Publications by authors named "NagaVaraKishore Pillarsetty"

49 Publications

Measuring Tumor Epichaperome Expression Using [I] PU-H71 Positron Emission Tomography as a Biomarker of Response for PU-H71 Plus Nab-Paclitaxel in HER2-Negative Metastatic Breast Cancer.

JCO Precis Oncol 2020 17;4. Epub 2020 Nov 17.

Oncology Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY.

Purpose: Epichaperome network maintenance is vital to survival of tumors that express it. PU-H71 is an epichaperome inhibitor that binds to the ATP-binding site of HSP90 and has demonstrated antitumor activity in breast cancer xenograft models and clinical safety in patients. PU-positron emission tomography (PET) is a theragnostic imaging tool that allows visualization of the epichaperome target. In this phase Ib trial, we present safety and tolerability for PU-H71 plus nab-paclitaxel in HER2-negative patients with metastatic breast cancer (MBC) and the utility of PU-PET as a noninvasive predictive biomarker.

Methods: We performed a 3 + 3 dose-escalation study with escalating PU-H71 doses and standard nab-paclitaxel. The primary objective was to establish safety and determine maximum tolerated dose (MTD)/recommended phase 2 dose. Secondary objectives were to assess pharmacokinetics and clinical efficacy. Patients could enroll in a companion PU-PET protocol to measure epichaperome expression before treatment initiation to allow exploratory correlation with treatment benefit.

Results: Of the 12 patients enrolled, dose-limiting toxicity occurred in one patient (G3 neutropenic fever) at dose level 1; MTD of PU-H71 was 300 mg/m plus nab-paclitaxel 260 mg/m administered every 3 weeks. Common toxicities included diarrhea, fatigue, peripheral neuropathy, and nausea. PU-H71 systemic exposure was not altered by nab-paclitaxel administration. Two of 12 patients had partial response (overall response rate, 17%) and the clinical benefit rate was 42% (5 of 12). Time to progression was associated with baseline epichaperome positivity and PU-H71 peak standard uptake value (SUV), with more durable disease control observed with high epichaperome levels.

Conclusion: The combination of PU-H71 and nab-paclitaxel was well tolerated, with evidence of clinical activity. More durable disease control without progression was observed in patients with high baseline epichaperome expression. A phase II trial of this combination with PU-PET as a companion diagnostic for patient selection is currently planned.
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http://dx.doi.org/10.1200/PO.20.00273DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7713524PMC
November 2020

The Unique Pharmacometrics of Small Molecule Therapeutic Drug Tracer Imaging for Clinical Oncology.

Cancers (Basel) 2020 Sep 22;12(9). Epub 2020 Sep 22.

Department of Radiology, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA.

Translational development of radiolabeled analogues or isotopologues of small molecule therapeutic drugs as clinical imaging biomarkers for optimizing patient outcomes in targeted cancer therapy aims to address an urgent and recurring clinical need in therapeutic cancer drug development: drug- and target-specific biomarker assays that can optimize patient selection, dosing strategy, and response assessment. Imaging the in vivo tumor pharmacokinetics and biomolecular pharmacodynamics of small molecule cancer drugs offers patient- and tumor-specific data which are not available from other pharmacometric modalities. This review article examines clinical research with a growing pharmacopoeia of investigational small molecule cancer drug tracers.
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http://dx.doi.org/10.3390/cancers12092712DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7563483PMC
September 2020

Oncology-Inspired Treatment Options for COVID-19.

J Nucl Med 2020 12 17;61(12):1720-1723. Epub 2020 Jul 17.

Department of Radiology, Memorial Sloan Kettering Cancer Center, New York, New York

CR3022 is a human antibody that binds to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we explore the use of CR3022 as a molecularly targeted radiotherapeutic. CR3022 was labeled with I and purified, yielding I-CR3022. Using a magnetic bead assay and a recombinant SARS-CoV-2 spike protein fragment, we tested binding of I-CR3022 in the presence and absence of CR3022. We conjugated the antibody CR3022 with a purity of more than 98% and a specific activity of more than 292 MBq/mg. Using a bead-based assay, we confirmed that binding of I-CR3022 is selective and is significantly reduced in the presence of unlabeled antibody (3.14% ± 0.14% specific uptake and 0.10% ± 0.01% specific uptake, respectively; < 0.0001). Our results confirm the potential of CR3022 as a molecularly targeted probe for SARS-CoV-2. A labeled version of CR3022 could potentially be used for Auger radiotherapy or noninvasive imaging.
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http://dx.doi.org/10.2967/jnumed.120.249748DOI Listing
December 2020

Predicting Gemcitabine Delivery by F-FAC PET in Murine Models of Pancreatic Cancer.

J Nucl Med 2021 02 9;62(2):195-200. Epub 2020 Jul 9.

Department of Medical Physics, Memorial Sloan Kettering Cancer Center, New York, New York

F-FAC (2'-deoxy-2'-F-fluoro-β-d-arabinofuranosylcytosine) has close structural similarity to gemcitabine and thus offers the potential to image drug delivery to tumors. We compared tumor F-FAC PET images with C-gemcitabine levels, established ex vivo, in 3 mouse models of pancreatic cancer. We further modified tumor gemcitabine levels with injectable PEGylated recombinant human hyaluronidase (PEGPH20) to test whether changes in gemcitabine would be tracked by F-FAC. F-FAC was synthesized as described previously. Three patient-derived xenograft (PDX) models were grown in the flanks of NSG mice. Mice were given PEGPH20 or vehicle intravenously 24 h before coinjection of F-FAC and C-gemcitabine. Animals were euthanized and imaged 1 h after tracer administration. Tumor and muscle uptake of both F-FAC and C-gemcitabine was obtained ex vivo. The efficacy of PEPGPH20 was validated through staining with hyaluronic acid binding protein. Additionally, an organoid culture, initiated from a KPC (Pdx-1 Cre LSL-Kras LSL-p53) tumor, was used to generate orthotopically growing tumors in C57BL/6J mice, and these tumors were then serially transplanted. Animals were injected with PEGPH20 and C-gemcitabine as described above to validate increased drug uptake by ex vivo assay. PET/MR images were obtained using a PET insert on a 7-T MR scanner. Animals were imaged immediately before injection with PEGPH20 and again 24 h later. Tumor-to-muscle ratios of C-gemcitabine and F-FAC correlated well across all PDX models and treatments ( = 0.78). There was a significant increase in the tumor PET signal in PEGPH20-treated PDX animals, and this signal was matched in ex vivo counts for 2 of 3 models. In KPC-derived tumors, PEGPH20 raised C-gemcitabine levels (tumor-to-muscle ratio of 1.9 vs. 2.4, control vs. treated, = 0.013). PET/MR F-FAC images showed a 12% increase in tumor F-FAC uptake after PEGPH20 treatment ( = 0.023). PEGPH20-treated animals uniformly displayed clear reductions in hyaluronic acid staining. F-FAC PET was shown to be a good surrogate for gemcitabine uptake and, when combined with MR, to successfully determine drug uptake in tumors growing in the pancreas. PEGPH20 had moderate effects on tumor uptake of gemcitabine.
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http://dx.doi.org/10.2967/jnumed.120.246926DOI Listing
February 2021

Improved radiosynthesis of I-MAPi, an auger theranostic agent.

