The association of mutant forms of Ras protein with a variety of human cancers has stimulated intense interest in therapies based on inhibiting oncogenic Ras signaling. Attachment of Ras proteins to the plasma membrane is required for effective Ras signaling and is initiated by the enzyme farnesyl protein transferase. We found that in the presence of potent farnesyl protein transferase inhibitors, Ras proteins in the human colon carcinoma cell line DLD-1 were alternatively prenylated by geranylgeranyl transferase-1. When H-Ras, N-Ras, K-Ras4A, and K-Ras4B were expressed individually in COS cells, H-Ras prenylation and membrane association were found to be uniquely sensitive to farnesyl transferase inhibitors; N-and K-Ras proteins incorporated the geranylgeranyl isoprene group and remained associated with the membrane fraction. The alternative prenylation of N-and K-Ras has significant implications for our understanding of the mechanism of action of farnesyl protein transferase inhibitors as anti-cancer chemotherapeutics.Newly synthesized Ras proteins are partitioned to the cytoplasmic face of the plasma membrane by a series of posttranslational modifications. The first step, catalyzed by the enzyme farnesyl protein transferase, is the addition of the 15-carbon isoprenyl group farnesyl to the sulfhydryl group of cysteine in the Ras carboxyl-terminal CAAX box (where C is cysteine, A is aliphatic, and X is typically Met or Ser) (1-3). Farnesylation is followed by proteolytic removal of the AAX amino acids and methylation of the carboxyl group of the farnesylated cysteine (4). Ras proteins at the plasma membrane cycle between an active GTP-bound state and an inactive GDPbound state. Mutations that stabilize the active GTP-bound state have been identified in over 30% of human tumors, with particularly high incidences in pancreatic (ϳ90%) and colon (ϳ50%) cancers. Four oncogenic Ras proteins have been described, H-Ras, N-Ras, K-Ras4A, and K-Ras4B. The majority of mutations associated with human cancer have been found in the K-Ras gene. The two K-Ras proteins are products of a single alternatively spliced transcript, with K-Ras4B the predominant isoform (Ͼ80%) (5, 6).Ras proteins that have been genetically modified so that they lack the isoprenylated cysteine do not associate with the plasma membrane and cannot transform fibroblasts (7). These genetic experiments provided the basis for the development of farnesyl transferase inhibitors (FTIs) 1 as anti-cancer agents. A number of reports have demonstrated that pharmacological inhibition of farnesyl protein transferase by CAAX analogs reduces anchorage-independent growth of Ras-transformed cells in soft agar (8) and slows growth of Ras-transformed cells in nude mice (9, 10). The FTIs appear relatively non-toxic in that they do not interfere with normal cell proliferation (11). This result was somewhat surprising because Ras function was shown to be necessary for normal growth factor signaling and cell proliferation (12). A mechanism through which cells may proliferate in...

The high frequency of activating RAS or BRAF mutations in cancer provides strong rationale for targeting the mitogen-activated protein kinase (MAPK) pathway. Selective BRAF and MAP-ERK kinase (MEK) inhibitors have shown clinical effi cacy in patients with melanoma. However, the majority of responses are transient, and resistance is often associated with pathway reactivation of the extracellular signal-regulated kinase (ERK) signaling pathway. Here, we describe the identifi cation and characterization of SCH772984, a novel and selective inhibitor of ERK1/2 that displays behaviors of both type I and type II kinase inhibitors. SCH772984 has nanomolar cellular potency in tumor cells with mutations in BRAF , NRAS , or KRAS and induces tumor regressions in xenograft models at tolerated doses. Importantly, SCH772984 effectively inhibited MAPK signaling and cell proliferation in BRAF or MEK inhibitor-resistant models as well as in tumor cells resistant to concurrent treatment with BRAF and MEK inhibitors. These data support the clinical development of ERK inhibitors for tumors refractory to MAPK inhibitors. SIGNIFICANCE: BRAF and MEK inhibitors have activity in MAPK-dependent cancers with BRAF or RAS mutations. However, resistance is associated with pathway alterations resulting in phospho-ERK reactivation. Here, we describe a novel ERK1/2 kinase inhibitor that has antitumor activity in MAPK inhibitor-naïve and MAPK inhibitor-resistant cells containing BRAF or RAS mutations. Cancer Discov; 3(7); 742-50.

Survivin is an inhibitor of apoptosis protein, which is over-expressed in most tumors. Aberrant expression of survivin and loss of wild-type p53 in many tumors prompted us to investigate a possible link between these two events. Here we show that wild-type p53 represses survivin expression at both mRNA and protein levels. Transient transfection analyses revealed that the expression of wild-type p53, but not mutant p53, was associated with strong repression of the survivin promoter in various cell types. The over-expression of exogenous survivin protein rescues cells from p53-induced apoptosis in a dose-dependent manner, suggesting that loss of survivin mediates, at least, in part the p53-dependent apoptotic pathway. In spite of the presence of two putative p53-binding sites in the survivin promoter, deletion and mutation analyses suggested that neither site is required for transcriptional repression of survivin expression. This was con®rmed by chromatin immunoprecipitation assays. Further analyses suggested that the modi®cation of chromatin within the survivin promoter could be a molecular explanation for silencing of survivin gene transcription by p53.

Hutchinson–Gilford progeria syndrome (HGPS) is an extremely rare, fatal, segmental premature aging syndrome caused by a mutation in LMNA that produces the farnesylated aberrant lamin A protein, progerin. This multisystem disorder causes failure to thrive and accelerated atherosclerosis leading to early death. Farnesyltransferase inhibitors have ameliorated disease phenotypes in preclinical studies. Twenty-five patients with HGPS received the farnesyltransferase inhibitor lonafarnib for a minimum of 2 y. Primary outcome success was predefined as a 50% increase over pretherapy in estimated annual rate of weight gain, or change from pretherapy weight loss to statistically significant on-study weight gain. Nine patients experienced a ≥50% increase, six experienced a ≥50% decrease, and 10 remained stable with respect to rate of weight gain. Secondary outcomes included decreases in arterial pulse wave velocity and carotid artery echodensity and increases in skeletal rigidity and sensorineural hearing within patient subgroups. All patients improved in one or more of these outcomes. Results from this clinical treatment trial for children with HGPS provide preliminary evidence that lonafarnib may improve vascular stiffness, bone structure, and audiological status.

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