Castration of Male Mice Induces Metabolic Remodeling of the Heart.

Androgen deprivation therapy of prostate cancer, which suppresses serum testosterone to castrate levels, is associated with increased risk of heart failure. Here we tested the hypothesis that castration alters cardiac energy substrate uptake, which is tightly coupled to the regulation of cardiac structure and function. Short-term (3-4 weeks) surgical castration of male mice reduced the relative heart weight. While castration did not affect cardiac function in unstressed conditions, we observed reductions in heart rate, stroke volume, cardiac output, and cardiac index during pharmacological stress with dobutamine in castrated vs sham-operated mice. Experiments using radiolabeled lipoproteins and glucose showed that castration shifted energy substrate uptake in the heart from lipids toward glucose, while testosterone replacement had the opposite effect. There was increased expression of fetal genes in the heart of castrated mice, including a strong increase in messenger RNA and protein levels of ß-myosin heavy chain (MHC), the fetal isoform of MHC. In conclusion, castration of male mice induces metabolic remodeling and expression of the fetal gene program in the heart, in association with a reduced cardiac performance during pharmacological stress. These findings may be relevant for the selection of treatment strategies for heart failure in the setting of testosterone deficiency.

Deficiency of mature B cells does not alter the atherogenic response to castration in male mice.

Testosterone deficiency in men is associated with increased atherosclerosis burden and increased cardiovascular risk. In male mice, testosterone deficiency induced by castration increases atherosclerosis as well as mature B cell numbers in spleen. As B cells are potentially pro-atherogenic, we hypothesized that there may be a link between these effects. To address whether mature B cell deficiency alter the atherogenic response to castration, we studied B cell-deficient µMT and genotype control male mice on an atherosclerosis-prone Apoe-/- background that were castrated or sham-operated pre-pubertally and fed a high-fat diet between 8 and 16 weeks of age to accelerate atherosclerosis development. Genotype did not affect the effects of castration on body weight or weights of fat depots and there were no differences in serum cholesterol levels across the four groups. Atherosclerosis assessed by quantification of lesion area in serial sections of the aortic root was significantly increased by castration and by the µMT mutation, with no significant interaction between genotype and surgery. In conclusion, castration evokes a similar atherogenic response in B cell-deficient µMT and control mice. These data suggest that atherogenesis following castration is unrelated to the effects of androgens on mature B cell numbers.

Liquid Biopsies or Therapeutic Drug Monitoring for CYP Activity Profile Determination.

During pharmacotherapy, knowledge about the actual drug and metabolite concentrations in plasma is often critical. Individual dose adjustments can be performed based on pre-emptive genotyping of certain absorption, distribution, metabolism, and excretion (ADME) genes but also using therapeutic drug monitoring (TDM). Analyses of liquid biopsies for tumor-derived components are well-established and have been found to be a good complement to biopsy examinations. Recently, liquid biopsy-based quantification of cell-free RNA (cfRNA) in plasma exosomes was proposed as a proxy measurement for the expression of different hepatic ADME genes and for the rate of drug metabolism, constituting an alternative to TDM. In this study, we validated these findings by examining the correlation between mRNA expression of eight different CYP genes in liver and the corresponding rate of enzyme-specific drug metabolism in 96 donor-matched liver samples. Analyses of CYP-dependent drug metabolism in liver microsomes in comparison to the level of mRNA expression for the different CYP genes revealed a mean Pearson correlation coefficient of 0.28. The highest correlations (0.33-0.34) were obtained for CYP2D6 and CYP3A4 and the weakest correlations were observed for CYP1A2 and CYP2B6 (0.18-0.21). In all cases, the correlations obtained were too weak to demonstrate a predictive relationship, likely due to different regulatory and post-translational events controlling the rate of enzyme activity. Our results reinforce the notion that, whilst liquid biopsy-based approaches might have utility for prediction of hepatic CYP protein expression, they are not currently an important substitute for TDM.

Toward predicting CYP2D6-mediated variable drug response from CYP2D6 gene sequencing data.

