Although mechanisms of acquired resistance of EGFR mutant non-small cell lung cancers to EGFR inhibitors have been identified, little is known about how resistant clones evolve during drug therapy. Here, we observe that acquired resistance caused by the T790M gatekeeper mutation can occur either by selection of pre-existing T790M clones or via genetic evolution of initially T790M-negative drug tolerant cells. The path to resistance impacts the biology of the resistant clone, as those that evolved from drug tolerant cells had a diminished apoptotic response to third generation EGFR inhibitors that target T790M EGFR; treatment with navitoclax, an inhibitor of BCL-XL and BCL-2 restored sensitivity. We corroborated these findings using cultures derived directly from EGFR inhibitor-resistant patient tumors. These findings provide evidence that clinically relevant drug resistant cancer cells can both pre-exist and evolve from drug tolerant cells, and point to therapeutic opportunities to prevent or overcome resistance in the clinic.

Nitroxoline shows promise as a potential therapeutic antiangiogenic agent.

Bioluminescence imaging (BLI) is becoming indispensable to the study of transgene expression during development and, in many in vivo models of disease such as cancer, for high throughput drug screening in vitro. Because reaction of D-luciferin with firefly luciferase ( fLuc) produces photons of sufficiently long wavelength to permit imaging in intact animals, use of this substrate and enzyme pair has become the method of choice for performing BLI in vivo. We now show that expression of the ATP-binding cassette (ABC) family transporter ABCG2/BCRP affects BLI signal output from the substrate D-luciferin. In vitro studies show that D-luciferin is a substrate for ABCG2/BCRP but not for the MDR1 P-glycoprotein (ABCB1/Pgp), multidrug resistance protein 1 (MRP1/ ABCC1), or multidrug resistance protein 2 (MRP2/ABCC2). D-Luciferin uptake within cells is shown to be modulated by ABC transporter inhibitors, including the potent and selective ABCG2/BCRP inhibitor fumitremorgin C. Images of xenografts engineered to express transgenic ABCG2/BCRP, as well as xenografts derived from the human prostate cancer cell line 22Rv1 that naturally express ABCG2/BCRP, show that ABCG2/ BCRP expression and function within regions of interest substantially influence D-luciferin-dependent bioluminescent output in vivo. These findings highlight the need to consider ABCG2/BCRP effects during D-luciferin-based BLI and suggest novel high throughput methods for identifying new ABCG2/

Molecular-genetic imaging is advancing from a valuable preclinical tool to guiding patient management. The strategy involves pairing an imaging reporter gene with a complementary imaging agent in a system that can be used to measure gene expression, protein interaction or track gene-tagged cells in vivo. Tissue-specific promoters can be used to delineate gene expression in certain tissues, particularly when coupled with an appropriate amplification mechanism. Here we show that the progression elevated gene-3 promoter (PEG-Prom), derived from a rodent gene mediating the malignant phenotype, can be used to drive imaging reporters selectively to enable detection of micrometastatic disease in murine models of human melanoma and breast cancer using bioluminescence and radionuclide-based molecular imaging techniques. Because of its strong promoter, tumor specificity and capacity for clinical translation, PEG-Prom-driven gene expression may represent a practical, new system for facilitating cancer imaging and therapy.

We report a strategy based on bioisosterism to improve the physicochemical properties of existing hydrophilic, urea-based GCPII inhibitors. Comprehensive structure-activity relationship studies of the P1' site of ZJ-43-and DCIBzL-based compounds identified several glutamate-free inhibitors with K i values below 20 nM. Among them, compound 32d (K i = 11 nM) exhibited selective uptake in GCPII-expressing tumors by SPECT-CT imaging in mice. A novel conformational change of amino acids in the S1' pharmacophore pocket was observed in the X-ray crystal structure of GCPII complexed with 32d. KeywordsPSMA; glutamate carboxypeptidase II; molecular imaging; radiopharmaceutical; SPECT Glutamate carboxypeptidase II (GCPII) is a type II zinc-dependent metalloprotease that cleaves N-acetylaspartylglutamate (NAAG) to release N-acetylaspartate (NAA) and glutamate (Glu) in the brain. 1, 2 Excessive production and release of Glu in the synaptic cleft may over-stimulate glutamate receptors, leading to Glu-associated neurotoxicity and neuronal death. A glutamatergic imbalance has been proposed in the pathophysiology of a variety of neurological diseases including ischemia, traumatic brain injury, neuropathic pain, amyotrophic lateral sclerosis, diabetic polyneuropathy, and schizophrenia.3 -8 Accordingly, maintaining Glu homeostasis at the synapse is a goal in the prevention and treatment of neuropsychiatric disease. Among the enzymes that modulate such Glu concentrations, GCPII is a potential target for effecting beneficial intrasynaptic Glu concentrations.GCPII has also been identified in non-neuronal tissues including kidney, small intestine, and prostate. Consequently GCPII has also been referred to as the prostate-specific membrane Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptBioorg Med Chem Lett. Author manuscript; available in PMC 2011 January 1. antigen (PSMA) in the prostate and as folate hydrolase (FOLH1) in the intestine. PSMA is over-expressed on the surface of androgen-independent prostate cancer cells and its active site is located in the extracellular region. Cell-surface expression and up-regulation in prostate tumors render PSMA an attractive target for the diagnosis and possibly therapy of prostate cancer. 9, 10 The FOLH1 activity of GCPII hydrolyzes γ-linked glutamates from poly-γ-glutamyl folate to produce folic acid, which can then be uptaken by folate receptors in the intestine.11 GCPII has also been identified in neovasculature of most solid tumors.12 That vascular expression enhances GCPII as a cancer imaging and therapeuti...