Ras Processing Research Articles

RAS specific protease induces irreversible growth arrest via p27 in several KRAS mutant colorectal cancer cell lines

Ras-specific proteases to degrade RAS within cancer cells are under active development as an innovative strategy to treat tumorigenesis. The naturally occurring biological toxin effector called RAS/RAP1-specific endopeptidase (RRSP) is known to cleave all RAS within a cell, including HRAS, KRAS, NRAS and mutant KRAS G13D. Yet, our understanding of the mechanisms by which RRSP drives growth inhibition are unknown. Here, we demonstrate, using isogenic mouse fibroblasts expressing a single isoform of RAS or mutant KRAS, that RRSP equally inactivates all isoforms of RAS as well as the major oncogenic KRAS mutants. To investigate how RAS processing might lead to varying outcomes in cell fate within cancer cells, we tested RRSP against four colorectal cancer cell lines with a range of cell fates. While cell lines highly susceptible to RRSP (HCT116 and SW1463) undergo apoptosis, RRSP treatment of GP5d and SW620 cells induces G1 cell cycle arrest. In some cell lines, growth effects were dictated by rescued expression of the tumor suppressor protein p27 (Kip1). The ability of RRSP to irreversibly inhibit cancer cell growth highlights the antitumor potential of RRSP, and further warrants investigation as a potential anti-tumor therapeutic.

Abstract C031: Ras processing by RRSP protease as a strategy to inhibit Ras-driven tumors

Abstract RAS genes are the most frequently mutated oncogenes in human cancers encoding four highly-related proteins that are critical molecular hubs for cancer cell proliferation and survival. Although promising results have been recently reported in early trials for KRas G12C inhibitors, there are no FDA-approved drugs that are effective against Ras-driven cancers, and Ras is still considered one of the most elusive targets in cancer research. We discovered the Ras-Rap1-Specific Protease (RRSP) from bacterium Vibrio vulnificus. RRSP is a naturally occurring endopeptidase that site-specifically cleaves the major Ras isoforms (H, N and K) and the most common mutated Ras oncoproteins (G12V, G13D and Q61R) within the Switch I region. In order to bring RRSP into cancer cells, RRSP was fused to diphtheria toxin binding subunit B (DTB). DTB delivers RRSP into the cytosol of targeted cells via binding to its receptor hHB-EGF (human heparin-binding epidermal growth factor-like growth factor). KRAS mutations occur in up to 50% of colorectal cancer (CRC) and in approximately 2% of triple-negative breast cancer (TNBC). However, 50% of TNBC show Ras upregulation via mutation-independent activation mechanisms. We investigated the anticancer potential of RRSP in CRC HCT-116 KRAS G13D as well as TNBC MDA-MB-231 KRAS G13D and MDA-MB-436 KRAS WT cell lines. RRSP was successfully delivered via DTB into CRC and TNBC cancer cell lines as indicated by Ras cleavage and pERK1/2 dephosphorylation at concentrations as low as 10 pM. As a consequence of Ras processing, a strong decrease in cell viability and proliferation was observed in all cell lines tested as per crystal violet staining and CellTiter Glo assay. A catalytically-dead mutant of RRSP carrying an amino acid substitution in its active site (RRSP*-DTB) did not show Ras processing nor reduced cell viability. Interestingly, our in vivo xenograft data demonstrated that, unlike RRSP*-DTB, RRSP-DTB effectively inhibited TNBC MDA-MB-436 and MDA-MB-231 tumor growth with no obvious toxicity. Proof of RRSP-target engagement was confirmed by loss of pERK1/2 phosphorylation via immunohistochemical analysis of tumor sections. We also found that RRSP-DTB strongly reduced the size and viability of 3-dimensional CRC HCT-116 spheroids in a time and dose-dependent manner. Moreover, an independent experiment at Charles River showed significant inhibition of tumor growth in a CRC HCT-116 xenograft model. Altogether our results revealed that, by physically processing Ras, RRSP strongly inhibits tumor growth in subcutaneously implanted human CRC and TNBC Ras-dependent tumors bearing either mutant or wild-type KRAS. This work supports further development of RRSP as an anticancer therapeutic for a broad spectrum of Ras-driven tumors and has the potential to take anti-Ras therapy to the next level. Citation Format: Vania Vidimar, Greg L Beilhartz, Roman A Melnyk, Karla JF Satchell. Ras processing by RRSP protease as a strategy to inhibit Ras-driven tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C031. doi:10.1158/1535-7163.TARG-19-C031