Pharmacogenomics is a key component of personalized medicine that promises safer and more effective drug treatment by individualizing drug choice and dose based on genetic profiles. In clinical practice, genetic biomarkers are used to categorize patients into *-alleles to predict CYP450 enzyme activity and adjust drug dosages accordingly. However, this approach leaves a large part of variability in drug response unexplained. Here, we present a proof-of-concept approach that uses continuous-scale (instead of categorical) assignments to predict enzyme activity. We used full CYP2D6 gene sequences obtained with long-read amplicon-based sequencing and cytochrome P450 (CYP) 2D6-mediated tamoxifen metabolism data from a prospective study of 561 patients with breast cancer to train a neural network. The model explained 79% of interindividual variability in CYP2D6 activity compared to 54% with the conventional *-allele approach, assigned enzyme activities to known alleles with previously reported effects, and predicted the activity of previously uncharacterized combinations of variants. The results were replicated in an independent cohort of tamoxifen-treated patients (model R 2 adjusted = 0.66 versus *-allele R 2 adjusted = 0.35) and a cohort of patients treated with the CYP2D6 substrate venlafaxine (model R 2 adjusted = 0.64 versus *-allele R 2 adjusted = 0.55). Human embryonic kidney cells were used to confirm the effect of five genetic variants on metabolism of the CYP2D6 substrate bufuralol in vitro. These results demonstrate the advantage of a continuous scale and a completely phased genotype for prediction of CYP2D6 enzyme activity and could potentially enable more accurate prediction of individual drug response.

Genomic profiling of the transcription factor Zfp148 and its impact on the p53 pathway.

Recent data suggest that the transcription factor Zfp148 represses activation of the tumor suppressor p53 in mice and that therapeutic targeting of the human orthologue ZNF148 could activate the p53 pathway without causing detrimental side effects. We have previously shown that Zfp148 deficiency promotes p53-dependent proliferation arrest of mouse embryonic fibroblasts (MEFs), but the underlying mechanism is not clear. Here, we showed that Zfp148 deficiency downregulated cell cycle genes in MEFs in a p53-dependent manner. Proliferation arrest of Zfp148-deficient cells required increased expression of ARF, a potent activator of the p53 pathway. Chromatin immunoprecipitation showed that Zfp148 bound to the ARF promoter, suggesting that Zfp148 represses ARF transcription. However, Zfp148 preferentially bound to promoters of other transcription factors, indicating that deletion of Zfp148 may have pleiotropic effects that activate ARF and p53 indirectly. In line with this, we found no evidence of genetic interaction between TP53 and ZNF148 in CRISPR and siRNA screen data from hundreds of human cancer cell lines. We conclude that Zfp148 deficiency, by increasing ARF transcription, downregulates cell cycle genes and cell proliferation in a p53-dependent manner. However, the lack of genetic interaction between ZNF148 and TP53 in human cancer cells suggests that therapeutic targeting of ZNF148 may not increase p53 activity in humans.

Androgen Receptors in Epithelial Cells Regulate Thymopoiesis and Recent Thymic Emigrants in Male Mice.

Androgens have profound effects on T cell homeostasis, including regulation of thymic T lymphopoiesis (thymopoiesis) and production of recent thymic emigrants (RTEs), i. e., immature T cells that derive from the thymus and continue their maturation to mature naïve T cells in secondary lymphoid organs. Here we investigated the androgen target cell for effects on thymopoiesis and RTEs in spleen and lymph nodes. Male mice with a general androgen receptor knockout (G-ARKO), T cell-specific (T-ARKO), or epithelial cell-specific (E-ARKO) knockout were examined. G-ARKO mice showed increased thymus weight and increased numbers of thymic T cell progenitors. These effects were not T cell-intrinsic, since T-ARKO mice displayed unaltered thymus weight and thymopoiesis. In line with a role for thymic epithelial cells (TECs), E-ARKO mice showed increased thymus weight and numbers of thymic T cell progenitors. Further, E-ARKO mice had more CD4+ and CD8+ T cells in spleen and an increased frequency of RTEs among T cells in spleen and lymph nodes. Depletion of the androgen receptor in epithelial cells was also associated with a small shift in the relative number of cortical (reduced) and medullary (increased) TECs and increased CCL25 staining in the thymic medulla, similar to previous observations in castrated mice. In conclusion, we demonstrate that the thymic epithelium is a target compartment for androgen-mediated regulation of thymopoiesis and consequently the generation of RTEs.

