Jason Thanh Lee

All Publications

• Publisher Correction: In vivo imaging of mitochondrial membrane potential in non-small-cell lung cancer. Nature Momcilovic, M., Jones, A., Bailey, S. T., Waldmann, C. M., Li, R., Lee, J. T., Abdelhady, G., Gomez, A., Holloway, T., Schmid, E., Stout, D., Fishbein, M. C., Stiles, L., Dabir, D. V., Dubinett, S. M., Christofk, H., Shirihai, O., Koehler, C. M., Sadeghi, S., Shackelford, D. B. 2020

  Abstract

  An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

  View details for DOI 10.1038/s41586-019-1890-z

  View details for PubMedID 31896820

• Multimodal imaging guides surgical management in a preclinical spinal implant infection model JCI INSIGHT Zoller, S. D., Park, H., Olafsen, T., Zamilpa, C., Burke, Z. C., Blumstein, G., Sheppard, W. L., Harnad, C. D., Hori, K. R., Tseng, J., Czupryna, J., McMannus, C., Lee, J. T., Bispo, M., Pastrana, F., Raineri, E. M., Miller, J. F., Miller, L. S., van Diji, J., Francis, K. P., Bernthal, N. M. 2019; 4 (3)

  Abstract

  Spine implant infections portend disastrous outcomes, as diagnosis is challenging and surgical eradication is at odds with mechanical spinal stability. Current imaging modalities can detect anatomical alterations and anomalies but cannot differentiate between infection and aseptic loosening, diagnose specific pathogens, or delineate the extent of an infection. Herein, a fully human monoclonal antibody 1D9, recognizing the immunodominant staphylococcal antigen A on the surface of Staphylococcus aureus, was assessed as a nuclear and fluorescent imaging probe in a preclinical model of S. aureus spinal implant infection, utilizing bioluminescently labeled bacteria to confirm the specificity and sensitivity of this targeting. Postoperative mice were administered 1D9 probe dual labeled with 89-zirconium (89Zr) and a bars represent SEM dye (NIR680) (89Zr-NIR680-1D9), and PET-CT and in vivo fluorescence and bioluminescence imaging were performed. The 89Zr-NIR680-1D9 probe accurately diagnosed both acute and subacute implant infection and permitted fluorescent image-guided surgery for selective debridement of infected tissue. Therefore, a single probe could noninvasively diagnose an infection and facilitate image-guided surgery to improve the clinical management of implant infections.

  View details for DOI 10.1172/jci.insight.124813

  View details for Web of Science ID 000458037000008

  View details for PubMedID 30728332

  View details for PubMedCentralID PMC6413782

• BIRC5 is a target for molecular imaging and detection of human pancreatic cancer. Cancer letters Liu, S. H., Hong, Y., Markowiak, S., Sanchez, R., Creeden, J., Nemunaitis, J., Kalinoski, A., Willey, J., Erhardt, P., Lee, J., van Dam, M., Brunicardi, F. C. 2019; 457: 10–19

  Abstract

  Pancreatic ductal adenocarcinoma (PDAC) is a major cause of cancer mortality with a dismal overall survival rate and an urgent need for detection of minute tumors. Current diagnostic modalities have high sensitivity and specificity for larger tumors, but not for minute PDAC. In this study, we test the feasibility of a precision diagnostic platform for detecting and localizing minute human PDAC in mice. This platform includes: 1) defining BIRC5 as an early PDAC-upregulated gene and utilizing an enhanced BIRC5 super-promoter to drive expression of dual Gaussia luciferase (GLuc) and sr39 thymidine kinase (sr39TK) reporter genes exponentially and specifically in PDAC; 2) utilizing a genetically-engineered AAV2RGD to ensure targeted delivery of GLuc and sr39TK specifically to PDAC; 3) using serologic GLuc and sr39TK microPET/CT imaging to detect and localize minute human PDAC in mice. The study demonstrates feasibility of a precision diagnostic platform using an integrated technology through a multiple-stage amplification strategy of dual reporter genes to enhance the specificity and sensitivity of detection and localization of minute PDAC tumors and currently undetectable disease.

  View details for DOI 10.1016/j.canlet.2019.04.036

  View details for PubMedID 31059751

• In vivo characterization of [18F]AVT-011 as a radiotracer for PET imaging of multidrug resistance. European journal of nuclear medicine and molecular imaging Kannan, P., Füredi, A., Dizdarevic, S., Wanek, T., Mairinger, S., Collins, J., Falls, T., van Dam, R. M., Maheshwari, D., Lee, J. T., Szakács, G., Langer, O. 2019

  Abstract

  Multidrug resistance (MDR) impedes cancer treatment. Two efflux transporters from the ATP-binding cassette (ABC) family, ABCB1 and ABCG2, may contribute to MDR by restricting the entry of therapeutic drugs into tumor cells. Although a higher expression of these transporters has been correlated with an unfavorable response to chemotherapy, transporter expression does not necessarily correlate with function. In this study, we characterized the pharmacological properties of [18F]AVT-011, a new PET radiotracer for imaging transporter-mediated MDR in tumors.AVT-011 was radiolabeled with 18F and evaluated with PET imaging in preclinical models. Transport of [18F]AVT-011 by ABCB1 and/or ABCG2 was assessed by measuring its uptake in the brains of wild-type, Abcb1a/b-/-, and Abcg2-/- mice at baseline and after administration of the ABCB1 inhibitor tariquidar (n = 5/group). Metabolism and biodistribution of [18F]AVT-011 were also measured. To measure ABCB1 function in tumors, we performed PET experiments using both [18F]AVT-011 and [18F]FDG in mice bearing orthotopic breast tumors (n = 7-10/group) expressing clinically relevant levels of ABCB1.At baseline, brain uptake was highest in Abcb1a/b-/- mice. After tariquidar administration, brain uptake increased 3-fold and 8-fold in wild-type and Abcg2-/- mice, respectively, but did not increase further in Abcb1a/b-/- mice. At 30 min after injection, the radiotracer was > 90% in its parent form and had highest uptake in organs of the hepatobiliary system. Compared with that in drug-sensitive tumors, uptake of [18F]AVT-011 was 32% lower in doxorubicin-resistant tumors with highest ABCB1 expression and increased by 40% with tariquidar administration. Tumor uptake of [18F]FDG did not significantly differ among groups.[18F]AVT-011 is a dual ABCB1/ABCG2 substrate radiotracer that can quantify transporter function at the blood-brain barrier and in ABCB1-expressing tumors, making it potentially suitable for clinical imaging of ABCB1-mediated MDR in tumors.

  View details for DOI 10.1007/s00259-019-04589-w

  View details for PubMedID 31729540

• In vivo imaging of mitochondrial membrane potential in non-small-cell lung cancer. Nature Momcilovic, M., Jones, A., Bailey, S. T., Waldmann, C. M., Li, R., Lee, J. T., Abdelhady, G., Gomez, A., Holloway, T., Schmid, E., Stout, D., Fishbein, M. C., Stiles, L., Dabir, D. V., Dubinett, S. M., Christofk, H., Shirihai, O., Koehler, C. M., Sadeghi, S., Shackelford, D. B. 2019

  Abstract

  Mitochondria are essential regulators of cellular energy and metabolism, and have a crucial role in sustaining the growth and survival of cancer cells. A central function of mitochondria is the synthesis of ATP by oxidative phosphorylation, known as mitochondrial bioenergetics. Mitochondria maintain oxidative phosphorylation by creating a membrane potential gradient that is generated by the electron transport chain to drive the synthesis of ATP1. Mitochondria are essential for tumour initiation and maintaining tumour cell growth in cell culture and xenografts2,3. However, our understanding of oxidative mitochondrial metabolism in cancer is limited because most studies have been performed in vitro in cell culture models. This highlights a need for in vivo studies to better understand how oxidative metabolism supports tumour growth. Here we measure mitochondrial membrane potential in non-small-cell lung cancer in vivo using a voltage-sensitive, positron emission tomography (PET) radiotracer known as 4-[18F]fluorobenzyl-triphenylphosphonium (18F-BnTP)4. By using PET imaging of 18F-BnTP, we profile mitochondrial membrane potential in autochthonous mouse models of lung cancer, and find distinct functional mitochondrial heterogeneity within subtypes of lung tumours. The use of 18F-BnTP PET imaging enabled us to functionally profile mitochondrial membrane potential in live tumours.

