Farnesyltransferase Inhibitors Target Research Articles

The CXCL12/CXCR4 Pathway As a Potential Target of Tipifarnib in Acute Myeloid Leukemia and Myelodysplastic Syndromes

BackgroundCXCL12 is a chemokine that is essential for homing of myeloid cells in the bone marrow (BM). It has been postulated that a CXCL12 receptor present in myeloid cells, CXCR4, signals in part through HRAS, a protein that is uniquely dependent on farnesylation for activity. Tipifarnib is a potent and selective inhibitor of the enzyme farnesyltransferase (FT) that had been shown to provide clinical benefit in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS); however, no molecular target that could explain the activity of tipifarnib in AML and MDS has been identified. Based on initial observations in T cell lymphoma (Witzig EHA 2017), we investigated a role for CXCL12 and myeloid cell homing in BM as biomarkers of the activity of tipifarnib in AML and MDS studies.MethodsCTEP20 was a phase 2, multicenter, open-label study that investigated the efficacy and safety of tipifarnib in 158 older adults with previously untreated, poor-risk AML. AML301 was a phase 3 (N=457), multicenter, open-label study that evaluated the efficacy and safety of tipifarnib compared with best supportive care (BSC), including hydroxyurea, as first-line therapy in elderly patients with newly diagnosed, de novo, or secondary AML. INT28 was a multicenter phase 2 study that evaluated tipifarnib in 82 pts with poor-risk MDS. Global gene expression data generated using the Affymetrix U133A gene chip from 34 pretreatment BM samples collected from CTEP20 pts (GSE8970) was analyzed with respect to pretreatment hematological parameters and study outcomes. The Kaplan-Meier method was employed to estimate survival and progression free survival (PFS) and the analysis of prognostic variables. Clinical trial information: NCT00027872, NCT00093990, NCT00050154.ResultsNine complete responses (CR) and 13 best responses of progression of disease (PD) were observed in 34 pts with available BM gene expression data in study CTEP20. Pts in the 3rd (upper) tertile of CXCL12 expression experienced 6 CRs and 2 PDs whereas 1 CR and 8 PDs were observed in the 1st (lower) tertile (p=0.015). BM homing of AML blasts correlated with CXCL12 expression: Median percentage of BM blasts (BMB) and peripheral blasts (PB) were, respectively, 45% and 0% in the upper CXCL12 tertile, and 30% and 26.5% in the lower tertile (p<0.001) (Figure, A). Pts with high tumor burden in BM (BMB blasts>40%) and low circulating blasts (PB<1%) (N=5) had high CXCL12 expression in BM (3423 vs 742 counts, P<0.001, N=27) and experienced prolonged clinical benefit, with 5 CRs and a median PFS of 431 days (431 vs 35 days, HR=0.23, P<0.001, N=27) (Figure, B). The effect of BM homing was then investigated in the phase 3 AML301 trial of tipifarnib vs BSC. While the overall study (N=457) failed to identify an increased efficacy of tipifarnib compared with BSC (median survival of 109 vs 107 days, hazards ratio, HR=1.02, ns), median survival approximately doubled in the tipifarnib am compared with BSC in the subset of patients with high BMB (>40%) and low PB (<10%) while no effect was observed in the control arm (201 vs 109 days, HR=0.58, p=0.02, N=82) (Figure, B). Since CXCL12 is also a potent chemotactic factor for normal neutrophils and syndromes of hyperactivation of the CXCL12/CXCR4 pathway present with neutropenia, we investigated whether neutropenia could be a surrogate of tipifarnib activity. In AML301 pts with WBC counts>LLN (no myelosuppression), increasing neutropenia correlated with increasing clinical benefit from tipifarnib that was due in part to a poorer prognosis of BSC in neutropenic pts (113 vs 80 days median OS, HR=0.81, p=0.036, N=109, for pts with baseline neutrophils<1 giga/L). Finally, we tested the effect of neutropenia on the activity of tipifarnib in INT28. Sixty seven of the 82 MDS pts were transfusion dependent (TD) at study entry. Overall conversion to transfusion independence (TI) and response rate (ORR: CR, CRp, PR) were respectively 16% and 7.5% in the TD pts. Pts with >1 giga/L neutrophils at study entry experienced 5% TI and no responses (N=37) while 30% TI, 2 CRs, 2 CRp and 1 PR were observed in the subset of neutropenic pts.ConclusionCXCL12 expression and BM homing constitute potential biomarkers of the activity of tipifarnib in AML and MDS supporting the hypothesis that the CXCL12 pathway is a potential target of FT inhibitors. [Display omitted] DisclosuresGualberto:Kura Oncology: Employment, Equity Ownership, Other: Chief Medical Officer, Patents & Royalties. Scholz:Kura Oncology: Employment, Equity Ownership, Patents & Royalties. Janes:Wellspring Biosciences: Employment. Kessler:Kura Oncology: Employment. Raza:Kura Oncology: Research Funding; Syros Pharmaceuticals: Research Funding; Novartis: Speakers Bureau; Celgene Inc.: Research Funding; Genoptix: Speakers Bureau; Onconova Therapeutics: Research Funding, Speakers Bureau; Janssen R&D: Research Funding.