Clinically Relevant Cytochrome P450 3A4 Induction Mechanisms and Drug Screening in Three-Dimensional Spheroid Cultures of Primary Human Hepatocytes.

Cytochrome P450 (CYP) 3A4 induction is an important cause of drug-drug interactions, making early identification of drug candidates with CYP3A4 induction liability in drug development a prerequisite. Here, we present three-dimensional (3D) spheroid cultures of primary human hepatocytes (PHHs) as a novel CYP3A4 induction screening model. Screening of 25 drugs (12 known CYP3A4 inducers in vivo and 13 negative controls) at physiologically relevant concentrations revealed a 100% sensitivity and 100% specificity of the system. Three of the in vivo CYP3A4 inducers displayed much higher CYP3A4 induction capacity in 3D spheroid cultures as compared with in two-dimensional (2D) monolayer cultures. Among those, we identified AZD1208, a proviral integration site for Moloney murine leukemia virus (PIM) kinase inhibitor terminated in phase I of development due to unexpected CYP3A4 autoinduction, as a CYP3A4 inducer only active in 3D spheroids but not in 2D monolayer cultures. Gene knockdown experiments revealed that AZD1208 requires pregnane X receptor (PXR) to induce CYP3A4. Rifampicin requires solely PXR to induce CYP3A4 and CYP2B6, while phenobarbital-mediated induction of these CYPs did not show absolute dependency on either PXR or constitutive androstane receptor (CAR), suggesting its ability to switch nuclear receptor activation. Mechanistic studies into AZD1208 uncovered an involvement of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway in CYP3A4 induction that is sensitive to the culture format used, as revealed by its inhibition of ERK1/2 Tyrosine 204 phosphorylation and sensitivity to epidermal growth factor (EGF) pressure. In line, we also identified lapatinib, a dual epidermal growth factor receptor/human epidermal growth factor receptor 2 (EGFR/HER2) inhibitor, as another CYP3A4 inducer only active in 3D spheroid culture. Our findings offer insights into the pathways involved in CYP3A4 induction and suggest PHH spheroids for preclinical CYP3A4 induction screening.

Human Liver Spheroids as a Model to Study Aetiology and Treatment of Hepatic Fibrosis.

Non-alcoholic fatty liver disease affects approximately one billion adults worldwide. Non-alcoholic steatohepatitis (NASH) is a progressive disease and underlies the advancement to liver fibrosis, cirrhosis, and hepatocellular carcinoma, for which there are no FDA-approved drug therapies. We developed a hetero-cellular spheroid system comprised of primary human hepatocytes (PHH) co-cultured with crude fractions of primary human liver non-parenchymal cells (NPC) from several matched or non-matched donors, to identify phenotypes with utility in investigating NASH pathogenesis and drug screening. Co-culture spheroids displayed stable expression of hepatocyte markers (albumin, CYP3A4) with the integration of stellate (vimentin, PDGFRß), endothelial (vWF, PECAM1), and CD68-positive cells. Several co-culture spheroids developed a fibrotic phenotype either spontaneously, primarily observed in PNPLA3 mutant donors, or after challenge with free fatty acids (FFA), as determined by COL1A1 and αSMA expression. This phenotype, as well as TGFß1 expression, was attenuated with an ALK5 inhibitor. Furthermore, CYP2E1, which has a strong pro-oxidant effect, was induced by NPCs and FFA. This system was used to evaluate the effects of anti-NASH drug candidates, which inhibited fibrillary deposition following 7 days of exposure. In conclusion, we suggest that this system is suitable for the evaluation of NASH pathogenesis and screening of anti-NASH drug candidates.

