Comprehensive Target Engagement by the EZH2 Inhibitor Tulmimetostat Allows for Targeting of ARID1A Mutant Cancers.

Recurrent somatic mutations in the BRG1/BRM-associated factor (BAF) chromatin remodeling complex subunit ARID1A occur frequently in advanced urothelial, endometrial, and ovarian clear cell carcinomas, creating an alternative chromatin state that may be exploited therapeutically. The histone methyltransferase EZH2 has been previously identified as targetable vulnerability in the context of ARID1A mutations. In this study, we describe the discovery of tulmimetostat, an orally available, clinical stage EZH2 inhibitor, and it elucidates the aspects of its application potential in ARID1A mutant tumors. Tulmimetostat administration achieved efficacy in multiple ARID1A mutant bladder, ovarian, and endometrial tumor models and improved cisplatin response in chemotherapy-resistant models. Consistent with its comprehensive and durable level of target coverage, tulmimetostat demonstrated greater efficacy than other PRC2-targeted inhibitors at comparable or lower exposures in a bladder cancer xenograft mouse model. Tulmimetostat mediated extensive changes in gene expression, in addition to a profound reduction in global H3K27me3 levels in tumors. Phase I clinical pharmacokinetic and pharmacodynamic data indicated that tulmimetostat exhibits durable exposure and profound target engagement. Importantly, a tulmimetostat controlled gene expression signature identified in whole blood from a cohort of 32 patients with cancer correlated with tulmimetostat exposure, representing a pharmacodynamic marker for the assessment of target coverage for PRC2-targeted agents in the clinic. Collectively, these data suggest that tulmimetostat has the potential to achieve clinical benefit in solid tumors as a monotherapy but also in combination with chemotherapeutic agents, and may be beneficial in various indications with recurrent ARID1A mutations. Significance: The EZH2 inhibitor tulmimetostat achieves comprehensive target inhibition in ARID1A mutant solid tumor models and cancer patients that can be assessed with a pharmacodynamic gene signature in peripheral blood.

Functional properties and substrate characterization of human CYP26A1, CYP26B1, and CYP26C1 expressed by recombinant baculovirus in insect cells.

INTRODUCTION: The cytochrome P450 CYP26 family of retinoic acid (RA) metabolizing enzymes, comprising CYP26A1, CYP26B1, and CYP26C1 is critical for establishing patterns of RA distribution during embryonic development and retinoid homeostasis in the adult. All three members of this family can metabolize all trans-RA. CYP26C1 has also been shown to efficiently metabolize the 9-cis isomer of RA. METHODS: We have co-expressed each of the CYP26 enzymes along with the NADPH-cytochrome P450 oxidoreductase using a baculovirus/Sf9 insect cell expression system to determine the enzymatic activities of these enzymes in cell free preparations and have established an in vitro binding assay to permit comparison of binding affinities of the three CYP26 enzymes. RESULTS: We demonstrated that the expressed enzymes can efficiently coordinate heme, as verified by spectral-difference analysis. All CYP26s efficiently metabolized all-trans-RA to polar aqueous-soluble metabolites, and in competition experiments exhibited IC(50) values of 16, 27, and 15nM for CYP26A1, B1, and C1 respectively for all-trans-RA. Furthermore, this metabolism was blocked with the CYP inhibitor ketoconazole. CYP26C1 metabolism of all trans-RA could also be effectively competed with 9-cis RA, with IC(50) of 62nM, and was sensitive to ketoconazole inhibition. DISCUSSION: CYP26 enzymes are functionally expressed in microsomal fractions of insect cells and stably bind radiolabeled RA isomers with affinities respecting their substrate specificities. We demonstrated that compared to CYP26A and CYP26B, only CYP26C1 was able to bind with high affinity to 9-cis-RA. These assays will be useful for the screening of synthetic substrates and inhibitors of CYP26 enzymes and may be applicable to other cytochrome P450s and their respective substrates.

CYP24, the enzyme that catabolizes the antiproliferative agent vitamin D, is increased in lung cancer.

