Elp3-mediated codon-dependent translation promotes mTORC2 activation and regulates macrophage polarization.

Macrophage polarization is a process whereby macrophages acquire distinct effector states (M1 or M2) to carry out multiple and sometimes opposite functions. We show here that translational reprogramming occurs during macrophage polarization and that this relies on the Elongator complex subunit Elp3, an enzyme that modifies the wobble uridine base U34 in cytosolic tRNAs. Elp3 expression is downregulated by classical M1-activating signals in myeloid cells, where it limits the production of pro-inflammatory cytokines via FoxO1 phosphorylation, and attenuates experimental colitis in mice. In contrast, alternative M2-activating signals upregulate Elp3 expression through a PI3K- and STAT6-dependent signaling pathway. The metabolic reprogramming linked to M2 macrophage polarization relies on Elp3 and the translation of multiple candidates, including the mitochondrial ribosome large subunit proteins Mrpl3, Mrpl13, and Mrpl47. By promoting translation of its activator Ric8b in a codon-dependent manner, Elp3 also regulates mTORC2 activation. Elp3 expression in myeloid cells further promotes Wnt-driven tumor initiation in the intestine by maintaining a pool of tumor-associated macrophages exhibiting M2 features. Collectively, our data establish a functional link between tRNA modifications, mTORC2 activation, and macrophage polarization.

P-glycoprotein, CYP3A, and Plasma Carboxylesterase Determine Brain Disposition and Oral Availability of the Novel Taxane Cabazitaxel (Jevtana) in Mice.

We aimed to clarify the roles of the multidrug-detoxifying proteins ABCB1, ABCG2, ABCC2, and CYP3A in oral availability and brain accumulation of cabazitaxel, a taxane developed for improved therapy of docetaxel-resistant prostate cancer. Cabazitaxel pharmacokinetics were studied in Abcb1a/1b, Abcg2, Abcc2, Cyp3a, and combination knockout mice. We found that human ABCB1, but not ABCG2, transported cabazitaxel in vitro. Upon oral cabazitaxel administration, total plasma levels were greatly increased due to binding to plasma carboxylesterase Ces1c, which is highly upregulated in several knockout strains. Ces1c inhibition and in vivo hepatic Ces1c knockdown reversed these effects. Correcting for Ces1c effects, Abcb1a/1b, Abcg2, and Abcc2 did not restrict cabazitaxel oral availability, whereas Abcb1a/1b, but not Abcg2, dramatically reduced cabazitaxel brain accumulation (>10-fold). Coadministration of the ABCB1 inhibitor elacridar completely reversed this brain accumulation effect. After correction for Ces1c effects, Cyp3a knockout mice demonstrated a strong (six-fold) increase in cabazitaxel oral availability, which was completely reversed by transgenic human CYP3A4 in intestine and liver. Cabazitaxel markedly inhibited mouse Ces1c, but human CES1 and CES2 only weakly. Ces1c upregulation can thus complicate preclinical cabazitaxel studies. In summary, ABCB1 limits cabazitaxel brain accumulation and therefore potentially therapeutic efficacy against (micro)metastases or primary tumors positioned wholly or partly behind a functional blood-brain barrier. This can be reversed with elacridar coadministration, and similar effects may apply to ABCB1-expressing tumors. CYP3A4 profoundly reduces the oral availability of cabazitaxel. This may potentially be greatly improved by coadministering ritonavir or other CYP3A inhibitors, suggesting the option of patient-friendly oral cabazitaxel therapy.

Increased oral availability and brain accumulation of the ALK inhibitor crizotinib by coadministration of the P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) inhibitor elacridar.

