Radiation Sensitization by Inhibition of Activated Ras

Strahlentherapieund Onkologie Review Article

731Strahlenther Onkol 2004 · No. 11 © Urban & Vogel

Radiation Sensitization by Inhibition of Activated Ras Thomas B. Brunner1, Stephen M. Hahn2, W. Gillies McKenna2, Eric J. Bernhard2

Background and Purpose: Ras has been identified as a significant contributor to radiation resistance. This article reviews pre-clinical and phase I clinical studies that reported on combining inhibition of activated Ras and downstream effectors of Ras with radiotherapy.Material and Methods: Transfection studies and RNA interference were used to check the role of the Ras isoforms for intrinsic ra-diation sensibility. Western blotting was used to control for prenylation inhibition of the respective Ras isoforms and for changes in activity of downstream proteins. Clonogenic assays with human and rodent tumor cell lines served for testing radiosensitiv-ity. In vivo, farnesyltransferase inhibitors (FTIs) and irradiation were used to treat xenograft tumors. Ex vivo plating efficiency measurements, regrowth of tumors, and EF5 staining for detection of hypoxia were endpoints in these studies. Simultaneous treatment with L-778,123 and irradiation was performed in non-small cell lung cancer, head and neck cancer, and pancreatic cancer patients.Results: Radiation sensitization was achieved in vitro and in vivo blocking the prenylation of Ras proteins in cell lines with Ras activated by mutations or receptor signaling. Among the many Ras downstream pathways the phosphoinositide 3 (PI3) kinase-Akt pathway was identified as a contributor to Ras-mediated radiation resistance. Furthermore, increased oxygenation was observed in xenograft tumors after FTI treatment. Combined treatment in a phase I study was safe and effective.Conclusion: The rational combination of FTIs with radiotherapy may improve the clinical results of patients with tumors who bear mutant or receptor-signaling activated Ras.

Key Words: Ras · Radiation · PI3 kinase · Farnesyltransferase

Strahlenther Onkol 2004;180:731–40 DOI 10.1007/s00066-004-9198-8

Strahlensensibilisierung durch Inhibition von aktiviertem Ras

Hintergrund und Ziel: Ras wurde als signifikanter Faktor der Strahlenresistenz erkannt. Dieser Artikel gibt einen Überblick über präklinische Studien und klinische Phase-I-Studien zur kombinierten Inhibierung von aktiviertem Ras und von Ras-Effektoren mit Radiotherapie.Material und Methodik: Transfektionsstudien und RNA-Interferenz kamen zur Anwendung, um die Rolle der Ras-Isoformen für die intrinsische Radiosensibilität zu untersuchen. Westernblots dienten der Kontrolle der Prenylierungsinhibierung der jeweiligen Ras-Isoformen und dem Screening von Änderungen der Aktivität von Downstream-Proteinen. Koloniebildungstests mit humanen und Nagertumorzelllinien wurden zur Testung der Radiosensitivität durchgeführt. In vivo wurden Farnesyltransferaseinhibitoren (FTIs) und Bestrahlung für die Behandlung von Xenografttumoren verwendet. Ex vivo waren Messungen der Platierungseffizienz, des Nachwachsens von Tumoren und der EF5-Färbung für die Detektion von Hypoxie Endpunkte dieser Studien. Patienten mit nichtkleinzelligem Bronchialkarzinom, Kopf-Hals-Tumoren und Pankreaskarzinom wurden simultan mit L-778,123 und Radiothe-rapie behandelt.Ergebnisse: Strahlensensibilisierung wurde in vitro and in vivo nach Blockade der Prenylierung von Ras-Proteinen in Zelllinien mit Ras-Aktivierung durch Mutationen oder Rezeptorsignalaktivität erreicht. Unter den vielen Ras-Downstream-Signalwegen wur-de die Bedeutung des Beitrags des Phosphoinositol-3-(PI3-)Kinase-Akt-Weges zur Strahlenresistenz identifiziert. Des Weiteren wurde nach FTI-Behandlung bei Xenografttumoren eine gesteigerte Oxygenierung beobachtet. Die Kombinationsbehandlung war in einer Phase-I-Studie sicher und wirksam.Schlussfolgerung: Die Kombination von FTIs mit Bestrahlung ist ein Ansatz, der die klinischen Ergebnisse von Patienten mit Tumoren verbessern könnte, bei denen Ras mutiert oder über Rezeptorsignale aktiviert ist.

Schlüsselwörter: Ras · Strahlen · PI3-Kinase · Farnesyltransferase

1 Department of Radiation Oncology, University Hospital, Erlangen, Germany, 2 Department of Radiation Oncology, University of Pennsylvania, Philadelphia, PA, USA.