Neoadjuvant Therapy with Drug Arglabin for Breast Cancer with Expression of H-Ras Oncoproteins.

Backgrounds:In breast cancer, blocking of Ras signaling and inhibition of H-Ras is quite promising. H-Ras may become a target for farnesyl transferase inhibitors, and in combination with other immunohistochemical factors it will contribute to the progression of a breast tumor. Purpose:The aim of this study was to evaluate the effectiveness of neoadjuvant therapy for breast cancer with the inclusion of farnesyl transferase inhibitor, arglabin interfering with the expression and concentration of H-Ras oncoproteins. Methods:Depending on the presence of H-Ras oncoproteins after Western-blot hybridization, the patients were divided a negative and positive expression of H-Ras groups. Results:Correlation analysis of methods used for determining the expression ability and concentration of H-Ras oncoproteins (immunohistochemistry and Western-blot analysis) demonstrated substantial statistical relationship Rs=0.71, p=0.03. The H-Ras oncoproteins were absent in patients receiving either “Arglabin” or standard AC regimen. However, in the AC + Arglabin group, there was a varying degrees of positive concentration of H-Ras oncoproteins (Kruskal-Wallis=6.92; p=0.03). Conclusion:These results indicate that Arglabin attenuates H-Ras oncoproteins expression which is a promising therapeutic target for breast cancer.

Inhibitors Of Farnesyltransferase and Everolimus Act Synergistically In Growth Inhibition Of Human T-NHL Cells By Involvement Of AMPK

