ABBV-319: a CD19-targeting glucocorticoid receptor modulator antibody-drug conjugate therapy for B-cell malignancies.

ABSTRACT: Glucocorticoids are key components of the standard-of-care treatment regimens for B-cell malignancy. However, systemic glucocorticoid treatment is associated with several adverse events. ABBV-319 is a CD19-targeting antibody-drug conjugate engineered to reduce glucocorticoid-associated toxicities while possessing 3 distinct mechanisms of action (MOA) to increase therapeutic efficacy: (1) antibody-mediated delivery of a glucocorticoid receptor modulator (GRM) payload to activate apoptosis, (2) inhibition of CD19 signaling, and (3) enhanced fragment crystallizable (Fc)-mediated effector function via afucosylation of the antibody backbone. ABBV-319 elicited potent GRM-driven antitumor activity against multiple malignant B-cell lines in vitro, as well as in cell line-derived xenografts and patient-derived xenografts (PDXs) in vivo. Remarkably, a single dose of ABBV-319 induced sustained tumor regression and enhanced antitumor activity compared with repeated dosing of systemic prednisolone at the maximum tolerated dose in mice. The unconjugated CD19 monoclonal antibody (mAb) also displayed antiproliferative activity in a subset of B-cell lymphoma cell lines through the inhibition of phosphoinositide 3-kinase signaling. Moreover, afucosylation of CD19 mAb enhanced Fc-mediated antibody-dependent cellular cytotoxicity. Notably, ABBV-319 displayed superior efficacy compared with afucosylated CD19 mAb in human CD34+ peripheral blood mononuclear cell-engrafted NSG-Tg(Hu-IL15) transgenic mice, demonstrating enhanced antitumor activity when multiple MOAs are enabled. ABBV-319 also showed durable antitumor activity across multiple B-cell lymphoma PDX models, including nongerminal center B-cell diffuse large B-cell lymphoma and relapsed lymphoma after R-CHOP treatment. Collectively, these data support the ongoing evaluation of ABBV-319 in a phase 1 clinical trial.

ABBV-011, A Novel, Calicheamicin-Based Antibody-Drug Conjugate, Targets SEZ6 to Eradicate Small Cell Lung Cancer Tumors.

In the past year, four antibody-drug conjugates (ADC) were approved, nearly doubling the marketed ADCs in oncology. Among other attributes, successful ADCs optimize targeting antibody, conjugation chemistry, and payload mechanism of action. Here, we describe the development of ABBV-011, a novel SEZ6-targeted, calicheamicin-based ADC for the treatment of small cell lung cancer (SCLC). We engineered a calicheamicin conjugate that lacks the acid-labile hydrazine linker that leads to systemic release of a toxic catabolite. We then screened a patient-derived xenograft library to identify SCLC as a tumor type with enhanced sensitivity to calicheamicin ADCs. Using RNA sequencing (RNA-seq) data from primary and xenograft SCLC samples, we identified seizure-related homolog 6 (SEZ6) as a surface-expressed SCLC target with broad expression in SCLC and minimal normal tissue expression by both RNA-seq and IHC. We developed an antibody targeting SEZ6 that is rapidly internalized upon receptor binding and, when conjugated to the calicheamicin linker drug, drives potent tumor regression in vitro and in vivo. These preclinical data suggest that ABBV-011 may provide a novel treatment for patients with SCLC and a rationale for ongoing phase I studies (NCT03639194).

Uncialamycin-based antibody-drug conjugates: Unique enediyne ADCs exhibiting bystander killing effect.

Antibody-drug conjugates (ADCs) have emerged as valuable targeted anticancer therapeutics with at least 11 approved therapies and over 80 advancing through clinical trials. Enediyne DNA-damaging payloads represented by the flagship of this family of antitumor agents, N-acetyl calicheamicin [Formula: see text], have a proven success track record. However, they pose a significant synthetic challenge in the development and optimization of linker drugs. We have recently reported a streamlined total synthesis of uncialamycin, another representative of the enediyne class of compounds, with compelling synthetic accessibility. Here we report the synthesis and evaluation of uncialamycin ADCs featuring a variety of cleavable and noncleavable linkers. We have discovered that uncialamycin ADCs display a strong bystander killing effect and are highly selective and cytotoxic in vitro and in vivo.

Design, Synthesis, and Biological Evaluation of Tubulysin Analogues, Linker-Drugs, and Antibody-Drug Conjugates, Insights into Structure-Activity Relationships, and Tubulysin-Tubulin Binding Derived from X-ray Crystallographic Analysis.