This paper is dedicated to Professor Rolf Sauer, MD, chair of the Department of Radiation Oncology, University of Erlangen, on the occasion of his 65th birthday.

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Brunner TB, et al. Radiation Sensitization by Inhibition of Activated Ras

732 Strahlenther Onkol 2004 · No. 11 © Urban & Vogel

IntroductionThe combination of irradiation with chemotherapy has been a major step forward to achieve responses and pro-longation of survival in many tumor en-tities. Combined treatment has become standard in tumors such as gastrointes-tinal tumors [76], lung cancer [69], head and neck cancer (HNC) [42], and blad-der cancer [60]. Nevertheless, failure rates in patients with the above quoted tumors are still high [33] and toxicity is limiting for more aggressive protocols [65]. Molecular-targeted combinations with radiation could increase efficacy of radiotherapy and circumvent increased toxicity. The first oncogenes to be im-plicated in human cancer were the ras genes. The growth-promoting effects in tumor cells of activating ras mutations are manifold, and ras activation is in-volved in about 30% of human tumors. Furthermore, ras takes a central place of many signal transduction pathways in the cell. Radiation resistance has been shown to be increased by ras activation in rodent and tumor cells [49].

The understanding of the posttrans-lational modifications of Ras together with the identification of the responsible enzymes involved led to the synthesis of farnesyltransferase inhibitors (FTIs), compounds that inhibit farnesyltransfer-ase (FTase). These drugs moved from preclinical studies to phase I through III clinical trials. Recently, the first phase I study of an FTI concurrent to radiother-apy was reported. This review will focus on the effects of combined FTI with ra-diotherapy.

The Ras Oncogene: Structure, Posttranslational Modifications, and Inhibition of Ras by Prenylation Inhibition

The three mammalian Ras genes yield four Ras proteins: H-Ras, N-Ras, K-Ras4A, and K-Ras4B. All Ras proteins are 188 amino acids in length, except K-Ras4B which is 189 amino acids long. The first 165 amino acids of the different Ras forms are largely homologous. The N-terminal conserved domains contain the effector, the exchange factor and the nu-cleotide-binding sites. The hypervariable region (HVR) after amino acid 165 (C-terminal region) can be divided into two domains: the linker domain and the membrane-targeting do-

main. The membrane-targeting domain consists of a C-termi-nal CAAX box (C: cysteine, A: aliphatic, X: any amino acid). It is common to all Ras proteins and serves as the primary membrane-targeting domain. A secondary membrane-target-ing domain, also situated in the HVR, contains cysteine palmi-toylation sites in H-, N-, and K-Ras4A or a polylysine domain in K-Ras4B [30, 31, 77] (Figure 1).

To be functional Ras has to be bound to the cell mem-brane [77]. Posttranslational prenylation at the cysteine resi-

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Figure 1. Ras posttranslational modification and transformation. Ras must be bound to the cell membrane to have transforming activity. Oncogenic Ras proteins lose their transforming ac-tivity when attachment to the plasma membrane is blocked by farnesyltransferase inhibitors (FTIs). The cysteine residue to which the isoprenyl group is attached is critical in this respect. After prenylation, the next steps in posttranslational modification are AAX proteolysis by Rce1 and then α-carboxymethylation of farnesylated cysteine residues by isoprenylcysteine carbox-yl methyltransferase. Cysteine palmitoylation sites are contained in H-Ras, N-Ras, and K-Ras4A or a polylysine domain in K-Ras4B as depicted by positive charges in the figure. H-Ras is shown as an example of the three palmitoylated forms.

Abbildung 1. Posttranslationelle Modifikation und Transformierung von Ras. Ras muss an die Zellmembran gebunden sein, um transformierende Aktivität zu besitzen. Onkogene Ras-Pro-teine verlieren ihre transformierende Aktivität, wenn die Anheftung an die Plasmamembran durch Farnesyltransferaseinhibitoren (FTIs) blockiert wird. Der Cysteinrest, an den die Isopre-nylgruppe gebunden ist, ist in dieser Hinsicht entscheidend. Nach der Prenylierung sind die nächsten Schritte der posttranslatorischen Modifikation eine AAX-Proteolyse durch Rce1 und dann eine α-Carboxymethylierung von farnesylierten Cysteinresten durch die Isoprenylcy-stein-Carboxyl-Methyltransferase. H-Ras, N-Ras, and K-Ras4A enthalten Cysteinpalmitoylie-rungsstellen bzw. eine Polylysindomäne in K-Ras4B, dargestellt anhand positiver Ladungen. H-Ras ist exemplarisch für die drei palmitoylierten Formen gezeigt.



due within the C-terminal CAAX box is necessary for mem-brane building. Prenyltransferases recognize the CAAX box [21], and the transforming activity of Ras proteins is lost when mutations in the CAAX sequence block prenylation and at-tachment to the plasma membrane [8, 35, 77, 78]. These find-ings led to the development of pharmacological compounds that inhibit prenyltransferases.