Structural basis of inactivation of Ras and Rap1 small GTPases by Ras/Rap1-specific endopeptidase from the sepsis-causing pathogen Vibrio vulnificus

Multifunctional autoprocessing repeats-in-toxin (MARTX) toxins are secreted by Gram-negative bacteria and function as primary virulence-promoting macromolecules that deliver multiple cytopathic and cytotoxic effector domains into the host cytoplasm. Among these effectors, Ras/Rap1-specific endopeptidase (RRSP) catalyzes the sequence-specific cleavage of the Switch I region of the cellular substrates Ras and Rap1 that are crucial for host innate immune defenses during infection. To dissect the molecular basis underpinning RRSP-mediated substrate inactivation, we determined the crystal structure of an RRSP from the sepsis-causing bacterial pathogen Vibrio vulnificus (VvRRSP). Structural and biochemical analyses revealed that VvRRSP is a metal-independent TIKI family endopeptidase composed of an N-terminal membrane-localization and substrate-recruitment domain (N lobe) connected via an inter-lobe linker to the C-terminal active site–coordinating core β-sheet–containing domain (C lobe). Structure-based mutagenesis identified the 2His/2Glu catalytic residues in the core catalytic domain that are shared with other TIKI family enzymes and that are essential for Ras processing. In vitro KRas cleavage assays disclosed that deleting the N lobe in VvRRSP causes complete loss of enzymatic activity. Endogenous Ras cleavage assays combined with confocal microscopy analysis of HEK293T cells indicated that the N lobe functions both in membrane localization via the first α-helix and in substrate assimilation by altering the functional conformation of the C lobe to facilitate recruitment of cellular substrates. Collectively, these results indicate that RRSP is a critical virulence factor that robustly inactivates Ras and Rap1 and augments the pathogenicity of invading bacteria via the combined effects of its N and C lobes.

Farnesyltransferase-Mediated Delivery of a Covalent Inhibitor Overcomes Alternative Prenylation to Mislocalize K-Ras.

Mutationally activated Ras is one of the most common oncogenic drivers found across all malignancies, and its selective inhibition has long been a goal in both pharma and academia. One of the oldest and most validated methods to inhibit overactive Ras signaling is by interfering with its post-translational processing and subsequent cellular localization. Previous attempts to target Ras processing led to the development of farnesyltransferase inhibitors, which can inhibit H-Ras localization but not K-Ras due to its ability to bypass farnesyltransterase inhibition through alternative prenylation by geranylgeranyltransferase. Here, we present the creation of a neo-substrate for farnesyltransferase that prevents the alternative prenlation by geranylgeranyltransferase and mislocalizes oncogenic K-Ras in cells.

Gene Essentiality Profiling Reveals Gene Networks and Synthetic Lethal Interactions with Oncogenic Ras

The genetic dependencies of human cancers widely vary. Here, we catalog this heterogeneity and use it to identify functional gene interactions and genotype-dependent liabilities in cancer. By using genome-wide CRISPR-based screens, we generate a gene essentiality dataset across 14 human acute myeloid leukemia (AML) cell lines. Sets of genes with correlated patterns of essentiality across the lines reveal new gene relationships, the essential substrates ofenzymes, and the molecular functions of uncharacterized proteins. Comparisons of differentially essential genes between Ras-dependent and -independent lines uncover synthetic lethal partners of oncogenic Ras. Screens in both human AML and engineered mouse pro-B cells converge on a surprisingly small number of genes in the Ras processing and MAPK pathways and pinpoint PREX1 as an AML-specific activator of MAPK signaling. Our findings suggest general strategies for defining mammalian gene networks and synthetic lethal interactions by exploiting the natural genetic and epigenetic diversity of human cancer cells.

Improving Prospects for Targeting RAS.

RAS mutations are among the most common oncogenic drivers in human cancers, affecting nearly a third of all solid tumors and around a fifth of common myeloid malignancies, but they have evaded therapeutic interventions, despite being the focus of intense research over the last three decades. Recent discoveries lend new understanding about the structure, function, and signaling of RAS and have opened new avenues for development of much needed new therapies. We discuss the various approaches under investigation to target mutant RAS proteins. The recent development of direct RAS inhibitors specific to KRAS G12C mutations represents a landmark discovery that promises to change the perception about RAS's druggability. Multiple clinical trials targeting synthetically lethal partners and/or downstream signaling partners of RAS are underway. Novel inhibitors targeting various arms of RAS processing and signaling have yielded encouraging results in the laboratory, but refinement of the drug-like properties of these molecules is required before they will be ready for the clinic.