Molecular design, synthesis, and biological evaluation of tubulysin analogues, linker-drugs, and antibody-drug conjugates are described. Among the new discoveries reported is the identification of new potent analogues within the tubulysin family that carry a C11 alkyl ether substituent, rather than the usual ester structural motif at that position, a fact that endows the former with higher plasma stability than that of the latter. Also described herein are X-ray crystallographic analysis studies of two tubulin-tubulysin complexes formed within the α/ß interface between two tubulin heterodimers and two highly potent tubulysin analogues, one of which exhibited a different binding mode to the one previously reported for tubulysin M. The X-ray crystallographic analysis-derived new insights into the binding modes of these tubulysin analogues explain their potencies and provide inspiration for further design, synthesis, and biological investigations within this class of antitumor agents. A number of these analogues were conjugated as payloads with appropriate linkers at different sites allowing their attachment onto targeting antibodies for cancer therapies. A number of such antibody-drug conjugates were constructed and tested, both in vivo and in vitro, leading to the identification of at least one promising ADC (Herceptin-LD3), warranting further investigations.

Evaluation of PNU-159682 antibody drug conjugates (ADCs).

PNU-159682 is a highly potent secondary metabolite of nemorubicin belonging to the anthracycline class of natural products. Due to its extremely high potency and only partially understood mechanism of action, it was deemed an interesting starting point for the development of a new suite of linker drugs for antibody drug conjugates (ADCs). Structure activity relationships were explored on the small molecule which led to six linker drugs being developed for conjugation to antibodies. Herein we describe the synthesis of novel PNU-159682 derivatives and the subsequent linker drugs as well as the corresponding biological evaluations of the small molecules and ADCs.

A PTK7-targeted antibody-drug conjugate reduces tumor-initiating cells and induces sustained tumor regressions.

Disease relapse after treatment is common in triple-negative breast cancer (TNBC), ovarian cancer (OVCA), and non-small cell lung cancer (NSCLC). Therapies that target tumor-initiating cells (TICs) should improve patient survival by eliminating the cells that can drive tumor recurrence and metastasis. We demonstrate that protein tyrosine kinase 7 (PTK7), a highly conserved but catalytically inactive receptor tyrosine kinase in the Wnt signaling pathway, is enriched on TICs in low-passage TNBC, OVCA, and NSCLC patient-derived xenografts (PDXs). To deliver a potent anticancer drug to PTK7-expressing TICs, we generated a targeted antibody-drug conjugate (ADC) composed of a humanized anti-PTK7 monoclonal antibody, a cleavable valine-citrulline-based linker, and Aur0101, an auristatin microtubule inhibitor. The PTK7-targeted ADC induced sustained tumor regressions and outperformed standard-of-care chemotherapy. Moreover, the ADC specifically reduced the frequency of TICs, as determined by serial transplantation experiments. In addition to reducing the TIC frequency, the PTK7-targeted ADC may have additional antitumor mechanisms of action, including the inhibition of angiogenesis and the stimulation of immune cells. Together, these preclinical data demonstrate the potential for the PTK7-targeted ADC to improve the long-term survival of cancer patients.

A DLL3-targeted antibody-drug conjugate eradicates high-grade pulmonary neuroendocrine tumor-initiating cells in vivo.

The high-grade pulmonary neuroendocrine tumors, small cell lung cancer (SCLC) and large cell neuroendocrine carcinoma (LCNEC), remain among the most deadly malignancies. Therapies that effectively target and kill tumor-initiating cells (TICs) in these cancers should translate to improved patient survival. Patient-derived xenograft (PDX) tumors serve as excellent models to study tumor biology and characterize TICs. Increased expression of delta-like 3 (DLL3) was discovered in SCLC and LCNEC PDX tumors and confirmed in primary SCLC and LCNEC tumors. DLL3 protein is expressed on the surface of tumor cells but not in normal adult tissues. A DLL3-targeted antibody-drug conjugate (ADC), SC16LD6.5, comprised of a humanized anti-DLL3 monoclonal antibody conjugated to a DNA-damaging pyrrolobenzodiazepine (PBD) dimer toxin, induced durable tumor regression in vivo across multiple PDX models. Serial transplantation experiments executed with limiting dilutions of cells provided functional evidence confirming that the lack of tumor recurrence after SC16LD6.5 exposure resulted from effective targeting of DLL3-expressing TICs. In vivo efficacy correlated with DLL3 expression, and responses were observed in PDX models initiated from patients with both limited and extensive-stage disease and were independent of their sensitivity to standard-of-care chemotherapy regimens. SC16LD6.5 effectively targets and eradicates DLL3-expressing TICs in SCLC and LCNEC PDX tumors and is a promising first-in-class ADC for the treatment of high-grade pulmonary neuroendocrine tumors.