  View details for DOI 10.1038/s41586-019-1715-0

  View details for PubMedID 31666695

• An HK2 Antisense Oligonucleotide Induces Synthetic Lethality in HK1-HK2+ Multiple Myeloma. Cancer research Xu, S., Zhou, T., Doh, H. M., Trinh, K. R., Catapang, A., Lee, J. T., Braas, D., Bayley, N. A., Yamada, R. E., Vasuthasawat, A., Sasine, J. P., Timmerman, J. M., Larson, S. M., Kim, Y., MacLeod, A. R., Morrison, S. L., Herschman, H. R. 2019; 79 (10): 2748–60

  Abstract

  Although the majority of adult tissues express only hexokinase 1 (HK1) for glycolysis, most cancers express hexokinase 2 (HK2) and many coexpress HK1 and HK2. In contrast to HK1+HK2+ cancers, HK1-HK2+ cancer subsets are sensitive to cytostasis induced by HK2shRNA knockdown and are also sensitive to synthetic lethality in response to the combination of HK2shRNA knockdown, an oxidative phosphorylation (OXPHOS) inhibitor diphenyleneiodonium (DPI), and a fatty acid oxidation (FAO) inhibitor perhexiline (PER). The majority of human multiple myeloma cell lines are HK1-HK2+. Here we describe an antisense oligonucleotide (ASO) directed against human HK2 (HK2-ASO1), which suppressed HK2 expression in human multiple myeloma cell cultures and human multiple myeloma mouse xenograft models. The HK2-ASO1/DPI/PER triple-combination achieved synthetic lethality in multiple myeloma cells in culture and prevented HK1-HK2+ multiple myeloma tumor xenograft progression. DPI was replaceable by the FDA-approved OXPHOS inhibitor metformin (MET), both for synthetic lethality in culture and for inhibition of tumor xenograft progression. In addition, we used an ASO targeting murine HK2 (mHK2-ASO1) to validate the safety of mHK2-ASO1/MET/PER combination therapy in mice bearing murine multiple myeloma tumors. HK2-ASO1 is the first agent that shows selective HK2 inhibition and therapeutic efficacy in cell culture and in animal models, supporting clinical development of this synthetically lethal combination as a therapy for HK1-HK2+ multiple myeloma. SIGNIFICANCE: A first-in-class HK2 antisense oligonucleotide suppresses HK2 expression in cell culture and in in vivo, presenting an effective, tolerated combination therapy for preventing progression of HK1-HK2+ multiple myeloma tumors. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/79/10/2748/F1.large.jpg.

  View details for DOI 10.1158/0008-5472.CAN-18-2799

  View details for PubMedID 30885978

  View details for PubMedCentralID PMC6522331

• Performance Evaluation of G8, a High-Sensitivity Benchtop Preclinical PET/CT Tomograph JOURNAL OF NUCLEAR MEDICINE Gu, Z., Taschereau, R., Vu, N. T., Prout, D. L., Silverman, R. W., Lee, J. T., Chatziioannou, A. F. 2019; 60 (1): 142–49

  Abstract

  G8 is a benchtop integrated PET/CT scanner dedicated to high-sensitivity and high-resolution imaging of mice. This work characterizes its National Electrical Manufacturers Association NU 4-2008 performance where applicable and also assesses the basic imaging performance of the CT subsystem. Methods: The PET subsystem in G8 consists of 4 flat-panel detectors arranged in a boxlike geometry. Each panel consists of 2 modules of a 26 × 26 pixelated bismuth germanate scintillator array with individual crystals measuring 1.75 × 1.75 × 7.2 mm. The crystal arrays are coupled to multichannel photomultiplier tubes via a tapered, pixelated glass lightguide. A cone-beam CT scanner consisting of a MicroFocus x-ray source and a complementary metal oxide semiconductor detector provides anatomic information. Sensitivity, spatial resolution, energy resolution, scatter fraction, count-rate performance, and the capability of performing phantom and mouse imaging were evaluated for the PET subsystem. Noise, dose level, contrast, and resolution were evaluated for the CT subsystem. Results: With an energy window of 350-650 keV, the peak sensitivity was 9.0% near the center of the field of view. The crystal energy resolution ranged from 15.0% to 69.6% in full width at half maximum (FWHM), with a mean of 19.3% ± 3.7%. The average intrinsic spatial resolution was 1.30 and 1.38 mm FWHM in the transverse and axial directions, respectively. The maximum-likelihood expectation maximization reconstructed image of a point source in air averaged 0.81 ± 0.11 mm FWHM. The peak noise-equivalent count rate for the mouse-sized phantom was 44 kcps for a total activity of 2.9 MBq (78 μCi), and the scatter fraction was 11%. For the CT subsystem, the value of the modulation transfer function at 10% was 2.05 cycles/mm. Conclusion: The overall performance demonstrates that the G8 can produce high-quality images for molecular imaging-based biomedical research.

  View details for DOI 10.2967/jnumed.118.208827

  View details for Web of Science ID 000454687800028

  View details for PubMedID 29903933

  View details for PubMedCentralID PMC6354226

• Utilizing F-18-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in Mouse Models of Lung Cancer JOVE-JOURNAL OF VISUALIZED EXPERIMENTS Momcilovic, M., Bailey, S. T., Lee, J. T., Zamilpa, C., Jones, A., Abdelhady, G., Mansfield, J., Francis, K. P., Shackelford, D. B. 2018

  Abstract

  A hallmark of advanced tumors is a switch to aerobic glycolysis that is readily measured by [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography (18F-FDG PET) imaging. Co-mutations in the KRAS proto-oncogene and the LKB1 tumor suppressor gene are frequent events in lung cancer that drive hypermetabolic, glycolytic tumor growth. A critical pathway regulating the growth and metabolism of these tumors is the mechanistic target of the rapamycin (mTOR) pathway, which can be effectively targeted using selective catalytic mTOR kinase inhibitors. The mTOR inhibitor MLN0128 suppresses glycolysis in mice bearing tumors with Kras and Lkb1 co-mutations, referred to as KL mice. The therapy response in KL mice is first measured by 18F-FDG PET and computed tomography (CT) imaging before and after the delivery of MLN0128. By utilizing 18F-FDG PET/CT, researchers are able to measure dynamic changes in the glucose metabolism in genetically engineered mouse models (GEMMs) of lung cancer following a therapeutic intervention with targeted therapies. This is followed by ex vivo autoradiography and a quantitative immunohistochemical (qIHC) analysis using morphometric software. The use of qIHC enables the detection and quantification of distinct changes in the biomarker profiles following treatment as well as the characterization of distinct tumor pathologies. The coupling of PET imaging to quantitative histology is an effective strategy to identify metabolic and therapeutic responses in vivo in mouse models of disease.

  View details for DOI 10.3791/57167

  View details for Web of Science ID 000444836300011

  View details for PubMedID 30080208

  View details for PubMedCentralID PMC6126521

• A precision therapeutic strategy for hexokinase 1-null, hexokinase 2-positive cancers CANCER & METABOLISM Xu, S., Catapang, A., Braas, D., Stiles, L., Doh, H. M., Lee, J. T., Graeber, T. G., Damoiseaux, R., Shirihai, O., Herschman, H. R. 2018; 6: 7