IntroductionThe outcome of patients with T cell lymphoma treated with standard chemoterapeutic substances remain poor, making the search for new active substances a highly medical need in this hematologic neoplasia. Recent phase II clinical trials showed very promising activity of farnesyltransferaseinhibitors (FTI) in relapsed/refractory T-NHL patients (Witzig et al. 2011). Regarding the molecular mechanisms behind this therapeutic effect, conflicting data regarding Ras as the initially proposed intracellular target of FTI and the involvement of MAP kinases in cellular effects of FTI in T cells exist (Marks et al. 2007, Ding et al. 2011). Together with observations in breast and ovarian cancer cells suggesting the GTPase Rheb as target for inhibition of farnesylation (Basso et al. 2005), the targets of FTI might vary according to the examined cell type. Interestingly, in breast cancer cells FTI mediatied inhibition of Rheb action resulted in reduced mTOR signaling. Nevertheless, as a putative additional targeted treatment approach in T-NHL, incubation with mTOR inhibitors showed not only substantial antiproliferative effects in normal T cells but also in malignant human T cell lymphoma lines in vitro (Huang et al. 2010). Since further clinical trials with both substances did not show severe side effects, adding everolimus as combination partner might even enhance clinical activity of FTI in T cell lymphomas. Therefore, in order to test this hypothesis and to analyse if both substances differ in their molecular mechanisms of action, FTI and everolimus were tested in vitro in T cell lymphoma lines (Karpas, Derl-2, Jurkat) to evaluate potential synergistic modes of action. Methods and ResultsIncubation of human T cell lymphoma lines Karpas and Derl-2 with the FTI SCH66336 (lonafarnib) or the mTOR inhibitor everolimus showed a reduction in proliferation in a dose dependent manner (EC50 for everolimus: 0.1nM, EC50 for lonafarnib: 0.5 µM). Combining both drugs resulted in synergistic inhibition of proliferation. This inhibitory effect correlated with increased p27KIP1 expression. In our experiments, Rheb appeared to be highly expressed in all examined T cell lymphoma lines with even additional increase of protein expression in Karpas cells after FTI incubation. Comparing FTI action to inhibition of mTOR by everolimus on a molecular level, in our experiments lonafarnib treatment of Karpas cells resulted in an unexpected reduction in AMPK-phosphorylation, implicating involvement of this metabolic pathway in FTI mediated inhibition of proliferation in malignant T cells. This effect could not be observed in everolimus treated Karpas cells. In contrast, naive human CD4+ T cells showed very little Rheb protein expression, which could be significantly increased after TCR stimulation by induction of Rheb mRNA transcription. While everolimus treatment of TCR-activated normal human CD4+ T cells resulted in AKT-hyperphosphorylation, FTI did not induce any changes in AKT. Contrary to the malignant T cells, FTI treatment had no impact of AMPK phosphorylation in activated T cells. Actually, naive T cells treated with FTI showed an hyperphosphorylated AMPK status. ConclusionLonafarnib and everolimus show synergistic antiproliferative effects in T cell lymphoma lines, most likely by interfering with mTOR and AMPK signalling, making this combination therapy interesting for clinical trials. In contrast, FTI does not mediate AMPK in activated normal T cells. This observations are in accordance with a differential targeting of Rheb by FTI in malignant or normal human T cells. Disclosures:No relevant conflicts of interest to declare.

Protein farnesylation inhibitors cause donut-shaped cell nuclei attributable to a centrosome separation defect

Despite the success of protein farnesyltransferase inhibitors (FTIs) in the treatment of certain malignancies, their mode of action is incompletely understood. Dissecting the molecular pathways affected by FTIs is important, particularly because this group of drugs is now being tested for the treatment of Hutchinson-Gilford progeria syndrome. In the current study, we show that FTI treatment causes a centrosome separation defect, leading to the formation of donut-shaped nuclei in nontransformed cell lines, tumor cell lines, and tissues of FTI-treated mice. Donut-shaped nuclei arise during chromatin decondensation in late mitosis; subsequently, cells with donut-shaped nuclei exhibit defects in karyokinesis, develop aneuploidy, and are often binucleated. Binucleated cells proliferate slowly. We identified lamin B1 and proteasome-mediated degradation of pericentrin as critical components in FTI-induced "donut formation" and binucleation. Reducing pericentrin expression or ectopic expression of nonfarnesylated lamin B1 was sufficient to elicit donut formation and binucleated cells, whereas blocking proteasomal degradation eliminated FTI-induced donut formation. Our studies have uncovered an important role of FTIs on centrosome separation and define pericentrin as a (indirect) target of FTIs affecting centrosome position and bipolar spindle formation, likely explaining some of the anticancer effects of these drugs.

Abstract P4-06-20: The Role of Aktl in RhoC GTPase-Mediated Inflammatory Breast Cancer Invasion