Anti-EFNA4 Calicheamicin Conjugates Effectively Target Triple-Negative Breast and Ovarian Tumor-Initiating Cells to Result in Sustained Tumor Regressions.

PURPOSE: Triple-negative breast cancer (TNBC) and ovarian cancer each comprise heterogeneous tumors, for which current therapies have little clinical benefit. Novel therapies that target and eradicate tumor-initiating cells (TIC) are needed to significantly improve survival. EXPERIMENTAL DESIGN: A panel of well-annotated patient-derived xenografts (PDX) was established, and surface markers that enriched for TIC in specific tumor subtypes were empirically determined. The TICs were queried for overexpressed antigens, one of which was selected to be the target of an antibody-drug conjugate (ADC). The efficacy of the ADC was evaluated in 15 PDX models to generate hypotheses for patient stratification. RESULTS: We herein identified E-cadherin (CD324) as a surface antigen able to reproducibly enrich for TIC in well-annotated, low-passage TNBC and ovarian cancer PDXs. Gene expression analysis of TIC led to the identification of Ephrin-A4 (EFNA4) as a prospective therapeutic target. An ADC comprising a humanized anti-EFNA4 monoclonal antibody conjugated to the DNA-damaging agent calicheamicin achieved sustained tumor regressions in both TNBC and ovarian cancer PDX in vivo. Non-claudin low TNBC tumors exhibited higher expression and more robust responses than other breast cancer subtypes, suggesting a specific translational application for tumor subclassification. CONCLUSIONS: These findings demonstrate the potential of PF-06647263 (anti-EFNA4-ADC) as a first-in-class compound designed to eradicate TIC. The use of well-annotated PDX for drug discovery enabled the identification of a novel TIC target, pharmacologic evaluation of the compound, and translational studies to inform clinical development.

Initiation and characterization of small cell lung cancer patient-derived xenografts from ultrasound-guided transbronchial needle aspirates.

Small cell lung cancer (SCLC) is a devastating disease with limited treatment options. Due to its early metastatic nature and rapid growth, surgical resection is rare. Standard of care treatment regimens remain largely unchanged since the 1980's, and five-year survival lingers near 5%. Patient-derived xenograft (PDX) models have been established for other tumor types, amplifying material for research and serving as models for preclinical experimentation; however, limited availability of primary tissue has curtailed development of these models for SCLC. The objective of this study was to establish PDX models from commonly collected fine needle aspirate biopsies of primary SCLC tumors, and to assess their utility as research models of primary SCLC tumors. These transbronchial needle aspirates efficiently engrafted as xenografts, and tumor histomorphology was similar to primary tumors. Resulting tumors were further characterized by H&E and immunohistochemistry, cryopreserved, and used to propagate tumor-bearing mice for the evaluation of standard of care chemotherapy regimens, to assess their utility as models for tumors in SCLC patients. When treated with Cisplatin and Etoposide, tumor-bearing mice responded similarly to patients from whom the tumors originated. Here, we demonstrate that PDX tumor models can be efficiently established from primary SCLC transbronchial needle aspirates, even after overnight shipping, and that resulting xenograft tumors are similar to matched primary tumors in cancer patients by both histology and chemo-sensitivity. This method enables physicians at non-research institutions to collaboratively contribute to the rapid establishment of extensive PDX collections of SCLC, enabling experimentation with clinically relevant tissues and development of improved therapies for SCLC patients.

Vascular endothelial growth factor receptor type 2-targeted contrast-enhanced US of pancreatic cancer neovasculature in a genetically engineered mouse model: potential for earlier detection.

