Chapter 35 K-ras Inhibitors and Pancreatic Cancer

Steven R. Alberts

1 Introduction

The ras family includes a group of five guanosine triphosphate-binding proteins (H-ras, K-ras, M-ras, N-ras, and R-ras). In mammals ras-proto-oncogenes encode for four related and highly conserved proteins, H-ras, N-ras, K-ras 4A, and K-ras 4B ( 1 ). Ras proteins serve as important components of signaling pathways involved in a variety of cellular functions, including cell cycle con-trol, cell adhesion, endocytosis, exocytosis, and apoptosis. In order for these proteins to perform their functions they need to bind guanosine triphosphate (GTP) ( 2 ). Guanosine triphosphate creates a conformational change allowing ras to attach more tightly to its intended target. Hydrolysis of GTP to guanosine diphosphate (GDP) inactivates ras. The ability of ras to exchange GDP for GTP is under the control of guanine nucleotide exchange factors (GEFs). The GEFs are activated by growth factors or cytokines and promote the release of GDP and therefore the binding of GTP. GTPase-activating proteins (GAPs) return ras to its inactive state.

Although a variety of genetic modifications have been identified in pancreatic carcinoma, mutations of K-ras are by far the most commonly occurring mutation. Mutations are seen in >85% of pancreatic ductal carcinomas ( 3 ). The develop-ment of mutations in K-ras appear early in the development of pancreatic cancer, having been observed in precursor lesions within the pancreatic duct ( 4 ). The mutations in K-ras in pancreatic cancer are also unique in that it typically involves codon 12, but may also rarely involve codons 13 or 61 ( 5 , 6 ). These mutations in K-ras make it resistant to GAP and as a result lead to constitutive activation of downstream pathways, resulting in altered regulation of cellular proliferation. In preclinical studies, using the pancreatic cancer cell lines Panc-1 and MiaPaca-2, blocking activated K-ras resulted in increased apoptosis and loss of other malignant features supporting a pivotal role for K-ras in the development and maintenance the malignant phenotype.

Based on the frequency and apparent critical role of K-ras in pancreatic cancer several approaches have been developed to block activated K-ras. This includes both farnesyl transferase inhibitors and antisense oligonucleotides.

A.M. Lowy et al. (eds.) Pancreatic Cancer. 601doi: 10.1007/978-0-387-69252-4, © Springer Science + Business Media, LLC 2008

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2 Farnesyltransferase Inhibitors

In order for ras proteins to properly associate to the cell membrane they must undergo prenylation ( 7 ). This process involves the addition of a farnesyl isoprenoid to ras by the enzyme farnesyltransferase (FTase) and is a critical step in the post-translational modification of ras. The use of farnesyltransferase inhibitors (FTIs) inhibited cell growth in 70% of cancer cell lines tested in one previously reported preclinical study. This effect was independent of ras mutation status, with both mutated and wild-type ras showing a response to the FTIs ( 8 ). The use of FTIs appears to result in a gradual cell cycle block with eventual cell cycle arrest consist-ent with the progressive depletion of activated ras ( 9 ).

The FTIs can be divided into three categories of agents based on their mecha-nism of action. These categories include competitive inhibitors of farnesyl pyro-phosphate (FPP), competitive inhibitors of CAAX, and analogs that have both of these properties ( 10 ). FPP is an enzyme involved in catalyzing protein prenylation by serving as the isoprenoid donor. The terminal amino acid sequence CAAX (C = cytosine, A = any aliphatic amino acid, X = serine or methionine) serves as the site of farnesylation and is present in all members of the ras family. For any of these inhibitors to appropriately exert their antineoplastic activity they must be given in a way that provides continuous exposure to the drug and thereby blocks the ongoing process of ras-related signal transduction.