Abstract Inflammatory breast cancer (IBC) is a highly aggressive form of locally advanced breast cancer, distinguished from other types of breast cancer by clinical, pathologic, and molecular features. It is clinically distinguished by rapid onset of primary skin changes, including redness of the skin, nipple retraction, and peau d’ aurange. Inflammatory breast cancer accounts for approximately 6-9% of new breast cancers in the United States annually and although the number of women affected by IBC is relatively modest, the overall the 5- and 10-year disease-free survival rates are less than 45% and 20%, respectively, making IBC the most lethal form of breast cancer. Evidence has suggested a role for the phosphoinositide 3-kinase/Akt (Protein Kinase B) signaling pathway in cellular motility, invasion, and metastasis. Recent evidence has shown activation of Akt2 (PKBβ) in breast cancer and its involvement in metastasis. However, the opposite appears to be true in inflammatory breast cancer (IBC), where Akt1 is involved in cellular motility and invasion. We have demonstrated a role for RhoC in conferring a highly invasive phenotype to IBC cells and are suggesting here that Akt1 phosphorylation of RhoC leads to cellular invasion. Farnesyltransferase inhibitors (FTIs) interfere with GTPase farnesylation and activity and we have shown that FTI treatment of SUM149 IBC cells leads to inhibition of the RhoC GTPase-induced invasive phenotype of IBC. RhoC is not a direct target of FTI action, however, its relative, RhoB, is a putative FTI target. It has been shown that FTI inhibition of farnesylated RhoB (fRhoB) leads to an accumulation of geranylgeranylated RhoB (ggRhoB) in the cell, which can interfere with GTPase signals and mimics the effects of FTI in IBC cells. RhoB GTPase, particularly ggRhoB, is an intracellular trafficking protein that regulates cellular localization of Akt1, thus affecting its activation. In this present study our primary objective was to examine the role of Akt1 and RhoC phosphorylation in IBC cell invasion as well as the localization of these various molecules within IBC cells upon FTI treatment. The SUM149 inflammatory breast cancer cell line overexpresses RhoC, lacks the PTEN gene, and consequently expresses a constitutively active Akt. Using the SUM149 cell line, we demonstrate that RhoC is a reputed substrate for Akt phosphorylation. We are able to decrease phospho-RhoC levels and inhibit SUM149 cellular invasion through inhibiting Akt1 activation with commercially available Akt inhibitors and siAkt1. Through immunofluorescence and protein localization experiments we are able to see a shift of phosphorylated Akt1 from the cell cytoplasm to the nucleus upon FTI treatment. We also observe accumulation of ggRhoB over time with localization of Akt1 away from the plasma membrane. These results suggest that Akt, specifically Akt1, phosphorylation of RhoC in inflammatory breast cancer cells is required for cellular invasion. We are also able to suggest that FTI treatment of IBC cells leads to increased ggRhoB levels, which may lead to changes in localization of proteins that will affect RhoC GTPase and the metastatic IBC phenotype. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-06-20.

Rho Family GTPase Modification and Dependence on CAAX Motif-signaled Posttranslational Modification

Rho GTPases (20 human members) comprise a major branch of the Ras superfamily of small GTPases, and aberrant Rho GTPase function has been implicated in oncogenesis and other human diseases. Although many of our current concepts of Rho GTPases are based on the three classical members (RhoA, Rac1, and Cdc42), recent studies have revealed the diversity of biological functions mediated by other family members. A key basis for the functional diversity of Rho GTPases is their association with distinct subcellular compartments, which is dictated in part by three posttranslational modifications signaled by their carboxyl-terminal CAAX (where C represents cysteine, A is an aliphatic amino acid, and X is a terminal amino acid) tetrapeptide motifs. CAAX motifs are substrates for the prenyltransferase-catalyzed addition of either farnesyl or geranylgeranyl isoprenoid lipids, Rce1-catalyzed endoproteolytic cleavage of the AAX amino acids, and Icmt-catalyzed carboxyl methylation of the isoprenylcysteine. We utilized pharmacologic, biochemical, and genetic approaches to determine the sequence requirements and roles of CAAX signal modifications in dictating the subcellular locations and functions of the Rho GTPase family. Although the classical Rho GTPases are modified by geranylgeranylation, we found that a majority of the other Rho GTPases are substrates for farnesyltransferase. We found that the membrane association and/or function of Rho GTPases are differentially dependent on Rce1- and Icmt-mediated modifications. Our results further delineate the sequence requirements for prenyltransferase specificity and functional roles for protein prenylation in Rho GTPase function. We conclude that a majority of Rho GTPases are targets for pharmacologic inhibitors of farnesyltransferase, Rce1, and Icmt.