Characterization of RAS Alterations in Myeloma: Why Direct Targeting of RAS May be the Most Appropriate Therapeutic Approach

Recent studies have shown that the RAS pathway is a major therapeutic target with which to develop personalized treatment strategies for myeloma patients. In order to translate this knowledge into patient benefit, we have taken a systematic approach by characterizing the mutational profile of the RAS pathway and examining its role in disease progression and patient outcome. Finally we investigated a targeted treatment approach using small molecules to inhibit the RAS MAPK signalling cascade at different levels.In order to gain a deeper understanding of RAS pathway alterations in myeloma, next-generation sequencing data was analyzed from 463 patients entered into the MRC Myeloma XI trial. Using published papers and online databases, a panel of 72 genes were identified as RAS MAPK family genes and were grouped into 5 categories: RAS GTPases, guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), downstream pathway or RAS processing genes. Thirty seven of these genes were mutated in 50% of patients, with novel mutations described for genes in each of these categories. The mutation frequency of KRAS (22.5%), NRAS (19.7%) and BRAF (7.8%) was in-line with other reports and KRAS was found mutated at more codons compared to NRAS. Additionally, recurrent mutations at the same codon were identified only in KRAS, NRAS and BRAF, attesting to their importance in this disease. A quarter of patients (n=59) had more than one RAS MAPK mutation. When more than one mutation was present, the majority were in two RAS GTPases (n=19) (most commonly KRAS and NRAS (n=8) or both in KRAS (n=6)) or in a RAS GTPase and a downstream pathway gene (n=12). One patient had mutations in KRAS, NRAS and BRAF but at sub-clonal levels, indicating they may be present in different cells.We also assessed the role of this pathway in myeloma by examining gene expression profiling data from plasma cells at various myeloma disease stages (GSE2113, GSE5900 and GSE6477). This analysis highlighted roles for KRAS, RAF1, MAPK1, MAP2K1, NF1, RASGRP1 and FNTA in disease progression from MGUS to PCL. Additionally, survival analysis suggested important roles for MAPK1 in determining outcome in newly diagnosed patients in the HOVON65/GMMG-HD4 (GSE19784) trial and FNTA, KRAS, MAP2K1 and RAF1 in determining outcome in relapsed patients in the APEX (GSE9782) trial. These results suggest the highlighted genes are important in mediating clinical outcome and could be therapeutic targets.Given the biological importance of this pathway, we have explored its therapeutic potential by using inhibitors that target the pathway at different levels. Inhibitors included those targeting RAS (n=2), RAF1 (n=2), RAF1/BRAF (n=2), MEK (n=2) and ERK (n=1). The effect of these inhibitors was explored in a panel of 8 myeloma cell lines chosen based on their differing RAS mutational status. Using Western blot analysis, all cell lines showed evidence of activation of the RAS MAPK pathway. No consistent pattern of expression was observed in terms of the levels of activated pathway proteins in KRAS mutant cells compared to NRAS or non-mutated cells. In contrast NRAS and BRAF mutant cells contained elevated levels of activated MEK compared to the other cell lines and were the most sensitive to a MEK inhibitor. Interestingly all cell lines, regardless of mutational status, were similarly sensitive to a novel KRAS inhibitor which prevents KRAS reaching its correct localization. It was shown to be effective at inducing apoptosis, even in the presence of the bone marrow stromal cells. Importantly, protein analysis demonstrated these effects were target-driven with Western blot analysis indicating inhibition of RAS signalling within two to six hours of treatment. Thus, the direct targeting of RAS early in the signalling cascade may be effective against myeloma, irrespective of the RAS mutation status. DisclosuresWalker:Onyx Pharmaceuticals: Consultancy, Honoraria.

National Cancer Institute focuses on RAS: Effort aims for better understanding, targeting of genes

National Cancer Institute focuses on <i>RAS</i>: Effort aims for better understanding, targeting of genes

Progress in Making Ras as a Druggable Target.

Progress in Making Ras as a Druggable Target.