Testosterone Protects Against Atherosclerosis in Male Mice by Targeting Thymic Epithelial Cells-Brief Report.

OBJECTIVE: Androgen deprivation therapy has been associated with increased cardiovascular risk in men. Experimental studies support that testosterone protects against atherosclerosis, but the target cell remains unclear. T cells are important modulators of atherosclerosis, and deficiency of testosterone or its receptor, the AR (androgen receptor), induces a prominent increase in thymus size. Here, we tested the hypothesis that atherosclerosis induced by testosterone deficiency in male mice is T-cell dependent. Further, given the important role of the thymic epithelium for T-cell homeostasis and development, we hypothesized that depletion of the AR in thymic epithelial cells will result in increased atherosclerosis. APPROACH AND RESULTS: Prepubertal castration of male atherosclerosis-prone apoE-/- mice increased atherosclerotic lesion area. Depletion of T cells using an anti-CD3 antibody abolished castration-induced atherogenesis, demonstrating a role of T cells. Male mice with depletion of the AR specifically in epithelial cells (E-ARKO [epithelial cell-specific AR knockout] mice) showed increased thymus weight, comparable with that of castrated mice. E-ARKO mice on an apoE-/- background displayed significantly increased atherosclerosis and increased infiltration of T cells in the vascular adventitia, supporting a T-cell-driven mechanism. Consistent with a role of the thymus, E-ARKO apoE-/- males subjected to prepubertal thymectomy showed no atherosclerosis phenotype. CONCLUSIONS: We show that atherogenesis induced by testosterone/AR deficiency is thymus- and T-cell dependent in male mice and that the thymic epithelial cell is a likely target cell for the antiatherogenic actions of testosterone. These insights may pave the way for new therapeutic strategies for safer endocrine treatment of prostate cancer.

Testosterone is an endogenous regulator of BAFF and splenic B cell number.

Testosterone deficiency in men is associated with increased risk for autoimmunity and increased B cell numbers through unknown mechanisms. Here we show that testosterone regulates the cytokine BAFF, an essential survival factor for B cells. Male mice lacking the androgen receptor have increased splenic B cell numbers, serum BAFF levels and splenic Baff mRNA. Testosterone deficiency by castration causes expansion of BAFF-producing fibroblastic reticular cells (FRCs) in spleen, which may be coupled to lower splenic noradrenaline levels in castrated males, as an α-adrenergic agonist decreases splenic FRC number in vitro. Antibody-mediated blockade of the BAFF receptor or treatment with the neurotoxin 6-hydroxydopamine revert the increased splenic B cell numbers induced by castration. Among healthy men, serum BAFF levels are higher in men with low testosterone. Our study uncovers a previously unrecognized regulation of BAFF by testosterone and raises important questions about BAFF in testosterone-mediated protection against autoimmunity.

Human Cytochrome P450 2W1 Is Not Expressed in Adrenal Cortex and Is Only Rarely Expressed in Adrenocortical Carcinomas.

Human cytochome P450 2W1 (CYP2W1) enzyme is expressed in fetal colon and in colon tumors. The level of expression is higher in colon metastases than in the parent tumors and the enzyme is a possible drug target for treatment of colorectal cancer, as demonstrated in mouse xenograft studies. A previous study published in this journal reported that CYP2W1 is highly expressed in normal and transformed adrenal tissue. However, adrenal expression of CYP2W1 protein was not seen in previous studies in our research group. To clarify this inconsistency, we have used qRT-PCR and Western blotting with CYP2W1-specific antibodies to probe a panel of 27 adrenocortical carcinomas and 35 normal adrenal cortex samples. CYP2W1 mRNA expression is seen in all samples. However, significant CYP2W1 protein expression was found in only one tumor sample (a testosterone-producing adrenocortical carcinoma) and not in any normal tissue. Differences in the specificity of the CYP2W1 antibodies used in the two studies may explain the apparent discrepancy. We conclude that normal adrenal tissue lacks P450 2W1 enzyme expression; also, adrenocortical carcinomas generally do not express the enzyme. This information thus underline the colon cancer specificity of CYP2W1 enzyme expression and has implications for the development of anti-colon cancer therapies based on CYP2W1 as a drug target, since 2W1-dependent bioactivation of prodrugs for CYP2W1 will not take place in normal adrenal tissue or other non-transformed tissues.

