Mechanisms of coordinating hyaluronan and glycosaminoglycan production by nucleotide sugars.

Hyaluronan is a versatile macromolecule capable of an exceptional range of functions from cushioning and hydration to dynamic signaling in development and disease. Because of its critical roles, hyaluronan production is regulated at multiple levels including epigenetic, transcriptional, and posttranslational control of the three hyaluronan synthase (HAS) enzymes. Precursor availability can dictate the rate and amount of hyaluronan synthesized and shed by the cells producing it. However, the nucleotide-activated sugar substrates for hyaluronan synthesis by HAS also participate in exquisitely fine-tuned cross-talking pathways that intersect with glycosaminoglycan production and central carbohydrate metabolism. Multiple UDP-sugars have alternative metabolic fates and exhibit coordinated and reciprocal allosteric control of enzymes within their biosynthetic pathways to preserve appropriate precursor ratios for accurate partitioning among downstream products, while also sensing and maintaining energy homeostasis. Since the dysregulation of nucleotide sugar and hyaluronan synthesis is associated with multiple pathologies, these pathways offer opportunities for therapeutic intervention. Recent structures of several key rate-limiting enzymes in the UDP-sugar synthesis pathways have offered new insights to the overall regulation of hyaluronan production by precursor fate decisions. The details of UDP-sugar control and the structural basis for underlying mechanisms are discussed in this review.

Inhibiting Hexamer Disassembly of Human UDP-Glucose Dehydrogenase by Photoactivated Amino Acid Cross-Linking.

The enzyme UDP-glucose dehydrogenase (UGDH) catalyzes the reaction of UDP-glucose to UDP-glucuronate through two successive NAD(+)-dependent oxidation steps. Human UGDH apoprotein is purified as a mixture of dimeric and hexameric species. Addition of substrate and cofactor stabilizes the oligomeric state to primarily the hexameric form. To determine if the dynamic conformations of hUGDH are required for catalytic activity, we used site-specific unnatural amino acid incorporation to facilitate cross-linking of monomeric subunits into predominantly obligate oligomeric species. Optimal cross-linking was achieved by encoding p-benzoyl-l-phenylalanine at position 458, normally a glutamine located within the dimer-dimer interface, and exposing the enzyme to long wavelength ultraviolet (UV) radiation in the presence of substrate and cofactor. Hexameric complexes were purified by gel filtration chromatography and found to contain significant fractions of dimer and trimer (approximately 50%) along with another 10% higher-molecular mass species. The activity of the cross-linked enzyme was reduced by almost 60% relative to that of the un-cross-linked UGDH mutant, and UV exposure had no effect on the activity of the wild-type enzyme. These results support a model for catalysis in which the ability to dissociate the dimer-dimer interface is as important for maximal enzyme function as has been previously shown for the formation of the hexamer.

Hyaluronidase Hyal1 Increases Tumor Cell Proliferation and Motility through Accelerated Vesicle Trafficking.

Hyaluronan (HA) turnover accelerates metastatic progression of prostate cancer in part by increasing rates of tumor cell proliferation and motility. To determine the mechanism, we overexpressed hyaluronidase 1 (Hyal1) as a fluorescent fusion protein and examined its impact on endocytosis and vesicular trafficking. Overexpression of Hyal1 led to increased rates of internalization of HA and the endocytic recycling marker transferrin. Live imaging of Hyal1, sucrose gradient centrifugation, and specific colocalization of Rab GTPases defined the subcellular distribution of Hyal1 as early and late endosomes, lysosomes, and recycling vesicles. Manipulation of vesicular trafficking by chemical inhibitors or with constitutively active and dominant negative Rab expression constructs caused atypical localization of Hyal1. Using the catalytically inactive point mutant Hyal1-E131Q, we found that enzymatic activity of Hyal1 was necessary for normal localization within the cell as Hyal1-E131Q was mainly detected within the endoplasmic reticulum. Expression of a HA-binding point mutant, Hyal1-Y202F, revealed that secretion of Hyal1 and concurrent reuptake from the extracellular space are critical for rapid HA internalization and cell proliferation. Overall, excess Hyal1 secretion accelerates endocytic vesicle trafficking in a substrate-dependent manner, promoting aggressive tumor cell behavior.

