- Email: [email protected]
K-ras as a Target for Lung Cancer Therapy
NOVEL AGENTS
IN THE
TREATMENT
OF
LUNG CANCER
K-ras as a Target for Lung Cancer Therapy Alex A. Adjei, MD, PhD
K-ras is currently accepted to be the most frequently mutated oncogene in non-small cell lung cancer. In addition, tumors harboring mutant K-ras seem to be refractory to most available systemic therapies, making K-ras an attractive target for cancer therapy. The complexity of K-ras signaling presents many opportunities for therapeutic targeting. A number of different approaches aimed at abrogating K-ras activity have been explored in clinical trials. Several of the putative K-ras-directed therapeutic agents tested have demonstrated clinical activity. However, many of these agents have multiple targets, and their antitumor effects may not be due to K-ras inhibition. To date, no selective, specific inhibitor of the K-ras pathway is available for routine clinical use. Key Words: K-ras, Mitogen-activated protein kinase, Raf kinase, NSCLC. (J Thorac Oncol. 2008;3: Suppl 2, S160 –S163)
A
ctivated Ras targets a number of downstream effectors, including Raf kinase, phosphoinositide 3⬘-kinase, and Ral guanine exchange factors (GEFs), to produce pleiotropic cellular effects. The importance of Ras signaling in cell growth and survival is evidenced by the importance of Ras in oncogenesis. Several K-ras point mutations have been identified that result in constitutive activation. These mutations are found at high frequency in a variety of human tumors, including 20 to 40% of non-small cell lung cancer (NSCLC), and are briefly described herein. In recent years, K-ras mutations have been correlated with nonresponsiveness to epidermal growth factor receptor (EGFR) inhibitors1,2 and systemic chemotherapy.3 Because the role of K-ras in oncogenesis is well established, various strategies have been developed to target K-ras for the treatment of human cancers. These strategies have targeted various stages of Ras signaling, ranging from inhibiting protein expression via antisense oligonucleotides to blocking posttranslational modification with farnesyltransferase inhibitors to inhibiting downstream effectors. Although some of these strategies have resulted in antitumor activity, it is still unclear what role K-ras inhibition will play Roswell Park Cancer Institute, Buffalo, New York. Disclosure: The authors declare no conflict of interest. Address for correspondence: Alex A. Adjei, MD, PhD, Department of Medicine Roswell Park Cancer Institute, Elm & Carlton Streets, Buffalo, NY 14263. E-mail: [email protected] Copyright © 2008 by the International Association for the Study of Lung Cancer ISSN: 1556-0864/08/0306-0160
S160
in cancer therapies, because the most well-characterized Ras inhibitors tested in clinical trials have alternative targets.
RAS MUTATIONS This subject has been comprehensively reviewed.1 The role of ras genes in inducing malignant transformation is supported by several lines of evidence. First, oncogenic ras but not normal ras transfected into rodent fibroblasts renders them tumorigenic.3 Second, transgenic mice harboring oncogenic ras mutations have an increased incidence of tumor formation.4 Finally, a high frequency of ras mutations has been found in a variety of tumor types, both naturally occurring and experimentally induced. Identified mutations are limited to a small number of sites (amino acids 12, 13, 59, and 61), all of which abolish guanosine triphosphatase (GTPase) activating protein (GAP)induced guanosine triphosphate (GTP) hydrolysis of the ras proteins. Such single-point mutations of the ras gene can lead to its constitutive activation of ras protein. These mutated forms of ras have impaired GTPase activity. Although they still bind GAP, there is no “off” sign since GTPase is no longer activated. This results in continuous stimulation of cellular proliferation. Mutations are frequently limited to only one of the ras genes, and frequency is dependent on tissue and tumor type. Thus, ras mutations are rare in cancers of the breast, ovary, stomach, esophagus, and prostate but are present in almost all adenocarcinomas of the pancreas and 50% of colon and thyroid cancers. Mutations in NSCLC are found only in the K-ras gene, with a frequency of approximately 40% in adenocarcinomas.5 Recent, emerging data suggest that in NSCLC, K-ras mutations may be particularly common in tumors from smokers, with a frequency of almost 60%.5 Based on the evidence presented herein, K-ras clearly plays a role in the development and maintenance of the malignant phenotype. The fact that Ras mutations are so prevalent in NSCLC, with K-ras mutations being the most common, makes this an attractive therapeutic target. For the above reasons, cancer therapy targeting K-ras is a rational approach that would be expected to produce a clinical benefit.
