Anderson Peck

18 F-FLT Positron Emission Tomography (PET) is a Pharmacodynamic Marker for EWS-FLI1 Activity and Ewing Sarcoma

Ewing sarcoma is a bone and soft-tissue tumor that depends on the activity of the EWS-FLI1 transcription factor for cell survival. Although a number of compounds have been shown to inhibit EWS-FLI1 in vitro, a clinical EWS-FLI1-directed therapy has not been achieved. One problem plaguing drug development efforts is the lack of a suitable, non-invasive, pharmacodynamic marker of EWS-FLI1 activity. Here we show that 18F-FLT PET (18F- 3′-deoxy-3′-fluorothymidine positron emission tomography) reflects EWS-FLI1 activity in Ewing sarcoma cells both in vitro and in vivo. 18F-FLT is transported into the cell by ENT1 and ENT2, where it is phosphorylated by TK1 and trapped intracellularly. In this report, we show that silencing of EWS-FLI1 with either siRNA or small-molecule EWS-FLI1 inhibitors suppressed the expression of ENT1, ENT2, and TK1 and thus decreased 18F-FLT PET activity. This effect was not through a generalized loss in viability or metabolic suppression, as there was no suppression of 18F-FDG PET activity and no suppression with chemotherapy. These results provide the basis for the clinical translation of 18F-FLT as a companion biomarker of EWS-FLI1 activity and a novel diagnostic imaging approach for Ewing sarcoma.

(99m)TC-Methylene diphosphonate uptake at injury site correlates with osteoblast differentiation and mineralization during bone healing in mice.

99mTc-Methylene diphosphonate (99mTc-MDP) is widely used in clinical settings to detect bone abnormalities. However, the mechanism of 99mTc-MDP uptake in bone is not well elucidated. In this study, we utilized a mouse tibia injury model, single-photon emission computed tomography (gamma scintigraphy or SPECT), ex vivo micro-computed tomography, and histology to monitor 99mTc-MDP uptake in injury sites during skeletal healing. In an ex vivo culture system, calvarial cells were differentiated into osteoblasts with osteogenic medium, pulsed with 99mTc-MDP at different time points, and quantitated for 99mTc-MDP uptake with a gamma counter. We demonstrated that 99mTc-MDP uptake in the injury sites corresponded to osteoblast generation in those sites throughout the healing process. The 99mTc-MDP uptake within the injury sites peaked on day 7 post-injury, while the injury sites were occupied by mature osteoblasts also starting from day 7. 99mTc-MDP uptake started to decrease 14 days post-surgery, when we observed the highest level of bony tissue in the injury sites. We also found that 99mTc-MDP uptake was associated with osteoblast maturation and mineralization in vitro. This study provides direct and biological evidence for 99mTc-MDP uptake in osteoblasts during bone healing in vivo and in vitro.

Abstract 1867: Near infrared fluorescent imaging of brain tumor with IR780 dye incorporated phospholipid nanoparticles

Background: Near-IR fluorescence (NIRF) imaging is becoming a promising approach in preclinical tumor detection and clinical image-guided oncological surgery. While heptamethine cyanine dye IR780 has excellent tumor targeting and imaging potential, its hydrophobic properties limit its clinical use. In this study, we developed nanoparticle formulations to facilitate the use of IR780 for fluorescent imaging of malignant brain tumor. Methods: Self-assembled IR780-liposomes and IR780-phospholipid micelles were prepared and their NIRF properties were characterized. The intracellular accumulation of IR780-nanoparticles in glioma cells were determined using confocal microscopy. The in vivo brain tumor targeting and NIRF imaging capacity of IR780-nanoparticles were evaluated using U87MG glioma ectopic and orthotopic xenograft models and a spontaneous glioma mouse model driven by RAS/RTK activation. Results: The loading of IR780 into liposomes or phospholipid micelles was efficient. The particle diameter of IR780-liposomes and IR780-phospholipid micelles were 95 nm and 26 nm, respectively. While stock solutions of each preparation were maintained at ready-to-use condition, the IR780-phospholipid micelles were more stable. In tissue culture cells, IR780-nanoparticles prepared by either method accumulated in mitochondria, however, in animals the IR780-phospholipid micelles showed enhanced intra-tumoral accumulation in U87MG ectopic tumors. Moreover, IR780-phospholipid micelles also showed preferred intracranial tumor accumulation and potent NIRF signal intensity in glioma orthotopic models at a real-time, non-invasive manner. Conclusion: The IR780-phospholipid micelles demonstrated tumor-specific NIRF imaging capacity in glioma preclinical mouse models, providing great potential for clinical imaging and image-guided surgery of brain tumors. Citation Format: Shihong Li, Jennifer Johnson, Anderson Peck, Qian Xie. Near infrared fluorescent imaging of brain tumor with IR780 dye incorporated phospholipid nanoparticles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1867. doi:10.1158/1538-7445.AM2017-1867