UDP-glucose dehydrogenase activity and optimal downstream cellular function require dynamic reorganization at the dimer-dimer subunit interfaces.

UDP-glucose dehydrogenase (UGDH) provides precursors for steroid elimination, hyaluronan production, and glycosaminoglycan synthesis. The wild-type UGDH enzyme purifies in a hexamer-dimer equilibrium and transiently undergoes dynamic motion that exposes the dimer-dimer interface during catalysis. In the current study we created and characterized point mutations that yielded exclusively dimeric species (obligate dimer, T325D), dimeric species that could be induced to form hexamers in the ternary complex with substrate and cofactor (T325A), and a previously described exclusively hexameric species (UGDHΔ132) to investigate the role of quaternary structure in regulation of the enzyme. Characterization of the purified enzymes revealed a significant decrease in the enzymatic activity of the obligate dimer and hexamer mutants. Kinetic analysis of wild-type UGDH and the inducible hexamer, T325A, showed that upon increasing enzyme concentration, which favors the hexameric species, activity was modestly decreased and exhibited cooperativity. In contrast, cooperative kinetic behavior was not observed in the obligate dimer, T325D. These observations suggest that the regulation of the quaternary assembly of the enzyme is essential for optimal activity and allosteric regulation. Comparison of kinetic and thermal stability parameters revealed structurally dependent properties consistent with a role for controlled assembly and disassembly of the hexamer in the regulation of UGDH. Finally, both T325A and T325D mutants were significantly less efficient in promoting downstream hyaluronan production by HEK293 cells. These data support a model that requires an operational dimer-hexamer equilibrium to function efficiently and preserve regulated activity in the cell.

UDP-glucose dehydrogenase polymorphisms from patients with congenital heart valve defects disrupt enzyme stability and quaternary assembly.

Cardiac valve defects are a common congenital heart malformation and a significant clinical problem. Defining molecular factors in cardiac valve development has facilitated identification of underlying causes of valve malformation. Gene disruption in zebrafish revealed a critical role for UDP-glucose dehydrogenase (UGDH) in valve development, so this gene was screened for polymorphisms in a patient population suffering from cardiac valve defects. Two genetic substitutions were identified and predicted to encode missense mutations of arginine 141 to cysteine and glutamate 416 to aspartate, respectively. Using a zebrafish model of defective heart valve formation caused by morpholino oligonucleotide knockdown of UGDH, transcripts encoding the UGDH R141C or E416D mutant enzymes were unable to restore cardiac valve formation and could only partially rescue cardiac edema. Characterization of the mutant recombinant enzymes purified from Escherichia coli revealed modest alterations in the enzymatic activity of the mutants and a significant reduction in the half-life of enzyme activity at 37 °C. This reduction in activity could be propagated to the wild-type enzyme in a 1:1 mixed reaction. Furthermore, the quaternary structure of both mutants, normally hexameric, was destabilized to favor the dimeric species, and the intrinsic thermal stability of the R141C mutant was highly compromised. The results are consistent with the reduced function of both missense mutations significantly reducing the ability of UGDH to provide precursors for cardiac cushion formation, which is essential to subsequent valve formation. The identification of these polymorphisms in patient populations will help identify families genetically at risk for valve defects.

Characterization of IRDye 800CW chlorotoxin as a targeting agent for brain tumors.