APPROACHES TO K-RAS TARGETING FOR LUNG CANCER THERAPY Because Ras signaling is complex, there are many steps at which to target therapies designed to interfere with Ras signaling. Although not specifically addressed in this review, upstream Ras activation by tyrosine kinase and other receptors is an active area of pharmaceutical development given the successes with agents targeting EGFR and vascular en-
Journal of Thoracic Oncology • Vol. 3, No. 6, Supplement 2, June 2008
Journal of Thoracic Oncology • Vol. 3, No. 6, Supplement 2, June 2008
dothelial growth factor receptor (VEGFR) families. It is likely that the antitumor activity of these agents is at least, in part, due to effects on the Ras signaling pathway. In support of this idea is the fact that tumors with ras mutations (and therefore constitutively active with lack of dependence on upstream signals) are resistant to EGFR inhibitors. Herein, we focus on strategies aimed at Ras or downstream effectors. Conceptually, targeting K-ras dysregulation could occur at many levels of the Ras signaling cascade, including inhibiting Ras protein expression, inhibiting membrane localization through posttranslational modification or trafficking, blocking Ras interaction with GEFs, enhancing Ras/GAP interactions, targeting oncogenic K-ras, or inhibiting Ras effectors. The ultimate test of Ras signaling as a valid cancer therapy would come from an agent designed to specifically target Ras dysregulation. Unfortunately, such an agent has not yet been developed. Considerable research went into the development of farnesyltransferase inhibitors, which were expected to inhibit posttranslational processing and membrane localization of Ras proteins and antisense oligonucleotides against ras. These efforts were unsuccessful and have been reviewed extensively elsewhere.6 The two promising approaches to inhibiting K-ras that are currently under investigation involve the inhibition of the downstream proteins raf kinase and mitogen-activated protein kinase (MAPK) kinase (MEK).
RAF KINASE INHIBITORS The raf family comprises three members: a-raf, b-raf, and c-raf or raf-1. These proteins enable converging of extracellular stimuli that then activate the components of the MAPK pathway. Although linearly located downstream of Ras, raf kinases are now known to be part of a branch point, controlling cell proliferation and other cellular functions through both the MAPK pathway and MAPK independent pathways. Of the three Raf proteins, C-Raf is the most widely expressed. C-Raf is located in the cytosol but once activated relocates to the membrane. Activating mutations of B-raf have been reported in ⬃70% of melanomas and in a number of other human tumor types, including ovarian and papillary thyroid carcinomas. A survey of 43 cancer cell lines demonstrated that all B-raf mutations resided in exons 11 or 15. Remarkably, 80% of these B-raf mutations represent a single nucleotide change of T-A at nucleotide 1796, resulting in a valine to glutamic acid substitution at residue 600 (V600E, exon 15) in the adenosine triphosphate binding and substrate recognition (CR3) domain. This substitution confers constitutive kinase activity. This activating B-raf allele can be detected, allowing tumor genotyping in the clinical setting. The activating mutations in K-ras and B-raf represent the first report of a tandem activating mutation in the same signaling pathway. Taking together, one or the other of these mutations may be present in approximately 50% of NSCLCs. Tumors such as NSCLC, which possess either K-ras or B-raf mutations, or overexpression receptors that signal through the ras-MAPK pathway such as EGFR may be amenable to inhibition of the downstream protein MEK. Strategies for targeting Raf include (1)
K-ras in Lung Cancer Therapy
antisense oligodeoxyribonucleotides that degrade the raf-1 messenger RNA and (2) inhibition of kinase activity, for example, BAY 43-9006. Sorafenib (Nexavar; BAY 43-9006; Bayer Pharmaceuticals, Montville, New Jersey) is a multikinase inhibitor of Raf-1; wild-type B-raf; oncogenic b-raf V600E; VEGFR-1, -2, and -3; platelet-derived growth factor receptor ; c-Kit; Flt-3; and RET in vitro.7,8 Sorafenib inhibited the growth of a variety of tumor xenograft models driven by up-regulation of Raf/MEK/ERK signaling.5 In vitro, sorafenib inhibited tumor proliferation and angiogenesis and promoted apoptosis in several cancer cell lines.9 Tolerability and preliminary efficacy of sorafenib were investigated in four dose-escalation phase 1 trials in patients with treatment-refractory, advanced solid tumors.10 –13 The optimum dose was established as sorafenib, 400 mg twice daily, given in an uninterrupted schedule. In a pivotal phase 3 trial (Treatment Approaches in Renal cancer Global Evaluation Trial [TARGETs]) involving patients with clear cell renal cell carcinoma, sorafenib significantly prolonged progression-free survival two-fold versus placebo and was generally well tolerated. Seven patients receiving sorafenib had partial responses, and 78% of patients had stable disease versus 55% receiving placebo.14 Based on these