Glioblastoma (GBM) is a lethal disease with no effective therapies available. We previously observed upregulation of the TAM (Tyro-3, Axl, and Mer) receptor tyrosine kinase family member AXL in mesenchymal GBM and showed that knockdown of AXL induced apoptosis of mesenchymal, but not proneural, glioma sphere cultures (GSC). In this study, we report that BGB324, a novel small molecule inhibitor of AXL, prolongs the survival of immunocompromised mice bearing GSC-derived mesenchymal GBM-like tumors. We show that protein S (PROS1), a known ligand of other TAM receptors, was secreted by tumor-associated macrophages/microglia and subsequently physically associated with and activated AXL in mesenchymal GSC. PROS1-driven phosphorylation of AXL (pAXL) induced NFκB activation in mesenchymal GSC, which was inhibited by BGB324 treatment. We also found that treatment of GSC-derived mouse GBM tumors with nivolumab, a blocking antibody against the immune checkpoint protein PD-1, increased intratumoral macrophages/microglia and activation of AXL. Combinatorial therapy with nivolumab plus BGB324 effectively prolonged the survival of mice bearing GBM tumors. Clinically, expression of AXL or PROS1 was associated with poor prognosis for patients with GBM. Our results suggest that the PROS1-AXL pathway regulates intrinsic mesenchymal signaling and the extrinsic immune microenvironment, contributing to the growth of aggressive GBM tumors. These findings suggest that development of combination treatments of AXL and immune checkpoint inhibitors may provide benefit to patients with GBM. .

In May 2001 a sample of Culex pipiens pipiens variety molestus Forskål from Marin County, California, collected as larvae and reared to adults, was found to show reduced resmethrin and permethrin knock-down responses in bottle bioassays relative to a standard susceptible Cx. pipiens quinquefasciatus Say colony (CQ1). Larval susceptibility tests, using CQ1 as standard susceptible, indicated that the Marin mosquitoes had LC50 resistance ratios of 18.3 for permethrin, 12 for deltamethrin and 3.3 for pyrethrum. A colony of Marin was established and rapidly developed higher levels of resistance in a few generations after exposure to permethrin as larvae. These selected larvae were shown to cross-resist to lambda-cyhalothrin as well as to DDT. However, adult knock-down time in the presence of permethrin, resmethrin and pyrethrum was not increased after increase in tolerance to pyrethroids as larvae. Partial and almost complete reversion to susceptibility as larvae was achieved with S, S, S-tributylphosphorotrithioate and piperonyl butoxide (PBO), respectively, suggesting the presence of carboxylesterase and P450 monooxygenase mediated resistance. Insensitive target site resistance (kdr) was also detected in some Marin mosquitoes by use of an existing PCR-based diagnostic assay designed for Cx. p. pipiens L mosquitoes. Carboxylesterase mediated resistance was supported by use of newly synthesized novel pyrethroid-selective substrates in activity assays. Bottle bioassays gave underestimates of the levels of tolerance to pyrethroids of Marin mosquitoes when compared with mortality rates in field trials using registered pyrethroid adulticides with and without PBO. This study represents the first report of resistance to pyrethroids in a feral population of a mosquito species in the USA.

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We have previously reported the synthesis of four α-cyano-containing ethers based on 2-naphthaldehyde (2-NA) as cytochrome P450 (P450) fluorescent substrates. Activity detection was based on the formation of fluorescent 2-NA following substrate hydrolysis. A major limitation of these substrates was the need to remove NADPH, a required cofactor for P450 oxidation, before measuring 2-NA fluorescence. In this article, we report the synthesis of a new series of novel P450 substrates using 6-dimethylamino-2-naphthaldehyde (6-DMANA), which has a green fluorescent emission that is well separated from the NADPH spectrum. A major advantage of the 6-DMANA substrates is that NADPH removal is not required before fluorescence detection. We used eight α-cyano ether-based substrates to determine the O-dealkylation activity of human, mouse, and rat liver microsomes. In addition, substrate activities were compared with the commercial substrate 7-ethoxyresorufin (7-ER). The catalytic turnover rates of both the 6-DMANA-and 2-NA-based substrates were in some cases threefold faster than the catalytic turnover rate of 7-ER. The 2-NAbased substrates had greater turnover than did the 6-DMANA-based substrates. Murine and rat liver microsomes prepared from animals that had been treated with various P450 inducers were used to examine for isozyme-selective turnover of the substrates. The vastly improved optical properties and synthetic flexibility of the α-cyano ether compounds suggest that they are possibly good general P450 substrates. KeywordsCytochrome P450; Fluorescent substrate; Liver microsomes; α-Cyanohydrin ether Cytochrome P450s (P450s or CYP) 1 are heme proteins that exist in variety of forms, with more than 500 different P450 isozymes reported. These enzymes play a significant role in the metabolism of a wide variety of xenobiotics, such as pesticides, food additives, and industrial chemicals, as well as endogenous compounds [1]. Because the P450s are among the most important enzyme families involved in the oxidative metabolism of drugs in mammalian systems [2], analysis of drug metabolism by P450s has become an essential part of the drug * Corresponding author. Fax: +530 752 1537. E-mail address: bdhammock@ucdavis.edu (B.D. Hammock).1 Abbreviations used: P450 or CYP, cytochrome P450; 2-NA, 2-naphthaldehyde; 6-DMANA, 6-dimethylamino-2-naphthaldehyde; 7-ER, 7-ethoxyresorufin; DMSO, dimethyl sulfoxide; BSA, bovine serum albumin; HMPA, hexamethylphosphoramide; TLC, thin-layer chromatography; BNF, β-naphthoflavone; PB, phenobarbital; MC, 3-methylcholanthene. [3,4]. P450 activity can also be responsible for insect resistance to many pesticides [5][6][7]. There are a number of different assay systems available for measuring P450 activity, including alkoxyresorufins, alkoxycoumarins, and their modified analogues [8][9][10][11]. NIH Public AccessOur laboratory has reported that α-cyano-containing esterase substrates have very low background fluorescence and are stable under most enzyme assay conditions [2]. The assays using th...