Crizotinib is an oral tyrosine kinase inhibitor approved for treating patients with non-small cell lung cancer (NSCLC) containing an anaplastic lymphoma kinase (ALK) rearrangement. We used knockout mice to study the roles of P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) in plasma pharmacokinetics and brain accumulation of oral crizotinib, and the feasibility of improving crizotinib kinetics using coadministration of the dual ABCB1/ABCG2 inhibitor elacridar. In vitro, crizotinib was a good transport substrate of human ABCB1, but not of human ABCG2 or murine Abcg2. With low-dose oral crizotinib (5 mg/kg), Abcb1a/1b(-/-) and Abcb1a/1b;Abcg2(-/-) mice had an approximately twofold higher plasma AUC than wild-type mice, and a markedly (~40-fold) higher brain accumulation at 24 hr. Also at 4 hr, crizotinib brain concentrations were ∼25-fold, and brain-to-plasma ratios ~14-fold higher in Abcb1a/1b(-/-) and Abcb1a/1b;Abcg2(-/-) mice than in wild-type mice. High-dose oral crizotinib (50 mg/kg) resulted in comparable plasma pharmacokinetics between wild-type and Abcb1a/1b(-/-) mice, suggesting saturation of intestinal Abcb1. Nonetheless, brain accumulation at 24 hr was still ~70-fold higher in Abcb1a/1b(-/-) than in wild-type mice. Importantly, oral elacridar coadministration increased the plasma and brain concentrations and brain-to-plasma ratios of crizotinib in wild-type mice, equaling the levels in Abcb1a/1b;Abcg2(-/-) mice. Our results indicate that crizotinib oral availability and brain accumulation were primarily restricted by Abcb1 at a non-saturating dose, and that coadministration of elacridar with crizotinib could substantially increase crizotinib oral availability and delivery to the brain. This principle might be used to enhance therapeutic efficacy of crizotinib against brain metastases in NSCLC patients.

Impact of P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) gene dosage on plasma pharmacokinetics and brain accumulation of dasatinib, sorafenib, and sunitinib.

Low brain accumulation of anticancer drugs due to efflux transporters may limit chemotherapeutic efficacy, necessitating a better understanding of the underlying mechanisms. P-glycoprotein (Abcb1a/1b) and breast cancer resistance protein (Abcg2) combination knockout mice often display disproportionately increased brain accumulation of shared drug substrates compared with single transporter knockout mice. Recently developed pharmacokinetic models could explain this phenomenon. To experimentally test these models and their wider relevance for tyrosine kinase inhibitors and other drugs, we selected dasatinib, sorafenib, and sunitinib because of their divergent oral availability and brain accumulation profiles: the brain accumulation of dasatinib is mainly restricted by Abcb1, that of sorafenib mainly by Abcg2, and that of sunitinib equally by Abcb1 and Abcg2. We analyzed the effect of halving the efflux activity of these transporters at the blood-brain barrier by generating heterozygous Abcb1a/1b;Abcg2 knockout mice and testing the plasma and brain levels of the drugs after oral administration at 10 mg/kg. Real-time reverse transcription-polymerase chain reaction analysis confirmed the ∼2-fold decreased expression of both transporters in brain. Interestingly, whereas complete knockout of the transporters caused 24- to 36-fold increases in brain accumulation of the drugs, the heterozygous mice only displayed 1.6- to 1.9-fold increases of brain accumulation relative to wild-type mice. These results are well in line with the predictions of the pharmacokinetic models and provide strong support for their validity for a wider range of drugs. Moreover, retrospective analysis of fetal accumulation of drugs across the placenta in Abcb1a/1b heterozygous knockout pups suggests that these models equally apply to the maternal-fetal barrier.

Brain accumulation of sunitinib is restricted by P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) and can be enhanced by oral elacridar and sunitinib coadministration.

Sunitinib is an orally active, multitargeted tyrosine kinase inhibitor which has been used for the treatment of metastatic renal cell carcinoma and imatinib-resistant gastrointestinal stromal tumors. We aimed to investigate the in vivo roles of the ATP-binding cassette drug efflux transporters ABCB1 and ABCG2 in plasma pharmacokinetics and brain accumulation of oral sunitinib, and the feasibility of improving sunitinib kinetics using oral coadministration of the dual ABCB1/ABCG2 inhibitor elacridar. We used in vitro transport assays and Abcb1a/1b(-/-) , Abcg2(-/-) and Abcb1a/1b/Abcg2(-/-) mice to study the roles of ABCB1 and ABCG2 in sunitinib disposition. In vitro, sunitinib was a good substrate of murine (mu)ABCG2 and a moderate substrate of human (hu)ABCB1 and huABCG2. In vivo, the systemic exposure of sunitinib after oral dosing (10 mg kg(-1) ) was unchanged when muABCB1 and/or muABCG2 were absent. Brain accumulation of sunitinib was markedly (23-fold) increased in Abcb1a/b/Abcg2(-/-) mice, but only slightly (2.3-fold) in Abcb1a/b(-/-) mice, and not in Abcg2(-/-) mice. Importantly, a clinically realistic coadministration of oral elacridar and oral sunitinib to wild-type mice resulted in markedly increased sunitinib brain accumulation, equaling levels in Abcb1a/1b/Abcg2(-/-) mice. This indicates complete inhibition of the blood-brain barrier (BBB) transporters. High-dose intravenous sunitinib could saturate BBB muABCG2, but not muABCB1A, illustrating a dose-dependent relative impact of the BBB transporters. Brain accumulation of sunitinib is effectively restricted by both muABCB1 and muABCG2 activity. Complete inhibition of both transporters, leading to markedly increased brain accumulation of sunitinib, is feasible and safe with a clinically realistic oral elacridar/sunitinib coadministration.