Functions and Pathways of RasThe binding of GTP (guanosine triphosphate) activates cel-lular Ras proteins for signaling (Figure 2). This is triggered by a signal initiated by the binding of growth factors to plasma membrane receptors with a tyrosine kinase activity under physiological conditions. Ligand binding by these receptors signals through the adapter protein GRB2 and the guanine nucleotide exchange factor SOS (son of sevenless). Rapid exchange of GTP for GDP (guanosine diphosphate) is cata-lyzed by SOS which is bound to Ras in the resting state. GTP hydrolysis interrupts signaling by Ras, and hydrolysis is ef-fected by Ras itself upon association with the GAP protein (GTPase-activating protein). Constitutive Ras signaling is the consequence of oncogenic mutations in Ras since they reduce GTP hydrolysis. Overexpression or activation of receptors such as the epidermal growth factor (EGF) receptor also in-creases Ras signaling. This is a common finding in many tu-mors including lung and breast cancers [51, 54].

Ras proteins are hubs in signal transduction from recep-tor tyrosine kinases. Many pathways are affected by Ras sig-naling. Two well-characterized pathways are the Raf-MAP (mitogen-activated protein) kinase pathway and the phos-phoinositide 3 (PI3) kinase pathway (reviewed in [28]). The Raf-MAP kinase pathway results in activation of the ETS (E26 transformation-specific) family of transcription factors. This complex of transcription factors regulates genes as for instance matrix metalloproteinases, a heparin-binding form of EGF and GM-CSF (granulocyte-macrophage colony-stim-ulating factor). Another subgroup of the ETS family interacts with the serum response factor (SRE). Within minutes after SRE interaction, the transcription of immediate response genes (e.g., the fos oncogene) is detectable. Thus, Ras signal-ing can induce genes involved in the process of tumor inva-sion, tumor stroma formation, and growth control.

Ras proteins also stimulate PI3 kinases, which have mul-tiple direct and indirect targets such as phosphoinositide-de-pendent kinases (PDKs), p70S6K, Rac, and guanine exchange factors [9]. Akts (protein kinase B or PKB) are activated by PDKs in cells exposed to diverse stimuli such as hormones, growth factors, and extracellular matrix components. Akt phosphorylates and regulates the function of many cellular proteins. These are involved in processes that include metab-olism, apoptosis, and proliferation through regulation of the cell cycle progression [53]. The PI3 kinase pathway also medi-ates the effects of Ras on the cytoskeleton (rearrangement of cortical filaments, formation of lamellopodia, and membrane

ruffling). Other downstream pathways of Ras lead, for ex-ample, to Ral, an activator of CDC42 and Rac. These in turn activate the various MEK kinase proteins that are involved in the regulation of the SAPK/JNK and p38 pathways [18, 59].

The Inhibition of Prenyltransferases Prenylation by FTase is the first and obligate step in H-Ras processing that results in a functional Ras protein (reviewed in [4]) (Figure 1). The 15-carbon farnesyl group is added to a cysteine residue at the fourth amino acid position from the carboxyl-terminal end of Ras. This residue is included within the CAAX recognition sequence for prenyltransferases. Since the addition of a prenyl side chain is required for Ras mem-brane localization, which is in turn necessary for oncogenic Ras-mediated transformation, several groups have developed inhibitors specific for one of the two prenyltransferases in-volved in Ras processing. Drug screens resulted in the isola-tion of these inhibitors as plant metabolites. In addition, FTIs and geranylgeranyltransferase inhibitors (GGTIs) were de-veloped. By inhibiting its posttranslational processing, these compounds inhibit H-Ras activity, whose processing is inhib-ited by FTIs alone.

By contrast, K-Ras is prenylated at its enzyme recogni-tion CAAX site with a 20-carbon geranylgeranyl group by the enzyme geranylgeranyltransferase-I when farnesylation is blocked. Specific GGTIs have also been developed. For the inhibition of K-Ras prenylation it is essential to combine the action of FTIs and GGTIs [2]. Specificity is very high for most FTIs and GGTIs for their respective targets. Therefore, a combination of FTI and GGTI is used to inhibit K-Ras pre-nylation. The inhibitors we have used to date include the FTIs L-744,832, L-779,575, and L-778,123 developed by Merck & Co. Inc., R115777 from Janssen Pharmaceuticals, FTI-276, and its methyl ester FTI-277, additionally, the GGTIs GGTI- 297 and its methyl ester GGTI-298 from Drs. Sebti and Hamilton.

