Selective Targeting of Ras Mutant Cancers via a New Small Molecule

ABSTRACTMutations in the Ras family of oncogenes are implicated in 33% of human cancers, making Ras an intensely pursued target in drug discovery. As an alternative to direct pharmacological inhibition of Ras, we looked for sensitivities in RAS mutant cells. Using a small molecule screen in cell lines with mutations in Ras and its effector Raf, we discovered 249C as a Ras-mutant selective cytotoxic agent against a spectrum of RAS-mutant cancers. By combining CRISPR chemical-genetic screening, comparative profiling and chemoproteomics, we identified that 249C binds to a unique subunit on vacuolar (V)-ATPase with nanomolar affinity, inhibiting its biochemical activity and, unexpectedly, altering V-ATPase translocation in Ras-induced macropinocytosis. Via binding to V-ATPase, 249C prevents lysosomal acidification and inhibits autophagy and macropinocytosis pathways that several Ras-driven cancers rely on for survival. In characterizing 249C’s mechanism, we show that potency varies with the identity of the RAS driver mutation highlighting a mutant-specific dependence on autophagy and macropinocytosis. Indeed, 249C potently inhibits tumor growth without adverse side effects in a mouse xenograft model of KRAS-driven non-small cell lung cancer. These data establish proof-of-concept for targeting V-ATPase as a way to indirectly target specific Ras mutants, and provide a fundamental link between V-ATPase localization and specific Ras mutant tumor-related activity.

Retinoschisin is linked to retinal Na/K-ATPase signaling and localization

Mutations in the RS1 gene cause X-linked juvenile retinoschisis (XLRS), a hereditary retinal dystrophy. We recently showed that retinoschisin, the protein encoded by RS1, regulates ERK signaling and apoptosis in retinal cells. In this study, we explored an influence of retinoschisin on the functionality of the Na/K-ATPase, its interaction partner at retinal plasma membranes. We show that retinoschisin binding requires the β2-subunit of the Na/K-ATPase, whereas the α-subunit is exchangeable. Our investigations revealed no effect of retinoschisin on Na/K-ATPase–mediated ATP hydrolysis and ion transport. However, we identified an influence of retinoschisin on Na/K-ATPase–regulated signaling cascades and Na/K-ATPase localization. In addition to the known ERK deactivation, retinoschisin treatment of retinoschisin-deficient (Rs1h-/Y) murine retinal explants decreased activation of Src, an initial transmitter in Na/K-ATPase signal transduction, and of Ca2+signaling marker Camk2. Immunohistochemistry on murine retinae revealed an overlap of the retinoschisin–Na/K-ATPase complex with proteins involved in Na/K-ATPase signaling, such as caveolin, phospholipase C, Src, and the IP3 receptor. Finally, retinoschisin treatment altered Na/K-ATPase localization in photoreceptors of Rs1h-/Yretinae. Taken together, our results suggest a regulatory effect of retinoschisin on Na/K-ATPase signaling and localization, whereas Na/K-ATPase-dysregulation caused by retinoschisin deficiency could represent an initial step in XLRS pathogenesis.

Altered Na+/K+-ATPase expression plays a role in rumen epithelium adaptation in sheep fed hay ad libitum or a mixed hay/concentrate diet