Abstract LB-047: Optimizing the trabectedin-mediated inhibition of EWS-FLI1 activity by schedule and fusion type

BACKGROUND: Ewing sarcoma (ES) is a pediatric malignancy characterized by the EWS-FLI1 transcription factor. ES tumors require the continuous transcriptional activity of EWS-FLI1 for the oncogenic phenotype, regardless of fusion type, which makes EWS-FLI1 an ideal therapeutic target. To date, no EWS-FLI1 directed therapies have been developed. Interestingly, there have been reports of ES patients as exceptional responders in clinical trials, including a complete response in a refractory, metastatic patient that was administered the natural product trabectedin in a Phase I trial. We have since established trabectedin as an EWS-FLI1 inhibitor that acts through a re-localization mechanism at concentrations that are clinically achievable. Intriguingly, trabectedin failed in the Phase II setting after a change in schedule that lead to a decrease in reported Cmax. In this report, we unify the disparate clinical trial conclusions by examining the schedule-dependency of trabectedin in ES cells. METHODS: Here, we model a broad range of exposures to trabectedin and investigate the cell viability kinetics using live cell imaging analysis software and traditional MTS. In parallel, we present a novel assay to quantitatively measure the kinetics of drug clearance in vitro. In addition, we use qPCR and western blotting to examine EWS-FLI1 target gene expression changes in response to different exposures to trabectedin. Finally, we investigated ES cell lines containing variant EWS-FLI1 fusion types and identify an increased sensitivity of type III/IV ES cell lines to trabectedin treatment. We recapitulated these findings in vivo using ES xenograft models. RESULTS: Sensitivity of ES cells to trabectedin treatment is schedule-dependent. Here, we demonstrate that cells treated with trabectedin at equivalent exposures but different Cmax exhibit profound differences in cell viability. The differences in viability correlate with Cmax that are necessary to inhibit EWS-FLI1 activity in vitro. By measuring the kinetics of drug clearance, we show that the inhibitory effects on EWS-FLI1 are durable even after trabectedin has been washed out of the culture medium. In addition, we examine the sensitivity of variant EWS-FLI1 fusion proteins such as type III/IV fusions both in vitro and in vivo. CONCLUSIONS: In this report, we use clinical trial data to develop a more clinically relevant cell culture system for ES drug treatments. Importantly, we demonstrate that equivalent exposures do not lead to equivalent outcomes. We believe that this observation explains the discordant results of the Phase I and II clinical trials, and suggests that the Cmax achieved in serum dictates whether a patient will respond. In addition, we show that type III/IV EWS-FLI1 fusion proteins are particularly sensitive to the drug. Citation Format: Matt Harlow, Elissa Boguslawski, Anderson Peck, Zachary Madaj, Maria J. Navarro, Pablo Aviles, Carlos Galmarini, Patrick Grohar. Optimizing the trabectedin-mediated inhibition of EWS-FLI1 activity by schedule and fusion type [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-047. doi:10.1158/1538-7445.AM2017-LB-047

Abstract A37: Complex crosstalk between MAPK signaling and energy metabolism in melanoma