results, sorafenib has been approved in several countries worldwide for the treatment of renal cell carcinoma and was granted orphan drug status in hepatocellular carcinoma (April 2006) by the Food and Drug Administration and European Commission. A phase 2 study in NSCLC was reported by Gatzeimeir et al., at the American Society of Clinical Oncology 2006 Annual Meeting. Fifty-two patients with refractory metastatic NSCLC were enrolled. Most of the patients had a performance status of 0 to 1 (44 of 52). Fifty had been previously treated with 1 to 2 chemotherapy regimens, including previous gefitinib therapy. Adenocarcinoma was the predominant histologic finding in 28 patients and squamous histologic findings were predominant in 16 patients. In this multicenter, open-labeled, single-arm phase 2 study, sorafenib was given at a dose of 400 mg twice daily continuously for a 28-day cycle until progression noted by Response Evaluation Criteria in Solid Tumors. No objective responses were seen. Stable disease was noted in 59% of patients. The progression-free survival was 2.7 months and the overall survival was 6.8 months. Two patients were continuing therapy at 2 years. The most common toxic effects were diarrhea (40%) and hand foot syndrome (37%). Four patients had a bleeding event (3 with epistaxis and 1 patient with squamous histologic findings and a cavitary lesion who developed fatal pulmonary hemorrhage after radiation, 30 days after stopping sorafenib therapy). Baseline lower plasma VEGF levels (⬍161 pg/mL) were predictive of improved median survival (292 days) compared with higher levels (⬎161 pg/mL) (184 days) (P ⫽ 0.05). Interestingly, the patients who showed a greater decrease (⬎78 pg/mL) with sorafenib treatment at day 15, cycle 1, had a shorter survival (168 days) compared with those who showed a lesser decrease (264 days) (P ⫽ 0.05). Currently, at least two phase 3 studies are under way (in Europe and North America). In addition to studies evaluating single-agent sor-
Copyright © 2008 by the International Association for the Study of Lung Cancer
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Adjei
afenib in the treatment of refractory NSCLC, other studies are looking at the combination of sorafenib with both chemotherapy and other targeted agents such as gefitinib. In a phase 1 study of sorafenib in combination with gefitinib, the combination of sorafenib at 400 mg orally twice daily and gefitinib at 250 mg orally once daily was well tolerated and led to tumor regression in 9 of 12 patients.15 In addition, a large randomized study is evaluating sorafenib and chemotherapy versus bevacizumab and chemotherapy in patients with metastatic NSCLC. Other more specific raf kinase inhibitors are currently in phase 1 trials and results are awaited with interest.
MEK INHIBITORS Three MEK inhibitors have been tested in the clinic. The first in this class, CI-1040, displayed encouraging antitumor activity in phase 1 studies and progressed to phase 2 testing in patients with metastatic or inoperable breast, colon, NSCLC, or pancreatic cancers.16 The study enrolled 67 patients, including 18 with NSCLC. Efficacy end points were not met and development was discontinued. A second-generation agent, PD 0325901, with higher potency and improved pharmacodynamic properties, was introduced into the clinic.17–19 Patients with advanced breast cancer, colon cancer, NSCLC, or melanoma were treated with doses ranging from 1 mg daily to 30 mg twice daily. Drug-related adverse events were similar to those reported for CI-1040, including rash, fatigue, nausea, diarrhea, and vomiting. Visual changes, including blurred vision and haloes, were reported in five patients receiving more than 15 mg twice daily. Dose-limiting toxic effects reported included rash (3 patients), cardiac events (2 patients), and anemia, diarrhea, and mucositis (1 patient). Encouraging evidence of antitumor activity was observed in this phase 1 trial with two partial responses in melanoma patients, and an additional eight patients (5 melanoma, 2 NSCLC, and 1 colon cancer) achieved stable disease lasting for 3 to 7 months.19 Development of this compound, however, has been stopped because of toxicity.20 The final MEK inhibitor to undergo clinical testing is AZD6244, which has been investigated in an open-label, multicenter, two-part phase 1 trial in patients with advanced solid malignancies. Although there were no objective responses, stable disease was observed in 14 of 31 patients (45%) who were assessable for response. Nine of these patients had stable disease that lasted longer than 5 months (6 melanoma, 1 breast cancer, 1 NSCLC, and 1 thyroid). Pretreatment and posttreatment tumor biopsy specimens obtained from 17 patients showed a mean 83% reduction in nuclear phospho-Erk staining. These results demonstrate AZD6244-induced target modulation within tumor tissue.21 Based on these results, multiple phase 2 studies are ongoing, including a study in NSCLC. In addition to the drugs described herein, there are three other MEK inhibitors, including a backup compound to AZD6244, that have entered the clinic.