The CYP2W1 enzyme: regulation, properties and activation of prodrugs.

CYP2W1 is expressed in the course of development of the gastrointestinal tract, silenced after birth in intestine and colon by epigenetic modifications, but activated following demethylation in colorectal cancer (CRC). The expression levels in CRC positively correlate with the degree of malignancy, are higher in metastases and are predictive of colon cancer survival. The CYP2W1 transcripts have been detected also in hepatocellular carcinoma, adrenocortical carcinoma, childhood rhabdomyosarcoma and breast cancer; however, here the protein expression remains to be confirmed. The CYP2W1 enzyme has an inverted orientation in the endoplasmic reticulum membrane, as compared to other cytochrome P450s and its immediate electron donor is unknown. Several lipid ligands have been proposed as endogenous substrates, among which retinol derivatives appear to have the highest affinities. However, the role of CYP2W1 in the endogenous and tumor localized metabolism of retinol derivatives has yet to be clarified. Indolines constitute high affinity exogenous compounds and specific chloromethylindolines have been shown to be activated by CYP2W1 into cytotoxic products in vitro and also in vivo, inhibiting the growth of human colon tumors in a mouse xenograft model. The CRC specific localization of CYP2W1 and its effective prodrug activation makes it a very promising target for future development of cancer therapeutics.

Membrane topology and search for potential redox partners of colon cancer-specific cytochrome P450 2W1.

Cytochrome P450 2W1 (CYP2W1) is a colon tumor-specific enzyme, suggested as a potential target for cancer therapy. In contrast to other endoplasmic reticulum P450s, we found completely inverted ER membrane topology of CYP2W1 using different approaches (redox sensitive luciferase assay and protease protection assay) and demonstrated that canonical CYP reductants, cytochrome P450 reductase, and cytochrome b5 cannot serve as electron donors for CYP2W1. Moreover, the reduced catalytic activity of the Asn177 mutant that is modified by glycan moieties in the wild-type enzyme indicates a functional relevance of CYP2W1 glycosylation.

Role of cytochrome P450 2C8*3 (CYP2C8*3) in paclitaxel metabolism and paclitaxel-induced neurotoxicity.

AIM: The CYP2C8*3 allele has been suggested as a risk factor for paclitaxel-induced neuropathy but the data hitherto published are conflicting. MATERIALS & METHODS: In total 435 patients were investigated with respect to maximum neuropathy grade and accumulated paclitaxel dose. The enzymatic properties of CYP2C8.3 variant were analyzed using heterologous mammalian HEK293 cell expression system. RESULTS: No significant association between CYP2C8*3 allele and neuropathy was found, although a trend was observed. The paclitaxel and amodiaquine metabolism by CYP2C8.3 were found similar to CYP2C8.1, whereas CYP2C8.3 was more efficient in the metabolism of rosiglitazone. CONCLUSION: These results indicate a difference in substrate specificity between CYP2C8.1 and CYP2C8.3; however, the CYP2C8*3 allele has no major impact on paclitaxel metabolism in vitro or of paclitaxel-induced neuropathy in vivo. Original submitted on 6 February 2015; revision submitted on 9 April 2015.

Developmental regulation and induction of cytochrome P450 2W1, an enzyme expressed in colon tumors.