Enhanced Antiproliferative and Apoptotic Response of HT-29 Adenocarcinoma Cells to Combination of Photoactivated Hypericin and Farnesyltransferase Inhibitor Manumycin A

Several photodynamically-active substances and farnesyltransferase inhibitors are currently being investigated as promising anticancer drugs. In this study, the combined effect of hypericin (the photodynamically-active pigment from Hypericum perforatum) and selective farnesyltransferase inhibitor manumycin (manumycin A; the selective farnesyltransferase inhibitor from Streptomyces parvulus) on HT-29 adenocarcinoma cells was examined. We found that the combination treatment of cells with photoactivated hypericin and manumycin resulted in enhanced antiproliferative and apoptotic response compared to the effect of single treatments. This was associated with increased suppression of clonogenic growth, S phase cell cycle arrest, elevated caspase-3/7 activity and time-dependent total cleavage of procaspase-3 and lamin B, cleavage of p21Bax into p18Bax and massive PARP cleavage. Moreover, we found that the apoptosis-inducing factor is implicated in signaling events triggered by photoactivated hypericin. Our results showed the relocalization of apoptosis-inducing factor (AIF) to the nuclei after hypericin treatment. In addition, we discovered that not only manumycin but also photoactivated hypericin induced the reduction of total Ras protein level. Manumycin decreased the amount of farnesylated Ras, and the combination treatment decreased the amount of both farnesylated and non-farnesylated Ras protein more dramatically. The present findings indicate that the inhibition of Ras processing may be the determining factor for enhancing the antiproliferative and apoptotic effects of combination treatment on HT-29 cells.

Processing the document flow at branch departments of scientific information at VINITI RAS and an interpretation of this work within the framework of physical ideas

This paper considers a document flow moving in the form of a continuum in virtual space. The methods of continuum dynamics are used to study it. A flow has various attributes, viz., volume, viscosity, and current velocity under pressure. The viscosity is determined by the laboriousness of processing the wide range of documents that are present in this virtual flow and the pressure is determined by the quantity of employees that are occupied with processing. The obtained mathematical model permits one to make conclusions about the necessary quantity of employees for the processing of the assigned document flow and the rate of document processing. It is noted that the departments for scientific information processing of VINITI RAS are staffed at 20---30% of the necessary level, which makes it impossible to accelerate the term of information processing and to increase the quality of the processing itself.

USP17 Regulates Ras Activation and Cell Proliferation by Blocking RCE1 Activity

The proto-oncogene Ras undergoes a series of post-translational modifications at its carboxyl-terminal CAAX motif that are essential for its proper membrane localization and function. One step in this process is the cleavage of the CAAX motif by the enzyme Ras-converting enzyme 1 (RCE1). Here we show that the deubiquitinating enzyme USP17 negatively regulates the activity of RCE1. We demonstrate that USP17 expression blocks Ras membrane localization and activation, thereby inhibiting phosphorylation of the downstream kinases MEK and ERK. Furthermore, we show that this effect is caused by the loss of RCE1 catalytic activity as a result of its deubiquitination by USP17. We also show that USP17 and RCE1 co-localize at the endoplasmic reticulum and that USP17 cannot block proliferation or Ras membrane localization in RCE1 null cells. These studies demonstrate that USP17 modulates Ras processing and activation, at least in part, by regulating RCE1 activity.

Targeting RAS Signaling Pathways in Juvenile Myelomonocytic Leukemia

The RAS proteins function as fundamental signaling switches that control normal cell growth and differentiation. Deregulated activation of RAS-dependent signaling pathways constitutes a potent mechanism of malignant cell transformation. Juvenile myelomonocytic leukemia (JMML) is a rapidly fatal myeloproliferative disorder of early childhood for which no effective treatment other than hematopoietic stem cell transplantation is currently available. Many aspects of JMML pathobiology are linked to deregulated RAS signaling. Hence, targeting RAS or its interactors on a molecular level is a promising strategy in the development of novel rational therapies for this menacing disease. Here we give an overview of current concepts on the pathogenesis of JMML, present important aspects of cellular RAS biology that can be exploited for pharmacologic manipulation, and discuss mouse models that have greatly advanced our understanding of the role RAS plays in JMML. In addition, we review recent approaches to develop agents that interfere with the RAS network at the level of the granulocyte-macrophage colony-stimulating factor receptor, posttranslational RAS processing (prenylation and endoprotease cleavage), RAF serine/threonine kinase, MEK mitogen-activated protein kinase, and target of rapamycin activity. Preclinical and clinical data of these pharmaceuticals in JMML and other myeloid malignancies is discussed.