1Alpha,25-dihydroxyvitamin D3 (1,25D3) displays potent antiproliferative activity in a variety of tumor model systems and is currently under investigation in clinical trials in cancer. Studies were initiated to explore its potential in nonsmall cell lung cancer (NSCLC), as effective approaches to the treatment of that disease are needed. In evaluating factors that may affect activity in NSCLC, the authors found that CYP24 (25-hydroxyvitamin D3-24-hydroxylase), the enzyme that catabolizes 1,25D3, is frequently expressed in NSCLC cell lines but not in the nontumorigenic bronchial epithelial cell line, Beas2B. CYP24 expression by RT-PCR was also detected in 10/18 primary lung tumors but in only 1/11 normal lung tissue specimens. Tumor-specific CYP24 upregulation was confirmed at the protein level via immunoblot analysis of patient-matched normal lung tissue and lung tumor extracts. Enzymatically active CYP24 is expected to desensitize NSCLC cells to 1,25D3. The authors therefore implemented a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay for 1,25D3 and its CYP24-generated metabolites to determine whether NSCLC cells express active enzyme. Analysis of NSCLC cell cultures revealed time-dependent loss of 1,25D3 coincident with the appearance of CYP24-generated metabolites. MK-24(S)-S(O)(NH)-Ph-1, a specific inhibitor of CYP24, slowed the loss of 1,25D3 and increased 1,25D3 half-life. Furthermore, combination of 1,25D3 with MK-24(S)-S(O)(NH)-Ph-1 resulted in a significant decrease in the concentration of 1,25D3 required to achieve maximum growth inhibition in NSCLC cells. These data suggest that increased CYP24 expression in lung tumors restricts 1,25D3 activity and support the preclinical evaluation of CYP24 inhibitors for lung cancer treatment.

A novel human cytochrome P450, CYP26C1, involved in metabolism of 9-cis and all-trans isomers of retinoic acid.

Retinoids are potent regulators of cell proliferation, cell differentiation, and morphogenesis and are important therapeutic agents in oncology and dermatology. The gene regulatory activity of endogenous retinoids is effected primarily by retinoic acid isomers (all-trans and 9-cis) that are synthesized from retinaldehyde precursors in a broad range of tissues and act as ligands for nuclear retinoic acid receptors. The catabolism of all-trans-retinoic acid (atRA) is an important mechanism of controlling RA levels in cell and tissues. We have previously identified two cytochrome P450s, P450RAI-1 and P450RAI-2 (herein named CYP26A1 and CYP26B1), which were shown to be responsible for catabolism of atRA both in the embryo and the adult. In this report, we describe the identification, molecular cloning, and substrate characterization of a third member of the CYP26 family, named CYP26C1. Transiently transfected cells expressing CYP26C1 convert atRA to polar water-soluble metabolites similar to those generated by CYP26A1 and -B1. Competition studies with all-trans, 13-cis, and 9-cis isomers of retinoic acid demonstrated that atRA was the preferred substrate for CYP26C1. Although CYP26C1 shares extensive sequence similarity with CYP26A1 and CYP26B1, its catalytic activity appears distinct from those of other CYP26 family members. Specifically, CYP26C1 can also recognize and metabolize 9-cis-RA and is much less sensitive than the other CYP26 family members to the inhibitory effects of ketoconazole. CYP26C1 is not widely expressed in the adult but is inducible by RA in HPK1a, transformed human keratinocyte cell lines. This third CYP26 member may play a specific role in catabolizing both all-trans and 9-cis isomers of RA.

CYP2U1, a novel human thymus- and brain-specific cytochrome P450, catalyzes omega- and (omega-1)-hydroxylation of fatty acids.

Long chain fatty acids have recently emerged as critical signaling molecules in neuronal, cardiovascular, and renal processes, yet little is presently known about the precise mechanisms controlling their tissue distribution and bioactivation. We have identified a novel cytochrome P450, CYP2U1, which may play an important role in modulating the arachidonic acid signaling pathway. Northern blot and real-time PCR analysis demonstrated that CYP2U1 transcripts were most abundant in the thymus and the brain (cerebellum), indicating a specific physiological role for CYP2U1 in these tissues. Recombinant human CYP2U1 protein, expressed in baculovirus-infected Sf9 insect cells, was found to metabolize arachidonic acid exclusively to two region-specific products as determined by liquid chromatography-mass spectrometry. These metabolites were identified as 19- and 20-hydroxy-modified arachidonic acids by liquid chromatography-tandem mass spectrometry analysis. In addition to omega/omega-1 hydroxylation of arachidonic acid, CYP2U1 protein also catalyzed the hydroxylation of structurally related long chain fatty acid (docosahexaenoic acid) but not fatty acids such as lauric acid or linoleic acid. This is the first report of the cloning and functional expression of a new human member of P450 family 2, CYP2U1, which metabolizes long chain fatty acids. Based on the ability of CYP2U1 to generate bioactive eicosanoid derivatives, we postulate that CYP2U1 plays an important physiological role in fatty acid signaling processes in both cerebellum and thymus.