Primary brain tumors present significant challenges for surgical resection because of their location and the frequent occurrence of malignant projections extending beyond the primary tumor. Visualization of the tumor margins during surgery is critical for a favorable outcome. We report the use of IRDye 800CW chlorotoxin (CLTX) as a targeted imaging agent for brain tumors in a spontaneous mouse model of medulloblastoma, ND2:SmoA1. Specificity and functionality of the targeted agent were confirmed in cell-based assays. Tumors were detected by magnetic resonance imaging and IRDye 800CW CLTX administered to individual animals for optical imaging at 1-month increments. The integrity of the blood-brain barrier (BBB) was measured by Evan's Blue perfusion prior to sacrifice. Results show that IRDye 800CW CLTX specifically targeted tumor tissue. The extravasation of Evan's Blue was observed in all tumors, suggesting that the presence of the tumors can introduce alterations in the permeability of the BBB. Because increased vascular permeability was observed early in the disease model, larger dye-labeled imaging agents that exceed current BBB size restrictions may warrant renewed consideration as candidates for tumor detection and surgical resection. Our study provides data characterizing in vitro and in vivo use of IRDye 800CW CLTX as a broadly applicable tumor imaging agent.

Systemic blockade of the hyaluronan receptor for endocytosis prevents lymph node metastasis of prostate cancer.

Tumor progression and metastasis are promoted by the remodeling of organized tissue architecture and engagement of molecular interactions that support tumor cell passage through endothelial barriers. Prostate tumor cells that secrete and turn over excessive quantities of pericellular hyaluronan (HA) exhibit accelerated growth kinetics and spontaneous lymph node metastasis in mice. The HA receptor for endocytosis (HARE) is an endocytic clearance receptor for HA in the liver that is also highly expressed in sinusoidal endothelium of lymph nodes and bone marrow, which are frequent sites of prostate cancer metastasis. In our study, we tested the hypothesis that HARE can act as an endothelial receptor for metastatic tumor cells with pericellular HA. In an orthotopic mouse model of prostate cancer, we delivered a monoclonal antibody against HARE that specifically blocks HA binding and internalization. This treatment fully blocked the formation of metastatic tumors in lymph nodes. No effects on primary tumor growth were observed and the antibody did not induce toxic outcomes in any other tissue. Our results implicate HARE for the first time in potentiation of tumor metastasis and suggest a novel mechanism by which tumor cell-associated HA could promote tissue-specific dissemination. "Published 2012 Wiley Periodicals, Inc. This article is a US Government work, and, as such, is in the public domain in the United States of America."

Hyaluronan suppresses prostate tumor cell proliferation through diminished expression of N-cadherin and aberrant growth factor receptor signaling.

Hyaluronan (HA) production has been functionally implicated in prostate tumorigenesis and metastasis. We previously used prostate tumor cells overexpressing the HA synthesizing enzyme HAS3 or the clinically relevant hyaluronidase Hyal1 to show that excess HA production suppresses tumor growth, while HA turnover accelerates spontaneous metastasis from the prostate. Here, we examined pathways responsible for effects of HAS3 and Hyal1 on tumor cell phenotype. Detailed characterization of cell cycle progression revealed that expression of Hyal1 accelerated cell cycle re-entry following synchronization, whereas HAS3 alone delayed entry. Hyal1 expressing cells exhibited a significant reduction in their ability to sustain ERK phosphorylation upon stimulation by growth factors, and in their expression of the cyclin-dependent kinase inhibitor p21. In contrast, HAS3 expressing cells showed prolonged ERK phosphorylation and increased expression of both p21 and p27, in asynchronous and synchronized cultures. Changes in cell cycle regulatory proteins were accompanied by HA-induced suppression of N-cadherin, while E-cadherin expression and ß-catenin expression and distribution remained unchanged. Our results are consistent with a model in which excess HA synthesis suppresses cell proliferation by promoting homotypic E-cadherin mediated cell-cell adhesion, consequently signaling to elevate cell cycle inhibitor expression and suppress G1- to S-phase transition.

Enzymatic defects underlying hereditary glutamate cysteine ligase deficiency are mitigated by association of the catalytic and regulatory subunits.