Effects of Farnesyltransferase Inhibitors in Preclinical Models

Initial studies of FTase inhibitor activity demonstrated rever-sion of morphological transformation and inhibition of tumor growth of cells transformed by oncogenic H-ras.

However, these inhibitors had no effect on the behav-ior of cells transformed by other oncogenes such as raf-1, or of cells transformed by H-ras genetically modified to be at-tached to the membrane via myristylation or by prenylation by a geranylgeranyl group [14, 56, 57]. The expected picture of FTI specificity was significantly changed when subsequent studies of human tumor cells showed that these inhibitors are effective in blocking the growth of a wide variety of tumor cell lines both with and without ras mutation. It was also shown that they block the growth of rodent tumors and human tumor xenografts in vivo, some of which express wild-type-(wt-)ras. Taken together, in spite of having been developed to specifi-



cally target Ras, it is apparent that this class of compounds has activity on prenylated proteins other than Ras that are involved in promoting transformed cell growth. It now ap-pears that growth inhibition by FTase inhibitors cannot be predicted, either by the mutation status of tumor cells, or by their tissue of origin. In this context it is the more surprising that FTI treatment had little effect on the growth of normal cells, and both preclinical data and phase I clinical trials have demonstrated a relatively low level of normal cell and tissue toxicity associated with FTI treatment [64].

Activated Ras Imparts Radiation ResistanceMany studies have shown that radiation resistance is influ-enced by Ras activation. The first description of the link be-tween ras oncogene expression and increased radioresistance was reported in N-ras-transfected NIH 3T3 cells [20]. This observation subsequently was expanded to other forms of ras in NIH 3T3 cells [68]. Primary rat embryo fibroblasts (REFs) transformed at early passage with H-ras confirmed these findings [49]. More evidence for the influence of the differ-ent isoforms of ras (H-, K-, and N-ras) on intrinsic radiation resistance came from transfection experiments with activating mutations in the respective genes coding for the isoforms of Ras in several human tumor cell lines [3, 5, 32]. Changes in cell cycle distribution were excluded to be relevant for these effects, because no differences in cell cycle distribution were observed between parental cells and cells with loss of the ac-tivated ras allele, but radiation resistance correlated with ex-pression of activated Ras [5].

Farnesyltransferase Inhibitors Diminish Ras-Induced Radiation Resistance

The discovery that activated Ras goes along with increased radiation resistance led to the question of whether Ras inhibi-tion would lead to specific radiation sensitization of cells with activation of Ras signaling. The fact that it is theoretically easier to shut off an activated oncogene than to restore lost activity of a tumor suppressor prompted molecular targeting of Ras and the development of prenyltransferase inhibitors.

In contrast to the aforementioned studies of tumor growth inhibition we have focused on the effects of prenyltransferase inhibitors on radiosensitivity in terms of survival while con-trolling for growth inhibition. In these studies, tumors with activated Ras were radiosensitized while tumors where Ras was not active were not. FTI-induced radiosensitization did not depend on the degree of growth inhibition observed in the different cell lines studied. In this respect it is important to point out the way we combined FTI and radiation treatment, because methodological problems could have attributed at least in part to negative results in recently reported studies where FTIs were combined with irradiation [46, 50]: to test the influence of Ras on radiation sensitivity, Ras function needs to be blocked at the time of irradiation and therefore cells were exposed to FTIs 24 h before radiation treatment. Successful

inhibition of Ras prenylation was consistently shown by im-munoblotting at the time of irradiation for a broad variety of cell lines. Drug exposure was continued without interrup-tion for another 24 h to maintain its influence on the early sequence of events that determines radiosensitivity. There-after, the drugs were washed out to avoid the growth inhibition effects of FTIs. Clonogenic assays were performed to measure survival after irradiation. Other assays like the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay [46] or apoptosis assays, while useful for determining growth inhibition, are not adequate for measuring clonogenic survival after irradiation [7].

