Joonyoung Kim

Dynamic visualization of a joint-specific autoimmune response through positron emission tomography

In the K/BxN mouse model of rheumatoid arthritis, the transfer of autoantibodies specific for glucose-6-phosphate isomerase (GPI) into naïve mice rapidly induces joint-specific inflammation similar to that seen in human rheumatoid arthritis. The ubiquitous expression of GPI and the systemic circulation of anti-GPI immunoglobulin G (IgG) seem incongruous with the tissue specificity of this disease. By using PET (positron emission tomography), we show here that purified anti-GPI IgG localizes specifically to distal joints in the front and rear limbs within minutes of intravenous injection, reaches saturation by 20 min and remains localized for at least 24 h. In contrast, control IgG does not localize to joints or cause inflammation. The rapid kinetics of anti-GPI IgG joint localization supports a model in which autoantibodies bind directly to pre-existing extracellular GPI in normal healthy mouse joints.

Activation of Signal Transducer and Activator of Transcription 3 through a Phosphomimetic Serine 727 Promotes Prostate Tumorigenesis Independent of Tyrosine 705 Phosphorylation

Aberrantly activated signal transducer and activator of transcription 3 (Stat3) is implicated in the development of various human cancers. Y705 phosphorylation is conventionally thought to be required for Stat3 signal-dependent activation and seems to play an essential role in some malignancies. Recently, it was shown that Stat3 is activated through novel and noncanonical mechanisms, including phosphorylation at S727. Here, we investigate S727 phosphorylation of Stat3 and its subsequent effects in prostate cancer development, independent of Y705 phosphorylation, using mutated Stat3 in the human prostate cancer cell line LNCaP. We show mutation of S727 to the phosphomimetic residue Glu, and inactivation of Y705 (Y705F/S727E) resulted in a remarkable growth advantage in low-serum, enhanced anchorage-independent growth in soft agar, and increased tumorigenicity in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice, possibly by direct activation of downstream proto-oncogenes c-myc, mcl-1, and survivin. Y705F/S727E mutant cells were more invasive than Y705F/S727A (inactivation of Y705 and S727) mutant cells, and more Y705F/S727E mutant Stat3 was localized in the nuclei relative to Y705F/S727A mutant Stat3 at the steady state. Furthermore, the Y705F/S727E but not the Y705F/S727A mutant induced anchorage-independent growth of noncancerous prostate epithelial cells (RWPE-1). We further show that Stat3 is phosphorylated at S727 in 65% of malignant prostate tissues (n = 20) relative to 25% of normal prostate tissues (n = 4). Moreover, there is a positive correlation between phosphoS727-Stat3 expression and Gleason score in these prostate cancer tissues (P = 0.05). Our data suggest for the first time that S727 phosphorylation is sufficient to activate Stat3, thereby driving prostate tumorigenesis independent of Y705 phosphorylation.

MicroPET assessment of androgenic control of glucose and acetate uptake in the rat prostate and a prostate cancer tumor model

PET has been used to monitor changes in tumor metabolism in breast cancer following hormonal therapy. This study was undertaken to determine whether PET imaging could evaluate early metabolic changes in prostate tumor following androgen ablation therapy. Studies were performed comparing two positron-emitting tracers, 18F-FDG and 11C-acetate, in Sprague-Dawley male rats to monitor metabolic changes in normal prostate tissue. Additional studies were performed in nude mice bearing the CWR22 androgen-dependent human prostate tumor to evaluate metabolic changes in prostate tumor. In rats, for the androgen ablation pretreatment, 1 mg diethylstilbestrol (DES) was injected subcutaneously 3 and 24 hours before tracer injection. For androgen pretreatment, 500 microg dihydrotestosterone (DHT) was injected intraperitoneally 2 and 6 hours before tracer injection. The rats were divided into three groups, Group A (no-DES, no-DHT, n = 18), Group B (DES, no-DHT, n = 18) and Group C (DES, DHT, n = 18). In each group, 10 animals received 18F-FDG, whereas the remaining eight animals were administered 11C-acetate. Rats were sacrificed at 120 min post-injection of 18F-FDG or 30 min post-injection of 11C-acetate. Pretreatment of the mouse model using DHT (200 microg of DHT in 0.1 mL of sunflower seed oil) or DES (200 microg of DES in 0.1 mL of sunflower seed oil) was conducted every 2 days for one week. Mice were imaged with both tracers in the microPET scanner (Concorde Microsystems Inc.). DES treatment caused a decrease in acetate and glucose metabolism in the rat prostate. Co-treatment with DHT maintained the glucose metabolism levels at baseline values. In the tumor bearing mice, similar effects were seen in 18F-FDG study, while there was no significant difference in 11C-acetate uptake. These results indicate that changes in serum testosterone levels influence 18F-FDG uptake in the prostate gland, which is closely tied to glucose metabolism, within 24 hours of treatment and in the prostate tumor within 1 week. These early metabolic changes could enable monitoring metabolic changes in prostate tumor following treatment by imaging using 18F-FDG PET. Further studies are needed to clarify the reason for the insensitivity of 11C-acetate for measuring metabolic change in prostate tumor.