  Abstract

  Precision medicine therapies require identification of unique molecular cancer characteristics. Hexokinase (HK) activity has been proposed as a therapeutic target; however, different hexokinase isoforms have not been well characterized as alternative targets. While HK2 is highly expressed in the majority of cancers, cancer subtypes with differential HK1 and HK2 expression have not been characterized for their sensitivities to HK2 silencing.HK1 and HK2 expression in the Cancer Cell Line Encyclopedia dataset was analyzed. A doxycycline-inducible shRNA silencing system was used to examine the effect of HK2 knockdown in cultured cells and in xenograft models of HK1-HK2+ and HK1+HK2+ cancers. Glucose consumption and lactate production rates were measured to monitor HK activity in cell culture, and 18F-FDG PET/CT was used to monitor HK activity in xenograft tumors. A high-throughput screen was performed to search for synthetically lethal compounds in combination with HK2 inhibition in HK1-HK2+ liver cancer cells, and a combination therapy for liver cancers with this phenotype was developed. A metabolomic analysis was performed to examine changes in cellular energy levels and key metabolites in HK1-HK2+ cells treated with this combination therapy. The CRISPR Cas9 method was used to establish isogenic HK1+HK2+ and HK1-HK2+ cell lines to evaluate HK1-HK2+ cancer cell sensitivity to the combination therapy.Most tumors express both HK1 and HK2, and subsets of cancers from a wide variety of tissues of origin express only HK2. Unlike HK1+HK2+ cancers, HK1-HK2+ cancers are sensitive to HK2 silencing-induced cytostasis. Synthetic lethality was achieved in HK1-HK2+ liver cancer cells, by the combination of DPI, a mitochondrial complex I inhibitor, and HK2 inhibition, in HK1-HK2+ liver cancer cells. Perhexiline, a fatty acid oxidation inhibitor, further sensitizes HK1-HK2+ liver cancer cells to the complex I/HK2-targeted therapeutic combination. Although HK1+HK2+ lung cancer H460 cells are resistant to this therapeutic combination, isogenic HK1KOHK2+ cells are sensitive to this therapy.The HK1-HK2+ cancer subsets exist among a wide variety of cancer types. Selective inhibition of the HK1-HK2+ cancer cell-specific energy production pathways (HK2-driven glycolysis, oxidative phosphorylation and fatty acid oxidation), due to the unique presence of only the HK2 isoform, appears promising to treat HK1-HK2+ cancers. This therapeutic strategy will likely be tolerated by most normal tissues, where only HK1 is expressed.

  View details for DOI 10.1186/s40170-018-0181-8

  View details for Web of Science ID 000437119600001

  View details for PubMedID 29988332

  View details for PubMedCentralID PMC6022704

• Effects of teriparatide on morphology of aortic calcification in aged hyperlipidemic mice AMERICAN JOURNAL OF PHYSIOLOGY-HEART AND CIRCULATORY PHYSIOLOGY Hsu, J. J., Lu, J., Umar, S., Lee, J. T., Kulkarni, R. P., Ding, Y., Chang, C., Hsiai, T. K., Hokugo, A., Gkouveris, I., Tetradis, S., Nishimura, I., Demer, L. L., Tintut, Y. 2018; 314 (6): H1203–H1213

  Abstract

  Calcific aortic vasculopathy correlates with bone loss in osteoporosis in an age-independent manner. Prior work suggests that teriparatide, the bone anabolic treatment for postmenopausal osteoporosis, may inhibit the onset of aortic calcification. Whether teriparatide affects the progression of preexisting aortic calcification, widespread among this patient population, is unknown. Female apolipoprotein E-deficient mice were aged for over 1 yr to induce aortic calcification, treated for 4.5 wk with daily injections of control vehicle (PBS), 40 µg/kg teriparatide (PTH40), or 400 µg/kg teriparatide (PTH400), and assayed for aortic calcification by microcomputed tomography (microCT) before and after treatment. In a followup cohort, aged female apolipoprotein E-deficient mice were treated with PBS or PTH400 and assayed for aortic calcification by serial microCT and micropositron emission tomography. In both cohorts, aortic calcification detected by microCT progressed similarly in all groups. Mean aortic 18F-NaF incorporation, detected by serial micropositron emission tomography, increased in the PBS-treated group (+14 ± 5%). In contrast, 18F-NaF incorporation decreased in the PTH400-treated group (-33 ± 20%, P = 0.03). Quantitative histochemical analysis by Alizarin red staining revealed a lower mineral surface area index in the PTH400-treated group compared with the PBS-treated group ( P = 0.04). Furthermore, Masson trichrome staining showed a significant increase in collagen deposition in the left ventricular myocardium of mice that received PTH400 [2.1 ± 0.6% vs. control mice (0.5 ± 0.1%), P = 0.02]. In summary, although teriparatide may not affect the calcium mineral content of aortic calcification, it reduces 18F-NaF uptake in calcified lesions, suggesting the possibility that it may reduce mineral surface area with potential impact on plaque stability. NEW & NOTEWORTHY Parathyroid hormone regulates bone mineralization and may also affect vascular calcification, which is an important issue, given that its active fragment, teriparatide, is widely used for the treatment of osteoporosis. To determine whether teriparatide alters vascular calcification, we imaged aortic calcification in mice treated with teriparatide and control mice. Although teriparatide did not affect the calcium content of cardiovascular deposits, it reduced their fluoride tracer uptake.

  View details for DOI 10.1152/ajpheart.00718.2017

  View details for Web of Science ID 000441143800010

  View details for PubMedID 29451816

  View details for PubMedCentralID PMC6032086

• Microscale radiosynthesis, preclinical imaging and dosimetry study of [F-18]AMBF(3)-TATE: A potential PET tracer for clinical imaging of somatostatin receptors NUCLEAR MEDICINE AND BIOLOGY Lisova, K., Sergeev, M., Evans-Axelsson, S., Stuparu, A. D., Beykan, S., Collins, J., Jones, J., Lassmann, M., Herrmann, K., Perrin, D., Lee, J. T., Slavik, R., van Dam, R. 2018; 61: 36–44

  Abstract

  Peptides labeled with positron-emitting isotopes are emerging as a versatile class of compounds for the development of highly specific, targeted imaging agents for diagnostic imaging via positron-emission tomography (PET) and for precision medicine via theranostic applications. Despite the success of peptides labeled with gallium-68 (for imaging) or lutetium-177 (for therapy) in the clinical management of patients with neuroendocrine tumors or prostate cancer, there are significant advantages of using fluorine-18 for imaging. Recent developments have greatly simplified such labeling: in particular, labeling of organotrifluoroborates via isotopic exchange can readily be performed in a single-step under aqueous conditions and without the need for HPLC purification. Though an automated synthesis has not yet been explored, microfluidic approaches have emerged for 18F-labeling with high speed, minimal reagents, and high molar activity compared to conventional approaches. As a proof-of-concept, we performed microfluidic labeling of an octreotate analog ([18F]AMBF3-TATE), a promising 18F-labeled analog that could compete with [68Ga]Ga-DOTATATE with the advantage of providing a greater number of patient doses per batch produced.Both [18F]AMBF3-TATE and [68Ga]Ga-DOTATATE were labeled, the former by microscale methods adapted from manual labeling, and were imaged in mice bearing human SSTR2-overexpressing, rat SSTR2 wildtype, and SSTR2-negative xenografts. Furthermore, a dosimetry analysis was performed for [18F]AMBF3-TATE.The micro-synthesis exhibited highly-repeatable performance with radiochemical conversion of 50 ± 6% (n = 15), overall decay-corrected radiochemical yield of 16 ± 1% (n = 5) in ~40 min, radiochemical purity >99%, and high molar activity. Preclinical imaging with [18F]AMBF3-TATE in SSTR2 tumor models correlated well with [68Ga]Ga-DOTATATE. The favorable biodistribution, with the highest tracer accumulation in the bladder followed distantly by gastrointestinal tissues, resulted in 1.26 × 10-2 mSv/MBq maximal estimated effective dose in human, a value lower than that reported for current clinical 18F- and 68Ga-labeled compounds.The combination of novel chemical approaches to 18F-labeling and microdroplet radiochemistry have the potential to serve as a platform for greatly simplified development and production of 18F-labeled peptide tracers. Favorable preclinical imaging and dosimetry of [18F]AMBF3-TATE, combined with a convenient synthesis, validate this assertion and suggest strong potential for clinical translation.

  View details for DOI 10.1016/j.nucmedbio.2018.04.001

  View details for Web of Science ID 000437551700005

  View details for PubMedID 29747035

  View details for PubMedCentralID PMC6015542

• The GSK3 Signaling Axis Regulates Adaptive Glutamine Metabolism in Lung Squamous Cell Carcinoma CANCER CELL Momcilovic, M., Bailey, S. T., Lee, J. T., Fishbein, M. C., Braas, D., Go, J., Graeber, T. G., Parlati, F., Demo, S., Li, R., Walser, T. C., Gricowski, M., Shuman, R., Ibarra, J., Fridman, D., Phelps, M. E., Badran, K., John, M., Bernthal, N. M., Federman, N., Yanagawa, J., Dubinett, S. M., Sadeghi, S., Christofk, H. R., Shackelford, D. B. 2018; 33 (5): 905-+

  Abstract

  Altered metabolism is a hallmark of cancer growth, forming the conceptual basis for development of metabolic therapies as cancer treatments. We performed in vivo metabolic profiling and molecular analysis of lung squamous cell carcinoma (SCC) to identify metabolic nodes for therapeutic targeting. Lung SCCs adapt to chronic mTOR inhibition and suppression of glycolysis through the GSK3α/β signaling pathway, which upregulates glutaminolysis. Phospho-GSK3α/β protein levels are predictive of response to single-therapy mTOR inhibition while combinatorial treatment with the glutaminase inhibitor CB-839 effectively overcomes therapy resistance. In addition, we identified a conserved metabolic signature in a broad spectrum of hypermetabolic human tumors that may be predictive of patient outcome and response to combined metabolic therapies targeting mTOR and glutaminase.