Farnesyltransferase Inhibitors and RadiosensitivitySynergistic radiation-induced cell killing after prenyltrans-ferase inhibitor treatment was observed in both rodent and human tumor cells with ras mutations [3, 12]. This was first shown in cells with activated H-Ras, because it only under-goes farnesylation and not alternative geranylgeranylation [2]. Compared with this, inhibition of activated K-Ras with subsequent radiation sensitization requires blocking of both of these prenylation reactions, FTase and GGTase. Radia-tion sensitization was not unique to certain tissues of origin. Testing cells from bladder (T24), breast (HS578T), colon (SW480), lung (A549), and fibroblasts (HT1080) gave consis-tent results of sensitization allowing a generalization of the observation for all cells independent of their origin with acti-vated Ras status. Radiosensitization could be achieved both in vitro and in vivo in the case of H-Ras-expressing tumors using various FTIs including FTI-276, FTI-277 [64], L-744,832 [41], L-778,123 [6], and R115777 [19]. The correct balance of FTase and GGTase inhibition for blocking K-Ras prenylation in vivo has not yet been established, and therefore in vivo radiosensi-tization of tumors expressing K-ras mutations has been more difficult to demonstrate. Moreover, a higher level of toxicity of GGTase inhibitors in vivo compared to FTIs has been re-ported [45]. More recent work implies that in some tumors with K-Ras activation by mutation, inhibition of H-Ras may be sufficient for radiosensitization (Brunner TB, manuscript in preparation, [63]).

Tumor cell lines with Ras activity stimulated by recep-tor tyrosine kinase activation were also radiosensitized by farnesyltransferase inhibition as demonstrated by Gupta et al. [26]. SQ20B tumor xenografts also showed enhanced tumor oxygenation after treatment with the FTI L-778,123 accom-panied by enhanced tumor apoptosis (unpublished observa-tions). Similarly, combined treatment with this drug in vitro in a non-small cell lung cancer (NSCLC) cell line led to ra-diosensitization. The line with wt-ras status was isolated from one of the responding patients participating in the phase I trial with combined L-778,123 and radiation therapy (see below, [29]). These findings indicate that a wider spectrum of tumor types than those in which ras mutations are frequent can po-tentially be treated by Ras inhibition strategies. Whereas in



our laboratory radiosensitization by prenyltransferase inhibi-tors in preclinical tests is specific to tumor cells with oncogenic or activated Ras, others have noted effects in cells where Ras is not thought to be activated [13]. More than 20 mammalian proteins are farnesylated (reviewed in [15]), and some of them have been implicated in the antitumor effects. Possible candi-date targets in the context of cancer treatment include RhoB [43], the phosphatases PRL-1, -2, and -3 [79], and CENP-E and -F centromeric proteins (reviewed in [72]). An important argument against the role of these proteins as targets for radio-sensitization is that radiosensitization would also be expected for normal cells and not only cells with Ras activation, which has not been the case in our studies [2, 12].

More confirmation for Ras as the relevant target of FTIs in the context of radiosensitization comes from different ap-proaches to inhibit Ras expression or activity. Expression of oncogenic Ras and Raf was inhibited by antisense DNA, re-sulting in radiation sensitization [55]. Single-chain antibodies encoded on adenoviruses were employed to block Ras activ-ity in another study [61, 62]. Recently, small interfering RNA (siRNA) experiments have been performed in our laboratory to shut down gene expression by RNA interference (RNAi; reviewed in [66], [17]). We have targeted specific Ras isoforms such as K-Ras (Figure 2) and shown that in the SW480 colon cancer cell line, downregulation of oncogenic K-Ras expres-sion by K-ras allele-specific siRNA treatment reduces ra-diation survival [40]. Taken together, the studies cited above strongly implicate Ras signaling in promoting the radiation survival of tumor cells and at the same time Ras as the target of prenyltransferase inhibitors treatment when combined with irradiation.

Since many of the human tumor models we have studied as xenografts contain extensive regions of hypoxia and hypox-ic cells are markedly more resistant to killing by radiation [73, 74], we investigated the effects of FTIs on tumor xenograft hy-poxia [11]. L-744,832 treatment resulted in a reduction in hy-poxia in tumors that expressed activated H-Ras (cell lines T24, 141-1), but not in tumors with normal ras (cell lines HT-29, RT-4). These results were confirmed in human prostate tumor xenografts [67]. Similar findings were obtained in U87 glio-blastoma cell xenografts [16]. In these tumors, EGF receptor is overexpressed leading in turn to PI3 kinase-driven VEGF (vascular endothelial growth factor) overexpression similar to that seen in cells with Ras mutations [47]. These results suggest that FTase inhibition might contribute to radiosensitization in vivo by increasing tumor oxygenation. This finding could be of significance to combined treatment with FTIs and chemo-therapeutics as well, since hypoxic tumors show resistance to these agents [73].