Targeted therapies in head and neck cancer

Over the past decade, the important role of different growth factors and their receptors and signal transduction pathways in the genesis and progression of tumors has been well recognized and their mechanism of action and interactions is gradually being unraveled. Epidermal growth factor receptor (EGFR) overexpression is present in the vast majority of squamous cell head and neck cancers and carries a worse prognosis. EGFR is the target of multiple specifically targeted monoclonal antibodies and tyrosine kinases, which are in various stages of clinical development in squamous cell carcinoma of the head and neck (SCCHN). The search for EGFR mutations is an area of active investigation. The incidence and impact of EGFR mutations in SCCHN has yet to be determined. EGFR downstream signaling pathways are the target of farnesyltransferase inhibitors (FTIs) and mammalian target of rapamycin (mTOR) inhibitors. Cyclooxygenase-2 (COX-2) is overexpressed in premalignant lesions (oral leukoplakia) and in squamous cell carcinoma of the head and neck. EGFR and COX-2 signaling pathways form a positive feedback loop. As their toxicity profiles are non-overlapping, combination of COX-2 inhibitors and EGFR targeted therapies looks attractive. The majority of the studies, although not all, examining the prognostic significance of vascular endothelial growth factor (VEGF) expressing did observe a worse outcome in patients with SCCHN expressing VEGF and VEGF receptor 2 (VEGFR-2). Anti-VEGF strategies include neutralizing antibodies to VEGF or VEGFR and VEGFR tyrosine kinase inhibitors. Aurora kinase inhibitors, insulin like growth factor inhibitors, and histone acetylase inhibitors have recently gained interest as potential new promising ways of tackling tumors including SCCHN.

HDJ-2 as a Target for Radiosensitization of Glioblastoma Multiforme Cells by the Farnesyltransferase Inhibitor R115777 and the Role of the p53/p21 Pathway

Resistance of glioblastoma multiforme to radiotherapy poses a major clinical challenge. Farnesyltransferase inhibitors (FTI), such as R115777, have potential to increase radiotherapeutic benefit in this disease, although their mechanism of action is unclear. In our study with eight glioblastoma multiforme cell lines, the most sensitive ones underwent cell cycle arrest in response to FTI treatment. Radiosensitization by FTIs, however, seemed to involve other pathways. If R115777 treatment was initiated < 6 hours before irradiation, all eight glioblastoma multiforme lines were radiosensitized. However, if the time between drug and radiation was extended to 24 hours, cells harboring wild type but not mutated p53 were able to counteract drug-induced radiosensitization. The involvement of the p53/p21 pathway in the development of resistance was confirmed by showing that U87 cells transfected with human papillomavirus E6 to block p53 or interfering RNA to inhibit p21 stayed radiosensitive for 24 hours after drug treatment. The time dependency of R115777-induced radiosensitization suggested that the initial FTI target for early radiosensitization was short-lived, and that a p21-directed pathway restored resistance. Consideration of prenylated molecules that could potentially be involved led us to consider HDJ-2, a co-chaperone of heat shock protein 70. This hypothesis was strengthened by finding that cellular radiosensitivity was increased by genetic inhibition of HDJ-2, whereas overexpression conferred radioresistance. Importantly, irradiation of cells caused HDJ-2 to migrate from the cytoplasm to the nucleus, and this migration was inhibited by prior FTI treatment. These results have clinical relevance in that they help explain the variability in responses to FTIs that occurs following radiotherapy and elucidate some of the reasons for the complexity underlying FTI-induced radiosensitization.

Chemical genetics identifies Rab geranylgeranyl transferase as an apoptotic target of farnesyl transferase inhibitors

A chemical genetics approach identified a cellular target of several proapoptotic farnesyl transferase inhibitors (FTIs). Treatment with these FTIs caused p53-independent apoptosis in Caenorhabditis elegans, which was mimicked by knockdown of endosomal trafficking proteins, including Rab5, Rab7, the HOPS complex, and notably the enzyme Rab geranylgeranyl transferase (RabGGT). These FTIs were found to inhibit mammalian RabGGT with potencies that correlated with their proapoptotic activity. Knockdown of RabGGT induced apoptosis in mammalian cancer cell lines, and both RabGGT subunits were overexpressed in several tumor tissues. These findings validate RabGGT, and by extension endosomal function, as a therapeutically relevant target for modulation of apoptosis, and enhance our understanding of the mechanism of action of FTIs.