Int J Radiat Biol 2020 Jul 2:1-7. Epub 2020 Jul 2.

Department of Radiology, Memorial Sloan Kettering Cancer Center, New York, NY, USA.

I-MAPi, a novel PARP1-targeted Auger radiotherapeutic has shown promising results in pre-clinical glioma model. Currently, I-MAPi is synthesized using multistep synthesis that results in modest yields and low molar activities (MA) that limits the ability to translate this technology for human studies where high doses are administered. Therefore, new methods are needed to synthesize I-MAPi in high activity yields (AY) and improved MA to facilitate clinical translation and multicenter trials.I-MAPi was prepared in a single step via I-iododetannylation of the corresponding tributylstannane precursor. In vitro internalization assay, subcellular fractionation and confocal microscopy where used to evaluate the performance of I-MAPi in a small cell lung cancer model.I-MAPi was synthesized in a single step from the corresponding stannane precursor in AY of 45 ± 2% and MA of 11.8 ± 4.8 GBq mol. In vitro in LX22 cells showed rapid internalization (5 min) with accumulation found predominantly in the membrane, nucleus and chromatin of the cell as determined by subcellular fractionation. Here, we have developed an improved radiosynthesis of I-MAPi, an Auger theranostic agent. This process was achieved using a single step, I-iododestannylation reaction from the corresponding stannane precursor in good AY and MA. I-MAPi was evaluated in vitro in a small cell lung cancer model with high PARP expression, rapid internalization and high nuclear uptake shown.
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http://dx.doi.org/10.1080/09553002.2020.1781283DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7775866PMC
July 2020

Synthesis of the PET Tracer I-Trametinib for MAPK/ERK Kinase Distribution and Resistance Monitoring.

J Nucl Med 2020 12 22;61(12):1845-1850. Epub 2020 May 22.

Department of Pharmacology, Weill Cornell Graduate School, New York, New York

Trametinib is an extremely potent allosteric inhibitor of mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated kinase (ERK) (MEK) 1/2, which has been approved for treatment of metastatic melanoma and anaplastic thyroid cancer in patients with confirmed BRAF/K mutations. Though trametinib is highly efficacious, adverse side effects, including skin, gastrointestinal, and hepatic toxicity, are dose-limiting and can lead to treatment termination. Development of a noninvasive tool to visualize and quantify the delivery and distribution of trametinib (either as a single agent or in combination with other therapeutics) to tumors and organs would be helpful in assessing therapeutic index, personalizing individual dose, and potentially predicting resistance to therapy. To address these issues, we have developed a radiolabeled trametinib and evaluated the in vitro and in vivo properties. I-, I-, and I-trametinib, pure tracer analogs to trametinib, were synthesized in more than 95% purity, with an average yield of 69.7% and more than 100 GBq/μmol specific activity. Overall, I-trametinib uptake in a panel of cancer cell lines can be blocked with cold trametinib, confirming specificity of the radiotracer in vitro and in vivo. I-trametinib was taken up at higher rates in KRAS and BRAF mutant cell lines than in wild-type KRAS cancer cell lines. In vivo, biodistribution revealed high uptake in the liver 2 h after injection, followed by clearance through the gastrointestinal tract over 4 d. Importantly, uptake higher than expected was observed in the lung and heart for up to 24 h. Peak uptake in the skin and gastrointestinal tract was observed between 6 and 24 h, whereas in B16F10 melanoma-bearing mice peak tumor concentrations were achieved between 24 and 48 h. Tumor uptake relative to muscle and skin was relatively low, peaking at 3.4- to 8.1-fold by 72 h, respectively. The biodistribution of I-trametinib was significantly reduced in mice on trametinib therapy, providing a quantitative method to observe MEK inhibition in vivo. I-trametinib serves as an in vivo tool to personalize the dose instead of using the current single-fixed-dose scheme and, when combined with radiomic data, to monitor the emergence of therapy resistance. In addition, the production of iodinated trametinib affords researchers the ability to measure drug distribution for improved drug delivery studies.
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http://dx.doi.org/10.2967/jnumed.120.241901DOI Listing
December 2020

A one-pot radiosynthesis of [ F]PARPi.

J Labelled Comp Radiopharm 2020 07 16;63(9):419-425. Epub 2020 Jun 16.

Department of Radiology, Memorial Sloan Kettering Cancer Center, New York, New York, USA.

In this paper, we disclose a new strategy for the radiosynthesis of [ F]PARPi from the corresponding, boc-protected, nitro-precursor. Using a two-step procedure, [ F]PARPi could be isolated in radiochemical yields up to 9.6%. The reaction proceeds via an efficient one-pot, two-step process, allowing for simplification over previous methods that require complex multi-step, multi-pot strategies to be implemented.
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http://dx.doi.org/10.1002/jlcr.3847DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7551923PMC
July 2020

First-in-Human Trial of Epichaperome-Targeted PET in Patients with Cancer.

Clin Cancer Res 2020 10 4;26(19):5178-5187. Epub 2020 May 4.

Department of Radiology, Memorial Sloan Kettering Cancer Center, New York, New York.

Purpose: I-PU-H71 is an investigational first-in-class radiologic agent specific for imaging tumor epichaperome formations. The intracellular epichaperome forms under cellular stress and is a clinically validated oncotherapeutic target. We conducted a first-in-human study of microdose I-PU-H71 for PET to study biodistribution, pharmacokinetics, metabolism, and safety; and the feasibility of epichaperome-targeted tumor imaging.

Experimental Design: Adult patients with cancer ( = 30) received I-PU-H71 tracer (201±12 MBq, <25 μg) intravenous bolus followed by PET/CT scans and blood radioassays.

Results: I-PU-H71 PET detected tumors of different cancer types (breast, lymphoma, neuroblastoma, genitourinary, gynecologic, sarcoma, and pancreas). I-PU-H71 was retained by tumors for several days while it cleared rapidly from bones, healthy soft tissues, and blood. Radiation dosimetry is favorable and patients suffered no adverse effects.

Conclusions: Our first-in-human results demonstrate the safety and feasibility of noninvasive detection of tumor epichaperomes using I-PU-H71 PET, supporting clinical development of PU-H71 and other epichaperome-targeted therapeutics.
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http://dx.doi.org/10.1158/1078-0432.CCR-19-3704DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7541604PMC
October 2020

Comparing the intra-tumoral distribution of Gemcitabine, 5-Fluorouracil, and Capecitabine in a murine model of pancreatic ductal adenocarcinoma.