P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) restrict brain accumulation of the active sunitinib metabolite N-desethyl sunitinib.

N-desethyl sunitinib is a major and pharmacologically active metabolite of the tyrosine kinase inhibitor and anticancer drug sunitinib. Because the combination of N-desethyl sunitinib and sunitinib represents total active drug exposure, we investigated the impact of several multidrug efflux transporters on plasma pharmacokinetics and brain accumulation of N-desethyl sunitinib after sunitinib administration to wild-type and transporter knockout mice. In vitro, N-desethyl sunitinib was a good transport substrate of human ABCB1 and ABCG2 and murine Abcg2, but not ABCC2 or Abcc2. At 5 µM, ABCB1 and ABCG2 contributed almost equally to N-desethyl sunitinib transport. In vivo, the systemic exposure of N-desethyl sunitinib after oral dosing of sunitinib malate (10 mg/kg) was unchanged when Abcb1 and/or Abcg2 were absent. However, brain accumulation of N-desethyl sunitinib was markedly increased (13.7-fold) in Abcb1a/1b(-/-)/Abcg2(-/-) mice, but not in Abcb1a/1b(-/-) or Abcg2(-/-) mice. In the absence of the ABCB1 and ABCG2 inhibitor elacridar, brain concentrations of N-desethyl sunitinib were detectable only in Abcb1a/1b(-/-)/Abcg2(-/-) mice after sunitinib administration. Combined elacridar plus N-desethyl sunitinib treatment increased N-desethyl sunitinib plasma and brain exposures, but not brain-to-plasma ratios in wild-type mice. In conclusion, brain accumulation of N-desethyl sunitinib is effectively restricted by both Abcb1 and Abcg2. The effect of elacridar treatment in improving brain accumulation of N-desethyl sunitinib in wild-type mice was limited compared with its effect on sunitinib brain accumulation.

Super-Enhancers, Phase-Separated Condensates, and 3D Genome Organization in Cancer.

3D chromatin organization plays an important role in transcription regulation and gene expression. The 3D genome is highly maintained by several architectural proteins, such as CTCF, Yin Yang 1, and cohesin complex. This structural organization brings regulatory DNA elements in close proximity to their target promoters. In this review, we discuss the 3D chromatin organization of super-enhancers and their relationship to phase-separated condensates. Super-enhancers are large clusters of DNA elements. They can physically contact with their target promoters by chromatin looping during transcription. Multiple transcription factors can bind to enhancer and promoter sequences and recruit a complex array of transcriptional co-activators and RNA polymerase II to effect transcriptional activation. Phase-separated condensates of transcription factors and transcriptional co-activators have been implicated in assembling the transcription machinery at particular enhancers. Cancer cells can hijack super-enhancers to drive oncogenic transcription to promote cell survival and proliferation. These dysregulated transcriptional programs can cause cancer cells to become highly dependent on transcriptional regulators, such as Mediator and BRD4. Moreover, the expression of oncogenes that are driven by super-enhancers is sensitive to transcriptional perturbation and often occurs in phase-separated condensates, supporting therapeutic rationales of targeting SE components, 3D genome organization, or dysregulated condensates in cancer.

The E3 ligase COP1 promotes ERα signaling and suppresses EMT in breast cancer.

