Dose and Schedule Determine Distinct Molecular Mechanisms Underlying the Efficacy of the p53-MDM2 Inhibitor HDM201.

Activation of p53 by inhibitors of the p53-MDM2 interaction is being pursued as a therapeutic strategy in p53 wild-type cancers. Here, we report distinct mechanisms by which the novel, potent, and selective inhibitor of the p53-MDM2 interaction HDM201 elicits therapeutic efficacy when applied at various doses and schedules. Continuous exposure of HDM201 led to induction of p21 and delayed accumulation of apoptotic cells. By comparison, high-dose pulses of HDM201 were associated with marked induction of PUMA and a rapid onset of apoptosis. shRNA screens identified PUMA as a mediator of the p53 response specifically in the pulsed regimen. Consistent with this, the single high-dose HDM201 regimen resulted in rapid and marked induction of PUMA expression and apoptosis together with downregulation of Bcl-xL in vivo Knockdown of Bcl-xL was identified as the top sensitizer to HDM201 in vitro, and Bcl-xL was enriched in relapsing tumors from mice treated with intermittent high doses of HDM201. These findings define a regimen-dependent mechanism by which disruption of MDM2-p53 elicits therapeutic efficacy when given with infrequent dosing. In an ongoing HDM201 trial, the observed exposure-response relationship indicates that the molecular mechanism elicited by pulse dosing is likely reproducible in patients. These data support the clinical comparison of daily and intermittent regimens of p53-MDM2 inhibitors.Significance: Pulsed high doses versus sustained low doses of the p53-MDM2 inhibitor HDM201 elicit a proapoptotic response from wild-type p53 cancer cells, offering guidance to current clinical trials with this and other drugs that exploit the activity of p53. Cancer Res; 78(21); 6257-67. ©2018 AACR.

Lung tumors on multimodal radiographs derived from grating-based X-ray imaging--a feasibility study.

PURPOSE: The purpose of this study was to assess whether grating-based X-ray imaging may have a role in imaging of pulmonary nodules on radiographs. MATERIALS AND METHODS: A mouse lung containing multiple lung tumors was imaged using a small-animal scanner with a conventional X-ray source and a grating interferometer for phase-contrast imaging. We qualitatively compared the signal characteristics of lung nodules on transmission, dark-field and phase-contrast images. Furthermore, we quantitatively compared signal characteristics of lung tumors and the adjacent lung tissue and calculated the corresponding contrast-to-noise ratios. RESULTS: Of the 5 tumors visualized on the transmission image, 3/5 tumors were clearly visualized and 1 tumor was faintly visualized in the dark-field image as areas of decreased small angle scattering. In the phase-contrast images, 3/5 tumors were clearly visualized, while the remaining 2 tumors were faintly visualized by the phase-shift occurring at their edges. No additional tumors were visualized in either the dark-field or phase-contrast images. Compared to the adjacent lung tissue, lung tumors were characterized by a significant decrease in transmission signal (median 0.86 vs. 0.91, p = 0.04) and increase in dark-field signal (median 0.71 vs. 0.65, p = 0.04). Median contrast-to-noise ratios for the visualization of lung nodules were 4.4 for transmission images and 1.7 for dark-field images (p = 0.04). CONCLUSION: Lung nodules can be visualized on all three radiograph modalities derived from grating-based X-ray imaging. However, our initial data suggest that grating-based multimodal X-ray imaging does not increase the sensitivity of chest radiographs for the detection of lung nodules.

Multiplexed lipid dip-pen nanolithography on subcellular scales for the templating of functional proteins and cell culture.

Molecular patterning processes taking place in biological systems are challenging to study in vivo because of their dynamic behavior, subcellular size, and high degree of complexity. In vitro patterning of biomolecules using nanolithography allows simplification of the processes and detailed study of the dynamic interactions. Parallel dip-pen nanolithography (DPN) is uniquely capable of integrating functional biomolecules on subcellular length scales due to its constructive nature, high resolution, and high throughput. Phospholipids are particularly well suited as inks for DPN since a variety of different functional lipids can be readily patterned in parallel. Here DPN is used to spatially pattern multicomponent micro- and nanostructured supported lipid membranes and multilayers that are fluid and contain various amounts of biotin and/or nitrilotriacetic acid functional groups. The patterns are characterized by fluorescence microscopy and photoemission electron microscopy. Selective adsorption of functionalized or recombinant proteins based on streptavidin or histidine-tag coupling enables the semisynthetic fabrication of model peripheral membrane bound proteins. The biomimetic membrane patterns formed in this way are then used as substrates for cell culture, as demonstrated by the selective adhesion and activation of T-cells.