Clostridium difficile Toxin A Induces Expression of the Stress-induced Early Gene Product RhoB

Clostridium difficile toxin A monoglucosylates the Rho family GTPases Rho, Rac, and Cdc42. Glucosylation leads to the functional inactivation of Rho GTPases and causes disruption of the actin cytoskeleton. A cDNA microarray revealed the immediate early gene rhoB as the gene that was predominantly up-regulated in colonic CaCo-2 cells after treatment with toxin A. This toxin A effect was also detectable in epithelial cells such as HT29 and Madin-Darby canine kidney cells, as well as NIH 3T3 fibroblasts. The expression of RhoB was time-dependent and correlated with the morphological changes of cells. The up-regulation of RhoB was approximately 15-fold and was based on the de novo synthesis of the GTPase because cycloheximide completely inhibited the toxin A effect. After 8 h, a steady state was reached, with no further increase in RhoB. The p38 MAPK inhibitor SB202190 reduced the expression of RhoB, indicating a participation of the p38 MAPK in this stress response. Surprisingly, newly formed RhoB protein was only partially glucosylated by toxin A, sparing a pool of potentially active RhoB, as checked by sequential C3bot-catalyzed ADP-ribosylation. A pull-down assay in fact revealed a significant amount of active RhoB in toxin A-treated cells that was not present in control cells. We demonstrate for the first time that toxin A has not only the property to inactivate the GTPases RhoA, Rac1, and Cdc42 by glucosylation, but it also has the property to generate active RhoB that likely contributes to the overall picture of toxin treatment.

Farnesyltransferase inhibitors

The farnesyltransferase inhibitors (FTIs) were designed to inhibit the post-translational processing of Ras proteins, which are mutated in 30% of all human cancers. Recent studies suggest, however, that the target of FTIs may be a protein other than Ras, and that these agents may be more appropriately used to treat tumors with activated wild-type ras signaling. Preliminary results from several phase II and phase III studies have been reported. The FTIs fail to show significant single-agent activity in non-small cell lung cancer, small cell lung cancer, pancreatic cancer, refractory colorectal cancer, and bladder cancer. Activity has been shown in hematologic malignancies (acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndrome), breast cancer, and glioma. Several combination studies of FTIs and standard cytotoxic agents are ongoing.

Preclinical and clinical evaluation of farnesyltransferase inhibitors.

Farnesylation of Ras, a protooncogene that is frequently mutated in a number of malignancies, is critical for its biologic function. This observation has led to the development of several agents that inhibit farnesyltransferase, known as farnesyltransferase inhibitors (FTIs). The antiproliferative and antitumor effects of these agents have been demonstrated in preclinical and clinical studies. Interestingly, FTI activity does not necessarily rely on ras mutational status, indicating that Ras is not the only FTI target. Clinical data suggest that FTIs, alone and in combination with other agents, have antitumor activity. Further study is needed to determine the precise mechanism of FTI antitumor activity as well as how and where FTIs will be best used clinically.

Farnesyl transferase inhibitors: one target may be found in another

The fact that proteins such as Ras require farnesylation to induce malignant transformation prompted many investigators to design farnesyl transferase inhibitors (FTI) as novel anticancer drugs. FTIs inhibit the growth of ras transformed cells in vitro and induce tumor regression in ras dependent tumor in vivo. Moreover, FTIs inhibit tumor progression in human tumor xenograft models. Currently, FTIs are undergoing phase I and II trials in various cancer types. They show impressive antitumour efficacy and they lack toxicity. Despite these promising results, the development of such molecules in hindered by the absence of appropriate clinical endpoints and of surrogate biological markers. Indeed, it seems likely that Ras is not the critical target of FTIs and that inhibition of the farnesylation of proteins such as RhoB, might also contribute to the observed antitumour properties. Identification of targets that underlie their biological effect is essential in order to predict and evaluate their efficacy.