PURPOSE: To test ultrasonographic (US) imaging with vascular endothelial growth factor receptor type 2 (VEGFR2)-targeted microbubble contrast material for the detection of pancreatic ductal adenocarcinoma (PDAC) in a transgenic mouse model of pancreatic cancer development. MATERIALS AND METHODS: Experiments involving animals were approved by the Institutional Administrative Panel on Laboratory Animal Care at Stanford University. Transgenic mice (n = 44; Pdx1-Cre, KRas(G12D), Ink4a(-/-)) that spontaneously develop PDAC starting at 4 weeks of age were imaged by using a dedicated small-animal US system after intravenous injection of 5 × 10(7) clinical-grade VEGFR2-targeted microbubble contrast material. The pancreata in wild-type (WT) mice (n = 64) were scanned as controls. Pancreatic tissue was analyzed ex vivo by means of histologic examination (with hematoxylin-eosin staining) and immunostaining of vascular endothelial cell marker CD31 and VEGFR2. The Wilcoxon rank sum test and linear mixed-effects model were used for statistical analysis. RESULTS: VEGFR2-targeted US of PDAC showed significantly higher signal intensities (26.8-fold higher; mean intensity ± standard deviation, 6.7 linear arbitrary units [lau] ± 8.5; P < .001) in transgenic mice compared with normal, control pancreata of WT mice (mean intensity, 0.25 lau ± 0.25). The highest VEGFR2-targeted US signal intensities were observed in smaller tumors, less than 3 mm in diameter (30.8-fold higher than control tissue with mean intensity of 7.7 lau ± 9.3 [P < .001]; and 1.7-fold higher than lesions larger than 3 mm in diameter with mean intensity of 4.6 lau ± 5.8 [P < .024]). Ex vivo quantitative VEGFR2 immunofluorescence demonstrated that VEGFR2 expression was significantly higher in pancreatic tumors (P < .001; mean fluorescent intensity, 499.4 arbitrary units [au] ± 179.1) compared with normal pancreas (mean fluorescent intensity, 232.9 au ± 83.7). CONCLUSION: US with clinical-grade VEGFR2-targeted microbubbles allows detection of small foci of PDAC in transgenic mice.

Differential regulation of cyclin D1 expression by protein kinase C α and ϵ signaling in intestinal epithelial cells.

Cellular accumulation of cyclin D1, a key regulator of cell proliferation and tumorigenesis, is subject to tight control. Our previous studies have identified PKCα as a negative regulator of cyclin D1 in the intestinal epithelium. However, treatment of non-transformed IEC-18 ileal crypt cells with PKC agonists has a biphasic effect on cyclin D1 expression. Initial PKCα-mediated down-regulation is followed by recovery and subsequent accumulation of the cyclin to levels markedly higher than those seen in untreated cells. Using protein overexpression strategies, siRNA, and pharmacological inhibitors, we now demonstrate that the recovery and hyperinduction of cyclin D1 reflect the combined effects of (a) loss of negative signals from PKCα due to agonist-induced PKCα down-regulation and (b) positive effects of PKCϵ. PKCϵ-mediated up-regulation of cyclin D1 requires sustained ERK stimulation and transcriptional activation of the proximal cyclin D1 (CCDN1) promoter, without apparent involvement of changes in protein stability or translation. PKCϵ also up-regulates cyclin D1 expression in colon cancer cells, through mechanisms that parallel those in IEC-18 cells. Although induction of cyclin D1 by PKCϵ is dependent on non-canonical NF-κB activation, the NF-κB site in the proximal promoter is not required. Instead, cyclin D1 promoter activity is regulated by a novel interaction between NF-κB and factors that associate with the cyclic AMP-response element adjacent to the NF-κB site. The differential effects of PKCα and PKCϵ on cyclin D1 accumulation are likely to contribute to the opposing tumor-suppressive and tumor-promoting activities of these PKC family members in the intestinal epithelium.

Ultrasound molecular imaging in a human CD276 expression-modulated murine ovarian cancer model.