Pancreatic Cancer Cell Radiation Survival and Prenyltransferase Inhibition: The Role of K-Ras

Activating K-ras mutations are found in approximately 90% of pancreatic carcinomas and may contribute to the poor prognosis of these tumors. Because radiotherapy is frequently used in pancreatic cancer treatment, we assessed the contribution of oncogenic K-ras signaling to pancreatic cancer radiosensitivity. Seven human pancreatic carcinoma lines with activated K-ras and two cell lines with wild-type ras were used to examine clonogenic cell survival after Ras inhibition. Ras inhibition was accomplished by small interfering RNA (siRNA) knockdown of K-ras expression and by blocking Ras processing using a panel of prenyltransferase inhibitors of differing specificity for the two prenyltransferases that modify K-Ras. K-ras knockdown by siRNA or inhibition of prenyltransferase activity resulted in radiation sensitization in vitro and in vivo in tumors with oncogenic K-ras mutations. Inhibition of farnesyltransferase alone was sufficient to radiosensitize most K-ras mutant tumors, although K-Ras prenylation was not blocked. These results show that inhibition of activated K-Ras can promote radiation killing of pancreatic carcinoma in a superadditive manner. The finding that farnesyltransferase inhibition alone radiosensitizes tumors with K-ras mutations implies that a farnesyltransferase inhibitor-sensitive protein other than K-Ras may contribute to survival in the context of mutant K-ras. Farnesyltransferase inhibitors could therefore be of use as sensitizers for pancreatic carcinoma radiotherapy.

Benzimidazolones and indoles as non-thiol farnesyltransferase inhibitors based on tipifarnib scaffold: synthesis and activity

A series of analogs of tipifarnib ( 1) has been synthesized as inhibitors of FTase by substituting the benzimidazolones and indoles for the 2-quinolone of tipifarnib. The novel benzimidazolones are potent and selective FTase inhibitors (FTIs) with IC 50 values of the best compounds close to that of tipifarnib. The current series demonstrate good cellular activity as measured in their inhibiting the Ras processing in NIH-3T3 cells, with compounds 2c and 2f displaying EC 50 values of 18 and 22 nM, respectively.

Lovastatin alters the isoprenoid biosynthetic pathway in acute myelogenous leukemia cells in vivo

Lovastatin, a competitive inhibitor of hydroxymethylglutaryl coenzyme A reductase (HMGR), is used therapeutically to lower plasma cholesterol levels and has garnered attention for its cytotoxic effects in leukemia cells. In this study, escalating doses of lovastatin were administered to nine patients with acute myelogenous leukemia. Peripheral blood leukemia cells were drawn pre- and post-lovastatin dosing. Plasma lovastatin bioactivity ranged up to 234 nM lovastatin equivalents. Our results show that in vivo lovastatin, at up to 200 mg/dose, induces an increased activity of leukemia cell HMGR and alters leukemia cell proliferation without discernibly altering Ras processing.

Développement des inhibiteurs de farnésyl transférase comme agents anticancéreux

Ras proteins belong to the monomeric GTPases familly. They control cell growth, differentiation, proliferation, and survival. Ras mutations are frequently found in human cancers and play a fundamental role in tumorigenesis. Ras requires localization to the plasma membrane to exert its oncogenic effects. This subcelllular localization is dependent of protein farnesylation which is a post translational modification catalysed by the farnesyl transferase enzyme. Farnesyl transferase Inhibitors (FTI) were then designed ten to twelve years ago to inhibit ras processing and consequently the growth of ras mutated tumor. Preclinical data show that FTIs inhibit cell proliferation and survival in vitro and in vivo of a wide range of cancer cell lines, many of which contain wild type ras suggesting that mutated Ras is not the only target of the FTIs effects. Four FTIs went then through clinical trials and three of then are still developed in the clinic. Phase I et II clinical trials confirmed a relevant antitumor activity and a low toxicity. Phase III clinical trials are currently undergoing for both solid and hematologic tumors. The expected results should allow to define the position of FTIs as anticancer drugs, particularly in combination with conventional chemotherapy, hormone therapy, radiotherapy or any other new targeted compound.

Targeting RAS Signaling in Multiple Myeloma (MM): Synergistic Inhibition by Co-Treatment with the MEK Inhibitor U0126 and Prenylation Inhibitors.