PLoS One 2020 16;15(4):e0231745. Epub 2020 Apr 16.

Department of Medical Physics, Memorial Sloan Kettering Cancer Center, New York, NY, United States of America.

Purpose: To develop a technique to compare the intra-tumoral distribution of the drug gemcitabine, its surrogate [18F]-fluoroarabinocytosine ([18F]-FAC) and related chemotherapeutics 5-FU and capecitabine in a pre-clinical model of pancreatic ductal adenocarcinoma (PDAC).

Experimental Design: Using a KPC-organoid derived model of PDAC, we obtained autoradiographic images of the tumor distribution of, [14C]-gemcitabine, [14C]-5-FU, [3H]-capecitabine. These were compared indirectly by co-administering [18F]-FAC, a close analog of gemcitabine with a proven equivalent intra-tumor distribution. The short half-life of 18F allows for clean separation of 3H/14C labeled drugs in specimens by dual isotope digital autoradiography. Autoradiographic images of [14C]-gemcitabine, [3H]-capecitabine and [14C]-5-FU were each correlated to [18F]-FAC on a pixel-by-pixel basis. The tumor drug penetration was compared using cumulative histograms.

Results: Gemcitabine distribution correlated strongly with FAC as expected. 5-FU also gave a similar microdistribution to that of FAC, whereas no correlation was found between capecitabine or its metabolic products and FAC distribution. Accumulation of Gemcitabine and 5-FU was lower in hypoxic regions of the tumor, whereas no such correlation was observed for capecitabine and its metabolites.

Conclusions: Gemcitabine and 5-FU target the same regions of the tumor, leaving hypoxic cells untreated. Capecitabine metabolites penetrate further into the tumor but it is yet to be determined whether these metabolites are the active form of the drug.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0231745PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7162455PMC
August 2020

The epichaperome is a mediator of toxic hippocampal stress and leads to protein connectivity-based dysfunction.

Nat Commun 2020 01 16;11(1):319. Epub 2020 Jan 16.

Department of Radiology, Memorial Sloan Kettering Cancer Center, New York, NY, 10065, USA.

Optimal functioning of neuronal networks is critical to the complex cognitive processes of memory and executive function that deteriorate in Alzheimer's disease (AD). Here we use cellular and animal models as well as human biospecimens to show that AD-related stressors mediate global disturbances in dynamic intra- and inter-neuronal networks through pathologic rewiring of the chaperome system into epichaperomes. These structures provide the backbone upon which proteome-wide connectivity, and in turn, protein networks become disturbed and ultimately dysfunctional. We introduce the term protein connectivity-based dysfunction (PCBD) to define this mechanism. Among most sensitive to PCBD are pathways with key roles in synaptic plasticity. We show at cellular and target organ levels that network connectivity and functional imbalances revert to normal levels upon epichaperome inhibition. In conclusion, we provide proof-of-principle to propose AD is a PCBDopathy, a disease of proteome-wide connectivity defects mediated by maladaptive epichaperomes.
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http://dx.doi.org/10.1038/s41467-019-14082-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6965647PMC
January 2020

Paradigms for Precision Medicine in Epichaperome Cancer Therapy.

Cancer Cell 2019 11 24;36(5):559-573.e7. Epub 2019 Oct 24.

Department of Radiology, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA; Program in Molecular Pharmacology, Sloan Kettering Institute, New York, NY 10065, USA.

Alterations in protein-protein interaction networks are at the core of malignant transformation but have yet to be translated into appropriate diagnostic tools. We make use of the kinetic selectivity properties of an imaging probe to visualize and measure the epichaperome, a pathologic protein-protein interaction network. We are able to assay and image epichaperome networks in cancer and their engagement by inhibitor in patients' tumors at single-lesion resolution in real time, and demonstrate that quantitative evaluation at the level of individual tumors can be used to optimize dose and schedule selection. We thus provide preclinical and clinical evidence in the use of this theranostic platform for precision medicine targeting of the aberrant properties of protein networks.
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http://dx.doi.org/10.1016/j.ccell.2019.09.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6996250PMC
November 2019

A rapid bead-based radioligand binding assay for the determination of target-binding fraction and quality control of radiopharmaceuticals.

Nucl Med Biol 2019 04 3;71:32-38. Epub 2019 May 3.

Department of Radiology, Memorial Sloan Kettering Cancer Center, NY, USA; Radiochemistry and Molecular Imaging Probes Core, Memorial Sloan Kettering Cancer Center, New York, NY, USA; Department of Pharmacology, Weill Cornell Medical College, New York, NY, USA; Molecular Pharmacology Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA; Department of Radiology, Weill Cornell Medical College, New York, NY, USA. Electronic address:

Introduction: Determination of the target-binding fraction (TBF) of radiopharmaceuticals using cell-based assays is prone to inconsistencies arising from several intrinsic and extrinsic factors. Here, we report a cell-free quantitative method of analysis to determine the TBF of radioligands.

Methods: Magnetic beads functionalized with Ni-NTA or streptavidin were incubated with 1 μg of histidine-tagged or biotinylated antigen of choice for 15 min, followed by incubating 1 ng of the radioligand for 30 min. The beads, supernatant and wash fractions were measured for radioactivity on a gamma counter. The TBF was determined by quantifying the percentage of activity associated with the magnetic beads.

Results: The described method works robustly with a variety of radioisotopes and class of molecules used as radioligands. The entire assay can be completed within 2 h.

Conclusion: The described method yields results in a rapid and reliable manner whilst improving and extending the scope of previously described bead-based radioimmunoassays.

Advances In Knowledge: Using a bead-based radioligand binding assay overcomes the limitations of traditional cell-based assays. The described method is applicable to antibody as well as non-antibody based radioligands and is independent of the effect of target antigen density on cells, the choice of radioisotope used for synthesis of the radioligand and the temperature at which the assay is performed.

Implications For Patient Care: The bead-based radioligand binding assay is significantly easier to perform and is ideally suited for adoption by the radiopharmacy as a quality control method of analysis to fulfill the criteria for release of radiopharmaceuticals in the clinic. The use of this assay is likely to ensure a more reliable validation of radiopharmaceutical quality and result in fewer failed doses, which could ultimately translate to an efficient release of radiopharmaceuticals for administration to patients in the clinic.
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http://dx.doi.org/10.1016/j.nucmedbio.2019.04.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6599726PMC
April 2019

In Vitro and In Vivo Comparison of Gemcitabine and the Gemcitabine Analog 1-(2'-deoxy-2'-fluoroarabinofuranosyl) Cytosine (FAC) in Human Orthotopic and Genetically Modified Mouse Pancreatic Cancer Models.

Mol Imaging Biol 2017 12;19(6):885-892

Department of Medical Physics, Memorial Sloan Kettering Cancer Center, New York, NY, USA.

