TET1 deficiency attenuates the DNA damage response and promotes resistance to DNA damaging agents

<p>Recent studies have shown that loss of TET1 may play a significant role in the formation of tumors. Because genomic instability is a hallmark of cancer, we examined the potential involvement of 10-11 translocation 1 (TET1) in the DNA damage response (DDR). Here we demonstrate that, in response to clinically relevant doses of ionizing radiation (IR), human glial cells made TET1-deficient with lentiviral vectors displayed greater numbers of colony forming units and lower levels of apoptotic markers compared with glial cells transduced with control vectors; yet, they harbored greater DNA strand breaks. The G₂/M check point and expression of cyclin B1 were greatly diminished in TET1-deficient cells, and TET1-deficient cells displayed lower levels of γH2A.x following exposure to IR. Levels of DNA-PKcs, which are DNA-PK complex members, were lower in TET1-deficient cells compared with control cell lines. However, levels of ATM were similar in both cell lines. Cyclin B1, DNA-PKcs, and γH2A.x levels were each rescued by reintroduction of the TET1 catalytic domain. Finally, cytosine methylation within intron 1 of <i>PRKDC</i>, the gene encoding DNA-PKcs, was significantly higher upon depletion of TET1. Taken together, this study illustrates the involvement of TET1 in the different arms of the DDR and suggests its loss results in the continued survival of cells with genomic instability.</p

Salicylic Acid Conjugated Dendrimers Are a Tunable, High Performance CEST MRI NanoPlatform

Chemical exchange saturation transfer (CEST) is a novel MRI contrast mechanism that is well suited for imaging, however, existing small molecule CEST agents suffer from low sensitivity. We have developed salicylic acid conjugated dendrimers as a versatile, high performance nanoplatform. In particular, we have prepared nanocarriers based on generation 5-poly­(amidoamine) (PAMAM) dendrimers with salicylic acid covalently attached to their surface. The resulting conjugates produce strong CEST contrast 9.4 ppm from water with the proton exchange tunable from ∼1000 s^–1 to ∼4500 s^–1 making these dendrimers well suited for sensitive detection. Furthermore, we demonstrate that these conjugates can be used for monitoring convection enhanced delivery into U87 glioblastoma bearing mice, with the contrast produced by these nanoparticles persisting for over 1.5 h and distributed over ∼50% of the tumors. Our results demonstrate that SA modified dendrimers present a promising new nanoplatform for medical applications

Bioreducible Polymeric Nanoparticles Containing Multiplexed Cancer Stem Cell Regulating miRNAs Inhibit Glioblastoma Growth and Prolong Survival

Despite our growing molecular-level understanding of glioblastoma (GBM), treatment modalities remain limited. Recent developments in the mechanisms of cell fate regulation and nanomedicine provide new avenues by which to treat and manage brain tumors via the delivery of molecular therapeutics. Here, we have developed bioreducible poly­(β-amino ester) nanoparticles that demonstrate high intracellular delivery efficacy, low cytotoxicity, escape from endosomes, and promotion of cytosol-targeted environmentally triggered cargo release for miRNA delivery to tumor-propagating human cancer stem cells. In this report, we combined this nanobiotechnology with newly discovered cancer stem cell inhibiting miRNAs to develop self-assembled miRNA-containing polymeric nanoparticles (nano-miRs) to treat gliomas. We show that these nano-miRs effectively intracellularly deliver single and combination miRNA mimics that inhibit the stem cell phenotype of human GBM cells in vitro. Following direct intratumoral infusion, these nano-miRs were found to distribute through the tumors, inhibit the growth of established orthotopic human GBM xenografts, and cooperatively enhance the response to standard-of-care γ radiation. Co-delivery of two miRNAs, miR-148a and miR-296-5p, within the bioreducible nano-miR particles enabled long-term survival from GBM in mice

Validation of newly identified signaling components.

<p>(a) Venn diagram of number of newly identified phosphoproteins between the <i>in vitro</i> and <i>in vivo</i> systems (b) A global view of known kinase-substrate and/or protein-protein interaction between 88 newly identified candidates and c-Met signaling components. Official gene symbols were used instead of common protein names to avoid ambiguity. (c) Over-represented pathways for 88 hits in Fig. 4b. Red line denotes p-value (-log₁₀). (d-e) Validation of newly identified signaling components associated with c-Met activation in HGF treated U373 HGF⁻/c-Met⁺ cells using antibodies that specifically recognize the phosphorylated forms of PKCD (pThr505), and eEF2K (pSer366), respectively. The activating phosphorylation of these kinases was elevated in comparison to untreated cells. The bar chart represents quantification analyses of the phosphorylation signals from triplicate experiments. (f) Activation of PKA catalytic subunit is also confirmed to be associated with HGF stimulation using antibodies against phosphorylated PKA substrates coupled with H89 treatment. Arrows indicate extra proteins phosphorylated by PKA upon HGF stimulation. (g) A known PKA substrate, TRIP10 was examined to confirm PKA activation. Activation of PKA upon HGF treatment results in decreased amount of TRIP10, which can be fully rescued by H89 pre-treatment in U373 HGF-/c⁻Met⁺ cells, indicating PKA-dependent effects on TRIP10.</p

Scheme for profiling human phosphoproteome patterns using protein microarrays as readout.

<p>Cells are collected from <i>in cellulo</i> or <i>xenograft</i> model systems that were untreated or treated with a c-Met agonist or antagonist. Total proteins in lysed cells were then diluted to a kinase reaction buffer and applied to the human protein microarray. After the reaction is completed, the protein microarray is washed under stringent conditions, exposed to X-ray film, and the phosphorylation signals were acquired and analyzed GenePix software. Comparison of phosphorylation signals between different assays results in identification of differentially phosphorylated proteins associated with HGF/c-Met activation. Candidate proteins were then validated via traditional methods.</p

Kinase phosphorylome profiling recovered many of the known c-Met associated signaling proteins.

<p>(a,b) Schematic and representative block of the protein microarray after conducting phosphorylation assays using HGF-/Met+ U373 glioma cells and HGF+/Met+ U87 subcutaneous glioma xenografts, respectively. From left to right, top to bottom those proteins are MAPK3, HSPA9, and TSSK3 for U373 and TRAP1, MAPK3, GTF3C2, and NRP2 for U87. Phosphoprotein identified 23 and 20 of the 29 known signaling components in the HGF/c-Met signaling in <i>in vitro</i> and <i>in vivo</i> model systems, respectively. Proteins were spotted in duplicate. Red box, histones; Yellow box, BSA; Green box, differentially phosphorylated proteins. (c) Venn diagram showing substantial overlap of known hits observed on the microarray between the two model systems. (d,e) Immunoblot analyses confirming the increased phosphorylation of known HGF-associated proteins recovered from the <i>in vitro</i> and <i>in vivo</i> screens.</p

Expanded view of newly identified downstream events of HGF/c-Met phosphorylation networks in one potential cascade.

<p>All of the proteins were identified as hits on both the <i>in cellulo</i> and <i>in vivo</i> lysates phosphorylation assays. Red border indicates newly identified signaling components that have been validated to be hyper-phosphorylated downstream of HGF/c-Met signaling.</p