In Search for the Missing Link(s) Connecting Ras to Radioresistance

Many downstream pathways of Ras signaling have been de-scribed and their implications for survival after radiation are

under investigation. We used pharmacological inhibitors of potential downstream targets of Ras to explore the down-stream pathway leading to radioresistance [24]. Pharmacolog-ical inhibitors of MEK (PD98059, in later studies U0126), PI3 kinase (LY294002), p70S6K (rapamycin), and p38 (SB203580, PD169316) and an FTI (L-744,832) were used in activated H-Ras T24 bladder cancer cells. This approach was used to confirm the effects of the specific inhibitors on the respective pathways by immunoblotting the activated proteins or phos-phorylated forms of the proteins MAPK, AKT, p70S6K, and p38. In parallel, their effect on the clonogenic survival after irradiation was determined. Whereas the PI3 kinase inhibitor LY294002 sensitized T24 cells to a similar extent as obtained after L-744,832 treatment, none of the other inhibitors modi-fied radiation sensitivity. To exclude that the observation of the significance of PI3 kinase in Ras-mediated radiation re-sistance was unique to this cell line, 3.7 REFs with activated H-Ras, DLD-1 colon carcinoma cells with activated K-Ras and two cells lines with wt-ras (RT4 bladder carcinoma and MR4 REF) were tested in the following experiments. Akt phosphorylation was successfully blocked with LY294002 in all cell lines. In correlation with T24 cells, the cell lines with activated Ras could be sensitized to radiation while the two cell lines with wt-ras status were not. Subsequently, RT4 and MR4 cell lines transfected with the constitutively active PI3 kinase p110 subunit under control of a dexamethasone-sen-sitive promoter were more radioresistant after addition of dexamethasone. This characteristic was reverted by treatment with LY294002 [24]. The significance of the PI3 kinase path-way in radiation resistance has been confirmed by transfecting HT1080-expressing activated N-Ras with a plasmid-encoding PTEN (phosphatase and tensin homoloque deleted on chro-mosome ten) phosphatase. Overexpression of this phospha-tase which antagonizes PI3 kinase activity by dephosphorylat-ing IP3 [52] rendered HT1080 cells more sensitive to radiation compared to the parental cells with normal PTEN expression (unpublished observations).

The contribution of PI3 kinase signaling to radiation sur-vival has been demonstrated in a number of cell lines and by several groups [23, 34, 44, 71]. Most recently, our group has inhibited specific isoforms of Akt by siRNA. Downregula-tion of Akt-1 resulted in reduced phospho-Akt and reduced clonogenic survival [40]. Probing signaling pathways with the highly specific tool of RNAi will probably allow to define new potential targets for molecular interventions in combination with radiotherapy.

Other Ras downstream pathways have been implicated in radiation survival. Several groups reported on the contri-bution of MAP kinase in this context. Induction of MAP ki-nase signaling by radiation exposure [39], radiosensitization of MDA MB231 breast carcinoma cells after MEK inhibition [58], and similar findings in prostate and myeloid cells [10, 27] have been reported. However, the data on the role for MAP kinase in radiation sensitivity are conflicting because other



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Figure 2. A simplified schematic figure of the Ras signal transduction pathway. Upstream of Ras receptor tyrosin kinases (RTK) like epidermal growth factor receptor (EGFR) or platelet-derived growth factor receptor (PDGFR) are stimulated by an extracellular signal leading to dimerization and autophosphorylation of Src-homology-2 (SH2) domains on the intracellular surface of the protein. Consequently, GRB, an adapter protein, binds via its SH2 domains. Association of GRB with son of sevenless (SOS) attaches SOS to the cell membrane surface where it is able to interact with Ras. This stimulates guanosine triphosphate (GTP)–guanoside diphosphate (GDP) exchange. Activated GTP-Ras stimulates proliferative cel-lular processes through the activation of multiple pathways (e.g., Raf; mitogen-activated ERK kinase [MEK]; Jun N-terminal kinase [JNK]; Rac/Rho, phospholipase C [PLC] and phosphoinositide 3 kinase [PI3K]). Multiple channels of cross-talk exist between the pathways creating a complex network. The PI3 kinase to Akt pathway was identified to play an important role in survival after irradiation. Akt has multiple downstream targets (e.g., GSK3; FKHR; p21Waf1; cyclin D/E; P27Kip1, NOS; BAD; P70S6K; mTOR).