Glutamate cysteine ligase (GCL) deficiency is a rare autosomal recessive trait that compromises production of glutathione, a critical redox buffer and enzymatic cofactor. Patients have markedly reduced levels of erythrocyte glutathione, leading to hemolytic anemia and, in some cases, impaired neurological function. Human glutamate cysteine ligase is a heterodimer comprised of a catalytic subunit (GCLC) and a regulatory subunit (GCLM), which catalyzes the initial rate-limiting step in glutathione production. Four clinical missense mutations have been identified within GCLC: Arg127Cys, Pro158Leu, His370Leu, and Pro414Leu. Here, we have evaluated the impacts of these mutations on enzymatic function in vivo and in vitro to gain further insight into the pathology. Embryonic fibroblasts from GCLC null mice were transiently transfected with wild-type or mutant GCLC, and cellular glutathione levels were determined. The four mutant transfectants each had significantly lower levels of glutathione relative to that of the wild type, with the Pro414Leu mutant being most compromised. The contributions of the regulatory subunit to GCL activity were investigated using a Saccharomyces cerevisiae model system. Mutant GCLC alone could not complement a glutathione deficient strain and required the concurrent addition of GCLM to restore growth. Kinetic characterizations of the recombinant GCLC mutants indicated that the Arg127Cys, His370Leu, and Pro414Leu mutants have compromised enzymatic activity that can largely be rescued by the addition of GCLM. Interestingly, the Pro158Leu mutant has kinetic constants comparable to those of wild-type GCLC, suggesting that heterodimer formation is needed for stability in vivo. Strategies that promote heterodimer formation and persistence would be effective therapeutics for the treatment of GCL deficiency.

Near-infrared-labeled tetracycline derivative is an effective marker of bone deposition in mice.

Bone-specific compounds have been used effectively for the detection of bone mineralization, growth, and morphological changes. These agents typically contain iminodiacetic acid groups that can form complexes with apatite and fluoresce in the visible spectrum. We exploited a subset of these chemical chelators to produce a near-infrared (NIR) optical bone marker for preclinical animal imaging. By conjugating target compounds to IRDye 800CW, we extended the effective fluorescence signal detection to the NIR region without affecting the compound's ability to function as a marker of the mineralization process. Calcein and a tetracycline derivative (BoneTag agent [BT]) bound specifically to differentiated mineralized osteoblast cultures, with the latter exhibiting 6-fold higher signal intensities. Subsequent in vivo testing demonstrated effective skeletal labeling with IRDye 800CW BT. We were able to identify a changing mineralization front in bone sections from (i) normal growing mice injected with IRDye 800CW BT 6weeks prior to the administration of IRDye 680 BT and (ii) an osteoporosis mouse model comparing cortical bone in sham-treated and ovariectomized mice. These results provide evidence that the NIR-labeled BT is effective as a general marker of skeletal features and an indicator of the bone mineralization and remodeling processes.

Integration of Sugar Metabolism and Proteoglycan Synthesis by UDP-glucose Dehydrogenase.

Regulation of proteoglycan and glycosaminoglycan synthesis is critical throughout development, and to maintain normal adult functions in wound healing and the immune system, among others. It has become increasingly clear that these processes are also under tight metabolic control and that availability of carbohydrate and amino acid metabolite precursors has a role in the control of proteoglycan and glycosaminoglycan turnover. The enzyme uridine diphosphate (UDP)-glucose dehydrogenase (UGDH) produces UDP-glucuronate, an essential precursor for new glycosaminoglycan synthesis that is tightly controlled at multiple levels. Here, we review the cellular mechanisms that regulate UGDH expression, discuss the structural features of the enzyme, and use the structures to provide a context for recent studies that link post-translational modifications and allosteric modulators of UGDH to its function in downstream pathways.

Altered glucuronidation deregulates androgen dependent response profiles and signifies castration resistance in prostate cancer.