Purpose: Although gemcitabine is a mainstay of pancreatic cancer therapy, it is only moderately effective, and it would be desirable to measure drug uptake in patients. 1-(2'-deoxy-2'-fluoroarabinofuranosyl) cytosine (FAC), is an analog of gemcitabine, and when labeled with F-18, it may be a potential surrogate PET tracer for the drug.

Procedures: [F]FAC was synthesized to a radiochemical purity of >96 %. The human tumor lines AsPC1, BxPC3, Capan-1, Panc1, and MiaPaca2 were grown orthotopically in nude mice. KPC mice that conditionally express oncogenic K-ras and p53 mutations in pancreatic tissue were also used. The intra-tumoral distributions of [C]gemcitabine and [F]FAC were mapped with autoradiography. The inter-tumor correlation between [C]gemcitabine and [F]FAC was established in the orthotopic tumors. Expression of the equilibrative and concentrative nucleoside transporters (ENT, CNT) in vitro was detected by western blotting. Drug uptake was characterized in vitro using [H]gemcitabine and the effect of transporter inhibition on gemcitabine and FAC uptake was investigated. The relative affinity of cells for gemcitabine and FAC was tested in competition assays. The cell lines differed in sensitivity to transport inhibitors and in competition studies. There was a good in vivo correlation between the total uptake of [F]FAC and [C]gemcitabine, measured across all orthotopic tumors. Using the KPC and BxPC3 models, we found that [C]gemcitabine and [F]FAC were largely co-localized.

Conclusions: In the lines examined here, [F]FAC uptake correlates well with gemcitabine in vivo, supporting the notion that [F]FAC can serve as a PET radiotracer surrogate to determine the uptake and distribution of gemcitabine within pancreatic tumors.
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http://dx.doi.org/10.1007/s11307-017-1078-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5696795PMC
December 2017

Sustained ERK inhibition maximizes responses of BrafV600E thyroid cancers to radioiodine.

J Clin Invest 2016 11 26;126(11):4119-4124. Epub 2016 Sep 26.

Radioiodide (RAI) therapy of thyroid cancer exploits the relatively selective ability of thyroid cells to transport and accumulate iodide. Iodide uptake requires expression of critical genes that are involved in various steps of thyroid hormone biosynthesis. ERK signaling, which is markedly increased in thyroid cancer cells driven by oncogenic BRAF, represses the genetic program that enables iodide transport. Here, we determined that a critical threshold for inhibition of MAPK signaling is required to optimally restore expression of thyroid differentiation genes in thyroid cells and in mice with BrafV600E-induced thyroid cancer. Although the MEK inhibitor selumetinib transiently inhibited ERK signaling, which subsequently rebounded, the MEK inhibitor CKI suppressed ERK signaling in a sustained manner by preventing RAF reactivation. A small increase in ERK inhibition markedly increased the expression of thyroid differentiation genes, increased iodide accumulation in cancer cells, and thereby improved responses to RAI therapy. Only a short exposure to the drug was necessary to obtain a maximal response to RAI. These data suggest that potent inhibition of ERK signaling is required to adequately induce iodide uptake and indicate that this is a promising strategy for the treatment of BRAF-mutant thyroid cancer.
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http://dx.doi.org/10.1172/JCI89067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5096947PMC
November 2016

The epichaperome is an integrated chaperome network that facilitates tumour survival.

Nature 2016 Oct 5;538(7625):397-401. Epub 2016 Oct 5.

Department of Epidemiology-Biostatistics, Memorial Sloan Kettering Cancer Center, New York, New York 10065, USA.

Transient, multi-protein complexes are important facilitators of cellular functions. This includes the chaperome, an abundant protein family comprising chaperones, co-chaperones, adaptors, and folding enzymes-dynamic complexes of which regulate cellular homeostasis together with the protein degradation machinery. Numerous studies have addressed the role of chaperome members in isolation, yet little is known about their relationships regarding how they interact and function together in malignancy. As function is probably highly dependent on endogenous conditions found in native tumours, chaperomes have resisted investigation, mainly due to the limitations of methods needed to disrupt or engineer the cellular environment to facilitate analysis. Such limitations have led to a bottleneck in our understanding of chaperome-related disease biology and in the development of chaperome-targeted cancer treatment. Here we examined the chaperome complexes in a large set of tumour specimens. The methods used maintained the endogenous native state of tumours and we exploited this to investigate the molecular characteristics and composition of the chaperome in cancer, the molecular factors that drive chaperome networks to crosstalk in tumours, the distinguishing factors of the chaperome in tumours sensitive to pharmacologic inhibition, and the characteristics of tumours that may benefit from chaperome therapy. We find that under conditions of stress, such as malignant transformation fuelled by MYC, the chaperome becomes biochemically 'rewired' to form a network of stable, survival-facilitating, high-molecular-weight complexes. The chaperones heat shock protein 90 (HSP90) and heat shock cognate protein 70 (HSC70) are nucleating sites for these physically and functionally integrated complexes. The results indicate that these tightly integrated chaperome units, here termed the epichaperome, can function as a network to enhance cellular survival, irrespective of tissue of origin or genetic background. The epichaperome, present in over half of all cancers tested, has implications for diagnostics and also provides potential vulnerability as a target for drug intervention.
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http://dx.doi.org/10.1038/nature19807DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5283383PMC
October 2016

Radioiodinated PARP1 tracers for glioblastoma imaging.

EJNMMI Res 2015 Dec 4;5(1):123. Epub 2015 Sep 4.

Department of Radiology, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY, 10065, USA.

Background: Although the understanding of the genetic and molecular basis of cancer has advanced significantly over the past several decades, imaging and treatment options for glioblastoma patients have been more limited (N Engl J Med 359:492-507, 2008). This is in part due to difficulties in diagnosing this disease early, combined with its diffuse, infiltrative growth. This study was aimed at the development of a novel diagnostic tool for glioblastoma through the synthesis of a small molecule based on radioiodinated poly(ADP-ribose)polymerase 1 (PARP1) targeted tracers. This PARP1 is a biomarker that is overexpressed in glioblastoma tissue, but has only low expression levels in the healthy brain (Neoplasia 16:432-40, 2014).

Methods: A library of PARP1 inhibitors (iodo-PARPis) was synthesized. Based on their pharmacokinetic properties and nuclear PARP1 binding, the most successful inhibitor was radiolabeled with (131)I and (124)I. Biodistribution as well as imaging experiments were performed in orthotopic and subcutaneous mouse models of glioblastoma.