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CONCLUSIONS Since the identification of K-ras as the transforming agent of the Kirsten murine sarcoma virus, more than 20 years of accumulated evidence lends extensive support to the importance of K-ras in cellular signaling, tumorigenesis, and maintenance of the malignant phenotype in NSCLC. The advent of EGFR inhibitors have further highlighted the importance of K-ras as a negative predictor of response to therapy. Therefore, K-ras has been an important focus for cancer therapy. Although a number of agents interrupting with signal transduction proteins downstream of K-ras continue to be investigated, ongoing research seeks to identify specific and potent inhibitors of K-ras. The validity of targeting Ras for lung cancer therapy will not be fully realized until clinical responses are observed using a strategy that selectively and effectively targets K-ras. Several candidate strategies that could answer this question have been described. Both antisense oligonucleotides and small interfering RNAs have the potential for specific inhibition, but they are currently limited by drug delivery. If effective delivery techniques were devised, then these may be suitable strategies. Peptidomimetics could also specifically block oncogenic signaling. Additionally, as high-throughput screening progresses, it is likely that small-molecule inhibitors will be identified that can specifically inhibit Ras function. REFERENCES 1. Adjei AA. Blocking oncogenic Ras signaling for cancer therapy. J Natl Cancer Inst 2001;93:1062–1074. 2. Pao W, Wang TY, Riely GJ, et al. KRAS mutations and primary resistance of lung adenocarcinomas to gefitinib or erlotinib. PLos Med 2005;2:e17. 3. Lowy DR, Willumsen BM. Function and regulation of ras. Annu Rev Biochem 1993;62:851– 891. 4. Barbacid M. Ras genes. Annu Rev Biochem 1987;56:779 – 827. 5. Suzuki Y, Orita M, Shiraishi M, Hayashi K, Sekiya T. Detection of ras gene mutations in human lung cancers by single-strand conformation polymorphism analysis of polymerase chain reaction products. Oncogene 1990;5:1037–1043. 6. Adjei AA. Farnesyl transferase inhibitors. Cancer Chemother Biol Response Modif 2003;21:127–144. 7. Wilhelm SM, Carter C, Tang L, et al. BAY 43-9006 exhibits broad spectrum oral anti-tumor activity and targets the Raf/MEK/ERK pathway and receptor tyrosine kinases involved in tumor progression and angiogenesis. Cancer Res 2004;64:7099 –7109. 8. Levy AP, Pauloski N, Braun D, et al. Analysis of transcription and protein expression changes in the 786-O human renal cell carcinoma tumor xenograft model in response to treatment with the multi-kinase inhibitor sorafenib (BAY 43-9006). Proc Am Assoc Cancer Res 2006; 47:213–214 (abstract and oral presentation). 9. Yu C, Bruzek LM, Meng XW, et al. The role of Mcl-1 downregulation in the proapoptotic activity of the multikinase inhibitor BAY 43-9006. Oncogene 2005;24:6861– 6869. 10. Clark JW, Eder JP, Ryan D, Lathia C, Lenz HJ. Safety and pharmacokinetics of the dual action Raf kinase and vascular endothelial growth factor receptor inhibitor, BAY 43-9006, in patients with advanced, refractory solid tumors. Clin Cancer Res 2005;11:5472–5480. 11. Awada A, Hendlisz A, Gil T, et al. Phase I safety and pharmacokinetics of BAY 43-9006 administered for 21 days on/7 days off in patients with advanced, refractory solid tumours. Br J Cancer 2005;92:1855–1861. 12. Moore M, Hirte HW, Siu L, et al. Phase I study to determine the safety and pharmacokinetics of the novel Raf kinase and VEGFR inhibitor BAY 43-9006, administered for 28 days on/7 days off in patients with advanced, refractory solid tumors. Ann Oncol 2005;16:1688 –1694.
Copyright © 2008 by the International Association for the Study of Lung Cancer
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13. Strumberg D, Richly H, Hilger RA, et al. Phase I clinical and pharmacokinetic study of the novel Raf kinase and vascular endothelial growth factor receptor inhibitor BAY 43-9006 in patients with advanced refractory solid tumors. J Clin Oncol 2005;23:965–972. 14. Escudier B, Szczylik C, Eisen T, et al. Randomized phase III trial of the multi-kinase inhibitor sorafenib (BAY 43-9006) in patients with advanced renal cell carcinoma (RCC). Eur J Cancer Suppl 2005;3:226. 15. Adjei AA, Molina JR, Marks R, et al. Phase I trial of sorafenib in combination with gefitinib in patients with refractory or recurrent nonsmall-cell lung cancer. Clin Cancer Res 2007;13:2684 –2691. 16. Rinehart J, Adjei AA, Lorusso PM, et al. Multicenter phase II study of the oral MEK inhibitor, CI-1040, in patients with advanced non-smallcell lung, breast, colon, and pancreatic cancer. J Clin Oncol 2004;22: 4456 – 4462. 17. Lorusso P, Krishnamurthi S, Rinehart JR, et al. A phase 1–2 clinical
18.