15-Deoxy-Δ12,14-Prostaglandin J2 Inhibits Transcriptional Activity of Estrogen Receptor-α via Covalent Modification of DNA-Binding Domain

The cyclopentenone 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) inhibits proliferation of cancer cells, including breast cancers, by peroxisome proliferator-activated receptor-γ (PPARγ)–dependent and PPARγ-independent mechanisms. However, little is known about its effect on the transcriptional activity of estrogen receptor-α (ERα) that plays vital roles in the growth of breast cancers. Here, we show that 15d-PGJ2 inhibits both 17β-estradiol (E2)–dependent and E2-independent ERα transcriptional activity by PPARγ-independent mechanism. In addition, 15d-PGJ2 directly modifies ERα protein via its reactive cyclopentenone moiety, evidenced by incorporation of biotinylated 15d-PGJ2 into ERα, both in vitro and in vivo . Nanoflow reverse-phase liquid chromatography tandem mass spectrometry analysis identifies two cysteines (Cys227 and Cys240) within the COOH-terminal zinc finger of ERα DNA-binding domain (DBD) as targets for covalent modification by 15d-PGJ2. Gel mobility shift and chromatin immunoprecipitation assays show that 15d-PGJ2 inhibits DNA binding of ERα and subsequent repression of ERα target gene expression, such as pS2 and c-Myc . Therefore, our results suggest that 15d-PGJ2 can block ERα function by covalent modification of cysteine residues within the vulnerable COOH-terminal zinc finger of ERα DBD, resulting in fundamental inhibition of both hormone-dependent and hormone-independent ERα transcriptional activity. [Cancer Res 2007;67(6):2595–602]

Monitoring the effect of mild hyperthermia on tumour hypoxia by Cu-ATSM PET scanning

Purpose: Mild hyperthermia can improve tumour oxygenation and enhance radiosensitivity. Imaging the hypoxic fraction of a tumour can guide hyperthermia treatment planning and facilitate treatment optimization. 64Cu-ATSM (Copper-diacetyl-bis(N4-methylthiosemicarbazone)) is a positron emitting compound that has been demonstrated to have rapid uptake and selective retention in hypoxic cells and has been used for imaging human and animal tumours. The purpose of the present report is to establish methodology that will allow one to use Cu-ATSM PET scanning to detect the impact of hyperthermia on tumour physiology in as little time as possible. Materials and methods: EMT6 tumours (mouse mammary carcinoma) were implanted into the subcutaneous tissue of both thighs of 10 BALB/c mice (one heated, one control tumour per animal). The target thermal dose was 41.5°C × 45 min. Without interrupting heating, 64Cu-ATSM (mean activity 1.8 mCi) was then injected and serial PET scans were obtained. In a sub-group of four animals, a low administered activity (∼0.3 mCi) 64Cu-ATSM scan was also conducted before heating to permit a direct comparison of the effects of hyperthermia on the same tumours. In another sub-group of five animals, a low activity (∼0.3 mCi) 64Cu-PTSM (pyruvaldehyde-bis(N*-methylthiosemicarbazone)) scan was conducted before heating, to confirm a posited correlation between perfusion and early 64Cu-ATSM uptake. Results: This study corrected for perfusion differences by dividing tumour uptake by the average early (first minute) uptake (‘self-normalized uptake’). The 10 heated tumours showed a significantly (p = 0.007) lower self-normalized uptake than control tumours by 2 min. For the four mice with low activity Cu-ATSM scans performed before hyperthermia, the tumours to be heated demonstrated self-normalized uptake consistent with the unheated control tumours and which departed significantly (p ≤ 0.02) from their post-hyperthermia scans by 5 min. Comparisons between scans and needle electrode surveys were performed in an additional four animals with eight tumours. For technical reasons electrode surveys were done after the end of hyperthermia—and, therefore, these animals also had comparison scans taken after hyperthermia. Reduced self-normalized uptake on scans was associated with increased pO2 on electrode surveys. These data also suggested a substantial degradation of the effect on tumour hypoxia by ∼15–45 min after the end of mild hyperthermia. Conclusion: Short imaging times of ∼5 min with modest (∼4–10) numbers of mice can discriminate the effects of mild hyperthermia on tumour physiology. The long-term objective is to use this tool to identify as short and mild a hyperthermia session as possible.