Abbildung 2. Vereinfachte schematische Darstellung des Ras-Signaltransduktionsweges. Oberhalb von Ras werden Rezeptortyrosinkinasen (RTK) wie z.B. EGFR („epidermal growth factor receptor“) oder PDGFR („platelet-derived growth factor receptor“) durch ein extrazelluläres Signal stimu-liert, was zur Bildung von Dimeren und Autophosphorylierung von Src-Homologie-2-(SH2-)Domänen an der intrazellulären Oberfläche des Prote-ins führt. Anschließend bindet GRB, ein Adapterprotein, über seine SH2-Domänen. Die Assoziierung von GRB mit „son of sevenless“ (SOS) macht SOS membranständig, wo es mit Ras interagieren kann. Dieses regt den Austausch von Guanosintriphosphat (GTP) zu Guanosindiphosphat (GDP) an. Aktiviertes GTP-Ras stimuliert proliferative zelluläre Prozesse durch die Aktivierung von multiplen Signalwegen (z.B. Raf; mitogenaktivierte ERK-Kinase [MEK]; Jun-N-terminale Kinase [JNK]; Rac/Rho, Phospholipase C [PLC] und Phosphoinositol-3-Kinase [PI3K]). Multiple Verbindungen gegenseitiger Beeinflussung existieren zwischen den Signalwegen, wodurch ein komplexes Netzwerk entsteht. Der Signalweg von PI3-Kinase zu Akt wurde als bedeutsam für das Überleben nach Radiotherapie identifiziert. Akt hat multiple Downstream-Zielproteine (z.B. GSK3; FKHR; p21Waf1; Cyclin D/E; p27Kip1, NOS; BAD; p70S6K; mTOR).



groups did not observe radiosensitization upon inhibition of this pathway [1, 23, 25].

Raf is another member within the signaling cascades of the Ras oncogene. An activated raf allele was shown to re-sult in radiation resistance, and antisense c-raf-1 was able to cause sensitization to radiation in vitro [36–38, 55, 70] and in vivo [22]. Similarly, contribution of raf to radiation survival was shown by a constitutively active form of Raf or expression of an oncogenic H-ras effector mutant that retains the abil-ity to promote Raf activation in a rat intestinal epithelial cell model [75]. This report also observed little effect of nuclear factor-(NF-)κB on radiation survival. The authors of this re-port concluded that the contribution of Raf is MAP kinase-in-dependent as MAP signaling did not contribute to radiation survival in this study [23].

Taking into consideration all these results, (1) Ras activa-tion by mutation or upstream signaling can increase intrinsic radiation resistance of tumor cells through activation of PI3 ki-nase. Raf may also play a role in this respect. (2) The influence of Ras activation on the resistance of tumors to radiation is ef-fective not only in vitro but also in vivo. (3) The relatively high activity of FTIs in combination with irradiation in vivo can be explained in part by enhancement of both apoptosis and oxy-genation in these tumors. These changes could contribute sig-nificantly to tumor radiosensitization, even if the mechanisms for these observations are not yet fully understood.

It is possible that differences in the signal transduction specificity of the different isoforms of ras could be significant in this regard. Since K-ras is the most frequently mutated form of ras in human malignancies, a perceived problem in the de-velopment of FTIs has been their poor activity against K-ras because of its ability to be alternately prenylated. However, if sensitivity to drugs and radiation is regulated via PI3 kinase activation, that is preferentially activated by H-ras, then the FTIs may turn out to be useful in this regard, since they have high activity against H-ras with little normal tissue toxicity.

Phase I Study of L-778,123 Combined with Radiation Therapy

The preclinical observation that FTI treatment could reverse intrinsic radioresistance was the basis for evaluating the FTI L-778,123 in combination with radiotherapy [29]. The FTI used in this trial, L-778,123 (Merck Research Laboratory, Rahway, NJ, USA), is a peptidomimetic inhibitor of farnes-yltransferase. The recommended phase II dose was found to be 560 mg/m2/day by continuous infusion over 2 weeks in a phase I trial of prolonged continuous infusion of L-778,123 alone. The dose-limiting toxicities (DLTs) of this regimen were grade 4 neutropenia and prolongation of the corrected QT interval (QTc) on electrocardiogram (ECG).

The primary endpoint of this phase I trial was to estab-lish the maximally tolerated dose and DLTs of L-778,123 in combination with radiation. Patients with a histologically documented NSCLC or HNC who in the opinion of the in-

vestigators required radiotherapy were eligible. The starting dose (dose level 1) of L-778,123 was 280 mg/m2/day for 7 days of each week during weeks 1, 2, 4, and 5 of radiotherapy. Dose escalations were planned in each patient group via a modi-fied Fibonacci scheme. The second dose level was 560 mg/m2/day for 7 days of each week during weeks 1, 2, 4, 5, and 7 of radiotherapy. Radiologic assessment of tumor response was first obtained 8 weeks after the completion of radiotherapy and then regularly thereafter. Response reporting was based upon the best response achieved observed in any follow-up scan.