  View details for DOI 10.1016/j.ccell.2018.04.002

  View details for Web of Science ID 000432494500014

  View details for PubMedID 29763624

  View details for PubMedCentralID PMC6451645

• The 4-N-acyl and 4-N-alkyl gemcitabine analogues with silicon-fluoride-acceptor: Application to F-18-Radiolabeling EUROPEAN JOURNAL OF MEDICINAL CHEMISTRY Gonzalez, C., Sanchez, A., Collins, J., Lisova, K., Lee, J. T., van Dam, R., Barbieri, M., Ramachandran, C., Wnuk, S. F. 2018; 148: 314–24

  Abstract

  The coupling of gemcitabine with functionalized carboxylic acids using peptide coupling conditions afforded 4-N-alkanoyl analogues with a terminal alkyne or azido moiety. Reaction of 4-N-tosylgemcitabine with azidoalkyl amine provided 4-N-alkyl gemcitabine with a terminal azido group. Click reaction with silane building blocks afforded 4-N-alkanoyl or 4-N-alkyl gemcitabine analogues suitable for fluorination. RP-HPLC analysis indicated better chemical stability of 4-N-alkyl gemcitabine analogues versus 4-N-alkanoyl analogues in acidic aqueous conditions. The 4-N-alkanoyl gemcitabine analogues showed potent cytostatic activity against L1210 cell line, but cytotoxicity of the 4-N-alkylgemcitabine analogues was low. However, 4-N-alkanoyl and 4-N-alkyl analogues had comparable antiproliferative activities in the HEK293 cells. The 4-N-alkyl analogue with a terminal azide group was shown to be localized inside HEK293 cells by fluorescence microscopy after labelling with Fluor 488-alkyne. The [18F]4-N-alkyl or alkanoyl silane gemcitabine analogues were successfully synthesized using microscale and conventional silane-labeling radiochemical protocols. Preliminary positron-emission tomography (PET) imaging in mice showed the biodistribution of [18F]4-N-alkyl to have initial concentration in the liver, kidneys and GI tract followed by increasing signal in the bone.

  View details for DOI 10.1016/j.ejmech.2018.02.017

  View details for Web of Science ID 000428824700025

  View details for PubMedID 29471120

  View details for PubMedCentralID PMC5841594

• Performing radiosynthesis in microvolumes to maximize molar activity of tracers for positron emission tomography COMMUNICATIONS CHEMISTRY Sergeev, M., Lazari, M., Morgia, F., Collins, J., Javed, M., Sergeeva, O., Jones, J., Phelps, M. E., Lee, J. T., Keng, P., van Dam, R. 2018; 1

  View details for DOI 10.1038/s42004-018-0009-z

  View details for Web of Science ID 000433892800002

• Ex Vivo Radiolabeling and In Vivo PET Imaging of T Cells Expressing Nuclear Reporter Genes REPORTER GENE IMAGING: METHODS AND PROTOCOLS Moroz, M. A., Zanzonico, P., Lee, J. T., Ponomarev, V., Dubey, P. 2018; 1790: 153–63

  Abstract

  Recent advances in T cell-based immunotherapies from bench to bedside have highlighted the need for improved diagnostic imaging of T cell trafficking in vivo and the means to noninvasively investigate failures in treatment response. T cells expressing tumor-associated T cell receptors (TCRs) or engineered with chimeric antigen receptors (CARs) face multiple challenges, including possible influence of genetic engineering on T cell efficacy, inhibitory effects of the tumor microenvironment, tumor checkpoint proteins and on-target, off-tissue toxicities (Kershaw et al., Nat Rev Cancer 13:525-541, 2013; Corrigan-Curay et al., Mol Ther 22:1564-1574, 2014; June et al., Sci Trans Med 7:280-287, 2015; Whiteside et al., Clin Cancer Res 22:1845-1855, 2016; Rosenberg and Restifo, Science 348:62-68, 2015). Positron emission tomography (PET) imaging with nuclear reporter genes is potentially one of the most sensitive and noninvasive methods to quantitatively track and monitor function of adoptively transferred cells in vivo. However, in vivo PET detection of T cells after administration into patients is limited by the degree of tracer accumulation per cell in situ and cell density in target tissues. We describe here a method for ex vivo radiolabeling of T cells, a reliable and robust technique for PET imaging of the kinetics of T cell biodistribution from the time of administration to subsequent localization in targeted tumors and other tissues of the body. This noninvasive technique can provide valuable information to monitor and identify the potential efficacy of adoptive cell therapies.

  View details for DOI 10.1007/978-1-4939-7860-1_12

  View details for Web of Science ID 000448886300013

  View details for PubMedID 29858790

• Imaging T Cell Dynamics and Function Using PET and Human Nuclear Reporter Genes REPORTER GENE IMAGING: METHODS AND PROTOCOLS Lee, J. T., Moroz, M. A., Ponomarev, V., Dubey, P. 2018; 1790: 165–80

  Abstract

  Adoptive cell transfer immunotherapy has demonstrated significant promise in the treatment of cancer, with long-term, durable responses. T cells expressing T cell receptors (TCRs) that recognize tumor antigens, or engineered with chimeric antigen receptors (CARs) can recognize and eliminate tumor cells even in advanced disease. Positron emission tomography (PET) imaging with nuclear reporter genes, a noninvasive method to track and monitor function of engineered cells in vivo, allows quantitative, longitudinal monitoring of these cells, including their expansion/contraction, migration, retention at target and off-target sites, and biological state. As an additional advantage, some reporter genes also exhibit "suicide potential" permitting the safe elimination of adoptively transferred T cells in instances of adverse reaction to therapy. Here, we describe the production of human nuclear reporter gene-expressing T cells and noninvasive PET imaging to monitor their cell fate in vivo.

  View details for DOI 10.1007/978-1-4939-7860-1_13

  View details for Web of Science ID 000448886300014

  View details for PubMedID 29858791

• Hexokinase 2 is targetable for HK1 negative, HK2 positive tumors from a wide variety of tissues of origin. Journal of nuclear medicine : official publication, Society of Nuclear Medicine Xu, S., Catapang, A., Doh, H. M., Bayley, N. A., Lee, J. T., Braas, D., Graeber, T. G., Herschman, H. R. 2018

  Abstract

  Although absent in most adult tissues, hexokinase 2 (HK2) is expressed in a majority of tumors and contributes to increased glucose consumption and to in vivo tumor 18F-FDG PET signaling. Methods: Both HK2 knockdown and knockout approaches were used to investigate the role of HK2 in cancer cell proliferation, in vivo xenograft tumor progression and 18F-FDG tumor accumulation. BioProfiler analysis monitored cell culture glucose consumption and lactate production; 18F-FDG PET/CT monitored in vivo tumor glucose accumulation. Cancer Cell Line Encyclopedia data were analyzed for HK1 and HK2 expression. Results: Neither cell proliferation in culture nor xenograft tumor progression are inhibited by HK2 knockdown or knockout in cancer cells that express HK1 and HK2. However, cancer subsets from a variety of tissues of origin express only HK2, but not HK1. In contrast to HK1+HK2+ cancers, HK2 knockdown in HK1-HK2+ cancer cells results in inhibition of cell proliferation, colony formation and xenograft tumor progression. Moreover, HK1KOHK2+ cancer cells are susceptible to HK2 inhibition, in contrast to their isogenic HK1+HK2+ parental cells. Conclusion: HK1 and HK2 expression are redundant in tumors; either can provide sufficient aerobic glycolysis for tumor growth; despite a reduction in 18F-FDG PET signal. Therapeutic HK2 inhibition is likely to be restricted to HK1-HK2+ tumor subsets, but stratification of tumors that express HK2, but not HK1, should identify tumors treatable with emerging HK2 specific inhibitors.