A Potential Dubin-Johnson Syndrome Imaging Agent: Synthesis, Biodistribution, and MicroPET Imaging

Dubin-Johnson syndrome (DJS) is caused by a deficiency of the human canalicular multispecific organic anion transporter (cMOAT). A new lipophilic copper-64 complex of 1,4,7-tris(carboxymethyl)-10-(tetradecyl)-1,4,7,10-tetraazadodecane (5) was prepared and evaluated for potential as a diagnostic tool for DJS. The prepared ligand was labeled with 64Cu citrate in high radiochemical purity. In vivo uptake and clearance of the complex was determined through biodistribution studies using normal Sprague-Dawley rats and mutant cMOAT-deficient (TR−) rats. In normal rats, the radioactive copper complex was cleared quickly from the body exclusively through the hepatic pathway. The 64Cu complex was taken up rapidly by the liver and quickly excreted into the small intestine and then the upper large intestine, whereas < 1% ID/organ was found in the kidney at all time points post injection. Whereas activity was accumulated continuously in the liver of TR− rats, it was not excreted into the small intestine. MicroPET studies of normal and TR rats were consistent with biodistribution data and showed dramatically different images. This study strongly suggests that cMOAT is involved in excretion of 64Cu-5. The significant difference between the biodistribution data and microPET images of the normal and TR− rats demonstrates that this new 64Cu complex may allow noninvasive diagnosis of DJS in humans.

Abstract 871: [18F]BMS-986192 as a novel PET imaging agent for assessment of PD-L1 expressionin vivo

Objectives Inhibition of the Programmed Death Ligand-1 (PD-L1)/PD-1 interaction allows for potent anti-tumor activity and antibodies that disrupt this interaction have been approved for the treatment of multiple cancer types. PD-L1 expression has been investigated clinically as a potential biomarker to predict response to anti-PD-1/PD-L1 therapy. BMS-986192, an Adnectin with high affinity and specificity for human PD-L1, was selected in vitro from a complex library. Here we report the discovery and first preclinical evaluation of [18F]BMS-986192 as a PET imaging agent to detect PD-L1 expression in vivo. Methods [18F]BMS-986192 was radiolabeled via copper-free click chemistry and assessed for its ability to detect PD-L1 expression. Tracer binding to human L2987 (PD-L1+) and HT-29 (PD-L1-) xenografts as well as human non-small cell lung cancer (NSCLC) tissue samples was assessed by autoradiography (ARG). Tracer binding was compared to PD-L1 expression assessed independently with anti-PD-L1 immunohistochemistry (IHC). In vivo performance of the tracer was also assessed by PET imaging in mice bearing bilateral L2987 and HT-29 xenografts, and tracer biodistribution was further assayed in these animals ex vivo by gamma counter. Finally, initial in vivo biodistribution and radiation dosimetry was measured by PET in cynomolgus monkey. Results ARG studies showed increased [18F]BMS-986192 total binding to PD-L1(+) L2987 xenograft compared to PD-L1(-) HT-29 xenograft tissue. Radiotracer binding was higher in all tested human NSCLC tissue samples compared to xenografts. Dose-dependent blockade was seen in all PD-L1(+) tissues co-incubated with cold BMS-986192, and binding was unaffected by co-incubation with cold non-PD-L1 binding control. Visual comparison of tracer binding aligns closely with PD-L1 IHC both spatially as well as in intensity. Preferential accumulation of [18F]BMS-986192 was noted in PD-L1(+) L2987 compared to PD-L1(-) HT-29 xenografts in tumor-bearing mice. PET studies in cynomolgus monkeys confirmed binding to PD-L1(+) tissue (e.g. spleen) with minimal nonspecific background signal exclusive of primary clearance organs. Radiation dosimetry of [18F]BMS-986192 indicates an estimated single administration dose limit of 6.2 mCi for an average human subject. Conclusions ARG, PET studies, and ex vivo measurements in rodent and cynomolgus monkey demonstrated sensitive and specific [18F]BMS-986192 binding to PD-L1. Low background signal in cynomolgus monkey in the context of endogenous PD-L1 expression further supports the potential of this tracer for sensitive detection of PD-L1(+) lesions in vivo. Radiation dosimetry suggests that [18F]BMS-986192 can be safely administered in human trials, with estimated absorbed radiation doses well within safe parameters for human administration. [18F]BMS-986192 has potential as a sensitive PD-L1 imaging agent for same-day imaging in patients. Citation Format: Ralph A. Smith, David Donnelly, Paul E. Morin, Dasa Lipovsek, Jochem Gokemeijer, Daniel Cohen, Joonyoung Kim, Adrienne Pena, Olufemi Adelakun, Xi-Tao Wang, Patrick Chow, Samuel J. Bonacorsi, Wendy Hayes. [18F]BMS-986192 as a novel PET imaging agent for assessment of PD-L1 expression in vivo [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 871. doi:10.1158/1538-7445.AM2017-871