Results: One member of our iodo-poly(ADP-ribose)polymerase 1 (PARP1) inhibitor library, I2-PARPi, shows promising biophysical properties for in vivo application. All synthesized tracers have IC50 values in the nanomolar range (9 ± 2-107 ± 4 nM) and were able to inhibit the uptake of a fluorescent PARP1 inhibitor analog (PARPi-FL). I2-PARPi was able to reduce the uptake of PARPi-FL by 78 ± 4 % in vivo. In mouse models of glioblastoma, we show that the radioiodinated inhibitor analog has high uptake in tumor tissue (U251 MG xenograft, tumor, 0.43 ± 0.06 %ID/g; brain, 0.01 ± 0.00 %ID/g; muscle, 0.03 ± 0.01 %ID/g; liver, 2.35 ± 0.57 %ID/g; thyroid, 0.24 ± 0.06 %ID/g). PET and SPECT imaging performed in orthotopic glioblastoma models with [(124)I]- and [(131)I]-I2-PARPi showed selective accumulation in the tumor tissue. These results were also verified using autoradiography of tumor sections, which displayed focal selective uptake of the tracer in the tumor regions as confirmed by histology. The uptake could be blocked through pre-injection of excess unlabeled PARP1 inhibitor (Olaparib).

Conclusions: We have successfully synthesized and radioiodinated the PARP1 selective tracer I2-PARPi. The novel tracer shows selective binding to tumor tissue, both in vitro and in models of glioblastoma, and has the potential to serve as a selective PET imaging agent for brain tumors.
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http://dx.doi.org/10.1186/s13550-015-0123-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4559561PMC
December 2015

Synthesis and evaluation of (18)F-labeled ATP competitive inhibitors of topoisomerase II as probes for imaging topoisomerase II expression.

Eur J Med Chem 2014 Oct 8;86:769-81. Epub 2014 Sep 8.

Department of Radiology and the Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA. Electronic address:

Type II topoisomerase (Topo-II) is an ATP-dependent enzyme that is essential in the transcription, replication, and chromosome segregation processes and, as such, represents an attractive target for cancer therapy. Numerous studies indicate that the response to treatment with Topo-II inhibitors is highly dependent on both the levels and the activity of the enzyme. Consequently, a non-invasive assay to measure tumoral Topo-II levels has the potential to differentiate responders from non-responders. With the ultimate goal of developing a radiofluorinated tracer for positron emission tomography (PET) imaging, we have designed, synthesized, and evaluated a set of fluorinated compounds based on the structure of the ATP-competitive Topo-II inhibitor QAP1. Compounds 18 and 19b showed inhibition of Topo-II in in vitro assays and exhibited moderate, Topo-II level dependent cytotoxicity in SK-BR-3 and MCF-7 cell lines. Based on these results, (18)F-labeled analogs of these two compounds were synthesized and evaluated as PET probes for imaging Topo-II overexpression in mice bearing SK-BR-3 xenografts. [(18)F]-18 and [(18)F]-19b were synthesized from their corresponding protected tosylated derivatives by fluorination and subsequent deprotection. Small animal PET imaging studies indicated that both compounds do not accumulate in tumors and exhibit poor pharmacokinetics, clearing from the blood pool very rapidly and getting metabolized over. The insights gained from the current study will surely aid in the design and construction of future generations of PET agents for the non-invasive delineation of Topo-II expression.
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http://dx.doi.org/10.1016/j.ejmech.2014.09.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4406421PMC
October 2014

Development of a minimal saponin vaccine adjuvant based on QS-21.

Nat Chem 2014 Jul 1;6(7):635-43. Epub 2014 Jun 1.

1] Molecular Pharmacology & Chemistry Program, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York 10065, USA [2] Pharmacology Program, Weill Cornell Graduate School of Medical Sciences, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York 10065, USA [3] Tri-Institutional Research Program, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York 10065, USA [4].

Adjuvants are materials added to vaccines to enhance the immunological response to an antigen. QS-21 is a natural product adjuvant under investigation in numerous vaccine clinical trials, but its use is constrained by scarcity, toxicity, instability and an enigmatic molecular mechanism of action. Herein we describe the development of a minimal QS-21 analogue that decouples adjuvant activity from toxicity and provides a powerful platform for mechanistic investigations. We found that the entire branched trisaccharide domain of QS-21 is dispensable for adjuvant activity and that the C4-aldehyde substituent, previously proposed to bind covalently to an unknown cellular target, is also not required. Biodistribution studies revealed that active adjuvants were retained preferentially at the injection site and the nearest draining lymph nodes compared with the attenuated variants. Overall, these studies have yielded critical insights into saponin structure-function relationships, provided practical synthetic access to non-toxic adjuvants, and established a platform for detailed mechanistic studies.
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http://dx.doi.org/10.1038/nchem.1963DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4215704PMC
July 2014

Building Blocks for the Construction of Bioorthogonally Reactive Peptides via Solid-Phase Peptide Synthesis.

ChemistryOpen 2014 Apr 6;3(2):48-53. Epub 2014 Apr 6.

Radiochemistry and Imaging Sciences Service, Department of Radiology, Memorial Sloan-Kettering Cancer Center 1275 York Avenue, New York, NY 10065 (USA).

The need for post-synthetic modifications and reactive prosthetic groups has long been a limiting factor in the synthesis and study of peptidic and peptidomimetic imaging agents. In this regard, the application of biologically and chemically orthogonal reactions to the design and development of novel radiotracers has the potential to have far-reaching implications in both the laboratory and the clinic. Herein, we report the synthesis and development of a series of modular and versatile building blocks for inverse electron-demand Diels-Alder copper-free click chemistry: tetrazine-functionalized artificial amino acids. Following the development of a novel peptide coupling protocol for peptide synthesis in the presence of tetrazines, we successfully demonstrated its effectiveness and applicability. This versatile methodology has the potential to have a transformational impact, opening the door for the rapid, facile, and modular synthesis of bioorthogonally reactive peptide probes.
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http://dx.doi.org/10.1002/open.201402000DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4000166PMC
April 2014

Increased KIT inhibition enhances therapeutic efficacy in gastrointestinal stromal tumor.

Clin Cancer Res 2014 May 28;20(9):2350-62. Epub 2014 Feb 28.

Authors' Affiliations: Departments of Surgery, Radiology, Developmental Biology, and Pathology, Memorial Sloan-Kettering Cancer Center, New York, New York.

Purpose: Gastrointestinal stromal tumor (GIST) is the most common human sarcoma and a model of targeted molecular therapy. GIST depends on oncogenic KIT signaling and responds to the tyrosine kinase inhibitor imatinib. However, imatinib is rarely curative. We hypothesized that PLX3397, which inhibits KIT and colony-stimulating-factor-1 receptor (CSF1R), would be more efficacious than imatinib in GIST by also depleting tumor-associated macrophages, which are generally thought to support tumor growth.

Experimental Design: We treated Kit(V558del/+) mice that develop GIST or mice with subcutaneous human GIST xenografts with imatinib or PLX3397 and analyzed tumor weight, cellular composition, histology, molecular signaling, and fibrosis. In vitro assays on human GIST cell lines were also performed.

Results: PLX3397 was more effective than imatinib in reducing tumor weight and cellularity in both Kit(V558del)(/+) murine GIST and human GIST xenografts. The superiority of PLX3397 did not depend on depletion of tumor-associated macrophages, because adding CSF1R inhibition did not improve the effects of imatinib. Instead, PLX3397 was a more potent KIT inhibitor than imatinib in vitro. PLX3397 therapy also induced substantial intratumoral fibrosis, which impaired the subsequent delivery of small molecules.