The notoriously high glucose uptake by metastatic melanoma (MM) lesions is commonly exploited clinically using PET/CT imaging to evaluate disease progression and treatment. The purpose of this study was to use patient-derived xenograft (PDX) MM models and a PDX derived (MM8.1) cell line, to determine how driver oncogene mutations and drugs targeting the RAS-RAF-MEK signaling pathways impact glucose uptake, ATP production and mitochondrial plasticity of MM cells both in vivo and in vitro and the implications on drug resistance. MM BRAF V600E+ patients are routinely treated targeting either mutant BRAF kinase (vemurafenib), or the MAPK downstream signaling intermediate; MEK. To begin exploring links between oncogenic BRAF and metabolism, we first conducted differential gene expression (DGE) profiling of 30 different MM lesions (n=15 patients: pre- and post-engraftment). Focusing on PGC1α; a master mitochondrial regulator promoting oxidative phosphorylation (OXPHOS) revealed lower expression levels in BRAF V600E+ tumors compared to BRAF V600V+ tumors. Further analysis of a BRAF V600E+ MM PDX model revealed strong glucose uptake by PET/CT imaging at baseline. However, 48 hrs. after treatment with vemurafenib triggered growth arrest, accompanied by increased PGC1α; mRNA levels and significant reduction in glucose uptake without change in tumor volume. Upon acquisition of acquired vemurafenib resistance in the PDX model (day 70), rapidly growing tumors displayed reversal to high glucose uptake; which was again diminished by targeting MEK (PD0325901). DGE analysis of vehicle vs. 48 hrs. day 50 and day 70 vemurafenib treated tumors revealed oscillation in the expression of genes regulating glycolysis and oxidative phosphorylation (OXPHOS) that is reflected in the PET/CT imaging. To gain deeper insight, a cell line derived from a vehicle treated tumor, was examined using the Seahorse metabolic analyzer. Vehicle treated MM8.1 cells display high glycolytic capacity and predominantly glycolytic-derived ATP production, which is reflected by only minor inhibition of ATP production with oligomycin (an inhibitor of ATP synthase, a component of the OXHPOS system). Upon treatment with PLX4032, there was rapid reduction in glycolytic capacity along with a concurrent increase in maximum oxygen consumption capacity. PLX4032 treatment additionally enhanced MM8.1 sensitivity to oligomycin, but only transiently (24 hrs. pre-treatment), as later exposure times did not induce oligomycin sensitivity despite DGE changes to indicate otherwise. In conclusion, caution is warranted in evaluating drug responses in patients using PET/CT depending on duration of treatment. Cross-talk involving MAPK signaling and energy metabolism is a dynamic process, with more of a rheostat than switch-type properties, requiring further evaluation to identify therapeutic approaches exploiting metabolic events within MM cells. Citation Format: Nathan J. Lanning, Pimiento Jose, Noel R. Monks, Paula J. Davidson, Andrew S. Borgman, Ting-Tung Chang, Anderson S. Peck, David J. Monsma, Chelsea A. Peterson, Mackeigan P. Jeff, Brian J. Nickoloff. Complex crosstalk between MAPK signaling and energy metabolism in melanoma. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A37. doi: 10.1158/1557-3125.RASONC14-A37

Abstract B54: Difluoromethylornithine treatment affects the LIN28/Let-7 axis resulting in reduced glycolytic metabolism in neuroblastoma

Background: Overexpression of LIN28 correlates with poor outcome in neuroblastoma (NB). The LIN28/Let-7 axis affects many cellular processes including cell differentiation and glycolytic metabolism. MYCN overexpression correlates with decreased Let-7 miRNA which results in an increase in LIN28 protein. Recent studies have shown that ODC inhibition decreases LIN28 levels. We propose that therapy targeting ODC will affect the LIN28/Let-7 axis, thus suppressing the glycolytic metabolic activity of NB tumor cells. We also propose that cells overexpressing LIN28 will have greater sensitivity to Difluoromethylornithine (DFMO) treatment which inhibits ODC and decreases cellular polyamines. Methods: Two MYCN high-expression cell lines, BE(2)-C and SMS-KCNR, and one MYCN low-expression cell line, CHLA90, were grown in RPMI-1640 medium with 10% fetal bovine serum for 24 hours prior to treatment with DFMO. Cells were treated with 5 mM or 10 mM DFMO from 48-96 hours followed by cell viability assay, ATP per cell, and western blot analysis and 6 hours for qPCR analysis. Cell viability was measured using Calcein AM fluorescence assay. IC 50 values were calculated using GraphPad software. ATP per cell was measured by combining Cell Titer GLO luminescence assay with CyQuant fluorescence assay. Western blot analysis was used to measure LIN28B and MYCN protein levels. TaqMan PCR reagents were used to measure Let-7 miRNA levels using qPCR analysis. SMS-KCNR cells were injected subcutaneously into nude mice for in vivo xenograft studies. Mice were drugged with 2% DFMO in drinking water when tumors reached 200mm 3 . Tumor volumes were measured using both caliper and micro-CT, and tumor glycolytic metabolism was determined by Maximum Standard Uptake Value (SUVMax) in the tumors through longitudinal 18 F-FDG micro PET/CT scans on days 19 and 32. Results: treatment with high and low dose DFMO resulted in decreased LIN28B protein levels in all three cell lines at 48, 72, and 96 hours timepoints. MYCN protein levels decreased in MYCN high-expression cell lines, BE(2)-C and SMS-KCNR, with high and low dose DFMO treatments, but did not change in MYCN low-expression cell line CHLA90. Let-7 miRNA levels were increased in both MYCN high-expression cell lines after 6 hours of high dose DFMO treatment and no change was seen in CHLA90 cells. Sensitivity to DFMO correlated with LIN28B expression levels in all three cell lines (BE(2)-C>SMS-KCNR>CHLA90). BE(2)-C cells were most sensitive to DFMO treatment with an IC 50 of 3.01 mM followed by SMS-KCNR cells (10.61 mM), and CHLA90 cells, which showed resistance (25.76 mM). In addition, ATP per cell levels were most significantly reduced in BE(2)-C cells after treatment with DFMO followed by SMS-KCNR and CHLA90 cells. In vivo 18 F-FDG PET/CT studies showed decreased SUVMax in the DFMO treatment group indicating reduced glycolytic metabolism. Conclusions: Treatment with DFMO reverses the LIN28/Let-7 axis with a decrease in LIN28B protein expression and an increase in Let-7 miRNA expression. This axis has been shown to play a role in metabolic activity of cells. Decreased metabolic activity and decreased tumor growth is seen in NB cells both in vitro and in vivo following DFMO treatment. NB cell lines with higher levels of LIN28B and MYCN expression are more sensitive to DFMO treatment. These studies suggest that targeting of the LIN28/Let-7 pathway may offer a new method of treating neuroblastoma. Citation Format: Ann Kendzicky, Maria Rich, Anderson Peck, Zhao Ping, Elizabeth VanSickle, Heather McClung, Anthony Chang, Giselle Sholler. Difluoromethylornithine treatment affects the LIN28/Let-7 axis resulting in reduced glycolytic metabolism in neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B54.

