Edited by Norma AllewellThe KRAS GTPase plays a critical role in the control of cellular growth. The activity of KRAS is regulated by guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and also post-translational modification. Lysine 104 in KRAS can be modified by ubiquitylation and acetylation, but the role of this residue in intrinsic KRAS function has not been well characterized. We find that lysine 104 is important for GEF recognition, because mutations at this position impaired GEF-mediated nucleotide exchange. Because the KRAS K104Q mutant has recently been employed as an acetylation mimetic, we conducted a series of studies to evaluate its in vitro and cellbased properties. Herein, we found that KRAS K104Q exhibited defects in both GEF-mediated exchange and GAP-mediated GTP hydrolysis, consistent with NMR-detected structural perturbations in localized regions of KRAS important for recognition of these regulatory proteins. Despite the partial defect in both GEF and GAP regulation, KRAS K104Q did not alter steady-state GTP-bound levels or the ability of the oncogenic KRAS G12V mutant to cause morphologic transformation of NIH 3T3 mouse fibroblasts and of WT KRAS to rescue the growth defect of mouse embryonic fibroblasts deficient in all Ras genes. We conclude that the KRAS K104Q mutant retains both WT and mutant KRAS function, probably due to offsetting defects in recognition of factors that up-regulate (GEF) and down-regulate (GAP) RAS activity.RAS proteins function as molecular switches that cycle between active GTP-and inactive GDP-bound states to regulate signal transduction pathways that modulate cellular growth control. In the unstimulated cell, RAS proteins are populated in their inactive GDP-bound state. However, in response to growth-stimulatory signals, guanine nucleotide exchange factors (GEFs) 2 co-localize and up-regulate RAS by facilitating exchange of GDP for GTP. Inactivation of RAS is achieved through GTPase-activating proteins (GAPs) that bind to GTPbound RAS and promote GTP hydrolysis (1, 2). Several point mutations in RAS have been identified that dysregulate RAS nucleotide exchange or hydrolysis, often leading to hyperactivation and promoting tumorigenesis. The most common RAS mutations identified in cancer occur at residues 12, 13, and 61 and render RAS GAP defective, thereby populating RAS in its active GTP-bound state (3). Constitutive hyperactivation of RAS promotes chronic stimulation of effector-mediated downstream pathways, causing deregulated growth and tumorigenic growth transformation.RAS contains two dynamic regions termed switch I (SWI; residues 30 -37) and switch II (SWII; residues 60 -76 with 66 -74 corresponding to helix 2 (H2)) that populate distinct conformations when the protein is bound to GDP versus GTP. Effectors and GAP proteins recognize specific conformations of the switch regions and bind with preferential affinity to the active GTP-bound state. Activated GTP-bound RAS can interact with multiple effectors (e.g. RAF kinase, RAL exchang...

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Results and discussions L-resistant clones were obtained Resistance was confirmed by MTT analysis. Data obtained by microarray were analysed by principal component analysis to determine the significant sources of variability in the data sets. Gene expression changes are clearly observed between two resistant clones versus parental line. Impressively, 132 genes were significant differentially expressed among resistant clones versus parental line. Unsupervised hierarchical clustering of 132 genes revealed a robust classification between three different groups. When analysed in details, it was possible to identify a large number of genes regulated by NFR2, a master transcriptional regulator that activates genes involved in oxidative stress response, detoxification, and drug resistance. NRF2 expression was evaluated by Western Blot analysis among both L and T resistant cells. After subcellular fractionation, it was possible to observe a nuclear overexpression among resistant cell lines, this data was confirmed by IF. When siRNA of NRF2 was performed, a decrease of cell growth among resistant cell lines was observed. When treatment with L was administered in knockdown cells, it was possible to restore sensitivity. Conclusion NRF2 is identified as a new mechanism of resistance to antiHER2 inhibition in GC. The evaluation of its expression among xenograft models and patients, who experienced a disease progression, are ongoing. Introduction Gastric cancer (GC) is the 3rd leading cause of cancer related deaths and the 5th commonest cancer worldwide, affecting~1 million individuals per year. For early-stage disease, surgical resection is potentially curative, however >80% of GC patients present advanced, unresectable and not curable disease, and an average overall survival (OS) of~1 year. Poor patient survival is justified by late stage diagnosis and by poor response to therapy. We hypothesised that some GC clones that intrinsically resist to chemotherapy overexpress a variant of CD44, the main cell surface receptor for hyaluronic acid. Supporting this hypothesis is our finding that CD44v6 becomes overexpressed in~70% of all GCs, as opposed to normal mucosa. Here, we aim to investigate whether CD44v6 overexpression influences response to cisplatin treatment. Material and methods We established isogenic GC cell lines overexpressing either CD44v6, CD44std or an empty vector (Mock), and CD44v6 RNAi-depleted GC cell lines that endogenously express CD44v6. These were all characterised by RT-PCR, western-blot, immunofluorescence and flow-cytometry. The effect of cisplatin on cell survival was evaluated by SRB and Annexin-V assays. The expression of signalling partners downstream of CD44 was evaluated by western-blot and immunofluorescence. PO-501Results and discussions CD44v6 overexpression increased cisplatin resistance and its depletion sensitised cells to cisplatin treatment. Moreover, when isogenic CD44v6-expressing cells were co-cultured with the non-expressing counterpart, treated with cisplatin and allowed to recover for...

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