RhoB controls the 24 kDa FGF-2-induced radioresistance in HeLa cells by preventing post-mitotic cell death.

Farnesylated Ras oncoprotein induces a cellular resistance to ionizing radiation that can be reversed by farnesyltransferase inhibitors (FTI). We previously demonstrated that, expression of the 24 kDa FGF2 isoform in wild type ras bearing HeLa cells, induced radioresistance which was also reversed by FTI. We tested the hypothesis that wild type Ras or RhoB, which has been proposed as a potential FTI target, could control the FGF-2-induced radioresistance mechanisms. For this, we expressed inducible dominant negative forms of Ras (RasN17) and Rho (RhoBN19) in 24 kDa FGF2 transfected HeLa cells and analysed their survival after irradiation. While no cell survival modification was observed after RasN17 induction, the expression of RhoBN19 induced a radiosensitization of FGF2 radioresistant HeLa cells in the same range as the one observed after a 48 h treatment with the specific FTI, R115777. Moreover, we showed that activated RhoB but not RhoA induced radioresistance in NIH3T3 cells. The radiosensitizer effect of RhoBN19 expression was due to the induction of the radiation induced post-mitotic cell death. Taken together, these data demonstrate that 24 kDa FGF-2-induced radioresistance is controlled by Rho pathways and suggest that RhoB should be a major determinant in cellular resistance to ionizing radiation.

Farnesyltransferase inhibitors: Potential therapeutic for inflammatory breast cancer?

Inflammatory breast cancer (IBC) is an aggressive and poorly managed disease that accounts for up to 6% of new breast cancer cases in the United States annually. Recent preclinical findings suggest that IBC might be treated with farnesyltransferase inhibitors (FTIs), a novel class of experimental therapeutics that are currently in clinical trials. FTIs exhibit considerable selectivity and efficacy against tumors in preclinical models, and they have been well-tolerated in Phase I human trials. How FTIs target cancer cells has emerged as an important question, with the somewhat disappointing results from initial Phase II efficacy trials. FTI development was predicated on Ras inhibition but there is considerable evidence that other mechanisms are important for mediating the antineoplastic activities of these drugs. One important mediator that has emerged is RhoB, a small GTPase that is recruited by FTIs to trigger growth inhibition and apoptosis. Recently, van Golen and Merajver and their colleagues have demonstrated that FTIs will recruit RhoB to suppress IBC cell phenotypes or neoplastic transformation of human mammary epithelial cells by RhoC, a key oncogenic driver in IBC. A mechanistic implication of this study is that RhoB can interfere with RhoC-dependent signals required for neoplastic pathophysiology. Further preclinical and clinical investigations to test the potential therapeutic utility of FTIs for treating RhoC-driven breast cancers such as IBC would seem warranted.

Farnesyltransferase inhibitor, R115777, reverses the resistance of human glioma cell lines to ionizing radiation.

We investigated for the first time the ability of farnesyltransferase inhibitors (FTI) to radiosensitize human glioma. For this, human glioma cell lines were treated with the specific FTI, R115777, 48 hr prior to a 2Gy irradiation. The treatment with R115777 decreased by 45% the SF2 value of the more radioresistant glioma cell lines (SF763 and U87) without any significant effect on the radioresistance of the radiosensitive ones (SF767 and U251-MG). This radiosensitizer effect was due to the induction of post-mitotic necrotic cell death. We then tested the hypothesis that wild-type Ras or RhoB, which has been proposed as potential FTI target, could control the glioma radioresistance. For this, we expressed inducible dominant negative forms of Ras (RasN17) and RhoB (RhoBN19) in radioresistant U87 glioma cell line and analyzed the survival after irradiation of the obtained clones. While blocking Ras pathways by expression of RasN17 did not affect the SF2 value of the U87 glioma cell line, the expression of RhoBN19 dramatically reduced the cell survival after irradiation of these cells. Taken together, these data demonstrated that RhoB, but not Ras, is implicated in glioma radioresistance. Furthermore, the R115777 differential radiosensitizer effect underlines the potential therapeutic interest of using this drug as a radiosensitizer of human glioma.