The CAAX inhibitor SCH66336 is an orally bioavailable agent that has been assessed in a series of phase I and II trials. In vitro it is a potent inhibitor of cell lines with K-ras mutations, other ras mutations, and wild-type ras ( 11 ). Phase I tri-als in patients with non-hematologic cancers have been performed. In a trial of a 7-day administration every 3 weeks gastrointestinal toxicity (nausea, vomiting, and diarrhea) and fatigue were dose limiting ( 12 ). This trial also showed evidence of inhibition of farnesylation in buccal cells from patients treated in the trial. In two other trials of continuous daily administration similar toxicity was noted together with reversible myelosuppression and neurocortical toxicity ( 13 , 14 ). In a subse-quent phase I trial SCH66336 was combined with gemcitabine and showed gas-trointestinal toxicity and moderate myelosuppression as the most common toxicities (15 ). Although no phase II or III trials with SCH66336 have been reported for pan-creatic cancer, preclinical assessment with xenografts have shown activity with this tumor type ( 11 ).

Another CAAX inhibitor, R115777, has been evaluated in a clinical trial for pancreatic cancer. Whereas SCH66336 is a tricyclic drug derived from an anti-his-tamine lead compound, R115777 is a quinoline that was originally developed as an anti-fungal agent ( 16 ). Initial evidence of activity was noted in preclinical studies including pancreatic cancer xenografts ( 17 ). In subsequent phase I trials a variety of dosing schedules have been evaluated, including twice daily for 5 days every 2 weeks, twice daily for 21 days every 4 weeks, and continuous dosing ( 18 – 21 ). Early evidence of activity in pancreatic cancer was noted in these trials. The dose limiting toxicities were similar to those noted for SCH66336. A subsequent phase

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I trial established a schedule for gemcitabine and R115777 ( 22 ). Evidence of activityin pancreatic cancer was also noted in this trial.

In a phase II trial of patients with previously untreated metastatic pancreatic cancer, R115777 was given as a twice daily dose of 300 mg for 21 days every 4 weeks.Twenty patients were accrued to this trial. No objective responses were seen. The median survival time was 19.7 weeks. Only a partial inhibition of farnesylation was observed in peripheral blood mononuclear cells obtained from patients enrolled in this trial ( 23 ). In a separate phase II trial of R115777 using the same dosing schedule,58 patients with previously untreated either locally advanced or metastatic pancreaticcancer enrolled ( 24 ). In 53 evaluable patients from this trial a median survival of 2.6 months was reported.

While the phase II trials were underway a phase III trial of gemcitabine and R115777 was also undertaken. Using a randomized, double-blind, placebo-controlled design patients with previously untreated locally advanced, unresectable or meta-static pancreatic cancer were randomized to either gemcitabine and placebo or gemcitabine and R115777 ( 25 ). The R115777 was given at a dose of 200 mg twice a day continuously. The gemcitabine was given at a dose of 1,000 mg/m 2 over 30 minutes weekly for 7 weeks followed by a 1-week break for the first cycle and then weekly for 3 weeks followed by a 1-week break for subsequent cycles. A total of 688 patients were enrolled from 126 sites in 14 countries. Of these patients, 341 received gemcitabine and R115777 and 347 received gemcitabine and placebo. Median overall survival was the primary endpoint, with those receiving R115777 living 193 days versus 182 days for those receiving placebo ( p = 0.75). No mean-ingful difference was seen in other endpoints including progression-free survival, response rate, or time-to-performance status deterioration.

Other FTIs are being evaluated for cancer in general. This includes L-778,123, a drug that blocks both FTase and geranylgernanyltransferase I ( 26). A combination of L-778,123 and radiation was recently evaluated in a phase I trial for patients with locally advanced pancreatic cancer ( 27 ). Although the combination was reasonably well tolerated, only limited efficacy was noted.

At this point the role of FTIs in the treatment of pancreatic cancer remains uncertain. The initial trials, including a phase III trial, do not show meaningful clinical results. Further work will be needed to determine if FTIs in combination with other targeted therapy may be of benefit.