Abstract 363: Development of computer-aided detection (CAD) tool for liver metastasis micro CT imaging using targeted contrast agent

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Introduction: Preclinical in-vivo micro CT studies of liver metastasis are difficult due to poor inherent soft tissue contrast and the need for highly technical, manual analysis of the data. Research has implicated that Kupffer cells in the liver encapsulate liver metastases providing an opportunity to deliver macrophage-specific contrast agents for the detection of small metastatic lesions. A new, long-acting preclinical CT contrast agent that targets Kupffer cells has been developed that may allow automated detection of liver lesions via CAD software. Method: A pancreatic cancer liver metastasis model was created by surgically implanting human pancreatic cancer cell line (L3.6pl) within the spleen. Mice were injected with the contrast agent and scanned to obtain baseline anatomy of the liver before implantation. The mice were scanned 1 day after implantation and weekly after that for 5 weeks to monitor the liver metastasis progression. The control group followed an identical protocol but with a sham surgery. Liver tissues were harvested and fixed in paraffin blocks after the last scan. Paraffin blocks were scanned using high resolution micro CT before IHC staining. Human Mitochondrial and F4/80 IHC were used to identify L3.6pl and Kupffer cells, respectively. The CT images were compared to the IHC images from the same block to verify that the locations of the contrast agent and the Kupffer cells were related. Once the pattern of contrast agent and metastatic tumors had been identified, CAD software was developed for automatic tumor detection. Results: The contrast agent was evenly distributed throughout the healthy liver tissue within 1 hour post injection. In healthy mice, the homogenous distribution of contrast remained unchanged for at least 6 weeks. In liver metastasis models, the contrast began to concentrate in various areas of the liver within 2 weeks post implantation. As tumors developed and grew, the contrast became highly concentrated on the borders of tumors creating a 3 dimensional outline of the lesion. IHC staining and micro CT imaging of the fixed tissue verified that the tumors are surrounded by Kupffer cells and that the distribution of concentrated contrast agent matched them. Software was able to detect the tumors based on these contrast outlines and compare them over successive weekly scans. Conclusion: Our new imaging method enables automated detection and evaluation of liver metastasis 1 mm or smaller from as early as 2 weeks. In addition to allowing better visualization, it provides new insight into macrophage motility within the liver. CAD software can take advantage of this unique capability to automate data analysis and allow for large scale longitudinal studies. This new imaging method could be a useful tool to facilitate longitudinal imaging of liver metastases in mice and has the potential for translation into clinical practice. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 363. doi:1538-7445.AM2012-363