Oncogenic activation of RAS genes via point mutations occurs in 20%–30% of human cancers. The development of effective RAS inhibitors has been challenging, necessitating new approaches to inhibit this oncogenic protein. Functional studies have shown that the switch region of RAS interacts with a large number of effector proteins containing a common RAS-binding domain (RBD). Because RBD-mediated interactions are essential for RAS signaling, blocking RBD association with small molecules constitutes an attractive therapeutic approach. Here, we present evidence that rigosertib, a styryl-benzyl sulfone, acts as a RAS-mimetic and interacts with the RBDs of RAF kinases, resulting in their inability to bind to RAS, disruption of RAF activation, and inhibition of the RAS-RAF-MEK pathway. We also find that rigosertib binds to the RBDs of Ral-GDS and PI3Ks. These results suggest that targeting of RBDs across multiple signaling pathways by rigosertib may represent an effective strategy for inactivation of RAS signaling.

Methylthio-DADMe-immucillin-A (MT-DADMeCellular proliferation is associated with increased levels of polyamine biosynthesis and polyamine pools (1). Target validation for the polyamine pathway as an anticancer approach has come from ␣-difluoromethylornithine (DFMO), 3 a suicide inhibitor of ornithine decarboxylase (ODC) and the committed step of polyamine biosynthesis (2, 3). ODC is a difficult cancer target because of its rapid turnover and the dose-limiting toxicity of anti-ODC agents (4). Because of these difficulties, DFMO has not gained wide use. But the polyamine pathway, through its close interaction with S-adenosylmethionine (AdoMet) recycling, remains a target for cancer therapy. We investigated the possibility that feedback inhibition by 5Ј-methylthioadenosine (MTA), induced by a transition state analogue inhibitor of 5Ј-methylthioadenosine phosphorylase (MTAP), could be used to block this pathway and initiate anticancer effects. The results indicate that blocking MTA recycling with transition state analogues of MTAP induces apoptosis through specific epigenetic changes in specific cultured cancer cell lines. Inhibition of MTAP is effective in treating a xenograft model of head and neck cancer in mice.MTA is a product of both spermidine and spermine synthases and provides product inhibition at two sequential sites in the polyamine pathway (Fig. 1A). In humans, MTA is degraded exclusively by MTAP (EC 2.4.2.28), expressed from a single gene locus at 9p21. MTAP produces adenine and 5-methylthio-␣-D-ribose-1-phosphate (Fig. 1B), and these products are recycled to AdoMet. Inhibitors of MTAP are therefore expected to increase intracellular MTA, cause feedback inhibition of polyamine biosynthesis, prevent AdoMet recycling, and disrupt AdoMet-dependent methylation activity. One or more of these activities is expected to be associated with antiproliferative activity (5-8).The transition state structure of human MTAP has been established by kinetic isotope effects and quantum chemical calculations. It is characterized by a late transition state with weak participation of the phosphate nucleophile, similar to that of human purine nucleoside phosphorylase but slightly more advanced (Fig. 1B) (9 -15). Analogues of the human MTAP transition state have been synthesized and are powerful and specific inhibitors (16 -18). Methylthio-DADMe-Immucillin-A (MT-DADMe-ImmA) is a chemically stable transition state analogue of human MTAP and is a slow onset tightly binding inhibitor with a dissociation constant of 86 pM (18). * This work was supported by National Institutes of Health Grants GM41916and CA85953 and a pilot project award from P30 CA013330. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The S-adenosylmethionine (AdoMet) salvage enzyme 5 -methylthioadenosine phosphorylase (MTAP) has been implicated as both a cancer target and a tumor suppressor. We tested these hypotheses in mouse xenografts of human lung cancers. AdoMet recycling from 5 -methylthioadenosine (MTA) was blocked by inhibition of MTAP with methylthioDADMe-Immucillin-A (MTDIA), an orally available, nontoxic, picomolar transition state analogue. Blood, urine, and tumor levels of MTA increased in response to MTDIA treatment. Disruption of pathways linked to polyamine synthesis and S-adenosylmethionine (AdoMet) 2 salvage provides metabolic targets in anticancer therapy based on the essential roles of these metabolites in cell growth. AdoMet is the major methyl donor for biosynthetic methylation reactions, a precursor for polyamine synthesis, and the source of methyl groups for DNA methylation. Targeting polyamine metabolism directly at L-ornithine decarboxylase by ␣,␣-difluoromethylornithine has had limited anticancer success (1). Two AdoMet molecules are converted to 5Ј-methylthioadenosine (MTA) in spermine synthesis, and 5Ј-methylthioadenosine phosphorylase (MTAP) recycles MTA by phosphorolysis to permit subsequent resynthesis of AdoMet (Fig. 1). Our working hypothesis was that inhibition of MTAP would affect cellular MTA and AdoMet metabolism with downstream effects on protein, DNA methylation, polyamine synthesis, and polyamine-dependent enzyme reactions. We targeted MTAP by transition state analysis and developed methylthio-DADMe-Immucillin-A (MTDIA), an orally available transition state analogue inhibitor of MTAP (2). Others have proposed that MTAP is a tumor suppressor gene (3), and experiments here explore the effects of MTAP inhibition in human lung cancer xenografts.We previously demonstrated that treatment of human FaDu head and neck tumors in mouse xenografts with MT-DIA prevented tumor growth with no apparent toxicity to the mice (4). Here, we report that both MTAP-positive (H358) and MTAP-deleted (A549) human lung cancer cell lines are also sensitive to MTAP inhibition in mouse xenografts. The mechanism is probed by the metabolic and genetic consequences of MTDIA administration. In culture, MTDIA in combination with MTA slows A549 cell growth but induces apoptosis in H358 cells.Lung cancer is the leading cause of cancer-related deaths worldwide (5). Patients diagnosed at an advanced stage have a median survival of less than 12 months (6 -8). Thus, development of novel therapeutics for lung cancer is a research priority. MTDIA demonstrated significant suppression of tumor growth with human lung cancer A549 and H358 cells in mouse xenografts. Low toxicity, oral availability, and significant tumor suppression by MTDIA all support additional evaluation as an agent for the treatment of lung cancers. EXPERIMENTAL PROCEDURESCell Lines-Human non-small cell lung adenocarcinoma (NSCLC) cell line A549 and prostate carcinoma cell line PC3 were obtained from the American Type Culture Collection (Manassas, VA). Human bronchioloalveolar ...