Glucuronidation controls androgen levels in the prostate and the dysregulation of enzymes in this pathway is associated with castration resistant prostate cancer. UDP-glucose dehydrogenase (UGDH) produces UDP-glucuronate, the essential precursor for glucuronidation, and its expression is elevated in prostate cancer. We compared protein and metabolite levels relevant to the glucuronidation pathway in five prostate cancer patient-derived xenograft models paired with their isogenic counterparts that were selected in vivo for castration resistant (CR) recurrence. All pairs showed changes in UGDH and associated enzymes and metabolites that were consistent with those we found in an isogenic androgen dependent (AD) and CR LNCaP prostate cancer model. Ectopic overexpression of UGDH in LNCaP AD cells blunted androgen-dependent gene expression, increased proteoglycan synthesis, significantly increased cell growth compared to controls, and eliminated dose responsive growth suppression with enzalutamide treatment. In contrast, the knockdown of UGDH diminished proteoglycans, suppressed androgen dependent growth irrespective of androgens, and restored androgen sensitivity in CR cells. Importantly, the knockdown of UGDH in both LNCaP AD and CR cells dramatically sensitized these cells to enzalutamide. These results support a role for UGDH in androgen responsiveness and a target for therapeutic strategies in advanced prostate cancer.

Udp-glucose dehydrogenase as a novel field-specific candidate biomarker of prostate cancer.

Uridine diphosphate (UDP)-glucose dehydrogenase (UGDH) catalyzes the oxidation of UDP-glucose to yield UDP-glucuronic acid, a precursor for synthesis of glycosaminoglycans and proteoglycans that promote aggressive prostate cancer (PC) progression. The purpose of our study was to determine if the UGDH expression in normal appearing acini (NAA) from cancerous glands is a candidate biomarker for PC field disease/effect assayed by quantitative fluorescence imaging analysis (QFIA). A polyclonal antibody to UGDH was titrated to saturation binding and fluorescent microscopic images acquired from fixed, paraffin-embedded tissue slices were quantitatively analyzed. Specificity of the assay was confirmed by Western blot analysis and competitive inhibition of tissue labeling with the recombinant UGDH. Reproducibility of the UGDH measurements was high within and across analytical runs. Quantification of UGDH by QFIA and Reverse-Phase Protein Array analysis were strongly correlated (r = 0.97), validating the QFIA measurements. Analysis of cancerous acini (CA) and NAA from PC patients vs. normal acini (NA) from noncancerous controls (32 matched pairs) revealed significant (p < 0.01) differences, with CA (increased) vs. NA, NAA (decreased) vs. NA and CA (increased) vs. NAA. Areas under the Receiver Operating Characteristic curves were 0.68 (95% CI: 0.59-0.83) for NAA and 0.71 (95% CI: 0.59-0.83) for CA (both vs. NA). These results support the UGDH content in prostatic acini as a novel candidate biomarker that may complement the development of a multi-biomarker panel for detecting PC within the tumor adjacent field on a histologically normal biopsy specimen.

Spontaneous metastasis of prostate cancer is promoted by excess hyaluronan synthesis and processing.

Accumulation of extracellular hyaluronan (HA) and its processing enzyme, the hyaluronidase Hyal1, predicts invasive, metastatic progression of human prostate cancer. To dissect the roles of hyaluronan synthases (HAS) and Hyal1 in tumorigenesis and metastasis, we selected nonmetastatic 22Rv1 prostate tumor cells that overexpress HAS2, HAS3, or Hyal1 individually, and compared these cells with co-transfectants expressing Hyal1 + HAS2 or Hyal1 + HAS3. Cells expressing only HAS were less tumorigenic than vector control transfectants on orthotopic injection into mice. In contrast, cells co-expressing Hyal1 + HAS2 or Hyal1 + HAS3 showed greater than sixfold and twofold increases in tumorigenesis, respectively. Fluorescence and histological quantification revealed spontaneous lymph node metastasis in all Hyal1 transfectant-implanted mice, and node burden increased an additional twofold when Hyal1 and HAS were co-expressed. Cells only expressing HAS were not metastatic. Thus, excess HA synthesis and processing in concert accelerate the acquisition of a metastatic phenotype by prostate tumor cells. Intratumoral vascularity did not correlate with either tumor size or metastatic potential. Analysis of cell cycle progression revealed shortened doubling times of Hyal1-expressing cells. Both adhesion and motility on extracellular matrix were diminished in HA-overproducing cells; however, motility was increased twofold by Hyal1 expression and fourfold to sixfold by Hyal1/HAS co-expression, in close agreement with observed metastatic potential. This is the first comprehensive examination of these enzymes in a relevant prostate cancer microenvironment.