19. 20. 21.
K-ras in Lung Cancer Therapy
study of a second generation oral MEK inhibitor, PD 0325901 in patients with advanced cancer. J Clin Oncol (Meeting Abstr) 2005;23:3011. Menon SS, Whitfield LR, Sadis S, et al. Pharmacokinetics (PK) and pharmacodynamics (PD) of PD 0325901, a second generation MEK inhibitor after multiple oral doses of PD 0325901 to advanced cancer patients. J Clin Oncol (Meeting Abstr). 2005;23:3066. Wang D, Boerner SA, Winkler JD, Lorusso PM. Clinical experience of MEK inhibitors in cancer therapy. Biochim Biophys Acta 2007;1773: 1248 –1255. Pfizer Pipeline. Available at: http://www.pfizer.com/research/pipeline. jsp. Accessed October 15, 2007. Adjei AA, Cohen RB, Franklin WA, Molina J. Phase IB and pharmacodynamic study of the MEK inhibitor AZD6244 (ARRY-142886) in patients with advanced solid malignancies. AACR-EORTC-NCI Molecular Targets Meeting, November 2006.
Copyright © 2008 by the International Association for the Study of Lung Cancer
S163
IN THE
TREATMENT
OF
LUNG CANCER
K-ras as a Target for Lung Cancer Therapy Alex A. Adjei, MD, PhD
K-ras is currently accepted to be the most frequently mutated oncogene in non-small cell lung cancer. In addition, tumors harboring mutant K-ras seem to be refractory to most available systemic therapies, making K-ras an attractive target for cancer therapy. The complexity of K-ras signaling presents many opportunities for therapeutic targeting. A number of different approaches aimed at abrogating K-ras activity have been explored in clinical trials. Several of the putative K-ras-directed therapeutic agents tested have demonstrated clinical activity. However, many of these agents have multiple targets, and their antitumor effects may not be due to K-ras inhibition. To date, no selective, specific inhibitor of the K-ras pathway is available for routine clinical use. Key Words: K-ras, Mitogen-activated protein kinase, Raf kinase, NSCLC. (J Thorac Oncol. 2008;3: Suppl 2, S160 –S163)
A
ctivated Ras targets a number of downstream effectors, including Raf kinase, phosphoinositide 3⬘-kinase, and Ral guanine exchange factors (GEFs), to produce pleiotropic cellular effects. The importance of Ras signaling in cell growth and survival is evidenced by the importance of Ras in oncogenesis. Several K-ras point mutations have been identified that result in constitutive activation. These mutations are found at high frequency in a variety of human tumors, including 20 to 40% of non-small cell lung cancer (NSCLC), and are briefly described herein. In recent years, K-ras mutations have been correlated with nonresponsiveness to epidermal growth factor receptor (EGFR) inhibitors1,2 and systemic chemotherapy.3 Because the role of K-ras in oncogenesis is well established, various strategies have been developed to target K-ras for the treatment of human cancers. These strategies have targeted various stages of Ras signaling, ranging from inhibiting protein expression via antisense oligonucleotides to blocking posttranslational modification with farnesyltransferase inhibitors to inhibiting downstream effectors. Although some of these strategies have resulted in antitumor activity, it is still unclear what role K-ras inhibition will play Roswell Park Cancer Institute, Buffalo, New York. Disclosure: The authors declare no conflict of interest. Address for correspondence: Alex A. Adjei, MD, PhD, Department of Medicine Roswell Park Cancer Institute, Elm & Carlton Streets, Buffalo, NY 14263. E-mail: [email protected] Copyright © 2008 by the International Association for the Study of Lung Cancer ISSN: 1556-0864/08/0306-0160
S160
in cancer therapies, because the most well-characterized Ras inhibitors tested in clinical trials have alternative targets.
RAS MUTATIONS This subject has been comprehensively reviewed.1 The role of ras genes in inducing malignant transformation is supported by several lines of evidence. First, oncogenic ras but not normal ras transfected into rodent fibroblasts renders them tumorigenic.3 Second, transgenic mice harboring oncogenic ras mutations have an increased incidence of tumor formation.4 Finally, a high frequency of ras mutations has been found in a variety of tumor types, both naturally occurring and experimentally induced. Identified mutations are limited to a small number of sites (amino acids 12, 13, 59, and 61), all of which abolish guanosine triphosphatase (GTPase) activating protein (GAP)induced guanosine triphosphate (GTP) hydrolysis of the ras proteins. Such single-point mutations of the ras gene can lead to its constitutive activation of ras protein. These mutated forms of ras have impaired GTPase activity. Although they still bind GAP, there is no “off” sign since GTPase is no longer activated. This results in continuous stimulation of cellular proliferation. Mutations are frequently limited to only one of the ras genes, and frequency is dependent on tissue and tumor type. Thus, ras mutations are rare in cancers of the breast, ovary, stomach, esophagus, and prostate but are present in almost all adenocarcinomas of the pancreas and 50% of colon and thyroid cancers. Mutations in NSCLC are found only in the K-ras gene, with a frequency of approximately 40% in adenocarcinomas.5 Recent, emerging data suggest that in NSCLC, K-ras mutations may be particularly common in tumors from smokers, with a frequency of almost 60%.5 Based on the evidence presented herein, K-ras clearly plays a role in the development and maintenance of the malignant phenotype. The fact that Ras mutations are so prevalent in NSCLC, with K-ras mutations being the most common, makes this an attractive therapeutic target. For the above reasons, cancer therapy targeting K-ras is a rational approach that would be expected to produce a clinical benefit.
