < .001) and did not alter the brain:plasma ratio. Conclusions: In summary, the ABCG2 inhibitor, botryllamide G, increases brain exposure to lapatinib in mice lacking Abcb1, although the combination of botryllamide G and tariquidar increases brain exposure in wild-type mice only briefly (1 h). Additional research is needed to find analogs of this compound that have better pharmacokinetics and pharmacodynamic effects on ABCG2 inhibition.

Synthesis, Structural Characterization, and Antiangiogenic Activity of Polyfluorinated Benzamides.

The introduction of fluorine into bioactive molecules is a matter of importance in medicinal chemistry. In this study, representatives of various chemical entities of fluoroaromatic compounds were synthesized. Depending on the reaction conditions, either tetrafluorophthalimides or ammonium tetrafluorophthalamates are accessible from tetrafluorophthalic anhydride and primary amines. Tetrafluorophthalamic acids undergo thermal decarboxylation to yield tetrafluorobenzamides. These could be successfully converted upon treatment with primary amines, in the course of an aromatic nucleophilic substitution, to 2,3,5-trifluorobenzamides with respective amino substituents at the 4-position. The five structure types were characterized by means of spectroscopic and crystallographic methods. The synthesized compounds were evaluated as inhibitors of angiogenesis by measuring microvessel outgrowth in a rat aortic ring assay. The biological activity was maintained throughout these different polyfluorinated chemotypes.

Differential Expression of OATP1B3 Mediates Unconjugated Testosterone Influx.

Castration-resistant prostate cancer (CRPC) has greater intratumoral testosterone concentrations than similar tumors from eugonadal men; simple diffusion does not account for this observation. This study was undertaken to ascertain the androgen uptake kinetics, functional, and clinical relevance of de novo expression of the steroid hormone transporter OATP1B3 (SLCO1B3). Experiments testing the cellular uptake of androgens suggest that testosterone is an excellent substrate of OATP1B3 (Km = 23.2 µmol/L; Vmax = 321.6 pmol/mg/minute), and cells expressing a doxycycline-inducible SLCO1B3 construct had greater uptake of a clinically relevant concentration of 3H-testosterone (50 nmol/L; 1.6-fold, P = 0.0027). When compared with Slco1b2 (-/-) mice, Slco1b2 (-/-)/hSLCO1B3 knockins had greater hepatic uptake (15% greater AUC, P = 0.0040) and lower plasma exposure to 3H-testosterone (17% lower AUC, P = 0.0030). Of 82 transporters genes, SLCO1B3 is the second-most differentially expressed transporter in CRPC cell lines (116-fold vs. androgen-sensitive cells), with a differentially spliced cancer-type ct-SLCO1B3 making up the majority of SLCO1B3 expression. Overexpression of SLCO1B3 in androgen-responsive cells results in 1.5- to 2-fold greater testosterone uptake, whereas siRNA knockdown of SLCO1B3 in CRPC cells did not change intracellular testosterone concentration. Primary human prostate tumors express SLCO1B3 to a greater extent than ct-SLCO1B3 (26% of total SLCO1B3 expression vs. 0.08%), suggesting that androgen uptake in these tumor cells also is greater. Non-liver tumors do not differentially express SLCO1B3.Implications: This study suggests that de novo OATP1B3 expression in prostate cancer drives greater androgen uptake and is consistent with previous observations that greater OATP1B3 activity results in the development of androgen deprivation therapy resistance and shorter overall survival. Mol Cancer Res; 15(8); 1096-105. ©2017 AACR.

Building a hit list for the fight against metastatic castration resistant prostate cancer.

Identification of genetic alterations in Metastatic Castration Resistant Prostate Cancer (mCRPC) may provide scientists and clinicians with a list of new targets for drug research and development. Recently published in Cell, Robinson et al. present the first look at genetic data from mCRPC lesions gathered over multiple years and from many institutions.

Alcohol and Aldehyde Dehydrogenases Contribute to Sex-Related Differences in Clearance of Zolpidem in Rats.

OBJECTIVES: The recommended zolpidem starting dose was lowered in females (5 mg vs. 10 mg) since side effects were more frequent and severe than those of males; the mechanism underlying sex differences in pharmacokinetics (PK) is unknown. We hypothesized that such differences were caused by known sex-related variability in alcohol dehydrogenase (ADH) expression. METHODS: Male, female, and castrated male rats were administered 2.6 mg/kg zolpidem, ± disulfiram (ADH/ALDH pathway inhibitor) to compare PK changes induced by sex and gonadal hormones. PK analyses were conducted in rat plasma and rat brain. KEY FINDINGS: Sex differences in PK were evident: females had a higher C MAX (112.4 vs. 68.1 ug/L) and AUC (537.8 vs. 231.8 h(∗)ug/L) than uncastrated males. Castration induced an earlier T MAX (0.25 vs. 1 h), greater C MAX (109.1 vs. 68.1 ug/L), and a corresponding AUC increase (339.7 vs. 231.8 h(∗)ug/L). Administration of disulfiram caused more drastic C MAX and T MAX changes in male vs. female rats that mirrored the effects of castration on first-pass metabolism, suggesting that the observed PK differences may be caused by ADH/ALDH expression. Brain concentrations paralleled plasma concentrations. CONCLUSION: These findings indicate that sex differences in zolpidem PK are influenced by variation in the expression of ADH/ALDH due to gonadal androgens.

Pharmacogenetics of membrane transporters: a review of current approaches.

This chapter provides a review of the pharmacogenetics of membrane transporters, including ABC transporters and OATPs. Membrane transporters are heavily involved in drug disposition, by actively transporting substrate drugs between organs and tissues. As such, polymorphisms in the genes encoding these proteins may have a significant effect on the absorption, distribution, metabolism, excretion, and activity of compounds. Although few drug transporter polymorphisms have transitioned from the bench to the bedside, this chapter discusses clinical development of transporter pharmacogenetic markers. Finally, development of SLCO1B1 genotyping to avoid statin induced adverse drug reactions is discussed as a model case for transporter pharmacogenetics clinical development.