Novel Radiotracer for ImmunoPET Imaging of PD-1 Checkpoint Expression on Tumor Infiltrating Lymphocytes.

Immune checkpoint signaling through the programmed death 1 (PD-1) axis to its ligand (PD-L1) significantly dampens anti-tumor immune responses. Cancer patients treated with checkpoint inhibitors that block this suppressive signaling have exhibited objective response rates of 20-40% for advanced solid tumors, lymphomas, and malignant melanomas. This represents a tremendous advance in cancer treatment. Unfortunately, all patients do not respond to immune checkpoint blockade. Recent findings suggest that patients with tumor infiltrating lymphocytes (TILs) expressing PD-1 may be most likely to respond to αPD-1/PD-L1 checkpoint inhibitors. There is a compelling need for diagnostic and prognostic imaging tools to assess the PD-1 status of TILs in vivo. Here we have developed a novel immunoPET tracer to image PD-1 expressing TILs in a transgenic mouse model bearing melanoma. A (64)Cu labeled anti-mouse antibody (IgG) PD-1 immuno positron emission tomography (PET) tracer was developed to detect PD-1 expressing murine TILs. Quality control of the tracer showed >95% purity by HPLC and >70% immunoreactivity in an in vitro cell binding assay. ImmunoPET scans were performed over 1-48 h on Foxp3+.LuciDTR4 mice bearing B16-F10 melanoma tumors. Mice receiving anti-PD-1 tracer (200 ± 10 µCi/10-12 µg/200 µL) revealed high tracer uptake in lymphoid organs and tumors. BLI images of FoxP3(+) CD4(+) Tregs known to express PD-1 confirmed lymphocyte infiltration of tumors at the time of PET imaging. Biodistribution measurements performed at 48 h revealed a high (11×) tumor to muscle uptake ratio of the PET tracer (p < 0.05). PD-1 tumors exhibited 7.4 ± 0.7%ID/g tracer uptake and showed a 2× fold signal decrease when binding was blocked by unlabeled antibody. To the best of our knowledge this data is the first report to image PD-1 expression in living subjects with PET. This radiotracer has the potential to assess the prognostic value of PD-1 in preclinical models of immunotherapy and may ultimately aid in predicting response to therapies targeting immune checkpoints.

Engineering domain fusion chimeras from I-OnuI family LAGLIDADG homing endonucleases.

Although engineered LAGLIDADG homing endonucleases (LHEs) are finding increasing applications in biotechnology, their generation remains a challenging, industrial-scale process. As new single-chain LAGLIDADG nuclease scaffolds are identified, however, an alternative paradigm is emerging: identification of an LHE scaffold whose native cleavage site is a close match to a desired target sequence, followed by small-scale engineering to modestly refine recognition specificity. The application of this paradigm could be accelerated if methods were available for fusing N- and C-terminal domains from newly identified LHEs into chimeric enzymes with hybrid cleavage sites. Here we have analyzed the structural requirements for fusion of domains extracted from six single-chain I-OnuI family LHEs, spanning 40-70% amino acid identity. Our analyses demonstrate that both the LAGLIDADG helical interface residues and the linker peptide composition have important effects on the stability and activity of chimeric enzymes. Using a simple domain fusion method in which linker peptide residues predicted to contact their respective domains are retained, and in which limited variation is introduced into the LAGLIDADG helix and nearby interface residues, catalytically active enzymes were recoverable for ≈ 70% of domain chimeras. This method will be useful for creating large numbers of chimeric LHEs for genome engineering applications.

A proteomic investigation of ligand-dependent HSP90 complexes reveals CHORDC1 as a novel ADP-dependent HSP90-interacting protein.

Structural studies of the chaperone HSP90 have revealed that nucleotide and drug ligands induce several distinct conformational states; however, little is known how these conformations affect interactions with co-chaperones and client proteins. Here we use tandem affinity purification and LC-MS/MS to investigate the proteome-wide effects of ATP, ADP, and geldanamycin on the constituents of the human HSP90 interactome. We identified 52 known and novel components of HSP90 complexes that are regulated by these ligands, including several co-chaperones. Interestingly, our results also show that geldanamycin treatment causes HSP90 complexes to become significantly enriched for core transcription machinery, suggesting that HSP90 inhibition may have broad based effects on transcription and RNA processing. We further characterized a novel ADP-dependent HSP90 interaction with the cysteine- and histidine-rich domain (CHORD)-containing protein CHORDC1. We show that this interaction is stimulated by high ADP:ATP ratios in cell lysates and in vitro with purified recombinant proteins. Furthermore, we demonstrate that this interaction is dependent upon the ability of HSP90 to bind nucleotides and requires the presence of a linker region between the CHORD domains in CHORDC1. Together these findings suggest that the HSP90 interactome is dynamic with respect to nucleotide and drug ligands and that pharmacological inhibition of HSP90 may stimulate the formation of specific complexes.

Analysis of gene expression in Egr-1 transfected human fibrosarcoma cells.

BACKGROUND AND OBJECTIVES: It has been shown previously that transcription factor Egr-1 functions as a tumor suppressor gene. This study was conducted to investigate the possible mechanisms responsible for this function. METHODS: A cDNA microarray technique was applied to study differential gene expression between Egr-1-transfected cells and control-transfected cells. RESULTS: A number of genes differentially expressed were identified. Among 5,184 genes examined by cDNA microarray technique, 53 were up-regulated in Egr-1 transfected cells, and 276 were down-regulated in Egr-1 transfected cells by at least two times or more; 13 genes were independently confirmed by reverse transcription polymerase chain reaction (RT-PCR). Of these 13 genes, 11 were in agreement with cDNA microarrays in terms of gene expression levels. CONCLUSIONS: Egr-1 regulated almost every category of genes according to gene ontology system, and repressed more genes than it induced. The identification and further characterization of genes regulated by Egr-1 should provide valuable information to understand Egr-1 tumor suppressor action and the role of Egr-1 as a transcription factor in general.

Connexin 43 suppresses human glioblastoma cell growth by down-regulation of monocyte chemotactic protein 1, as discovered using protein array technology.

Previously, we demonstrated that connexin 43 (cx43) suppressed the growth of human glioblastoma cells. To investigate the molecular mechanisms involved in tumor suppression by cx43, we developed a human cytokine array system, which simultaneously detects the expression of 43 cytokines. By using this new technology, we analyzed the cx43-regulated genes in cx43-transfected cells. The cytokine arrays showed that expression of monocyte chemotactic protein-1 (MCP-1) was profoundly reduced in cx43-transfected cells. RT-PCR, immuno-Western blot, and cDNA microarrays further confirmed this observation. Addition of conditioned medium from control-transfected cells and recombinant MCP-1 to cx43-transfected cells significantly enhanced cx43-transfected cell proliferation and colony formation in soft agar. In contrast, addition of neutralization antibody against MCP-1 significantly inhibited cell proliferation in control-transfected cells. Our results suggested that MCP-1 is involved in the suppression of human glioblastoma cell growth by cx43.