  View details for DOI 10.2967/jnumed.118.212365

  View details for PubMedID 29880505

• Cytoplasmic p53 couples oncogene-driven glucose metabolism to apoptosis and is a therapeutic target in glioblastoma NATURE MEDICINE Mai, W. X., Gosa, L., Daniels, V. W., Ta, L., Tsang, J. E., Higgins, B., Gilmore, W., Bayley, N. A., Harati, M., Lee, J. T., Yong, W. H., Kornblum, H. I., Bensinger, S. J., Mischel, P. S., Rao, P., Clark, P. M., Cloughesy, T. F., Letai, A., Nathanson, D. A. 2017; 23 (11): 1342-+

  Abstract

  Cross-talk among oncogenic signaling and metabolic pathways may create opportunities for new therapeutic strategies in cancer. Here we show that although acute inhibition of EGFR-driven glucose metabolism induces only minimal cell death, it lowers the apoptotic threshold in a subset of patient-derived glioblastoma (GBM) cells. Mechanistic studies revealed that after attenuated glucose consumption, Bcl-xL blocks cytoplasmic p53 from triggering intrinsic apoptosis. Consequently, targeting of EGFR-driven glucose metabolism in combination with pharmacological stabilization of p53 with the brain-penetrant small molecule idasanutlin resulted in synthetic lethality in orthotopic glioblastoma xenograft models. Notably, neither the degree of EGFR-signaling inhibition nor genetic analysis of EGFR was sufficient to predict sensitivity to this therapeutic combination. However, detection of rapid inhibitory effects on [18F]fluorodeoxyglucose uptake, assessed through noninvasive positron emission tomography, was an effective predictive biomarker of response in vivo. Together, these studies identify a crucial link among oncogene signaling, glucose metabolism, and cytoplasmic p53, which may potentially be exploited for combination therapy in GBM and possibly other malignancies.

  View details for DOI 10.1038/nm.4418

  View details for Web of Science ID 000414548300016

  View details for PubMedID 29035366

  View details for PubMedCentralID PMC5683421

• Detection of immune responses after immunotherapy in glioblastoma using PET and MRI PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Antonios, J. P., Soto, H., Everson, R. G., Moughon, D. L., Wang, A. C., Orpilla, J., Radu, C., Ellingson, B. M., Lee, J. T., Cloughesy, T., Phelps, M. E., Czernin, J., Liau, L. M., Prins, R. M. 2017; 114 (38): 10220–25

  Abstract

  Contrast-enhanced MRI is typically used to follow treatment response and progression in patients with glioblastoma (GBM). However, differentiating tumor progression from pseudoprogression remains a clinical dilemma largely unmitigated by current advances in imaging techniques. Noninvasive imaging techniques capable of distinguishing these two conditions could play an important role in the clinical management of patients with GBM and other brain malignancies. We hypothesized that PET probes for deoxycytidine kinase (dCK) could be used to differentiate immune inflammatory responses from other sources of contrast-enhancement on MRI. Orthotopic malignant gliomas were established in syngeneic immunocompetent mice and then treated with dendritic cell (DC) vaccination and/or PD-1 mAb blockade. Mice were then imaged with [18F]-FAC PET/CT and MRI with i.v. contrast. The ratio of contrast enhancement on MRI to normalized PET probe uptake, which we term the immunotherapeutic response index, delineated specific regions of immune inflammatory activity. On postmortem examination, FACS-based enumeration of intracranial tumor-infiltrating lymphocytes directly correlated with quantitative [18F]-FAC PET probe uptake. Three patients with GBM undergoing treatment with tumor lysate-pulsed DC vaccination and PD-1 mAb blockade were also imaged before and after therapy using MRI and a clinical PET probe for dCK. Unlike in mice, [18F]-FAC is rapidly catabolized in humans; thus, we used another dCK PET probe, [18F]-clofarabine ([18F]-CFA), that may be more clinically relevant. Enhanced [18F]-CFA PET probe accumulation was identified in tumor and secondary lymphoid organs after immunotherapy. Our findings identify a noninvasive modality capable of imaging the host antitumor immune response against intracranial tumors.

  View details for DOI 10.1073/pnas.1706689114

  View details for Web of Science ID 000411157100075

  View details for PubMedID 28874539

  View details for PubMedCentralID PMC5617282

• Automatic concentration and reformulation of PET tracers via microfluidic membrane distillation LAB ON A CHIP Chao, P. H., Collins, J., Argus, J. P., Tseng, W., Lee, J. T., van Dam, R. 2017; 17 (10): 1802–16

  Abstract

  Short-lived radiolabeled tracers for positron emission tomography (PET) must be rapidly synthesized, purified, and formulated into injectable solution just prior to imaging. Current radiosynthesizers are generally designed for clinical use, and the HPLC purification and SPE formulation processes often result in a final volume that is too large for preclinical and emerging in vitro applications. Conventional technologies and techniques for reducing this volume tend to be slow, resulting in radioactive decay of the product, and often require manual handling of the radioactive materials. We present a fully-automated microfluidic system based on sweeping gas membrane distillation to rapidly perform the concentration and formulation process. After detailed characterization of the system, we demonstrate fast and efficient concentration and formulation of several PET tracers, evaluate residual solvent content to establish the safety of the formulated tracers for injection, and show that the formulated tracer can be used for in vivo imaging.

  View details for DOI 10.1039/c6lc01569g

  View details for Web of Science ID 000401551300011

  View details for PubMedID 28443841

  View details for PubMedCentralID PMC5497730

• Radiochemistry on electrodes: Synthesis of an F-18-labelled and in vivo stable COX-2 inhibitor PLOS ONE Lebedev, A., Jiao, J., Lee, J., Yang, F., Allison, N., Herschman, H., Sadeghi, S. 2017; 12 (5): e0176606

  Abstract

  New radiochemistry techniques can yield novel PET tracers for COX-2 and address the shortcomings in in vivo stability and specificity, which have held back clinical translation of tracers to image COX-2 expression. Current techniques limit radiosynthesis to analogs of the COX-2 inhibitors with fluorine-18 added via a carbon chain, or on an aromatic position which renders the radiolabeled analog less specific towards COX-2, resulting in tracers with low in vivo stability or specificity. To solve this problem, we have developed a new high affinity, 18F-labelled COX-2 inhibitor that is radiolabeled directly on a heteroaromatic ring. This molecule exhibits favorable biodistribution and increased metabolic stability. Synthesis of this molecule cannot be achieved by traditional means; consequently, we have developed an automated electrochemical radiosynthesis platform to synthesize up to 5 mCi of radiochemically pure 18F-COX-2ib in 4 hours (2% decay-corrected radiochemical yield). In vitro studies demonstrated clear correlation between COX-2 expression and uptake of the tracer. PET imaging of healthy animals confirmed that the molecule is excreted from blood within an hour, mainly through the hepatobiliary excretion pathway. In vivo metabolism data demonstrated that > 95% of the injected radioactivity remains in the form of the parent molecule 1 hour after injection.

  View details for DOI 10.1371/journal.pone.0176606

  View details for Web of Science ID 000400646300037

  View details for PubMedID 28464017

  View details for PubMedCentralID PMC5413030

• Rigor and Reproducibility in Analysis of Vascular Calcification CIRCULATION RESEARCH Demer, L. L., Tintut, Y., Nguyen, K., Hsiai, T., Lee, J. T. 2017; 120 (8): 1240–42

  View details for DOI 10.1161/CIRCRESAHA.116.310326

  View details for Web of Science ID 000399020500012

  View details for PubMedID 28408452

  View details for PubMedCentralID PMC5407390

• Cardiac Fibroblasts Adopt Osteogenic Fates and Can Be Targeted to Attenuate Pathological Heart Calcification CELL STEM CELL Pillai, I. L., Li, S., Romay, M., Lam, L., Lu, Y., Huang, J., Dillard, N., Zemanova, M., Rubbi, L., Wang, Y., Lee, J., Xia, M., Liang, O., Xie, Y., Pellegrini, M., Lusis, A. J., Deb, A. 2017; 20 (2): 218-+

  Abstract

  Mammalian tissues calcify with age and injury. Analogous to bone formation, osteogenic cells are thought to be recruited to the affected tissue and induce mineralization. In the heart, calcification of cardiac muscle leads to conduction system disturbances and is one of the most common pathologies underlying heart blocks. However the cell identity and mechanisms contributing to pathological heart muscle calcification remain unknown. Using lineage tracing, murine models of heart calcification and in vivo transplantation assays, we show that cardiac fibroblasts (CFs) adopt an osteoblast cell-like fate and contribute directly to heart muscle calcification. Small-molecule inhibition of ENPP1, an enzyme that is induced upon injury and regulates bone mineralization, significantly attenuated cardiac calcification. Inhibitors of bone mineralization completely prevented ectopic cardiac calcification and improved post injury heart function. Taken together, these findings highlight the plasticity of fibroblasts in contributing to ectopic calcification and identify pharmacological targets for therapeutic development.