Evolving therapies: farnesyltransferase inhibitors.

Farnesyltransferase inhibitors (FTIs) are compounds designed to interfere with the signal transduction of cancer cells containing ras gene mutations. Specifically, FTIs were designed to prevent the farnesylation of Ras and other intracellular proteins, and they have been shown to have an effect on malignant cell proliferation and survival. However, the actual intracellular target of FTIs and the cellular determinants of drug action that correlate with antitumor effects currently are unknown. The following are key questions relating to FTI cell biology and clinical development: 1) Are ras gene mutations required for FTIs to be effective? 2) Does the effect of FTI therapy depend on which ras isoform is active (ie, H-ras, K-ras4A/B, or N-ras) in cancer cells? 3) What level of farnesyltransferase inhibition is required for clinical effect? 4) What surrogate biomarkers can be used to evaluate the biologic effect of FTIs in ongoing clinical trials? 5) What is the mechanism of FTI antitumor activity? Although the preliminary results are encouraging, more understanding of the intracellular mechanism of the FTIs is needed to determine how best to administer them in patients with cancer and to test their antitumor effects.

The Farnesyltransferase Inhibitor, FTI-2153, Blocks Bipolar Spindle Formation and Chromosome Alignment and Causes Prometaphase Accumulation during Mitosis of Human Lung Cancer Cells

Even though farnesyltransferase inhibitors (FTIs), a novel class of therapeutic agents presently in clinical trials, have preclinically outstanding anticancer activity and impressive lack of toxicity, their mechanism of action is not well understood. To enhance our understanding of how FTIs inhibit the growth of tumors, we have investigated their effects on cell cycle progression of two human lung cancer cell lines, A-549 and Calu-1. In this report, we show in synchronized A-549 and Calu-1 cells that FTI-2153 treatment resulted in a large accumulation of cells in the mitosis phase of the cell division cycle, with some cells in the G(0)/G(1) phase. Furthermore, microtubule immunostaining and 4,6-diamidino-2-phenylindole DNA staining demonstrated that the FTI-2153-induced accumulation in mitosis is due to the inability of these cells to progress from prophase to metaphase. FTI-2153 inhibited the ability of A-549 and Calu-1 cells to form bipolar spindles and caused formation of monoasteral spindles. Furthermore, FTI-2153 induced a ring-shaped chromosome morphology and inhibited chromosome alignment. Time-lapse videomicroscopy confirmed this result by showing that FTI-2153-treated cells are unable to align their chromosomes at the metaphase plate. FTI-2153 did not affect the localization to the kinetochores of two farnesylated centromeric proteins, CENP-E and CENP-F. Thus, a mechanism by which FTIs inhibit progression through mitosis and tumor growth is by blocking bipolar spindle formation and chromosome alignment.

Both farnesylated and geranylgeranylated RhoB inhibit malignant transformation and suppress human tumor growth in nude mice.

Whereas the GTPase RhoA has been shown to promote proliferation and malignant transformation, the involvement of RhoB in these processes is not well understood. In this manuscript RhoB is shown to be a potent suppressor of transformation and human tumor growth in nude mice. In several human cancer cell lines, RhoA promotes focus formation whereas RhoB is as potent as the tumor suppressor p53 at inhibiting transformation in this assay. RhoB is both farnesylated (F) and geranylgeranylated (GG), and RhoB-F has been suggested as a target for the antitumor activity of farnesyltransferase inhibitors. Here we demonstrate that both RhoB-F and RhoB-GG inhibit anchorage-dependent and -independent growth, induce apoptosis, inhibit constitutive activation of Erk and insulin-like growth factor-1 stimulation of Akt, and suppress tumor growth in nude mice. The data demonstrate that RhoB is a potent suppressor of human tumor growth and that RhoB-F is not a target for farnesyltransferase inhibitors.