ERα signaling drives proliferation, survival and cancer initiation in the mammary gland. Therefore, it is critical to elucidate mechanisms by which ERα expression is regulated. We show that the tumor suppressor E3 ligase COP1 promotes the degradative polyubiquitination of the microtubule-associated protein HPIP. As such, COP1 negatively regulates estrogen-dependent AKT activation in breast cancer cells. However, COP1 also induces ERα expression and ERα-dependent gene transcription, at least through c-Jun degradation. COP1 and ERα levels are positively correlated in clinical cases of breast cancer. COP1 also supports the metabolic reprogramming by estrogens, including glycolysis. On the other hand, COP1 suppresses EMT in breast cancer cells. COP1 deficiency also contributes to Tamoxifen resistance, at least through protective autophagy. Therefore, COP1 acts as an oncogenic E3 ligase by promoting ERα signaling but also acts as a tumor suppressor candidate by preventing EMT, which reflects a dual role of COP1 in breast cancer.

Double-transduced MDCKII cells to study human P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) interplay in drug transport across the blood-brain barrier.

P-glycoprotein (P-gp/ABCB1) and breast cancer resistance protein (BCRP/ABCG2) combination knockout mice display disproportionately increased brain penetration of shared substrates, including topotecan and several tyrosine kinase inhibitors, compared to mice deficient for only one transporter. To better study the interplay of both transporters also in vitro, we generated a transduced polarized MDCKII cell line stably coexpressing substantial levels of human ABCB1 and ABCG2 (MDCKII-ABCB1/ABCG2). Next, we measured concentration-dependent transepithelial transport of topotecan, sorafenib and sunitinib. By blocking either one or both of the transporters simultaneously, using specific inhibitors, we aimed to mimic the ABCB1-ABCG2 interplay at the blood-brain barrier in wild-type, single or combination knockout mice. ABCB1 and ABCG2 contributed to similar extents to topotecan transport, which was only partly saturable. For sorafenib transport, ABCG2 was the major determinant at low concentrations. However, saturation of ABCG2-mediated transport occurred at higher sorafenib concentrations, where ABCB1 was still fully active. Furthermore, sunitinib was transported equally by ABCB1 and ABCG2 at low concentrations, but ABCG2-mediated transport became saturated at lower concentrations than ABCB1-mediated transport. The relative impact of these transporters can thus be affected by the applied drug concentrations. A comparison of the in vitro observed (inverse) transport ratios and cellular accumulation of the drugs at low concentrations with in vivo brain penetration data from corresponding Abcb1a/1b⁻/⁻, Abcg2⁻/⁻ and Abcb1a/1b;Abcg2⁻/⁻ mouse strains revealed very similar qualitative patterns for each of the tested drugs. MDCKII-ABCB1/ABCG2 cells thus present a useful in vitro model to study the interplay of ABCB1 and ABCG2.

The Endosomal Protein CEMIP Links WNT Signaling to MEK1-ERK1/2 Activation in Selumetinib-Resistant Intestinal Organoids.

MAPK signaling pathways are constitutively active in colon cancer and also promote acquired resistance to MEK1 inhibition. Here, we demonstrate that BRAFV600E -mutated colorectal cancers acquire resistance to MEK1 inhibition by inducing expression of the scaffold protein CEMIP through a ß-catenin- and FRA-1-dependent pathway. CEMIP was found in endosomes and bound MEK1 to sustain ERK1/2 activation in MEK1 inhibitor-resistant BRAFV600E-mutated colorectal cancers. The CEMIP-dependent pathway maintained c-Myc protein levels through ERK1/2 and provided metabolic advantage in resistant cells, potentially by sustaining amino acids synthesis. CEMIP silencing circumvented resistance to MEK1 inhibition, partly, through a decrease of both ERK1/2 signaling and c-Myc. Together, our data identify a cross-talk between Wnt and MAPK signaling cascades, which involves CEMIP. Activation of this pathway promotes survival by potentially regulating levels of specific amino acids via a Myc-associated cascade. Targeting this node may provide a promising avenue for treatment of colon cancers that have acquired resistance to targeted therapies.Significance: MEK1 inhibitor-resistant colorectal cancer relies on the scaffold and endosomal protein CEMIP to maintain ERK1/2 signaling and Myc-driven transcription. Cancer Res; 78(16); 4533-48. ©2018 AACR.