PURPOSE: To develop a mouse ovarian cancer model that allows modulating the expression levels of human vascular targets in mouse xenograft tumors and to test whether expression of CD276 during tumor angiogenesis can be visualized by molecularly targeted ultrasound in vivo. EXPERIMENTAL DESIGN: CD276-expressing MILE SVEN 1 (MS1) mouse endothelial cells were engineered and used for coinjection with 2008 human ovarian cancer cells for subcutaneous xenograft tumor induction in 15 nude mice. Fourteen control mice were injected with 2008 cells only. After confirming their binding specificity in flow chamber cell attachment studies, anti-CD276 antibody-functionalized contrast microbubbles were used for in vivo CD276-targeted contrast-enhanced ultrasound imaging. RESULTS: CD276-targeted ultrasound imaging signal was significantly higher (P = 0.006) in mixed MS1/2008 tumors than in control tumors. Compared with control microbubbles, the ultrasound signal using CD276-targeted microbubbles was significantly higher (P = 0.002), and blocking with purified anti-CD276 antibody significantly decreased (P = 0.0096) the signal in mixed MS1/2008 tumors. Immunofluorescence analysis of the tumor tissue confirmed higher quantitative immunofluorescence signal in mixed MS1/2008 tumors than in control 2008 only tumors, but showed not significantly different (P = 0.54) microvessel density. CONCLUSIONS: Our novel small animal model allows for modulating the expression of human tumor-associated vascular endothelial imaging targets in a mouse host and these expression differences can be visualized noninvasively by ultrasound molecular imaging. The animal model can be applied to other human vascular targets and may facilitate the preclinical development of new imaging probes such as microbubbles targeted at human vascular markers not expressed in mice.

Detection of pancreatic ductal adenocarcinoma in mice by ultrasound imaging of thymocyte differentiation antigen 1.

BACKGROUND & AIMS: Early detection of pancreatic ductal adenocarcinoma (PDAC) allows for surgical resection and increases patient survival times. Imaging agents that bind and amplify the signal of neovascular proteins in neoplasms can be detected by ultrasound, enabling accurate detection of small lesions. We searched for new markers of neovasculature in PDAC and assessed their potential for tumor detection by ultrasound molecular imaging. METHODS: Thymocyte differentiation antigen 1 (Thy1) was identified as a specific biomarker of PDAC neovasculature by proteomic analysis. Up-regulation in PDAC was validated by immunohistochemical analysis of pancreatic tissue samples from 28 healthy individuals, 15 with primary chronic pancreatitis tissues, and 196 with PDAC. Binding of Thy1-targeted contrast microbubbles was assessed in cultured cells, in mice with orthotopic PDAC xenograft tumors expressing human Thy1 on the neovasculature, and on the neovasculature of a genetic mouse model of PDAC. RESULTS: Based on immunohistochemical analyses, levels of Thy1 were significantly higher in the vascular of human PDAC than chronic pancreatitis (P = .007) or normal tissue samples (P < .0001). In mice, ultrasound imaging accurately detected human Thy1-positive PDAC xenografts, as well as PDACs that express endogenous Thy1 in genetic mouse models of PDAC. CONCLUSIONS: We have identified and validated Thy1 as a marker of PDAC that can be detected by ultrasound molecular imaging in mice. The development of a specific imaging agent and identification of Thy1 as a new biomarker could aid in the diagnosis of this cancer and management of patients.

Ultrasound-mediated gene delivery with cationic versus neutral microbubbles: effect of DNA and microbubble dose on in vivo transfection efficiency.

OBJECTIVE: To assess the effect of varying microbubble (MB) and DNA doses on the overall and comparative efficiencies of ultrasound (US)-mediated gene delivery (UMGD) to murine hindlimb skeletal muscle using cationic versus neutral MBs. MATERIALS AND METHODS: Cationic and control neutral MBs were characterized for size, charge, plasmid DNA binding, and ability to protect DNA against endonuclease degradation. UMGD of a codon optimized firefly luciferase (Fluc) reporter plasmid to endothelial cells (1 MHz, 1 W/cm², 20% duty cycle, 1 min) was performed in cell culture using cationic, neutral, or no MBs. In vivo UMGD to mouse hindlimb muscle was performed by insonation (1 MHz, 2 W/cm², 50% duty cycle, 5 min) after intravenous administration of Fluc combined with cationic, neutral, or no MBs. Gene delivery efficiency was assessed by serial in vivo bioluminescence imaging. Efficiency of in vivo UMGD with cationic versus neutral MBs was systematically evaluated by varying plasmid DNA dose (10, 17.5, 25, 37.5, and 50 µg) while maintaining a constant MB dose of 1x10(8) MBs and by changing MB dose (1x10(7), 5x10(7), 1x10(8), or 5x10(8) MBs) while keeping a constant DNA dose of 50 µg. RESULTS: Cationic and size-matched control neutral MBs differed significantly in zeta potential with cationic MBs being able to bind plasmid DNA (binding capacity of 0.03 pg/MB) and partially protect DNA from nuclease degradation while neutral MBs could not. Cationic MBs enhanced UMGD compared to neutral MBs as well as no MB and no US controls both in cell culture (P < 0.001) and in vivo (P < 0.05). Regardless of MB type, in vivo UMGD efficiency increased dose-dependently with DNA dose and showed overall maximum transfection with 50 µg DNA. However, there was an inverse correlation (ρ = -0.90; P = 0.02) between DNA dose and the degree of enhanced UMGD efficiency observed with using cationic MBs instead of neutral MBs. The delivery efficiency advantage associated with cationic MBs was most prominent at the lowest investigated DNA dose (7.5-fold increase with cationic versus neutral MBs at a DNA dose of 10 µg; P = 0.02) compared to only a 1.4-fold increase at a DNA dose of 50 µg (P < 0.01). With increasing MB dose, overall in vivo UMGD efficiency increased dose-dependently with a maximum reached at a dose of 1x10(8) MBs with no further significant increase with 5x10(8) MBs (P = 0.97). However, compared to neutral MBs, cationic MBs enhanced UMGD efficiency the most at low MB doses. Relative enhancement of UMGD efficiency using cationic over neutral MBs decreased from a factor of 27 for 1x10(7) MBs (P = 0.02) to a factor of 1.4 for 1x10(8) MBs (P < 0.01) and no significant difference for 5x10(8) MBs. CONCLUSIONS: Cationic MBs enhance UMGD to mouse skeletal muscle relative to neutral MBs but this is dependent on MB and DNA dose. The enhancement effect of cationic MBs on UMGD efficiency is more evident when lower doses of MBs or DNA are used, whereas the advantage of cationic MBs over neutral MBs is substantially reduced in the presence of excess MBs or DNA.

