Sandra Sexton

Nrf2 Amplifies Oxidative Stress via Induction of Klf9

Reactive oxygen species (ROS) activate NF-E2-related transcription factor 2 (Nrf2), a key transcriptional regulator driving antioxidant gene expression and protection from oxidant injury. Here, we report that in response to elevation of intracellular ROS above a critical threshold, Nrf2 stimulates expression of transcription Kruppel-like factor 9 (Klf9), resulting in further Klf9-dependent increases in ROS and subsequent cell death. We demonstrated that Klf9 independently causes increased ROS levels in various types of cultured cells and in mouse tissues and is required for pathogenesis of bleomycin-induced pulmonary fibrosis in mice. Mechanistically, Klf9 binds to the promoters and alters the expression of several genes involved in the metabolism of ROS, including suppression of thioredoxin reductase 2, an enzyme participating in ROS clearance. Our data reveal an Nrf2-dependent feedforward regulation of ROS and identify Klf9 as a ubiquitous regulator of oxidative stress and lung injury.

Conditional deletion of p53 and Rb in the renin-expressing compartment of the pancreas leads to a highly penetrant metastatic pancreatic neuroendocrine carcinoma

Efforts to model human pancreatic neuroendocrine tumors (PanNETs) in animals have been moderately successful, with minimal evidence for glucagonomas or metastatic spread. The renin gene, although classically associated with expression in the kidney, is also expressed in many other extrarenal tissues including the pancreas. To induce tumorigenesis within rennin-specific tissues, floxed alleles of p53 and Rb were selectively abrogated using Cre-recombinase driven by the renin promoter. The primary neoplasm generated is a highly metastatic islet cell carcinoma of the pancreas. Lineage tracing identifies descendants of renin-expressing cells as pancreatic alpha cells despite a lack of active renin expression in the mature pancreas. Both primary and metastatic tumors express high levels of glucagon; furthermore, an increased level of glucagon is found in the serum, identifying the pancreatic cancer as a functional glucagonoma. This new model is highly penetrant and exhibits robust frequency of metastases to the lymph nodes and the liver, mimicking human disease, and provides a useful platform for better understanding pancreatic endocrine differentiation and development, as well as islet cell carcinogenesis. The use of fluorescent reporters for lineage tracing of the cells contributing to disease initiation and progression provides an unique opportunity to dissect the timeline of disease, examining mechanisms of the metastatic process, as well as recovering primary and metastatic cells for identifying cooperating mutations that are necessary for progression of disease.

Robot‐assisted approach to ‘W’‐configuration urinary diversion: a step‐by‐step technique

To describe a detailed step‐by‐step approach of our technique for robot‐assisted intracorporeal ‘W’‐configuration orthotopic ileal neobladder.

Spatiotemporal Optoacoustic Mapping of Tumor Hemodynamics in a Clinically Relevant Orthotopic Rabbit Model of Head and Neck Cancer

The purpose of this study was to investigate the usefulness of photoacoustic imaging (PAI) for spatiotemporal mapping of tumor hemodynamics in a rabbit model of head and neck carcinoma. Shope cottontail rabbit papilloma virus associated VX2 carcinomas were established in adult male New Zealand White rabbits (n = 9) by surgical transplantation of tumor tissue in the neck. Noninvasive PAI with co-registered ultrasound (US) was performed to longitudinally monitor tumor growth, oxygen saturation (%sO2), and hemoglobin concentration (HbT). PAI findings were validated with Doppler sonography measures of percent vascularity (PV). Differences in tumor volumes, %sO2, HbT, and PV values over time were analyzed using repeated-measures analysis of variance with multiple comparisons. Two-tailed Spearman correlation analysis was performed to determine the correlation coefficient (r) for comparisons between %sO2, HbT, and tumor volume. US revealed a significant (P < .0001) increase in tumor volume over the 3-week period from 549 ± 260 mm3 on day 7 to 5055 ± 438 mm3 at 21 days postimplantation. Consistent with this aggressive tumor growth, PAI revealed a significant (P < .05) and progressive reduction in %sO2 from day 7 (37.6 ± 7.4%) to day 21 (9.5 ± 2.1%). Corresponding Doppler images also showed a decrease in PV over time. PAI revealed considerable intratumoral spatial heterogeneity with the tumor rim showing two- to three-fold higher %sO2 values compared to the core. Noninvasive PAI based on endogenous contrast provides a label-free method for longitudinal monitoring of temporal changes and spatial heterogeneity in thick head and neck tumors.