A total of nine patients were enrolled, six patients had NSCLC: two with unresectable stage IIIa disease and four with stage IIIb disease. All three patients with HNC had unresect-able stage IV disease. Six patients were enrolled on dose level 1 and three patients on dose level 2. Acute toxicity in the NSCLC group was esophagitis in one patient treated at dose level 1 with grade 2 esophagitis. All other patients had ≤ grade 1 esophagi-tis (one patient at dose level 1 and two patients at dose level 2). This is in contrast to studies of combined concurrent cytotoxic chemotherapy and radiotherapy in NSCLC, in which substan-tial rates of moderate and severe esophagitis are reported. Mucositis in the HNC patients was also mild. No grade 3 or 4 radiation skin reaction was observed in any treatment group. Other toxicities were mild and included fatigue, anorexia, nau-sea, and diarrhea. Two dose levels of L-778,123 were evaluated in combination with radiotherapy. No DLTs were observed at the 280-mg/m2/day dose level. One DLT, grade 4 neutropenia and thrombocytopenia and interruptions of radiotherapy, was observed in one patient treated at the 560-mg/m2/day level. Overall, however, hematologic toxicity was mild.

In summary, the combination of FTI and radiation ther-apy was well tolerated. Comparison of the observed toxicities with those seen with concurrent chemo-radiation treatment at these sites was favorable.

Response assessment was not the primary endpoint of this trial, but nevertheless responses were evaluated after the completion of therapy. In stage IV HNC, two of the three pa-tients completed treatment and underwent restaging. Both patients had no evidence of disease on follow-up CT scans or nasopharyngoscopy. One patient remains alive and is without evidence of disease. The second patient had a stroke and was lost to follow-up.

One of the six patients with NSCLC had DLTs and distant metastatic disease and, therefore, was taken off study. One patient completed treatment but did not have measurable disease and subsequently died of metastatic disease. Three of the remaining four patients had complete responses to ir-radiation and L-778,123; the fourth patient had a partial re-sponse. No evidence of progression of the primary lesions was observed for a period of 7–12 months after treatment. Three of the four patients with evaluable disease developed distant metastases. The fourth patient developed a malignant pleural effusion.



Activating mutations of ras were not required for inclusion but ras mutational status of the patients was assessed. None of the four patients who responded to treatment with FTI and ir-radiation were found to have a ras mutation. As reported and discussed above, despite this, L-778,123 led to radiosensitiza-tion in the cell line isolated from one of these patients.

A second stratum of this phase I trial enrolled twelve patients with pancreatic cancer treated with L-778,123 at the same two dose levels than above and concomitant radio-therapy to 59.4 Gy in standard fractions [48]. No DLTs were observed in the eight patients treated on dose level 1. Two of the four patients on dose level 2 experienced DLTs. In one case grade 3 diarrhea and in the other case grade 3 gastro-intestinal hemorrhage associated with grade 3 thrombocyto-penia and neutropenia were observed. The hemorrhage from a gastric ulcer was believed to be outside the radiation field. Other common toxicities were mild neutropenia, dehydration, hyperglycemia, and nausea/vomiting.

Therapy was completed in eight patients and all of them had evaluable disease by radiologic measurements within 12 weeks of completion of radiotherapy. Two of the four patients who discontinued the study discontinued for disease progres-sion at distant sites and two for toxicities consisting of hem-orrhagic gastritis and bacterial infection, respectively. One patient on dose level 1 showed a partial response of 6 months duration. Stable disease was observed in five of the eight pa-tients completing therapy for at least 2 months (range 2–6 months). Two patients had progressive disease at their first post-therapy assessment, both at 4 months. Radiosensitization of a study patient-derived cell line was demonstrated in the presence of L-778,123 and went along with reduction of high baseline phospho-Akt and phospho-MAP kinase levels after FTI treatment.

ConclusionIn summary, Ras activation has been identified as a contribu-tor to radiation resistance of tumor cells in a series of studies from multiple laboratories. Likewise, inhibiting Ras by a va-riety of techniques including prenylation inhibition has been shown to sensitize tumors cells with mutant or wild-type but activated ras through upstream signaling for radiation in vitro and in vivo. One of the factors that contributes to increased in vivo efficacy compared to in vitro experiments could be in-creased tumor oxygenation after FTI treatment. The effects of FTIs on radiation sensitivity may be mediated by inhibition of PI3 kinase activation and Akt signaling. The combination of FTIs with irradiation is, in our experience, well tolerated and no additional radiation toxicity to normal tissues was ob-served. The phase I trial of L-778,123 with irradiation yielded encouraging results for this combination consistent with our preclinical findings. Further preclinical work will have to show if different tumors share one common signaling pathway or if variants of signaling exist that require specific targeting for radiosensitization.

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Address for CorrespondenceThomas B. Brunner, MDDepartment of Radiation OncologyUniversity HospitalUniversitätsstraße 2791054 ErlangenGermanyPhone (+49/9131) 853-3405, Fax -9335e-mail: [email protected]

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Radiation Sensitization by Inhibition of Activated Ras