  View details for PubMedID 27867037

  View details for PubMedCentralID PMC5291784

• Comparative Analysis of Human Nucleoside Kinase-Based Reporter Systems for PET Imaging MOLECULAR IMAGING AND BIOLOGY Lee, J. T., Zhang, H., Moroz, M. A., Likar, Y., Shenker, L., Sumzin, N., Lobo, J., Zurita, J., Collins, J., van Dam, R., Ponomarev, V. 2017; 19 (1): 100–108

  Abstract

  Radionuclide-based reporter gene imaging has the sensitivity to monitor gene- and cell-based therapies in human subjects. Potential immunogenicity of current viral transgenes warrants development of human-based reporter systems. We compared human nucleoside kinase reporters to a panel of nucleoside analogs of FEAU, FMAU, and FIAU, including the first in vivo assessment of L-[18F]FEAU.Human isogenic U87 cell lines were transduced to express different human reporter genes including dCK-R104M/D133A (dCKDM), dCK-R104Q/D133N (dCKep16A), dCK-A100V/R104M/D133A (dCK3M), and TK2-N93D/L109F (TK2DM), and wild-type dCK (dCK) and herpes simplex virus type-1 (HSVTK) reporter gene as references. In vitro cell uptake assays were performed with [18F]FEAU, L-[18F]FEAU, [14C]FMAU, L-[18F]FMAU, and [124I]FIAU. Micro-positron emission tomography/X-ray computed tomography imaging of xenograft-bearing nu/nu mice was conducted with [18F]FEAU, L-[18F]FEAU, L-[18F]FMAU, and [124I]FIAU on consecutive days. A cell viability assay was also performed to assess sensitivities to gemcitabine and bromovinyldeoxyuridine (BVdU).In vitro, dCKep16A and dCKDM with [18F]FEAU exhibited the highest sensitivity and selectivity of the human reporters, second only to HSVTK/[18F]FEAU. L-[18F]FEAU biodistribution in mice was on par with [18F]FEAU and L-[18F]FMAU. L-[18F]FMAU uptake in isogenic xenografts was highest for all human reporter genes. However, [18F]FEAU was the most selective of the short half-life reporter probes due to its minimal recognition by human dCK and relative sensitivity, whereas [124I]FIAU permitted imaging at a later time point, improving signal-to-background ratio. Of the human reporter genes, dCKep16A consistently outperformed the other tested reporters. Reporter genes of interest increased potency to the nucleoside analog prodrugs gemcitabine and BVdU.We demonstrate that human nucleoside kinase reporter systems vary significantly in their sensitivity and selectivity for in vivo imaging. The sufficiently high signal-to-background ratios and enhanced suicide gene potential support clinical translation.

  View details for DOI 10.1007/s11307-016-0981-6

  View details for Web of Science ID 000392205000012

  View details for PubMedID 27393689

  View details for PubMedCentralID PMC5345744

• Targeted Inhibition of EGFR and Glutaminase Induces Metabolic Crisis in EGFR Mutant Lung Cancer CELL REPORTS Momcilovic, M., Bailey, S. T., Lee, J. T., Fishbein, M. C., Magyar, C., Braas, D., Graeber, T., Jackson, N. J., Czernin, J., Emberley, E., Gross, M., Janes, J., Mackinnon, A., Pan, A., Rodriguez, M., Works, M., Zhang, W., Parlati, F., Demo, S., Garon, E., Krysan, K., Walser, T. C., Dubinett, S. M., Sadeghi, S., Christofk, H. R., Shackelford, D. B. 2017; 18 (3): 601–10

  Abstract

  Cancer cells exhibit increased use of nutrients, including glucose and glutamine, to support the bioenergetic and biosynthetic demands of proliferation. We tested the small-molecule inhibitor of glutaminase CB-839 in combination with erlotinib on epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer (NSCLC) as a therapeutic strategy to simultaneously impair cancer glucose and glutamine utilization and thereby suppress tumor growth. Here, we show that CB-839 cooperates with erlotinib to drive energetic stress and activate the AMP-activated protein kinase (AMPK) pathway in EGFR (del19) lung tumors. Tumor cells undergo metabolic crisis and cell death, resulting in rapid tumor regression in vivo in mouse NSCLC xenografts. Consistently, positron emission tomography (PET) imaging with 18F-fluoro-2-deoxyglucose (18F-FDG) and 11C-glutamine (11C-Gln) of xenografts indicated reduced glucose and glutamine uptake in tumors following treatment with CB-839 + erlotinib. Therefore, PET imaging with 18F-FDG and 11C-Gln tracers can be used to non-invasively measure metabolic response to CB-839 and erlotinib combination therapy.

  View details for DOI 10.1016/j.celrep.2016.12.061

  View details for Web of Science ID 000396470600003

  View details for PubMedID 28099841

  View details for PubMedCentralID PMC5260616

• Synthesis and preclinical evaluation of an (AlF)-F-18 radiofluorinated GLU-UREA-LYS(AHX)-HBED-CC PSMA ligand EUROPEAN JOURNAL OF NUCLEAR MEDICINE AND MOLECULAR IMAGING Boschi, S., Lee, J. T., Beykan, S., Slavik, R., Wei, L., Spick, C., Eberlein, U., Buck, A. K., Lodi, F., Cicoria, G., Czernin, J., Lassmann, M., Fanti, S., Herrmann, K. 2016; 43 (12): 2122–30

  Abstract

  The aim of this study was to synthesize and preclinically evaluate an 18F-PSMA positron emission tomography (PET) tracer. Prostate-specific membrane antigen (PSMA) specificity, biodistribution, and dosimetry in healthy and tumor-bearing mice were determined.Several conditions for the labeling of 18F-PSMA-11 via 18F-AlF-complexation were screened to study the influence of reaction temperature, peptide amount, ethanol volume, and reaction time. After synthesis optimization, biodistribution and dosimetry studies were performed in C57BL6 mice. For proof of PSMA-specificity, mice were implanted with PSMA-negative (PC3) and PSMA-positive (LNCaP) tumors in contralateral flanks. Static and dynamic microPET/computed tomography (CT) imaging was performed.Quantitative labeling yields could be achieved with >97 % radiochemical purity. The 18F-PSMA-11 uptake was more than 24-fold higher in PSMA-high LNCaP than in PSMA-low PC3 tumors (18.4 ± 3.3 %ID/g and 0.795 ± 0.260 %ID/g, respectively; p < 4.2e-5). Results were confirmed by ex vivo gamma counter analysis of tissues after the last imaging time point. The highest absorbed dose was reported for the kidneys. The maximum effective dose for an administered activity of 200 MBq was 1.72 mSv.18F-PSMA-11 using direct labeling of chelate-attached peptide with aluminum-fluoride detected PSMA-expressing tumors with high tumor-to-liver ratios. The kidneys were the dose-limiting organs. Even by applying the most stringent dosimetric calculations, injected activities of up to 0.56 GBq are feasible.