Cytochrome P450 2W1 (CYP2W1) is expressed predominantly in colorectal and also in hepatic tumors, whereas the levels are insignificant in the corresponding normal human adult tissues. CYP2W1 has been proposed as an attractive target for colorectal cancer (CRC) therapy by exploiting its ability to activate duocarmycin prodrugs to cytotoxic metabolites. However, its endogenous function, regulation and developmental pattern of expression remain unexplored. Here we report the CYP2W1 developmental expression in the murine and human gastrointestinal tissues. The gene expression in the colon and small intestine commence at early stages of embryonic life and is completely silenced shortly after the birth. Immunohistochemical analysis of human fetal colon revealed that CYP2W1 expression is restricted to the crypt cells. The silencing of CYP2W1 after birth correlates with the increased methylation of CpG-rich regions in both murine and human CYP2W1 genes. Analysis of CYP2W1 expression in the colon adenocarcinoma cell line HCC2998 revealed that the gene expression can be induced by e.g. the antitumor agent imatinib, linoleic acid and its derivatives. The imatinib mediated induction of CYP2W1 suggests an adjuvant therapy to treatment with duocarmycins that thus would involve induction of tumor CYP2W1 levels followed by the CYP2W1 activated duocarmycin prodrugs. Taken together these data strongly support further exploration of CYP2W1 as a specific drug target in CRC.

Whole-exome sequencing reveals defective CYP3A4 variants predictive of paclitaxel dose-limiting neuropathy.

PURPOSE: Paclitaxel, a widely used chemotherapeutic drug, can cause peripheral neuropathies leading to dose reductions and treatment suspensions and decreasing the quality of life of patients. It has been suggested that genetic variants altering paclitaxel pharmacokinetics increase neuropathy risk, but the major causes of interindividual differences in susceptibility to paclitaxel toxicity remain unexplained. We carried out a whole-exome sequencing (WES) study to identify genetic susceptibility variants associated with paclitaxel neuropathy. EXPERIMENTAL DESIGN: Blood samples from 8 patients with severe paclitaxel-induced peripheral neuropathy were selected for WES. An independent cohort of 228 cancer patients with complete paclitaxel neuropathy data was used for variant screening by DHPLC and association analysis. HEK293 cells were used for heterologous expression and characterization of two novel CYP3A4 enzymes. RESULTS: WES revealed 2 patients with rare CYP3A4 variants, a premature stop codon (CYP3A4*20 allele) and a novel missense variant (CYP3A4*25, p.P389S) causing reduced enzyme expression. Screening for CYP3A4 variants in the independent cohort revealed three additional CYP3A4*20 carriers, and two patients with missense variants exhibiting diminished enzyme activity (CYP3A4*8 and the novel CYP3A4*27 allele, p.L475V). Relative to CYP3A4 wild-type patients, those carrying CYP3A4 defective variants had more severe neuropathy (2- and 1.3-fold higher risk of neuropathy for loss-of-function and missense variants, respectively, P = 0.045) and higher probability of neuropathy-induced paclitaxel treatment modifications (7- and 3-fold higher risk for loss-of-function and missense variants, respectively, P = 5.9 × 10(-5)). CONCLUSION: This is the first description of a genetic marker associated with paclitaxel treatment modifications caused by neuropathy. CYP3A4 defective variants may provide a basis for paclitaxel treatment individualization.

The expression of CYP2W1 in colorectal primary tumors, corresponding lymph node metastases and liver metastases.

INTRODUCTION: Metastatic disease is a major cause of death in patients with colorectal cancer (CRC). We have previously investigated expression of an orphan cytochrome P450 (CYP) enzyme, CYP2W1, and found high expression in about one third of colorectal tumors. CYP2W1 has proven to metabolize duocarmycin analogs into cytotoxic substances, compounds that in xenografts of CRC cells expressing CYP2W1 completely inhibit tumor growth. This study was designed to evaluate whether the enzyme is expressed in primary CRC and corresponding metastases. MATERIAL AND METHODS: Samples from primary tumors, corresponding lymph node metastases and liver metastases from 96 patients were collected and analyzed by immunohistochemistry. Data regarding patient's demographics, tumor characteristics and survival were also collected. RESULTS: Out of 96 patients, 25 (26%) had high CYP2W1 expression in the primary tumor and 46 (48%) showed high levels in the liver metastasis. In total 59 patients had lymph node metastases, and 31% of them had high CYP2W1 expression. When comparing the expression in primary tumor with that of the first liver metastasis, the increase in expression was statistically significant (p = 0.005). CONCLUSION: High CYP2W1 expression is seen in 26% of primary CRC and in 48% of corresponding liver metastases. This opens possibilities for new targeted therapies to metastatic CRC in the future.