Conclusions: PLX3397 therapy has greater efficacy than imatinib in preclinical GIST models and warrants study in patients with GIST. The resultant intratumoral fibrosis may represent one of the barriers to achieving complete tumor eradication.
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http://dx.doi.org/10.1158/1078-0432.CCR-13-3033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4008656PMC
May 2014

Influence of free fatty acids on glucose uptake in prostate cancer cells.

Nucl Med Biol 2014 Mar 17;41(3):254-8. Epub 2013 Dec 17.

Radiochemistry & Imaging Sciences Services, Department of Radiology, Memorial Sloan-Kettering Cancer Center, New York, NY, USA.

Introduction: The study focuses on the interaction between glucose and free fatty acids (FFA) in malignant human prostate cancer cell lines by an in vitro observation of uptake of fluoro-2-deoxy-D-glucose (FDG) and acetate.

Methods: Human prostate cancer cell lines (PC3, CWR22Rv1, LNCaP, and DU145) were incubated for 2 h and 24 h in glucose-containing (5.5 mM) Dulbecco's Modified Eagle's Medium (DMEM) with varying concentrations of the free fatty acid palmitate (0-1.0 mM). Then the cells were incubated with [(18)F]-FDG (1 μCi/mL; 0.037 MBq/mL) in DMEM either in presence or absence of glucose and in presence of varying concentrations of palmitate for 1 h. Standardized procedures regarding cell counting and measuring for (18)F radioactivity were applied. Cell uptake studies with (14)C-1-acetate under the same conditions were performed on PC3 cells.

Results: In glucose containing media there was significantly increased FDG uptake after 24 h incubation in all cell lines, except DU145, when upper physiological levels of palmitate were added. A 4-fold increase of FDG uptake in PC3 cells (15.11% vs. 3.94%/10(6) cells) was observed in media with 1.0 mM palmitate compared to media with no palmitate. The same tendency was observed in PC3 and CWR22Rv1 cells after 2 h incubation. In glucose-free media no significant differences in FDG uptake after 24 h incubation were observed. The significant differences after 2 h incubation all pointed in the direction of increased FDG uptake when palmitate was added. Acetate uptake in PC3 cells was significantly lower when palmitate was added in glucose-free DMEM. No clear tendency when comparing FDG or acetate uptake in the same media at different time points of incubation was observed.

Conclusions: Our results indicate a FFA dependent metabolic boost/switch of glucose uptake in PCa, with patterns reflecting the true heterogeneity of the disease.
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http://dx.doi.org/10.1016/j.nucmedbio.2013.12.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4959437PMC
March 2014

(18)F-labeled-bioorthogonal liposomes for in vivo targeting.

Bioconjug Chem 2013 Nov 7;24(11):1784-9. Epub 2013 Nov 7.

Department of Radiology, ‡Department of Medical Physics, §Molecular Imaging and Therapy Service, Department of Radiology, and ∥Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center , New York, New York, 10065, United States.

Liposomes are attractive vehicles for the controlled release of drugs and cytotoxins and have a long-standing history in medical research and clinical practice. In addition to established therapeutic indications, liposomes have several favorable properties for molecular imaging, including high stability and the ability to be labeled with radioisotopes, as well as paramagnetic and fluorescent contrast agents. However, long circulation times and difficulties in creating targeted liposomes have proven challenges for imaging. In this study, we have addressed these limitations using a recently developed strategy for bioorthogonal conjugation, the reaction between tetrazines and trans-cyclooctenes. By coating radiolabeled liposomes with trans-cyclooctene and pretargeting with a tetrazine coupled to a targeted peptide, we were able to selectively enhance the retention of liposomes and bind them to tumor tissue in live animals. The rapid reaction between tetrazines and trans-cyclooctenes allowed imaging to be performed with the short-lived PET tracer (18)F, yielding signal-to-background activity ratios of 7:1. The covalent, bioorthogonally driven tumor-targeting of liposomes by in vivo click chemistry is promising and should be explored for more selective and rapid delivery of radiodiagnostics and radiotherapeutics, two classes of drugs which particularly benefit from fast clearance, low nonspecific binding, and the associated reduced toxicity to kidneys and bone marrow.
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http://dx.doi.org/10.1021/bc400322hDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3903177PMC
November 2013

Synthesis and evaluation of 18F-labeled benzylguanidine analogs for targeting the human norepinephrine transporter.

Eur J Nucl Med Mol Imaging 2014 Feb 31;41(2):322-32. Epub 2013 Oct 31.

Department of Radiology, Memorial Sloan-Kettering Cancer Center (MSKCC), New York, NY, 10065, USA.

Purpose: Both (131)I- and (123)I-labeled meta-iodobenzylguanidine (MIBG) have been widely used in the clinic for targeted imaging of the norepinephrine transporter (NET). The human NET (hNET) gene has been imaged successfully with (124)I-MIBG positron emission tomography (PET) at time points of >24 h post-injection (p.i.). (18)F-labeled MIBG analogs may be ideal to image hNET expression at time points of <8 h p.i. We developed improved methods for the synthesis of known MIBG analogs, [(18)F]MFBG and [(18)F]PFBG and evaluated them in hNET reporter gene-transduced C6 rat glioma cells and xenografts.

Methods: [(18)F]MFBG and [(18)F]PFBG were synthesized manually using a three-step synthetic scheme. Wild-type and hNET reporter gene-transduced C6 rat glioma cells and xenografts were used to comparatively evaluate the (18)F-labeled analogs with [(123)I]/[(124)I]MIBG.

Results: The fluorination efficacy on benzonitrile was predominantly determined by the position of the trimethylammonium group. The para-isomer afforded higher yields (75 ± 7%) than meta-isomer (21 ± 5%). The reaction of [(18)F]fluorobenzylamine with 1H-pyrazole-1-carboximidamide was more efficient than with 2-methyl-2-thiopseudourea. The overall radiochemical yields (decay-corrected) were 11 ± 2% (n = 12) for [(18)F]MFBG and 41 ± 12% (n = 5) for [(18)F]PFBG, respectively. The specific uptakes of [(18)F]MFBG and [(18)F]PFBG were similar in C6-hNET cells, but 4-fold less than that of [(123)I]/[(124)I]MIBG. However, in vivo [(18)F]MFBG accumulation in C6-hNET tumors was 1.6-fold higher than that of [(18)F]PFBG at 1 h p.i., whereas their uptakes were similar at 4 h. Despite [(18)F]MFBG having a 2.8-fold lower affinity to hNET and approximately 4-fold lower cell uptake in vitro compared to [(123)I]/[(124)I]MIBG, PET imaging demonstrated that [(18)F]MFBG was able to visualize C6-hNET xenografts better than [(124)I]MIBG. Biodistribution studies showed [(18)F]MFBG and (123)I-MIBG had a similar tumor accumulation, which was lower than that of no-carrier-added [(124)I]MIBG, but [(18)F]MFBG showed a significantly more rapid body clearance and lower uptake in most non-targeting organs.