APPROACHES TO K-RAS TARGETING FOR LUNG CANCER THERAPY Because Ras signaling is complex, there are many steps at which to target therapies designed to interfere with Ras signaling. Although not specifically addressed in this review, upstream Ras activation by tyrosine kinase and other receptors is an active area of pharmaceutical development given the successes with agents targeting EGFR and vascular en-
Journal of Thoracic Oncology • Vol. 3, No. 6, Supplement 2, June 2008
Journal of Thoracic Oncology • Vol. 3, No. 6, Supplement 2, June 2008
dothelial growth factor receptor (VEGFR) families. It is likely that the antitumor activity of these agents is at least, in part, due to effects on the Ras signaling pathway. In support of this idea is the fact that tumors with ras mutations (and therefore constitutively active with lack of dependence on upstream signals) are resistant to EGFR inhibitors. Herein, we focus on strategies aimed at Ras or downstream effectors. Conceptually, targeting K-ras dysregulation could occur at many levels of the Ras signaling cascade, including inhibiting Ras protein expression, inhibiting membrane localization through posttranslational modification or trafficking, blocking Ras interaction with GEFs, enhancing Ras/GAP interactions, targeting oncogenic K-ras, or inhibiting Ras effectors. The ultimate test of Ras signaling as a valid cancer therapy would come from an agent designed to specifically target Ras dysregulation. Unfortunately, such an agent has not yet been developed. Considerable research went into the development of farnesyltransferase inhibitors, which were expected to inhibit posttranslational processing and membrane localization of Ras proteins and antisense oligonucleotides against ras. These efforts were unsuccessful and have been reviewed extensively elsewhere.6 The two promising approaches to inhibiting K-ras that are currently under investigation involve the inhibition of the downstream proteins raf kinase and mitogen-activated protein kinase (MAPK) kinase (MEK).
RAF KINASE INHIBITORS The raf family comprises three members: a-raf, b-raf, and c-raf or raf-1. These proteins enable converging of extracellular stimuli that then activate the components of the MAPK pathway. Although linearly located downstream of Ras, raf kinases are now known to be part of a branch point, controlling cell proliferation and other cellular functions through both the MAPK pathway and MAPK independent pathways. Of the three Raf proteins, C-Raf is the most widely expressed. C-Raf is located in the cytosol but once activated relocates to the membrane. Activating mutations of B-raf have been reported in ⬃70% of melanomas and in a number of other human tumor types, including ovarian and papillary thyroid carcinomas. A survey of 43 cancer cell lines demonstrated that all B-raf mutations resided in exons 11 or 15. Remarkably, 80% of these B-raf mutations represent a single nucleotide change of T-A at nucleotide 1796, resulting in a valine to glutamic acid substitution at residue 600 (V600E, exon 15) in the adenosine triphosphate binding and substrate recognition (CR3) domain. This substitution confers constitutive kinase activity. This activating B-raf allele can be detected, allowing tumor genotyping in the clinical setting. The activating mutations in K-ras and B-raf represent the first report of a tandem activating mutation in the same signaling pathway. Taking together, one or the other of these mutations may be present in approximately 50% of NSCLCs. Tumors such as NSCLC, which possess either K-ras or B-raf mutations, or overexpression receptors that signal through the ras-MAPK pathway such as EGFR may be amenable to inhibition of the downstream protein MEK. Strategies for targeting Raf include (1)
K-ras in Lung Cancer Therapy
antisense oligodeoxyribonucleotides that degrade the raf-1 messenger RNA and (2) inhibition of kinase activity, for example, BAY 43-9006. Sorafenib (Nexavar; BAY 43-9006; Bayer Pharmaceuticals, Montville, New Jersey) is a multikinase inhibitor of Raf-1; wild-type B-raf; oncogenic b-raf V600E; VEGFR-1, -2, and -3; platelet-derived growth factor receptor ; c-Kit; Flt-3; and RET in vitro.7,8 Sorafenib inhibited the growth of a variety of tumor xenograft models driven by up-regulation of Raf/MEK/ERK signaling.5 In vitro, sorafenib inhibited tumor proliferation and angiogenesis and promoted apoptosis in several cancer cell lines.9 Tolerability and preliminary efficacy of sorafenib were investigated in four dose-escalation phase 1 trials in patients with treatment-refractory, advanced solid tumors.10 –13 The optimum dose was established as sorafenib, 400 mg twice daily, given in an uninterrupted schedule. In a pivotal phase 3 trial (Treatment Approaches in Renal cancer Global Evaluation Trial [TARGETs]) involving patients with clear cell renal cell carcinoma, sorafenib significantly prolonged progression-free survival two-fold versus placebo and was generally well tolerated. Seven patients receiving sorafenib had partial responses, and 78% of patients had stable disease versus 55% receiving placebo.14 Based on these results, sorafenib has been approved