CYP2W1 polymorphism: functional aspects and relation to risk for colorectal cancer.

AIM: This study aims to investigate the possible association between the risk of colorectal cancer (CRC) and allelic variants of CYP2W1 and their functional properties. MATERIALS & METHODS: The distribution of three different CYP2W1 alleles (CYP2W1*1, CYP2W1*2 and CYP2W1*6) in 1785 CRC patients and 1761 healthy blood donors was determined using the TaqMan(®) (Applied Biosystems, CA, USA) allelic discrimination assay or allele-specific amplification. Corresponding gene products (CYP2W1.1, CYP2W1.2 and CYP2W1.6) were expressed in human colon cancer SW480 cells and their activities towards two different substrates, the duocarmycin analogs ICT2706 and ICT2726, were monitored. RESULTS: No significant differences in the distribution of CYP2W1*1, CYP2W1*2 and CYP2W1*6 alleles were found between CRC patients and controls. The CYP2W1.1, CYP2W1.2 and CYP2W1.6 variant enzymes were expressed at the similar levels in the transfected SW480 cells and had comparable kinetics in terms of the metabolism of the duocarmycin ICT2726, as well as in the bioactivation of ICT2706 into a cytotoxic product. CONCLUSION: These epidemiological data obtained from a large population of CRC patients and controls cannot confirm the previously suggested decreased risk for CRC among carriers of CYP2W1*2. On the molecular level, this conclusion is further supported by the similar catalytic characteristics of the CYP2W1.1, CYP2W1.2 and CYP2W1.6 variants of CYP2W1.

Re-engineering of the duocarmycin structural architecture enables bioprecursor development targeting CYP1A1 and CYP2W1 for biological activity.

A library of duocarmycin bioprecursors based on the CPI and CBI scaffolds was synthesized and used to probe selective activation by cells expressing CYP1A1 and 2W1, CYPs known to be expressed in high frequency in some tumors. Several CPI-based compounds were pM-nM potent in CYP1A1 expressing cells. CYP2W1 was also shown to sensitize proliferating cells to several compounds, demonstrating its potential as a target for tumor selective activation of duocarmycin bioprecursors.

Colon cancer-specific cytochrome P450 2W1 converts duocarmycin analogues into potent tumor cytotoxins.

PURPOSE: Cytochrome P450 2W1 (CYP2W1) is a monooxygenase detected in 30% of colon cancers, whereas its expression in nontransformed adult tissues is absent, rendering it a tumor-specific drug target for development of novel colon cancer chemotherapy. Previously, we have identified duocarmycin synthetic derivatives as CYP2W1 substrates. In this study, we investigated whether two of these compounds, ICT2705 and ICT2706, could be activated by CYP2W1 into potent antitumor agents. EXPERIMENTAL DESIGN: The cytotoxic activity of ICT2705 and ICT2706 in vitro was tested in colon cancer cell lines expressing CYP2W1, and in vivo studies with ICT2706 were conducted on severe combined immunodeficient mice bearing CYP2W1-positive colon cancer xenografts. RESULTS: Cells expressing CYP2W1 suffer rapid loss of viability following treatment with ICT2705 and ICT2706, whereas the CYP2W1-positive human colon cancer xenografts display arrested growth in the mice treated with ICT2706. The specific cytotoxic metabolite generated by CYP2W1 metabolism of ICT2706 was identified in vitro. The cytotoxic events were accompanied by an accumulation of phosphorylated H2A.X histone, indicating DNA damage as a mechanism for cancer cell toxicity. This cytotoxic effect is most likely propagated by a bystander killing mechanism shown in colon cancer cells. Pharmacokinetic analysis of ICT2706 in mice identified higher concentration of the compound in tumor than in plasma, indicating preferential accumulation of drug in the target tissue. CONCLUSION: Our findings suggest a novel approach for treatment of colon cancer that uses a locoregional activation of systemically inactive prodrug by the tumor-specific activator enzyme CYP2W1.