P-glycoprotein, CYP3A, and plasma carboxylesterase determine brain and blood disposition of the mTOR Inhibitor everolimus (Afinitor) in mice.

PURPOSE: To clarify the role of ABCB1, ABCG2, and CYP3A in blood and brain exposure of everolimus using knockout mouse models. EXPERIMENTAL DESIGN: We used wild-type, Abcb1a/1b(-/-), Abcg2(-/-), Abcb1a/1b;Abcg2(-/-), and Cyp3a(-/-) mice to study everolimus oral bioavailability and brain accumulation. RESULTS: Following everolimus administration, brain concentrations and brain-to-liver ratios were substantially increased in Abcb1a/1b(-/-)and Abcb1a/1b;Abcg2(-/-), but not Abcg2(-/-)mice. The fraction of everolimus located in the plasma compartment was highly increased in all knockout strains. In vitro, everolimus was rapidly degraded in wild-type but not knockout plasma. Carboxylesterase 1c (Ces1c), a plasma carboxylesterase gene, was highly upregulated (∼80-fold) in the liver of knockout mice relative to wild-type mice, and plasma Ces1c likely protected everolimus from degradation by binding and stabilizing it. This binding was prevented by preincubation with the carboxylesterase inhibitor BNPP. In vivo knockdown experiments confirmed the involvement of Ces1c in everolimus stabilization. Everolimus also markedly inhibited the hydrolysis of irinotecan and p-nitrophenyl acetate by mouse plasma carboxylesterase and recombinant human CES2, respectively. After correcting for carboxylesterase binding, Cyp3a(-/-), but not Abcb1a/1b(-/-), Abcg2(-/-), or Abcb1a/1b;Abcg2(-/-)mice, displayed highly (>5-fold) increased oral availability of everolimus. CONCLUSIONS: Brain accumulation of everolimus was restricted by Abcb1, but not Abcg2, suggesting the use of coadministered ABCB1 inhibitors to improve brain tumor treatment. Cyp3a, but not Abcb1a/1b, restricted everolimus oral availability, underscoring drug-drug interaction risks via CYP3A. Upregulated Ces1c likely mediated the tight binding and stabilization of everolimus, causing higher plasma retention in knockout strains. This Ces upregulation might confound other pharmacologic studies.

Genetically modified mouse models for oral drug absorption and disposition.

Intestinal absorption is an essential step in the therapeutic use of most orally administered drugs and often mediated by enterocyte transmembrane transporters. Here we discuss several of these drug transport systems and knockout mouse models to study them. These studies showed that Multidrug resistance-associated protein 2 (Mrp2) can limit intestinal drug absorption. Organic cation transporter n1 (Octn1) and Octn2 might also facilitate intestinal drug absorption, although direct in vivo evidence is lacking. On the other hand, intestinal uptake of drugs is facilitated by the Equilibrative nucleoside transporter 1 (Ent1), Mrp3 and possibly Mrp4. No significant role in intestinal absorption for Oct1 and Oct2 or for Organic anion-transporting polypeptides (Oatp) 1a and 1b was found so far.

Liquid chromatography-tandem mass spectrometric assay for the ALK inhibitor crizotinib in mouse plasma.

A quantitative bioanalytical liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay for the ALK inhibitor crizotinib was developed and validated. Plasma samples were pre-treated using protein precipitation with acetonitrile containing crizotinib-(13)C(2)-(2)H(5) as internal standard. The extract was directly injected into the chromatographic system after dilution with water. This system consisted of a sub-2 µm particle, trifunctional bonded octadecyl silica column with a gradient using 0.1% (v/v) of ammonium hydroxide in water and methanol. The eluate was transferred into the electrospray interface with positive ionization and the analyte was detected in the selected reaction monitoring mode of a triple quadrupole mass spectrometer. The assay was validated in a 10-10,000 ng/ml calibration range with r(2)=0.99980±0.00014 for double logarithmic linear regression (n=5). Within day precisions (n=6) were 3.4-4.8%, between day (3 days; n=18) precisions 3.6-4.9%. Accuracies were between 107% and 112% for the whole calibration range. The drug was sufficiently stable under all relevant analytical conditions. Oxidative metabolites of crizotinib were monitored semi-quantitatively. Finally, the assay was successfully used to assess drug pharmacokinetics in mice.