Characterization and performance of a near-infrared 2-deoxyglucose optical imaging agent for mouse cancer models.

Malignant neoplasms exhibit an elevated rate of glycolysis over normal cells. This characteristic can be exploited for optical imaging of tumors in mice. A near-infrared fluorophore, IRDye 800CW, emission maximum 794 nm, was conjugated to 2-deoxyglucose (2-DG). An immunofluorescent cell-based assay was used to evaluate specificity and sensitivity of the conjugate in cultured cell monolayers. Dose-dependent uptake was established with increasing concentrations of IRDye 800CW 2-DG for epithelial and prostate carcinomas. IRDye 800CW 2-DG was specifically blocked by an antibody against GLUT1 glucose transporter, and by excess unlabeled 2-DG or d-glucose. Signal was increased by a phorbol ester activator of glucose transport. Fluorescence microscopy data confirmed localization of the conjugate in the cytoplasm. Subsequent in vivo studies optimized dose, clearance, and timing for signal capture in nude mouse xenografts. In all cases, tumors were clearly imaged with good signal-to-noise characteristics. These data indicate that IRDye 800CW 2-DG is a broadly applicable optical imaging agent for in vivo imaging of neoplasms in mice.

Melanoma chondroitin sulfate proteoglycan enhances FAK and ERK activation by distinct mechanisms.

Melanoma chondroitin sulfate proteoglycan (MCSP) is an early cell surface melanoma progression marker implicated in stimulating tumor cell proliferation, migration, and invasion. Focal adhesion kinase (FAK) plays a pivotal role in integrating growth factor and adhesion-related signaling pathways, facilitating cell spreading and migration. Extracellular signal-regulated kinase (ERK) 1 and 2, implicated in tumor growth and survival, has also been linked to clinical melanoma progression. We have cloned the MCSP core protein and expressed it in the MCSP-negative melanoma cell line WM1552C. Expression of MCSP enhances integrin-mediated cell spreading, FAK phosphorylation, and activation of ERK1/2. MCSP transfectants exhibit extensive MCSP-rich microspikes on adherent cells, where it also colocalizes with alpha4 integrin. Enhanced activation of FAK and ERK1/2 by MCSP appears to involve independent mechanisms because inhibition of FAK activation had no effect on ERK1/2 phosphorylation. These results indicate that MCSP may facilitate primary melanoma progression by enhancing the activation of key signaling pathways important for tumor invasion and growth.

Hyaluronan Pericellular Matrix: Particle Exclusion Assay.

Particle exclusion assays are used to visualize pericellular envelopes with a high content of hyaluronan. Pericellular hyaluronan is basally abundant in certain cell types while in others it is deposited in a highly dynamic manner in response to specific conditions and its presence may indicate cellular status. This assay, described here, is a quick semiquantitative approach to detecting pericellular hyaluronan using the hyaluronan-binding proteoglycan, aggrecan, to stabilize and amplify the surface matrix. Hyaluronan matrix can then be observed and quantified by microscopic image analysis of clear zones around individual cells, from which exogenously added fixed red blood cell particles are excluded.

Prostate tumor cell exosomes containing hyaluronidase Hyal1 stimulate prostate stromal cell motility by engagement of FAK-mediated integrin signaling.