Conclusion: [(18)F]MFBG and [(18)F]PFBG were synthesized in reasonable radiochemical yields under milder conditions. [(18)F]MFBG is a better PET ligand to image hNET expression in vivo at 1-4 h p.i. than both [(18)F]PFBG and [(123)I]/[(124)I]MIBG.
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http://dx.doi.org/10.1007/s00259-013-2558-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3947152PMC
February 2014

Antilipolytic drug boosts glucose metabolism in prostate cancer.

Nucl Med Biol 2013 May 28;40(4):524-8. Epub 2013 Feb 28.

Department of Clinical Physiology & Nuclear Medicine, Herlev Hospital, University Hospital of Copenhagen, Herlev, Denmark.

Introduction: The antilipolytic drug Acipimox reduces free fatty acid (FFA) levels in the blood stream. We examined the effect of reduced FFAs on glucose metabolism in androgen-dependent (CWR22Rv1) and androgen-independent (PC3) prostate cancer (PCa) xenografts.

Methods: Subcutaneous tumors were produced in nude mice by injection of PC3 and CWR22Rv1 PCa cells. The mice were divided into two groups (Acipimox vs. controls). Acipimox (50mg/kg) was administered by oral gavage 1h before injection of tracers. 1h after i.v. co-injection of 8.2MBq (222 ± 6.0 μCi) (18)F-FDG and~0.0037 MBq (0.1 μCi) (14)C-acetate, (18)F-FDG imaging was performed using a small-animal PET scanner. Counting rates in reconstructed images were converted to activity concentrations. Quantification was obtained by region-of-interest analysis using dedicated software. The mice were euthanized, and blood samples and organs were harvested. (18)F radioactivity was measured in a calibrated γ-counter using a dynamic counting window and decay correction. (14)C radioactivity was determined by liquid scintillation counting using external standard quench corrections. Counts were converted into activity, and percentage of the injected dose per gram (%ID/g) tissue was calculated.

Results: FDG biodistribution data in mice with PC3 xenografts demonstrated doubled average %ID/g tumor tissue after administration of Acipimox compared to controls (7.21 ± 1.93 vs. 3.59 ± 1.35, P=0.02). Tumor-to-organ ratios were generally higher in mice treated with Acipimox. This was supported by PET imaging data, both semi-quantitatively (mean tumor FDG uptake) and visually (tumor-to-background ratios). In mice with CWR22Rv1 xenografts there was no effect of Acipimox on FDG uptake, either in biodistribution or PET imaging. (14)C-acetate uptake was unaffected in PC3 and CWR22Rv1 xenografts.

Conclusions: In mice with PC3 PCa xenografts, acute administration of Acipimox increases tumor uptake of (18)F-FDG with general improvements in tumor-to-background ratios. Data indicate that administration of Acipimox prior to (18)F-FDG PET scans has potential to improve sensitivity and specificity in patients with castration-resistant advanced PCa.
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http://dx.doi.org/10.1016/j.nucmedbio.2013.01.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3662867PMC
May 2013

Fluorine-labeled dasatinib nanoformulations as targeted molecular imaging probes in a PDGFB-driven murine glioblastoma model.

Neoplasia 2012 Dec;14(12):1132-43

Department of Radiology, Sloan Kettering Institute for Cancer Research, New York, NY 10065, USA.

Dasatinib, a new-generation Src and platelet-derived growth factor receptor (PDGFR) inhibitor, is currently under evaluation in high-grade glioma clinical trials. To achieve optimum physicochemical and/or biologic properties, alternative drug delivery vehicles may be needed. We used a novel fluorinated dasatinib derivative (F-SKI249380), in combination with nanocarrier vehicles and metabolic imaging tools (microPET) to evaluate drug delivery and uptake in a platelet-derived growth factor B (PDGFB)-driven genetically engineered mouse model (GEMM) of high-grade glioma. We assessed dasatinib survival benefit on the basis of measured tumor volumes. Using brain tumor cells derived from PDGFB-driven gliomas, dose-dependent uptake and time-dependent inhibitory effects of F-SKI249380 on biologic activity were investigated and compared with the parent drug. PDGFR receptor status and tumor-specific targeting were non-invasively evaluated in vivo using (18)F-SKI249380 and (18)F-SKI249380-containing micellar and liposomal nanoformulations. A statistically significant survival benefit was found using dasatinib (95 mg/kg) versus saline vehicle (P < .001) in tumor volume-matched GEMM pairs. Competitive binding and treatment assays revealed comparable biologic properties for F-SKI249380 and the parent drug. In vivo, Significantly higher tumor uptake was observed for (18)F-SKI249380-containing micelle formulations [4.9 percentage of the injected dose per gram tissue (%ID/g); P = .002] compared to control values (1.6%ID/g). Saturation studies using excess cold dasatinib showed marked reduction of tumor uptake values to levels in normal brain (1.5%ID/g), consistent with in vivo binding specificity. Using (18)F-SKI249380-containing micelles as radiotracers to estimate therapeutic dosing requirements, we calculated intratumoral drug concentrations (24-60 nM) that were comparable to in vitro 50% inhibitory concentration values. (18)F-SKI249380 is a PDGFR-selective tracer, which demonstrates improved delivery to PDGFB-driven high-grade gliomas and facilitates treatment planning when coupled with nanoformulations and quantitative PET imaging approaches.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3540940PMC
http://dx.doi.org/10.1593/neo.121750DOI Listing
December 2012

Fluorine-labeled dasatinib nanoformulations as targeted molecular imaging probes in a PDGFB-driven murine glioblastoma model.

Neoplasia 2012 Dec;14(12):1132-43

Department of Radiology, Sloan Kettering Institute for Cancer Research, New York, NY 10065, USA.