Fast microbubble dwell-time based ultrasonic molecular imaging approach for quantification and monitoring of angiogenesis in cancer.

PURPOSE: To develop and test a fast ultrasonic molecular imaging technique for quantification and monitoring of angiogenesis in cancer. MATERIALS AND METHODS: A new software algorithm measuring the dwell time of contrast microbubbles in near real-time (henceforth, fast method) was developed and integrated in a clinical ultrasound system. In vivo quantification and monitoring of tumor angiogenesis during anti-VEGF antibody therapy was performed in human colon cancer xenografts in mice (n=20) using the new fast method following administration of vascular endothelial growth factor receptor 2 (VEGFR2)-targeted contrast microbubbles. Imaging results were compared with a traditional destruction/replenishment approach (henceforth, traditional method) in an intra-animal comparison. RESULTS: There was excellent correlation (R(2)=0.93; P<0.001) between the fast method and the traditional method in terms of VEGFR2-targeted in vivo ultrasonic molecular imaging with significantly higher (P=0.002) imaging signal in colon cancer xenografts using VEGFR2-targeted compared to control non-targeted contrast microbubbles. The new fast method was highly reproducible (ICC=0.87). Following anti-angiogenic therapy, ultrasonic molecular imaging signal decreased by an average of 41±10%, whereas imaging signal increased by an average of 54±8% in non-treated tumors over a 72-hour period. Decreased VEGFR2 expression levels following anti-VEGF therapy were confirmed on ex vivo immunofluorescent staining. CONCLUSIONS: Fast ultrasonic molecular imaging based on dwell time microbubble signal measurements correlates well with the traditional measurement method, and allows reliable in vivo monitoring of anti-angiogenic therapy in human colon cancer xenografts. The improved work-flow afforded by the new quantification approach may facilitate clinical translation of ultrasonic molecular imaging.

ß-Catenin regulates hepatic mitochondrial function and energy balance in mice.

BACKGROUND & AIMS: Wnt signaling regulates hepatic function and nutrient homeostasis. However, little is known about the roles of ß-catenin in cellular respiration or mitochondria of hepatocytes. METHODS: We investigated ß-catenin's role in the metabolic function of hepatocytes under homeostatic conditions and in response to metabolic stress using mice with hepatocyte-specific deletion of ß-catenin and their wild-type littermates, given either saline (sham) or ethanol (as a model of binge drinking and acute ethanol intoxication). RESULTS: Under homeostatic conditions, ß-catenin-deficient hepatocytes demonstrated mitochondrial dysfunctions that included impairments to the tricarboxylic acid cycle and oxidative phosphorylation (OXPHOS) and decreased production of adenosine triphosphate (ATP). There was no evidence for redox imbalance or oxidative cellular injury in the absence of metabolic stress. In mice with ß-catenin-deficient hepatocytes, ethanol intoxication led to significant redox imbalance in the hepatocytes and further deterioration in mitochondrial function that included reduced OXPHOS, fatty acid oxidation (FAO), and ATP production. Ethanol feeding significantly increased liver steatosis and oxidative damage, compared with wild-type mice, and disrupted the ratio of nicotinamide adenine dinucleotide. ß-catenin-deficient hepatocytes also had showed disrupted signaling of Sirt1/peroxisome proliferator-activated receptor-α signaling. CONCLUSIONS: ß-catenin has an important role in the maintenance of mitochondrial homeostasis, regulating ATP production via the tricarboxylic acid cycle, OXPHOS, and fatty acid oxidation; ß-catenin function in these systems is compromised under conditions of nutrient oxidative stress. Reagents that alter Wnt-ß-catenin signaling might be developed as a useful new therapeutic strategy for treatment of liver disease.