Irradiance controls photodynamic efficacy and tissue heating in experimental tumours: implication for interstitial PDT of locally advanced cancer

BackgroundCurrently delivered light dose (J/cm2) is the principal parameter guiding interstitial photodynamic therapy (I-PDT) of refractory locally advanced cancer. The aim of this study was to investigate the impact of light dose rate (irradiance, mW/cm2) and associated heating on tumour response and cure.MethodsFinite-element modeling was used to compute intratumoural irradiance and dose to guide Photofrin® I-PDT in locally advanced SCCVII in C3H mice and large VX2 neck tumours in New Zealand White rabbits. Light-induced tissue heating in mice was studied with real-time magnetic resonance thermometry.ResultsIn the mouse model, cure rates of 70–90% were obtained with I-PDT using 8.4–245 mW/cm2 and ≥45 J/cm2 in 100% of the SCCVII tumour. Increasing irradiance was associated with increase in tissue heating. I-PDT with Photofrin® resulted in significantly (p < 0.05) higher cure rate compared to light delivery alone at same irradiance and light dose. Local control and/or cures of VX2 were obtained using I-PDT with 16.5–398 mW/cm2 and ≥45 J/cm2 in 100% of the tumour.ConclusionIn Photofrin®-mediated I-PDT, a selected range of irradiance prompts effective photoreaction with tissue heating in the treatment of locally advanced mouse tumour. These irradiances were translated for effective local control of large VX2 tumours.

Novel mouse models of hepatic artery infusion

BACKGROUND The liver has unique anatomy in that most blood flow to normal hepatocytes is derived from the portal venous system, whereas liver tumors obtain their nutrient blood supply exclusively from the hepatic artery. The focused arterial delivery of anticancer agents to liver tumors has been performed for decades; however, preclinical models to standardize drug regimens and examine novel agents have been lacking. The purpose of this study was to establish preclinical hepatic artery infusion (HAI) models in a mouse and to evaluate the safety and delivery capability of the models. MATERIAL AND METHODS C57BL/6 and BALB/c mice were used to develop models of HAI via the hepatic artery (HA), superior pancreaticoduodenal artery (SPDA), or lienogastric artery (LGA). Success rates, distribution of perfusion, and associated morbidity and mortality were analyzed between groups. RESULTS All three models were feasible and reproducible in mice, and there was no statistical difference on body weight change between models. The HA model had a 13.3% mortality from acute liver failure, and the SPDA model demonstrated duodenal and pancreatic toxicity. SPDA and LGA routes had the highest success rates (96.7% and 91.4%, respectively) with low mortality, better drug delivery, and preserved physiologic liver function compared with the HA model. CONCLUSIONS The optimal route of HAI was mouse breed specific; SPDA access in BALB/c mice, and the LGA access in C57BL/6 mice. The described techniques serve as a reproducible platform for the identification and characterization of therapeutics for diverse metastatic liver tumors.

Abstract 5144: Low dose sorafenib delays hepatocellular cancer (HCC) development in the woodchuck model of hepatitis B related HCC

Introduction: The eastern woodchuck is an established model of human hepatitis B viral infection and spontaneously develops HCC in the context of chronic woodchuck hepatitis viral infection (WHV) at a median age of 24 months. In this translational model of angiogenesis driven HCC, we evaluated the impact of sorafenib on delaying HCC development. Methods: Woodchucks were bred and inoculated at birth with titered infectious WHV pools obtained from chronic WHV carriers. By 12 months of age, the rate of chronic WHV infection was >60%. Animals were trained to receive drug orally mixed in a liquid diet. After an initial single dose woodchuck PK study it was predicted that sorafenib given orally M-F at 2.5 mg/kg once daily or 5mg/kg daily would achieve exposure in woodchucks ∼ 2x IC50 and 5X IC50 for all targets of sorafenib. This is within the exposure achieved in humans where the starting dose achieves exposures that are ∼ 10-12X IC50. We hypothesized that for both long term tolerability and delaying of HCC development, lower doses maybe ideal. Viral titers of WHV DNA were serially assessed, PK exposure of sorafenib and serial ultrasound (USG) was done q2 weeks to assess HCC development. Six WHV+ animals of similar age per arm were randomized to Placebo (P), low or high dose sorafenib. Time to tumor development (TTD) was measured from start of therapy to first occurrence of at least one 2 cm HCC was confirmed on 2 serial USGs. Tumor volume of all tumors seen was assessed as tumor burden. Results: Median TTD was 393 days, 490 and 498 days in the P, low and high dose sorafenib groups respectively. No toxicity was seen and PK results showed exposures as predicted in the two sorafenib groups. The tumor burden adjusted for the duration of therapy was significantly lower in the low dose group than the high dose and P groups. We evaluated three possible mechansims for this delay in HCC. WHV titers decreased as tumor burden increased as viral replication halts when malignant transformation occurs. The delay in TTD was not secondary to an antiviral effect that is known in this model and in humans to delay HCC occurrence. Anti-angiogenic effect was measured on H/E following necropsy as tumor necrosis and involved 7% of the total tumor volume in low dose and 13% of total tumor volume in high dose animals and 0% in P animals. High dose sorafenib inhibited the ex-vivo mixed lymphocyte reaction of woodchuck PBMC whereas low dose sorafenib did not which mechanistically may explain the difference seen in tumor burden with low compared to high dose sorafenib. Conclusions: The delay in HCC by 100 days seen in woodchucks would translate into a 3-9 years delay in HCC occurrence in humans. The tolerability at low dose for over 2 years, lower tumor burden and differential immune effects seen at low dose vs higher dose have great translational significance in the light of the recently completed STORM trial. Support: Sorafenib: Bayer. Funding: ACS- MSRG 08-096-01-CCE Citation Format: Renuka V. Iyer, Sandra Sexton, Leslie Curtin, Gerald Fetterly, Orla Maguire, Hans Minderman, Ilia Toshkov, Bud Tennant, Alan Hutson, Donald L. Trump, Candace Johnson. Low dose sorafenib delays hepatocellular cancer (HCC) development in the woodchuck model of hepatitis B related HCC. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5144. doi:10.1158/1538-7445.AM2015-5144