The hyaluronidase Hyal1 is clinically and functionally implicated in prostate cancer progression and metastasis. Elevated Hyal1 accelerates vesicular trafficking in prostate tumor cells, thereby enhancing their metastatic potential in an autocrine manner through increased motility and proliferation. In this report, we found Hyal1 protein is a component of exosomes produced by prostate tumor cell lines overexpressing Hyal1. We investigated the role of exosomally shed Hyal1 in modulating tumor cell autonomous functions and in modifying the behavior of prostate stromal cells. Catalytic activity of Hyal1 was necessary for enrichment of Hyal1 in the exosome fraction, which was associated with increased presence of LC3BII, an autophagic marker, in the exosomes. Hyal1-positive exosome contents were internalized from the culture medium by WPMY-1 prostate stromal fibroblasts. Treatment of prostate stromal cells with tumor exosomes did not affect proliferation, but robustly stimulated their migration in a manner dependent on Hyal1 catalytic activity. Increased motility of exosome-treated stromal cells was accompanied by enhanced adhesion to a type IV collagen matrix, as well as increased FAK phosphorylation and integrin engagement through dynamic membrane residence of ß1 integrins. The presence of Hyal1 in tumor-derived exosomes and its ability to impact the behavior of stromal cells suggests cell-cell communication via exosomes is a novel mechanism by which elevated Hyal1 promotes prostate cancer progression.

Hyaluronan and hyaluronidase in genitourinary tumors.

Genitourinary cancers are the most frequently diagnosed cancers in men and the fifth most common in women. Management of disease through accurate and cost effective early diagnostic markers, as well as identification of valid prognostic indicators, has contributed significantly to improved treatment outcomes. In this review, we will discuss the function, regulation and clinical utility of hyaluronan (HA), genes encoding its metabolic enzymes and receptors that mediate its cellular effects. Specific HA synthase (HAS) and hyaluronidase (HAase) genes encode the enzymes that produce HA polymers and oligosaccharides, respectively. Differential effects of these enzymes in progression of genitourinary tumors are determined by the relative balance between HAS and HAase levels, as well as the distribution of receptors. The genes are regulated in a complex fashion at the transcriptional and post-translational levels, but also by epigenetic events, alternative mRNA splicing, and subcellular localization. Importantly, the major tumor-derived HAase enzyme, HYAL-1, either alone or together with HA, is an accurate diagnostic and prognostic marker for genitourinary tumors.

Molecular Pharmacodynamics-Guided Scheduling of Biologically Effective Doses: A Drug Development Paradigm Applied to MET Tyrosine Kinase Inhibitors.

The development of molecularly targeted agents has benefited from use of pharmacodynamic markers to identify "biologically effective doses" (BED) below MTDs, yet this knowledge remains underutilized in selecting dosage regimens and in comparing the effectiveness of targeted agents within a class. We sought to establish preclinical proof-of-concept for such pharmacodynamics-based BED regimens and effectiveness comparisons using MET kinase small-molecule inhibitors. Utilizing pharmacodynamic biomarker measurements of MET signaling (tumor pY1234/1235MET/total MET ratio) in a phase 0-like preclinical setting, we developed optimal dosage regimens for several MET kinase inhibitors and compared their antitumor efficacy in a MET-amplified gastric cancer xenograft model (SNU-5). Reductions in tumor pY1234/1235MET/total MET of 95%-99% were achievable with tolerable doses of EMD1214063/MSC2156119J (tepotinib), XL184 (cabozantinib), and XL880/GSK1363089 (foretinib), but not ARQ197 (tivantinib), which did not alter the pharmacodynamic biomarker. Duration of kinase suppression and rate of kinase recovery were specific to each agent, emphasizing the importance of developing customized dosage regimens to achieve continuous suppression of the pharmacodynamic biomarker at the required level (here, ≥90% MET kinase suppression). The customized dosage regimen of each inhibitor yielded substantial and sustained tumor regression; the equivalent effectiveness of customized dosage regimens that achieve the same level of continuous molecular target control represents preclinical proof-of-concept and illustrates the importance of proper scheduling of targeted agent BEDs. Pharmacodynamics-guided biologically effective dosage regimens (PD-BEDR) potentially offer a superior alternative to pharmacokinetic guidance (e.g., drug concentrations in surrogate tissues) for developing and making head-to-head comparisons of targeted agents. Mol Cancer Ther; 17(3); 698-709. ©2018 AACR.