3 Ras-Directed Antisense Therapy for Pancreatic Cancer

As the molecular changes leading to the development and progression of cancer have become more clearly understood a greater focus on molecular or targeted therapy has developed, with a large variety of approaches currently under evalua-tion. One potential approach of interest focuses on the use of oligonucleotides to interfere with the expression of RNA ( 28 ). Specifically, antisense oligonucleotides have been created that are able to bind to a complimentary target RNA and inhibits

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its expression, thereby limiting the synthesis of proteins believed to be important in the development and progression of cancer. In general, this approach has led to a reduction of protein overexpression, but not complete elimination of protein syn-thesis ( 29 ). In this manner normal physiologic expression is preserved, thereby limiting potentially therapy-related toxicities. Other potential approaches with oli-gonucleotides have used the oligonucleotides as decoy binding sites ( 30 ). Support for this approach comes from both preclinical studies as well as the recognition of naturally occurring antisense RNAs that serve to regulate gene expression ( 31 ).

Early development of antisense oligonucleotides began in the late 1970s with the creation of an oligonucleotide sequence complementary to a portion of the Rous sarcoma virus that effectively blocked viral replication in fibroblasts ( 32 , 33 ). Over the next two decades a large amount of preclinical data was generated on the use of this approach in many different settings. Based on a review of this work and its potential importance, the use of antisense oligonucleotides and their role in post-transcriptional gene silencing was recognized as the breakthrough of the year by Science in 2002 ( 34 ). In particular, the results of research in this field were recog-nized for the surprising ability of small RNA molecules to control DNA expression by shutting down genes or altering their levels of expression. The application of this technology to a number of medical fields is currently being investigated. However, only one antisense-based therapy has received full approval by the Food and Drug Administration (FDA). Fomivirsen (Vitravene™) was developed to target the major immediate-early gene of cytomegalovirus (CMV) as a means of treating CMV-induced retinitis in patients with acquired immunodeficiency syndrome ( 35 ).

A variety of the antisense oligonucleotides developed that have potential appli-cability in pancreatic cancer, including several directed at ras. Given the high fre-quency of K-ras mutation, particularly in one codon, therapeutic approaches that are very specific may be of greater benefit. Although an antisense oligonucleotide against K-ras has been developed ( 36 – 38 ), no antisense oligonucleotides directed specifically against K-ras have entered clinical trials for pancreatic cancer. However, an antisense oligonucleotide directed against H-ras has been evaluated in a phase II trial, based on in part on the preclinical observation that H-ras modulates mutated K-ras. The H-ras antisense inhibitor ISIS 2503 (phosphorothioate 29-oligodeoxyri-bonucleotide) is 20 nucleotides long and is designed to hybridize to a sequence in the initiation translation region of the human H-ras mRNA ( 39 ). Once hybridized to its target ISIS 2503 renders the hybridized mRNA amenable to degradation by RNaseH. Initial preclinical evaluation of ISIS 2503 showed activity inhibiting pro-liferation of cultured T24 bladder cells ( 40 ). Subsequent evaluation of ISIS 2503 in xenograft models showed activity against Mia-PaCa-2, a tumor known to possess K-ras mutations ( 41 ).

Following the establishment of appropriate schedules of administration for ISIS 2,503 as a single agent in phase I trials ( 41 , 42 ), a phase I trial of ISIS 2503 com-bined with gemcitabine was performed ( 43 ). This trial established a schedule of gemcitabine 1,000 mg/m 2 on days 1 and 8 and ISIS 2503 6 mg/kg/day as a 14-day continuous infusion starting on day 1. Although neutropenia and thrombocytopenia were frequently encountered with this combination, no dose-limiting toxicity was

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incurred in this trial. Building on this phase I trial, a phase II trial of ISIS 2503 with gemcitabine for patients with locally advanced or metastatic pancreatic cancer was undertaken ( 44 ). A total of 48 patients were enrolled, of which 43 had metastatic disease. The median overall survival was 6.7 months, with one patient having a complete response and four patients having partial responses. These measures of outcomes were similar to those expected from gemcitabine alone indicating that ISIS 2503 provided no additional benefit.

4 Conclusion

Mutations in ras, particularly K-ras, are an early and apparent critical step in the development of pancreatic cancer. Therapeutic approaches directed at ras, with either FTIs or antisense oligonucleotides, have not made a meaningful change in patient outcomes. The reason for the lack of benefit remains unclear, although it is likely that other molecular changes may be able to overcome selective attempts to block overexpression of ras. Further work is needed to determine the potential role of ras-directed agents in combination with other targeted therapies for a cancer in which little progress has been made.

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