Multiple myeloma (MM) is a fatal hematological malignancy associated with disruption of RAS-to-MAP kinase (MAPK/ERK) signaling. Prenylation inhibitors such as farnesyltransferase inhibitors (FTIs) and the competitive HMG-CoA reductase inhibitor lovastatin have been shown to block RAS post-translational modification and thus transforming ability. Targeting down-stream members of the RAS signaling cascade with compounds such as the MEK inhibitor U0126 is another strategy used to successfully disrupt oncogenic RAS signal transduction. In order to assess the effects of co-treating MM cells with U0126 and prenylation inhibitors (e.g. FTI L-744,832 and lovastatin), six MM cell lines were assayed for viability using the MTT assay, induction of apoptosis by TUNEL assay, and MEK activation by a phospho-MEK-specific FACS assay. Treated cells were also subjected to Western blotting to determine levels of phosphorylated MAP kinase as well as processed and unprocessed RAS and RAS-related proteins. Co-treating MM cells with U0126 and FTI L-744,832 or lovastatin synergistically inhibited MM cell proliferation. Incubation of NCI-H929 cells with U0126 caused a concentration dependent increase of cells in the G0/G1 phase of the cell cycle. Accordingly, 48 h treatment of NCI-H929 cells with 20 μM U0126 caused a reduction in activated MEK in both G0/G1 and G2/M cell cycle phases (41.9 and 39.5%, respectively) while 50 μM reduced activated MEK levels by 64.8% in G0/G1 and 74.5% in G2/M. Lovastatin treatment (48 h, 10 μM) of NCI-H929 led to an accumulation of cells at the G1/S boundary of the cell cycle and >90% loss of activated MEK in both G0/G1 and G2/M. Lovastatin-induced changes in cell cycle distribution and loss of MEK activation could not be rescued by farnesyl pyrophosphate, but were restored to levels similar to those measured in untreated cells by geranylgeranyl pyrophosphate or mevalonate. Co-treating OPM-2 cells 48 h with 10 μM lovastatin and 50 μM U0126 led to >96% decrease in activated MEK in G0/G1 and >76% reduction in G2/M. Lovastatin treatment also caused a loss of prenylated RAS and RAS-family proteins as demonstrated by Western blot analyses. Additionally, Western blotting showed that U0126 treatment (5 μM, 30 min) alone or in combination with lovastatin (5 μM, 30 min) blocked IL-6 stimulated activation of MAP kinase in OPM-2 cells but had no effect on pAKT levels. Our results support that inhibition of RAS processing and down-stream signaling are the major mechanisms of action through which U0126 and lovastatin synergistically inhibit MM cell growth. Furthermore, these data demonstrate the potential therapeutic usefulness of these compounds in the treatment of multiple myeloma.

Design and synthesis of o-trifluoromethylbiphenyl substituted 2-amino-nicotinonitriles as inhibitors of farnesyltransferase

A non-methionine FT inhibitor lead structure ( 1) was designed through computer modeling of the peptidomimetic FT inhibitor ABT839. Optimization of this lead resulted in compounds 2e and 2g, with FT IC 50 values of 1.3 and 1.8 nM, GGT IC 50 of 1400 nM, and EC 50 ( Ras processing) values of 13 and 11 nM, respectively.

Apoptotic and cytostatic farnesyltransferase inhibitors have distinct pharmacology and efficacy profiles in tumor models.

BMS-214662 and BMS-225975 are tetrahydrobenzodiazepine-based farnesyltransferase inhibitors (FTIs) that have nearly identical structures and very similar pharmacological profiles associated with farnesyltransferase (FT) inhibition. Despite their similar activity against FT in vitro and in cells, these compounds differ dramatically in their apoptotic potency and tumor-regressing activity in vivo. BMS-214662 is the most potent apoptotic FTI known and exhibits curative responses in mice bearing a variety of staged human tumor xenografts such as HCT-116 human colon tumor. By contrast, BMS-225975 does not cause tumor regression and at best causes partial tumor growth inhibition in staged HCT-116 human colon tumor xenografts. Lack of tumor regression activity in BMS-225975 was attributable to its relatively weak apoptotic potency, not to poor cell permeability or pharmacokinetics. Both compounds were equally effective in inhibiting Ras processing and causing accumulation of a variety of nonfarnesylated substrates of FT in HCT-116 cells. Because BMS-225975 has poor apoptotic activity compared with BMS-214662 but inhibits FT to the same extent as BMS-214662, it is very unlikely that FT inhibition alone can account for the apoptotic potency of BMS-214662. Clearly distinct patterns of sensitivities in a cell line panel were obtained for the apoptotic FTI BMS-214662 and the cytostatic FTI BMS-225975. Activation of the c-Jun-NH(2)-terminal kinase pathway was readily observed with BMS-214662 but not with BMS-225975. We developed a highly sensitive San-1 murine xenograft tumor model that is particularly useful for evaluating the in vivo activity of cytostatic FTIs such as BMS-225975.