in several countries worldwide for the treatment of renal cell carcinoma and was granted orphan drug status in hepatocellular carcinoma (April 2006) by the Food and Drug Administration and European Commission. A phase 2 study in NSCLC was reported by Gatzeimeir et al., at the American Society of Clinical Oncology 2006 Annual Meeting. Fifty-two patients with refractory metastatic NSCLC were enrolled. Most of the patients had a performance status of 0 to 1 (44 of 52). Fifty had been previously treated with 1 to 2 chemotherapy regimens, including previous gefitinib therapy. Adenocarcinoma was the predominant histologic finding in 28 patients and squamous histologic findings were predominant in 16 patients. In this multicenter, open-labeled, single-arm phase 2 study, sorafenib was given at a dose of 400 mg twice daily continuously for a 28-day cycle until progression noted by Response Evaluation Criteria in Solid Tumors. No objective responses were seen. Stable disease was noted in 59% of patients. The progression-free survival was 2.7 months and the overall survival was 6.8 months. Two patients were continuing therapy at 2 years. The most common toxic effects were diarrhea (40%) and hand foot syndrome (37%). Four patients had a bleeding event (3 with epistaxis and 1 patient with squamous histologic findings and a cavitary lesion who developed fatal pulmonary hemorrhage after radiation, 30 days after stopping sorafenib therapy). Baseline lower plasma VEGF levels (⬍161 pg/mL) were predictive of improved median survival (292 days) compared with higher levels (⬎161 pg/mL) (184 days) (P ⫽ 0.05). Interestingly, the patients who showed a greater decrease (⬎78 pg/mL) with sorafenib treatment at day 15, cycle 1, had a shorter survival (168 days) compared with those who showed a lesser decrease (264 days) (P ⫽ 0.05). Currently, at least two phase 3 studies are under way (in Europe and North America). In addition to studies evaluating single-agent sor-
Copyright © 2008 by the International Association for the Study of Lung Cancer
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Journal of Thoracic Oncology • Vol. 3, No. 6, Supplement 2, June 2008
Adjei
afenib in the treatment of refractory NSCLC, other studies are looking at the combination of sorafenib with both chemotherapy and other targeted agents such as gefitinib. In a phase 1 study of sorafenib in combination with gefitinib, the combination of sorafenib at 400 mg orally twice daily and gefitinib at 250 mg orally once daily was well tolerated and led to tumor regression in 9 of 12 patients.15 In addition, a large randomized study is evaluating sorafenib and chemotherapy versus bevacizumab and chemotherapy in patients with metastatic NSCLC. Other more specific raf kinase inhibitors are currently in phase 1 trials and results are awaited with interest.
MEK INHIBITORS Three MEK inhibitors have been tested in the clinic. The first in this class, CI-1040, displayed encouraging antitumor activity in phase 1 studies and progressed to phase 2 testing in patients with metastatic or inoperable breast, colon, NSCLC, or pancreatic cancers.16 The study enrolled 67 patients, including 18 with NSCLC. Efficacy end points were not met and development was discontinued. A second-generation agent, PD 0325901, with higher potency and improved pharmacodynamic properties, was introduced into the clinic.17–19 Patients with advanced breast cancer, colon cancer, NSCLC, or melanoma were treated with doses ranging from 1 mg daily to 30 mg twice daily. Drug-related adverse events were similar to those reported for CI-1040, including rash, fatigue, nausea, diarrhea, and vomiting. Visual changes, including blurred vision and haloes, were reported in five patients receiving more than 15 mg twice daily. Dose-limiting toxic effects reported included rash (3 patients), cardiac events (2 patients), and anemia, diarrhea, and mucositis (1 patient). Encouraging evidence of antitumor activity was observed in this phase 1 trial with two partial responses in melanoma patients, and an additional eight patients (5 melanoma, 2 NSCLC, and 1 colon cancer) achieved stable disease lasting for 3 to 7 months.19 Development of this compound, however, has been stopped because of toxicity.20 The final MEK inhibitor to undergo clinical testing is AZD6244, which has been investigated in an open-label, multicenter, two-part phase 1 trial in patients with advanced solid malignancies. Although there were no objective responses, stable disease was observed in 14 of 31 patients (45%) who were assessable for response. Nine of these patients had stable disease that lasted longer than 5 months (6 melanoma, 1 breast cancer, 1 NSCLC, and 1 thyroid). Pretreatment and posttreatment tumor biopsy specimens obtained from 17 patients showed a mean 83% reduction in nuclear phospho-Erk staining. These results demonstrate AZD6244-induced target modulation within tumor tissue.21 Based on these results, multiple phase 2 studies are ongoing, including a study in NSCLC. In addition to the drugs described herein, there are three other MEK inhibitors, including a backup compound to AZD6244, that have entered the clinic.