Cationic versus neutral microbubbles for ultrasound-mediated gene delivery in cancer.

PURPOSE: To test whether plasmid-binding cationic microbubbles (MBs) enhance ultrasound-mediated gene delivery efficiency relative to control neutral MBs in cell culture and in vivo tumors in mice. MATERIALS AND METHODS: Animal studies were approved by the institutional animal care committee. Cationic and neutral MBs were characterized in terms of size, charge, circulation time, and DNA binding. Click beetle luciferase (CBLuc) reporter plasmids were mixed with cationic or neutral MBs. The ability of cationic MBs to protect bound plasmids from nuclease degradation was tested by means of a deoxyribonuclease (DNase) protection assay. Relative efficiencies of ultrasound-mediated transfection (ultrasound parameters: 1 MHz, 1 W/cm(2), 20% duty cycle, 1 minute) of CBLuc to endothelial cells by using cationic, neutral, or no MBs were compared in cell culture. Ultrasound-mediated gene delivery to mouse hind limb tumors was performed in vivo (n = 24) with insonation (1 MHz, 2 W/cm(2), 50% duty cycle, 5 minutes) after intravenous administration of CBLuc with cationic, neutral, or no MBs. Tumor luciferase activity was assessed by means of serial in vivo bioluminescence imaging and ex vivo analysis. Results were compared by using analysis of variance. RESULTS: Cationic MBs (+15.8 mV; DNA binding capacity, 0.03 pg per MB) partially protected bound DNA from DNase degradation. Mean CBLuc expression of treated endothelial cells in culture was 20-fold higher with cationic than with neutral MBs (219.0 relative light units [RLUs]/µg protein ± 92.5 [standard deviation] vs 10.9 RLUs/µg protein ± 2.7, P = .001) and was significantly higher (P < .001) than that in the no MB and no ultrasound control groups. Serial in vivo bioluminescence of mouse tumors was significantly higher with cationic than with neutral MBs ([5.9 ± 2.2] to [9.3 ± 5.2] vs [2.4 ± 0.8] to [2.9 ± 1.1] × 10(4) photons/sec/cm(2)/steradian, P < .0001) and versus no MB and no ultrasound controls (P < .0001). Results of ex vivo analysis confirmed these results (ρ = 0.88, P < .0001). CONCLUSION: Plasmid-binding cationic MBs enhance ultrasound-mediated gene delivery efficiency relative to neutral MBs in both cell culture and mouse hind limb tumors.

Quantitative assessment of tumor angiogenesis using real-time motion-compensated contrast-enhanced ultrasound imaging.

PURPOSE: To develop and test a real-time motion compensation algorithm for contrast-enhanced ultrasound imaging of tumor angiogenesis on a clinical ultrasound system. MATERIALS AND METHODS: The Administrative Institutional Panel on Laboratory Animal Care approved all experiments. A new motion correction algorithm measuring the sum of absolute differences in pixel displacements within a designated tracking box was implemented in a clinical ultrasound machine. In vivo angiogenesis measurements (expressed as percent contrast area) with and without motion compensated maximum intensity persistence (MIP) ultrasound imaging were analyzed in human colon cancer xenografts (n = 64) in mice. Differences in MIP ultrasound imaging signal with and without motion compensation were compared and correlated with displacements in x- and y-directions. The algorithm was tested in an additional twelve colon cancer xenograft-bearing mice with (n = 6) and without (n = 6) anti-vascular therapy (ASA-404). In vivo MIP percent contrast area measurements were quantitatively correlated with ex vivo microvessel density (MVD) analysis. RESULTS: MIP percent contrast area was significantly different (P < 0.001) with and without motion compensation. Differences in percent contrast area correlated significantly (P < 0.001) with x- and y-displacements. MIP percent contrast area measurements were more reproducible with motion compensation (ICC = 0.69) than without (ICC = 0.51) on two consecutive ultrasound scans. Following anti-vascular therapy, motion-compensated MIP percent contrast area significantly (P = 0.03) decreased by 39.4 ± 14.6 % compared to non-treated mice and correlated well with ex vivo MVD analysis (Rho = 0.70; P = 0.05). CONCLUSION: Real-time motion-compensated MIP ultrasound imaging allows reliable and accurate quantification and monitoring of angiogenesis in tumors exposed to breathing-induced motion artifacts.