  View details for DOI 10.1007/s00259-016-3437-y

  View details for Web of Science ID 000385161100004

  View details for PubMedID 27329046

  View details for PubMedCentralID PMC5050145

• [18F]CFA as a clinically translatable probe for PET imaging of deoxycytidine kinase activity. Proceedings of the National Academy of Sciences of the United States of America Kim, W., Le, T. M., Wei, L., Poddar, S., Bazzy, J., Wang, X., Uong, N. T., Abt, E. R., Capri, J. R., Austin, W. R., Van Valkenburgh, J. S., Steele, D., Gipson, R. M., Slavik, R., Cabebe, A. E., Taechariyakul, T., Yaghoubi, S. S., Lee, J. T., Sadeghi, S., Lavie, A., Faull, K. F., Witte, O. N., Donahue, T. R., Phelps, M. E., Herschman, H. R., Herrmann, K., Czernin, J., Radu, C. G. 2016; 113 (15): 4027–32

  Abstract

  Deoxycytidine kinase (dCK), a rate-limiting enzyme in the cytosolic deoxyribonucleoside (dN) salvage pathway, is an important therapeutic and positron emission tomography (PET) imaging target in cancer. PET probes for dCK have been developed and are effective in mice but have suboptimal specificity and sensitivity in humans. To identify a more suitable probe for clinical dCK PET imaging, we compared the selectivity of two candidate compounds-[(18)F]Clofarabine; 2-chloro-2'-deoxy-2'-[(18)F]fluoro-9-β-d-arabinofuranosyl-adenine ([(18)F]CFA) and 2'-deoxy-2'-[(18)F]fluoro-9-β-d-arabinofuranosyl-guanine ([(18)F]F-AraG)-for dCK and deoxyguanosine kinase (dGK), a dCK-related mitochondrial enzyme. We demonstrate that, in the tracer concentration range used for PET imaging, [(18)F]CFA is primarily a substrate for dCK, with minimal cross-reactivity. In contrast, [(18)F]F-AraG is a better substrate for dGK than for dCK. [(18)F]CFA accumulation in leukemia cells correlated with dCK expression and was abrogated by treatment with a dCK inhibitor. Although [(18)F]CFA uptake was reduced by deoxycytidine (dC) competition, this inhibition required high dC concentrations present in murine, but not human, plasma. Expression of cytidine deaminase, a dC-catabolizing enzyme, in leukemia cells both in cell culture and in mice reduced the competition between dC and [(18)F]CFA, leading to increased dCK-dependent probe accumulation. First-in-human, to our knowledge, [(18)F]CFA PET/CT studies showed probe accumulation in tissues with high dCK expression: e.g., hematopoietic bone marrow and secondary lymphoid organs. The selectivity of [(18)F]CFA for dCK and its favorable biodistribution in humans justify further studies to validate [(18)F]CFA PET as a new cancer biomarker for treatment stratification and monitoring.

  View details for DOI 10.1073/pnas.1524212113

  View details for PubMedID 27035974

  View details for PubMedCentralID PMC4839461

• Comparative Analysis of T Cell Imaging with Human Nuclear Reporter Genes. Journal of nuclear medicine : official publication, Society of Nuclear Medicine Moroz, M. A., Zhang, H., Lee, J., Moroz, E., Zurita, J., Shenker, L., Serganova, I., Blasberg, R., Ponomarev, V. 2015; 56 (7): 1055–60

  Abstract

  Monitoring genetically altered T cells is an important component of adoptive T cell therapy in patients, and the ability to visualize their trafficking/targeting, proliferation/expansion, and retention/death using highly sensitive reporter systems that do not induce an immunologic response would provide useful information. Therefore, we focused on human reporter gene systems that have the potential for translation to clinical studies. The objective of the in vivo imaging studies was to determine the minimum number of T cells that could be visualized with the different nuclear reporter systems. We determined the imaging sensitivity (lower limit of T cell detection) of each reporter using appropriate radiolabeled probes for PET or SPECT imaging.Human T cells were transduced with retroviral vectors encoding for the human norepinephrine transporter (hNET), human sodium-iodide symporter (hNIS), a human deoxycytidine kinase double mutant (hdCKDM), and herpes simplex virus type 1 thymidine kinase (hsvTK) reporter genes. After viability and growth were assessed, 10(5) to 3 × 10(6) reporter T cells were injected subcutaneously on the shoulder area. The corresponding radiolabeled probe was injected intravenously 30 min later, followed by sequential PET or SPECT imaging. Radioactivity at the T cell injection sites and in the thigh (background) was measured.The viability and growth of experimental cells were unaffected by transduction. The hNET/meta-(18)F-fluorobenzylguanidine ((18)F-MFBG) reporter system could detect less than 1 × 10(5) T cells because of its high uptake in the transduced T cells and low background activity. The hNIS/(124)I-iodide reporter system could detect approximately 1 × 10(6) T cells; (124)I-iodide uptake at the T cell injection site was time-dependent and associated with high background. The hdCKDM/2'-(18)F-fluoro-5-ethyl-1-β-d-arabinofuranosyluracil ((18)F-FEAU) and hsvTK/(18)F-FEAU reporter systems detected approximately 3 × 10(5) T cells, respectively. (18)F-FEAU was a more efficient probe (higher uptake, lower background) than (124)I-1-(2-deoxy-2-fluoro-1-d-arabinofuranosyl)-5-iodouracil for both hdCKDM and hsvTK.A comparison of different reporter gene-reporter probe systems for imaging of T cell number was performed, and the hNET/(18)F-MFBG PET reporter system was found to be the most sensitive and capable of detecting approximately 35-40 × 10(3) T cells at the site of T cell injection in the animal model.

  View details for DOI 10.2967/jnumed.115.159855

  View details for PubMedID 26025962

  View details for PubMedCentralID PMC4511596

• Co-targeting of convergent nucleotide biosynthetic pathways for leukemia eradication. The Journal of experimental medicine Nathanson, D. A., Armijo, A. L., Tom, M., Li, Z., Dimitrova, E., Austin, W. R., Nomme, J., Campbell, D. O., Ta, L., Le, T. M., Lee, J. T., Darvish, R., Gordin, A., Wei, L., Liao, H. I., Wilks, M., Martin, C., Sadeghi, S., Murphy, J. M., Boulos, N., Phelps, M. E., Faull, K. F., Herschman, H. R., Jung, M. E., Czernin, J., Lavie, A., Radu, C. G. 2014; 211 (3): 473–86

  Abstract

  Pharmacological targeting of metabolic processes in cancer must overcome redundancy in biosynthetic pathways. Deoxycytidine (dC) triphosphate (dCTP) can be produced both by the de novo pathway (DNP) and by the nucleoside salvage pathway (NSP). However, the role of the NSP in dCTP production and DNA synthesis in cancer cells is currently not well understood. We show that acute lymphoblastic leukemia (ALL) cells avoid lethal replication stress after thymidine (dT)-induced inhibition of DNP dCTP synthesis by switching to NSP-mediated dCTP production. The metabolic switch in dCTP production triggered by DNP inhibition is accompanied by NSP up-regulation and can be prevented using DI-39, a new high-affinity small-molecule inhibitor of the NSP rate-limiting enzyme dC kinase (dCK). Positron emission tomography (PET) imaging was useful for following both the duration and degree of dCK inhibition by DI-39 treatment in vivo, thus providing a companion pharmacodynamic biomarker. Pharmacological co-targeting of the DNP with dT and the NSP with DI-39 was efficacious against ALL models in mice, without detectable host toxicity. These findings advance our understanding of nucleotide metabolism in leukemic cells, and identify dCTP biosynthesis as a potential new therapeutic target for metabolic interventions in ALL and possibly other hematological malignancies.

  View details for DOI 10.1084/jem.20131738

  View details for PubMedID 24567448

  View details for PubMedCentralID PMC3949575

• Development of new deoxycytidine kinase inhibitors and noninvasive in vivo evaluation using positron emission tomography. Journal of medicinal chemistry Murphy, J. M., Armijo, A. L., Nomme, J., Lee, C. H., Smith, Q. A., Li, Z., Campbell, D. O., Liao, H. I., Nathanson, D. A., Austin, W. R., Lee, J. T., Darvish, R., Wei, L., Wang, J., Su, Y., Damoiseaux, R., Sadeghi, S., Phelps, M. E., Herschman, H. R., Czernin, J., Alexandrova, A. N., Jung, M. E., Lavie, A., Radu, C. G. 2013; 56 (17): 6696–6708

  Abstract

  Combined inhibition of ribonucleotide reductase and deoxycytidine kinase (dCK) in multiple cancer cell lines depletes deoxycytidine triphosphate pools leading to DNA replication stress, cell cycle arrest, and apoptosis. Evidence implicating dCK in cancer cell proliferation and survival stimulated our interest in developing small molecule dCK inhibitors. Following a high throughput screen of a diverse chemical library, a structure-activity relationship study was carried out. Positron Emission Tomography (PET) using (18)F-L-1-(2'-deoxy-2'-FluoroArabinofuranosyl) Cytosine ((18)F-L-FAC), a dCK-specific substrate, was used to rapidly rank lead compounds based on their ability to inhibit dCK activity in vivo. Evaluation of a subset of the most potent compounds in cell culture (IC50 = ∼1-12 nM) using the (18)F-L-FAC PET pharmacodynamic assay identified compounds demonstrating superior in vivo efficacy.