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CONCLUSIONS Since the identification of K-ras as the transforming agent of the Kirsten murine sarcoma virus, more than 20 years of accumulated evidence lends extensive support to the importance of K-ras in cellular signaling, tumorigenesis, and maintenance of the malignant phenotype in NSCLC. The advent of EGFR inhibitors have further highlighted the importance of K-ras as a negative predictor of response to therapy. Therefore, K-ras has been an important focus for cancer therapy. Although a number of agents interrupting with signal transduction proteins downstream of K-ras continue to be investigated, ongoing research seeks to identify specific and potent inhibitors of K-ras. The validity of targeting Ras for lung cancer therapy will not be fully realized until clinical responses are observed using a strategy that selectively and effectively targets K-ras. Several candidate strategies that could answer this question have been described. Both antisense oligonucleotides and small interfering RNAs have the potential for specific inhibition, but they are currently limited by drug delivery. If effective delivery techniques were devised, then these may be suitable strategies. Peptidomimetics could also specifically block oncogenic signaling. Additionally, as high-throughput screening progresses, it is likely that small-molecule inhibitors will be identified that can specifically inhibit Ras function. REFERENCES 1. Adjei AA. Blocking oncogenic Ras signaling for cancer therapy. J Natl Cancer Inst 2001;93:1062–1074. 2. Pao W, Wang TY, Riely GJ, et al. KRAS mutations and primary resistance of lung adenocarcinomas to gefitinib or erlotinib. PLos Med 2005;2:e17. 3. Lowy DR, Willumsen BM. Function and regulation of ras. Annu Rev Biochem 1993;62:851– 891. 4. Barbacid M. Ras genes. Annu Rev Biochem 1987;56:779 – 827. 5. Suzuki Y, Orita M, Shiraishi M, Hayashi K, Sekiya T. Detection of ras gene mutations in human lung cancers by single-strand conformation polymorphism analysis of polymerase chain reaction products. Oncogene 1990;5:1037–1043. 6. Adjei AA. Farnesyl transferase inhibitors. Cancer Chemother Biol Response Modif 2003;21:127–144. 7. Wilhelm SM, Carter C, Tang L, et al. BAY 43-9006 exhibits broad spectrum oral anti-tumor activity and targets the Raf/MEK/ERK pathway and receptor tyrosine kinases involved in tumor progression and angiogenesis. Cancer Res 2004;64:7099 –7109. 8. Levy AP, Pauloski N, Braun D, et al. Analysis of transcription and protein expression changes in the 786-O human renal cell carcinoma tumor xenograft model in response to treatment with the multi-kinase inhibitor sorafenib (BAY 43-9006). Proc Am Assoc Cancer Res 2006; 47:213–214 (abstract and oral presentation). 9. Yu C, Bruzek LM, Meng XW, et al. The role of Mcl-1 downregulation in the proapoptotic activity of the multikinase inhibitor BAY 43-9006. Oncogene 2005;24:6861– 6869. 10. Clark JW, Eder JP, Ryan D, Lathia C, Lenz HJ. Safety and pharmacokinetics of the dual action Raf kinase and vascular endothelial growth factor receptor inhibitor, BAY 43-9006, in patients with advanced, refractory solid tumors. Clin Cancer Res 2005;11:5472–5480. 11. Awada A, Hendlisz A, Gil T, et al. Phase I safety and pharmacokinetics of BAY 43-9006 administered for 21 days on/7 days off in patients with advanced, refractory solid tumours. Br J Cancer 2005;92:1855–1861. 12. Moore M, Hirte HW, Siu L, et al. Phase I study to determine the safety and pharmacokinetics of the novel Raf kinase and VEGFR inhibitor BAY 43-9006, administered for 28 days on/7 days off in patients with advanced, refractory solid tumors. Ann Oncol 2005;16:1688 –1694.
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