Dasatinib, a new-generation Src and platelet-derived growth factor receptor (PDGFR) inhibitor, is currently under evaluation in high-grade glioma clinical trials. To achieve optimum physicochemical and/or biologic properties, alternative drug delivery vehicles may be needed. We used a novel fluorinated dasatinib derivative (F-SKI249380), in combination with nanocarrier vehicles and metabolic imaging tools (microPET) to evaluate drug delivery and uptake in a platelet-derived growth factor B (PDGFB)-driven genetically engineered mouse model (GEMM) of high-grade glioma. We assessed dasatinib survival benefit on the basis of measured tumor volumes. Using brain tumor cells derived from PDGFB-driven gliomas, dose-dependent uptake and time-dependent inhibitory effects of F-SKI249380 on biologic activity were investigated and compared with the parent drug. PDGFR receptor status and tumor-specific targeting were non-invasively evaluated in vivo using (18)F-SKI249380 and (18)F-SKI249380-containing micellar and liposomal nanoformulations. A statistically significant survival benefit was found using dasatinib (95 mg/kg) versus saline vehicle (P < .001) in tumor volume-matched GEMM pairs. Competitive binding and treatment assays revealed comparable biologic properties for F-SKI249380 and the parent drug. In vivo, Significantly higher tumor uptake was observed for (18)F-SKI249380-containing micelle formulations [4.9 percentage of the injected dose per gram tissue (%ID/g); P = .002] compared to control values (1.6%ID/g). Saturation studies using excess cold dasatinib showed marked reduction of tumor uptake values to levels in normal brain (1.5%ID/g), consistent with in vivo binding specificity. Using (18)F-SKI249380-containing micelles as radiotracers to estimate therapeutic dosing requirements, we calculated intratumoral drug concentrations (24-60 nM) that were comparable to in vitro 50% inhibitory concentration values. (18)F-SKI249380 is a PDGFR-selective tracer, which demonstrates improved delivery to PDGFB-driven high-grade gliomas and facilitates treatment planning when coupled with nanoformulations and quantitative PET imaging approaches.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3540940PMC
http://dx.doi.org/10.1593/neo.121750DOI Listing
December 2012

An improved strategy for the synthesis of [¹⁸F]-labeled arabinofuranosyl nucleosides.

Nucl Med Biol 2012 Nov 18;39(8):1182-8. Epub 2012 Jul 18.

Department of Radiology, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA.

The expression of the herpes simplex virus type-1 thymidine kinase (HSV1-tk) gene can be imaged efficaciously using a variety of 2'-[(18)F]fluoro-2'-deoxy-1-b-D-arabinofuranosyl-uracil derivatives [[(18)F]-FXAU, X=I(iodo), E(ethyl), and M(methyl)]. However, the application of these derivatives in clinical and translational studies has been impeded by their complicated and long syntheses (3-5h). To remedy these issues, in the study at hand we have investigated whether microwave or combined catalysts could facilitate the coupling reaction between sugar and nucleobase and, further, have probed the feasibility of establishing a novel approach for [(18)F]-FXAU synthesis. We have demonstrated that the rate of the trimethylsilyl trifluoromethanesulfonate (TMSOTf)-catalyzed coupling reaction between the 2-deoxy-sugar and uracil derivatives at 90 °C can be significantly accelerated by microwave-driven heating or by the addition of Lewis acid catalyst (SnCl(4)). Further, we have observed that the stability of the α- and β-anomers of [(18)F]-FXAU derivatives differs during the hydrolysis step. Using the microwave-driven heating approach, overall decay-corrected radiochemical yields of 19%-27% were achieved for [(18)F]-FXAU in 120min at a specific activity of >22MBq/nmol (595Ci/mmol). Ultimately, we believe that these high yielding syntheses of [(18)F]-FIAU, [(18)F]-FMAU and [(18)F]-FEAU will facilitate routine production for clinical applications.
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http://dx.doi.org/10.1016/j.nucmedbio.2012.06.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3517724PMC
November 2012

Efficient (18)F-labeling of large 37-amino-acid pHLIP peptide analogues and their biological evaluation.

Bioconjug Chem 2012 Aug 30;23(8):1557-66. Epub 2012 Jul 30.

Department of Radiology, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA.

Solid tumors often develop an acidic microenvironment, which plays a critical role in tumor progression and is associated with increased level of invasion and metastasis. The 37-residue pH (low) insertion peptide (pHLIP) is under study as an imaging platform because of its unique ability to insert into cell membranes at a low extracellular pH (pH(e) < 7). Labeling of peptides with [(18)F]-fluorine is usually performed via prosthetic groups using chemoselective coupling reactions. One of the most successful procedures involves the alkyne-azide copper(I) catalyzed cycloaddition (CuAAC). However, none of the known "click" methods have been applied to peptides as large as pHLIP. We designed a novel prosthetic group and extended the use of the CuAAC "click chemistry" for the simple and efficient (18)F-labeling of large peptides. For the evaluation of this labeling approach, a D-amino acid analogue of WT-pHLIP and an L-amino acid control peptide K-pHLIP, both functionalized at the N-terminus with 6-azidohexanoic acid, were used. The novel 6-[(18)F]fluoro-2-ethynylpyridine prosthetic group, was obtained via nucleophilic substitution on the corresponding bromo-precursor after 10 min at 130 °C with a radiochemical yield of 27.5 ± 6.6% (decay corrected) with high radiochemical purity ≥98%. The subsequent Cu(I)-catalyzed "click" reaction with the azido functionalized pHLIP peptides was quantitative within 5 min at 70 °C in a mixture of water and ethanol using Cu-acetate and sodium L-ascorbate. [(18)F]-D-WT-pHLIP and [(18)F]-L-K-pHLIP were obtained with total radiochemical yields of 5-20% after HPLC purification. The total reaction time was 85 min including formulation. In vitro stability tests revealed high stability of the [(18)F]-D-WT-pHLIP in human and mouse plasma after 120 min, with the parent tracer remaining intact at 65% and 85%, respectively. PET imaging and biodistribution studies in LNCaP and PC-3 xenografted mice with the [(18)F]-D-WT-pHLIP and the negative control [(18)F]-L-K-pHLIP revealed pH-dependent tumor retention. This reliable and efficient protocol promises to be useful for the (18)F-labeling of large peptides such as pHLIP and will accelerate the evaluation of numerous [(18)F]-pHLIP analogues as potential PET tracers.
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http://dx.doi.org/10.1021/bc3000222DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3529145PMC
August 2012

Affinity-based proteomics reveal cancer-specific networks coordinated by Hsp90.

Nat Chem Biol 2011 Sep 25;7(11):818-26. Epub 2011 Sep 25.

Molecular Pharmacology and Chemistry Program, Sloan-Kettering Institute, New York, New York, USA.

Most cancers are characterized by multiple molecular alterations, but identification of the key proteins involved in these signaling pathways is currently beyond reach. We show that the inhibitor PU-H71 preferentially targets tumor-enriched Hsp90 complexes and affinity captures Hsp90-dependent oncogenic client proteins. We have used PU-H71 affinity capture to design a proteomic approach that, when combined with bioinformatic pathway analysis, identifies dysregulated signaling networks and key oncoproteins in chronic myeloid leukemia. The identified interactome overlaps with the well-characterized altered proteome in this cancer, indicating that this method can provide global insights into the biology of individual tumors, including primary patient specimens. In addition, we show that this approach can be used to identify previously uncharacterized oncoproteins and mechanisms, potentially leading to new targeted therapies. We further show that the abundance of the PU-H71-enriched Hsp90 species, which is not dictated by Hsp90 expression alone, is predictive of the cell's sensitivity to Hsp90 inhibition.
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http://dx.doi.org/10.1038/nchembio.670DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3265389PMC
September 2011