  View details for DOI 10.1021/jm400457y

  View details for PubMedID 23947754

  View details for PubMedCentralID PMC3789385

• Structure-guided engineering of human thymidine kinase 2 as a positron emission tomography reporter gene for enhanced phosphorylation of non-natural thymidine analog reporter probe. The Journal of biological chemistry Campbell, D. O., Yaghoubi, S. S., Su, Y., Lee, J. T., Auerbach, M. S., Herschman, H., Satyamurthy, N., Czernin, J., Lavie, A., Radu, C. G. 2012; 287 (1): 446–54

  Abstract

  Positron emission tomography (PET) reporter gene imaging can be used to non-invasively monitor cell-based therapies. Therapeutic cells engineered to express a PET reporter gene (PRG) specifically accumulate a PET reporter probe (PRP) and can be detected by PET imaging. Expanding the utility of this technology requires the development of new non-immunogenic PRGs. Here we describe a new PRG-PRP system that employs, as the PRG, a mutated form of human thymidine kinase 2 (TK2) and 2'-deoxy-2'-18F-5-methyl-1-β-L-arabinofuranosyluracil (L-18F-FMAU) as the PRP. We identified L-18F-FMAU as a candidate PRP and determined its biodistribution in mice and humans. Using structure-guided enzyme engineering, we generated a TK2 double mutant (TK2-N93D/L109F) that efficiently phosphorylates L-18F-FMAU. The N93D/L109F TK2 mutant has lower activity for the endogenous nucleosides thymidine and deoxycytidine than wild type TK2, and its ectopic expression in therapeutic cells is not expected to alter nucleotide metabolism. Imaging studies in mice indicate that the sensitivity of the new human TK2-N93D/L109F PRG is comparable with that of a widely used PRG based on the herpes simplex virus 1 thymidine kinase. These findings suggest that the TK2-N93D/L109F/L-18F-FMAU PRG-PRP system warrants further evaluation in preclinical and clinical applications of cell-based therapies.

  View details for DOI 10.1074/jbc.M111.314666

  View details for PubMedID 22074768

  View details for PubMedCentralID PMC3249097

• Stratification of nucleoside analog chemotherapy using 1-(2'-deoxy-2'-18F-fluoro-β-D-arabinofuranosyl)cytosine and 1-(2'-deoxy-2'-18F-fluoro-β-L-arabinofuranosyl)-5-methylcytosine PET. Journal of nuclear medicine : official publication, Society of Nuclear Medicine Lee, J. T., Campbell, D. O., Satyamurthy, N., Czernin, J., Radu, C. G. 2012; 53 (2): 275–80

  Abstract

  The ability to measure tumor determinants of response to nucleoside analog (NA) chemotherapy agents such as gemcitabine and related compounds could significantly affect the management of several types of cancer. Previously we showed that the accumulation in tumors of the new PET tracer 1-(2'-deoxy-2'-(18)F-fluoro-β-d-arabinofuranosyl)cytosine ((18)F-FAC) is predictive of responses to gemcitabine. (18)F-FAC retention in cells requires deoxycytidine kinase (dCK), a rate-limiting enzyme in the deoxyribonucleoside salvage metabolism and in gemcitabine conversion from an inactive prodrug to a cytotoxic compound. The objectives of the current study were to determine whether (18)F-FAC tumor uptake is also influenced by cytidine deaminase (CDA), a determinant of resistance to gemcitabine; to develop a new PET assay using (18)F-FAC and the related probe 1-(2'-deoxy-2'-(18)F-fluoro-β-l-arabinofuranosyl)-5-methylcytosine (l-(18)F-FMAC) to profile tumor lesions for both dCK and CDA enzymatic activities; and to determine whether this PET assay can identify the most effective NA chemotherapy against tumors with differential expression of dCK and CDA.Isogenic murine leukemic cell lines with defined dCK and CDA activities were generated by retroviral transduction. A cell viability assay was used to determine the sensitivity of the isogenic cell lines to the dCK-dependent NA prodrugs gemcitabine and clofarabine. In vitro enzymatic and cell-based tracer uptake assays and in vivo PET with (18)F-FAC and l-(18)F-FMAC were used to predict tumor responses to gemcitabine and clofarabine.dCK and CDA activities measured by kinase and tracer uptake assays correlated with the sensitivity of isogenic cell lines to gemcitabine and clofarabine. Coexpression of CDA decreased the sensitivity of dCK-positive cells to gemcitabine treatment in vitro by 15-fold but did not affect responses to clofarabine. Coexpression of CDA decreased (18)F-FAC but not l-(18)F-FMAC, phosphorylation, and uptake by dCK-positive cells. (18)F-FAC and l-(18)F-FMAC PET estimates of the enzymatic activities of dCK and CDA in tumor implants in mice were predictive of responses to gemcitabine and clofarabine treatment in vivo.These findings support the utility of PET-based phenotyping of tumor nucleoside metabolism for guiding the selection of NA prodrugs.

  View details for DOI 10.2967/jnumed.111.090407

  View details for PubMedID 22302964

  View details for PubMedCentralID PMC3431619

• Imaging in Immunology Research SMALL ANIMAL IMAGING: BASICS AND PRACTICAL GUIDE Lee, J. T., Nair-Gill, E. D., Rabinovich, B. A., Radu, C. G., Wittie, O. N., Kiessling, F., Pichler, B. J., Hauff, P. 2011: 565–83

  View details for DOI 10.1007/978-3-642-12945-2_36

  View details for Web of Science ID 000289758700036

• Novel PET probes specific for deoxycytidine kinase. Journal of nuclear medicine : official publication, Society of Nuclear Medicine Shu, C. J., Campbell, D. O., Lee, J. T., Tran, A. Q., Wengrod, J. C., Witte, O. N., Phelps, M. E., Satyamurthy, N., Czernin, J., Radu, C. G. 2010; 51 (7): 1092–98

  Abstract

  Deoxycytidine kinase (dCK) is a rate-limiting enzyme in the deoxyribonucleoside salvage pathway and a critical determinant of therapeutic activity for several nucleoside analog prodrugs. We have previously reported the development of 1-(2'-deoxy-2'-(18)F-fluoro-beta-D-arabinofuranosyl)cytosine ((18)F-FAC), a new probe for PET of dCK activity in immune disorders and certain cancers. The objective of the current study was to develop PET probes with improved metabolic stability and specificity for dCK. Toward this goal, several candidate PET probes were synthesized and evaluated in vitro and in vivo.High-pressure liquid chromatography was used to analyze the metabolic stability of (18)F-FAC and several newly synthesized analogs with the natural D-enantiomeric sugar configuration or the corresponding unnatural L-configuration. In vitro kinase and uptake assays were used to determine the affinity of the (18)F-FAC L-nucleoside analogs for dCK. The biodistribution of selected L-analogs in mice was determined by small-animal PET/CT.Candidate PET probes were selected using the following criteria: low susceptibility to deamination, high affinity for purified recombinant dCK, high uptake in dCK-expressing cell lines, and biodistribution in mice reflective of the tissue-expression pattern of dCK. Among the 10 newly developed candidate probes, 1-(2'-deoxy-2'-(18)F-fluoro-beta-L-arabinofuranosyl)cytosine (L-(18)F-FAC) and 1-(2'-deoxy-2'-(18)F-fluoro-beta-L-arabinofuranosyl)-5-methylcytosine (L-(18)F-FMAC) most closely matched the selection criteria. The selection of L-(18)F-FAC and L-(18)F-FMAC was validated by showing that these two PET probes could be used to image animal models of leukemia and autoimmunity.Promising in vitro and in vivo data warrant biodistribution and dosimetry studies of L-(18)F-FAC and L-(18)F-FMAC in humans.

  View details for DOI 10.2967/jnumed.109.073361

  View details for PubMedID 20554721

  View details for PubMedCentralID PMC3119947