Pharmacokinetically stabilized cystine knot peptides that bind alpha-v-beta-6 integrin with single-digit nanomolar affinities for detection of pancreatic cancer.

PURPOSE: Detection of pancreatic cancer remains a high priority and effective diagnostic tools are needed for clinical applications. Many cancer cells overexpress integrin α(v)ß(6), a cell surface receptor being evaluated as a novel clinical biomarker. EXPERIMENTAL DESIGN: To validate this molecular target, several highly stable cystine knot peptides were engineered by directed evolution to bind specifically and with high affinity (3-6 nmol/L) to integrin α(v)ß(6). The binders do not cross-react with related integrin α(v)ß(5), integrin α(5)ß(1), or tumor-angiogenesis-associated integrin, α(v)ß(3). RESULTS: Positron emission tomography showed that these disulfide-stabilized peptides rapidly accumulate at tumors expressing integrin α(v)ß(6). Clinically relevant tumor-to-muscle ratios of 7.7 ± 2.4 to 11.3 ± 3.0 were achieved within 1 hour after radiotracer injection. Minimization of off-target dosing was achieved by reformatting α(v)ß(6)-binding activities across various natural and pharmacokinetically stabilized cystine knot scaffolds with different amino acid content. We show that the primary sequence of a peptide scaffold directs its pharmacokinetics. Scaffolds with high arginine or glutamic acid content suffered high renal retention of more than 75% injected dose per gram (%ID/g). Substitution of these amino acids with renally cleared amino acids, notably serine, led to significant decreases in renal accumulation of less than 20%ID/g 1 hour postinjection (P < 0.05, n = 3). CONCLUSIONS: We have engineered highly stable cystine knot peptides with potent and specific integrin α(v)ß(6)-binding activities for cancer detection. Pharmacokinetic engineering of scaffold primary sequence led to significant decreases in off-target radiotracer accumulation. Optimization of binding affinity, specificity, stability, and pharmacokinetics will facilitate translation of cystine knots for cancer molecular imaging.

Protein kinase Cα signaling regulates inhibitor of DNA binding 1 in the intestinal epithelium.

Increasing evidence supports a role for PKCα in growth arrest and tumor suppression in the intestinal epithelium. In contrast, the Id1 transcriptional repressor has pro-proliferative and tumorigenic properties in this tissue. Here, we identify Id1 as a novel target of PKCα signaling. Using a highly specific antibody and a combined morphological/biochemical approach, we establish that Id1 is a nuclear protein restricted to proliferating intestinal crypt cells. A relationship between PKCα and Id1 was supported by the demonstration that (a) down-regulation of Id1 at the crypt/villus junction coincides with PKCα activation, and (b) loss of PKCα in intestinal tumors is associated with increased levels of nuclear Id1. Manipulation of PKCα activity in IEC-18 nontransformed intestinal crypt cells determined that PKCα suppresses Id1 mRNA and protein via an Erk-dependent mechanism. PKCα, but not PKCδ, also inhibited Id1 expression in colon cancer cells. Id1 was found to regulate cyclin D1 levels in IEC-18 and colon cancer cells, pointing to a role for Id1 suppression in the antiproliferative/tumor suppressive activities of PKCα. Notably, Id1 expression was elevated in the intestinal epithelium of PKCα-knock-out mice, confirming that PKCα regulates Id1 in vivo. A wider role for PKCα in control of inhibitor of DNA binding factors is supported by its ability to down-regulate Id2 and Id3 in IEC-18 cells, although their suppression is more modest than that of Id1. This study provides the first demonstrated link between a specific PKC isozyme and inhibitor of DNA binding factors, and it points to a role for a PKCα → Erk ⊣ Id1 → cyclin D1 signaling axis in the maintenance of intestinal homeostasis.