Abstract 4647: Investigation of the pharmacokinetic profile of the novel PIM2 inhibitor, JP_11646

PIM2 is a serine/threonine protein kinase that has roles in cell growth, proliferation, and apoptosis via the regulation of multiple signal transduction cascades. JP_11646 is a novel PIM2 inhibitor that has shown antitumor activity against breast, colon, liver, lung, and pancreatic solid tumors, as well as multiple myeloma and leukemia. The pharmacokinetics (PK) of JP_11646 has not been described. Therefore, a PK study was conducted to characterize JP_11646 disposition in plasma and various tissues. Normal ICR (CD-1) mice received either single day or five consecutive days of 15 - 25 mg/kg JP_11646 via intraperitoneal (IP) or intravenous (IV) administration. Plasma samples were collected at serial time points (0.5, 1, 2, 4, 6, and 8 hrs) following a single dose of 20 mg/kg IV or 25 mg/kg IP JP_11646 administration. Plasma and various tissues (brain, heart, kidney, lung, liver, muscle, colon, pancreas, and spleen) were collected at serial time points following multiple daily doses of 15 mg/kg IP JP_11646 on day five. Three mice were euthanized per time point on each collection day. JP_11646 concentrations were determined by a validated LC-MS/MS method, with a LLOQ of 1 ng/ml. Noncompartmental PK analysis was performed using Phoenix 64 (Pharsight, WinNonlin 6.3). Following single dose administration of JP_11646 of 20 (IV) and 25 mg/kg (IP), mean plasma Cmax was 6,951 and 6,630 ng/ml, respectively. Mean plasma drug exposures (AUCinf) were 11,065 and 11,024 ng-hr/ml, respectively, resulting in similar drug exposure between the IV and IP route. To compare plasma and tissue exposure following multiple dose administration, JP_11646 was given at 15 mg/kg IP. The mean plasma Cmax was 4,601 ng/ml., while the mean Cmax in colon, pancreas, and spleen were 24,523, 77,811, and 18,835 ng/ml, respectively. The half-life of JP_11646 in plasma and tissues ranged from 0.8 - 3 hrs. Tissue exposure in colon, pancreas, and spleen was 48,792, 157,273, and 40,511 ng-hr/ml, respectively. The pancreas to plasma partition coefficient (Kp) of 20.6 demonstrates that the pancreas receives the highest exposure to JP_11646. The drug also distributes to the colon and spleen, with Kp values of 6.4 and 5.3, respectively. JP_11646 PK appears linear and the half-life ranges from 0.8 - 3.0 hrs. JP_11646 distributes widely to tissues, with the highest drug exposure in the pancreas, spleen, and colon. These results give insight into tumor types with potential for optimal antitumor therapy with JP_11646. Citation Format: Laura B. Pitzonka, Allison Gaudy, Sarah Schihl, Leslie Curtin, Sandra Sexton, Carmen M. Baldino, Justin Caserta, Yvonne Flanders, Stephane Dumas, Gerald Fetterly. Investigation of the pharmacokinetic profile of the novel PIM2 inhibitor, JP_11646. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4647. doi:10.1158/1538-7445.AM2014-4647