In this study we investigated rumen papillae morphology and the localization and expression of the<br />Na^+/K⁺-ATPase&nbsp;in eight sheep fed hay ad libitum (h) or hay ad libitum plus additional concentrate (h/c). Four sheep were provided with the ad libitum h-diet for the complete three-week experimental period. The second group of four sheep received the h-diet for only one week and was fed the mixed hay/concentrate (h/c) diet for another two weeks. The amount of concentrate supplement was stepwise increased from 150 to 1000 g/day and given in two meals. Following slaughter rumen papillae from the atrium ruminis (AR), the rumen ventralis (RV) and the ventral blind sac (BSV) were fixed and examined for morphological changes and Na⁺/K⁺-ATPase localization by morphometric methods and immunohistochemistry. Ruminal epithelial cells (REC) originating from the strata basale to granulosum were also isolated. Cellular Na⁺/K⁺-ATPase expression (mRNA and protein) and differentiation state were determined by RT-PCR, Western blot, and flow cytometry. Compared with data from h-fed sheep, morphometric analysis revealed an increased length and width of rumen papillae in h/c-fed sheep, resulting in a marked 41% and 62% increase in rumen papillae surface in AR and RV, respectively. The rumen mucosa of h/c-fed sheep was characterized by a predominant stratum corneum (42 &plusmn; 0.7 &micro;m vs. 28 &plusmn; 0.5 &micro;m), but the thickness of the metabolically active cell layers remained unchanged. REC suspensions from sheep fed the h/c diet generally contained more cells (7.30 &plusmn; 0.83 vs. 3.49 &plusmn; 0.52 &times; 10⁷/ml; P &lt; 0.001) and an increased proportion of REC positive for basal cytokeratin and for the differentiation marker cytokeratin 10 (P &lt; 0.05). Cellular (cell membrane) and epithelial (stratum basale to stratum granulosum) Na⁺/K⁺-ATPase localization was similar between rumen regions and was not changed by concentrate feeding. After two weeks on the h/c-diet, a 96% increase in the absolute number of Na⁺/K⁺-ATPase-positive REC (6.56 &plusmn; 0.84 vs. 3.35 &plusmn; 0.51 &times; 10⁷/ml; P = 0.003) and a 61% elevation (P = 0.043) in Na⁺/K⁺-ATPase protein expression in REC from the upper third of the suprabasal cell layers were found. Moreover, a two-fold (P = 0.001) elevation in cell membrane surface area accompanied by a reduction (1.19 &times; 10^&ndash;7 &plusmn; 1.72 &times; 10^&ndash;9 arbitrary units (AU)/cm2 vs. 1.73 &times; 10^&ndash;7 &plusmn; 8.16 &times; 10^&ndash;9 AU/cm² in the h-group; P &lt; 0.001) in specific Na⁺/K⁺-ATPase fluorescence per cm² of cell membrane surface area was observed after h/c-feeding. Na⁺/K⁺-ATPase &alpha; subunit mRNA expression was also reduced (P &lt; 0.0001) from 0.154 &plusmn; 0.013 to 0.057 &plusmn; 0.004 pg per pg S18 mRNA control in the h/c-compared with the h-group. Thus, the h/c-diet led to a rapid increase in REC number and total cell membrane surface area in metabolically active and resorptive cell layers and was accompanied by a reduction in Na⁺/K⁺-ATPase mRNA expression and abundance per cell membrane surface area.

Immunochemical analysis of the vacuolar proton-ATPase B-subunit in the gills of a euryhaline stingray (Dasyatis sabina): effects of salinity and relation to Na+/K+-ATPase

SUMMARY In the gills of freshwater teleost fishes, vacuolar proton-ATPase (V-H+-ATPase) is found on the apical membrane of pavement and chloride (Na+/K+-ATPase-rich) cells, and is an important transporter for energizing Na+ uptake and H+ excretion. In the gills of elasmobranch fishes, the V-H+-ATPase has not been extensively studied and its expression in freshwater individuals has not been examined. The goals of this study were to examine the effects of environmental salinity on the expression of V-H+-ATPase in the gills of an elasmobranch (the Atlantic stingray, Dasyatis sabina) and determine if V-H+-ATPase and Na+/K+-ATPase are expressed in the same cells. We found that gills from freshwater stingrays had the highest relative abundance of V-H+-ATPase and greatest number of V-H+-ATPase-rich cells, using immunoblotting and immunohistochemistry, respectively. When freshwater animals were acclimated to sea water for 1 week, V-H+-ATPase abundance and the number of V-H+-ATPase-rich cells decreased significantly. Atlantic stingrays from seawater environments were characterized by the lowest expression of V-H+-ATPase and least number of V-H+-ATPase-rich cells. In contrast to teleost fishes, localization of V-H+-ATPase in freshwater stingray gills was not found in pavement cells and occurred on the basolateral membrane in cells that are presumably rich in mitochondria. In freshwater stingrays acclimated to sea water and seawater stingrays, V-H+-ATPase localization appeared qualitatively to be stronger in the cytoplasm, which may suggest the transporter was stored in vesicles. Using a double-immunolabeling technique, we found that V-H+-ATPase and Na+/K+-ATPase occurred in distinct cells, which suggests there may be two types of mitochondrion-rich cells in the elasmobranch gill epithelium. Based on these findings, we propose a unique model of NaCl and acid–base regulation where the V-H+-ATPase-rich cells and Na+/K+-ATPase-rich cells are the sites of Cl– uptake/HCO3– excretion and Na+ uptake/H+ excretion, respectively.