Write an essay on targeting Epidermal growth factor receptor (EGFR) with tyrosine kinase inhibitors or TKIs to treat Patients with Lung cancer and the role of mutations developing resistance and sensitization

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Home >Biology homework help >Write an essay on targeting Epidermal growth factor receptor (EGFR) with tyrosine kinase inhibitors or TKIs to treat Patients with Lung cancer and the role of mutations developing resistance and sensitization

Lung cancer is the leading cause of cancer death, accounting for one third of all deaths from cancer worldwide. Like most cancers, lung cancer is a con- glomeration of diseases of diverse aetiology, broadly divided into small-cell lung cancer (SCLC, comprising 20% of lung cancers), and non-small-cell lung cancer (NSCLC, comprising 80% of lung cancers). SCLC is a tumour of neural crest origin and initially responds well to chemotherapy, but commonly recurs with resistant disease. NSCLC is thought to originate in lung epithe- lial cells, and comprises diverse histological subtypes including adenocarcinoma, bronchioloalveolar, squa- mous, anaplastic and large-cell carcinomas1. Most patients with advanced NSCLC present with metastatic disease and, if left untreated, have a median survival after diagnosis of 4–5 months and a 1-year survival of less than 10% (REF. 2). Combination cytotoxic chemo- therapy, the treatment of choice in these cases, results in a modest increase in survival at the cost of signifi- cant toxicity to the patient3. The advent of molecular- targeted therapeutics has therefore generated much optimism, given the perception that the limits of chemotherapy in NSCLC have been reached and that further advances in the treatment of NSCLC will have to involve radically different approaches (reviewed in REF. 4). Against this backdrop, the approval of small- molecule inhibitors of the epidermal growth factor receptor (EGFR) kinase for the treatment of NSCLC in 2003 was heralded with much fanfare, although the limitations of their efficacy have become readily apparent (reviewed in REF. 5).

The deregulation of EGFR in NSCLC The receptor tyrosine kinase (RTK) super-family of cell- surface receptors serve as mediators of cell signalling by extra-cellular growth factors6. Members of the ErbB family of RTKs, such as EGFR (also known as ERBB1 or HER1), ERBB2 (also known as HER2), ERBB3 (also known as HER3) and ERBB4 (also known as HER4) have received much attention, given their strong asso- ciation with malignant proliferation (reviewed in REF. 7). Increased levels of EGFR gene expression are observed in cancers of the head and neck, ovary, cervix, bladder, oesophagus, stomach, brain, breast, endometrium, colon and lung, and frequently seem to confer an adverse prognosis (reviewed in REFS 6,8). Extending previous observations of almost two decades ago9,10, recent retrospective analyses have reported EGFR over- expression in 62% of NSCLC cases, and its expression is correlated with a poor prognosis8,11,12. In some cases, genomic analyses documented the amplification of chromosomal region 7p12, where the EGFR gene is located13. In addition to EGFR overexpression, its cognate ligands, epidermal growth factor (EGF) and transforming growth factor-α (TGFα) are also fre- quently expressed in NSCLCs, and can establish autocrine loops that lead to receptor hyperactivity14,15. The disrup- tion of these autocrine loops is the primary rationale for antibody-based EGFR-targeted therapeutics16.

Various strategies involving small-molecule inhibi- tors have also been developed to target EGFR and/or its family members, and these are in various stages of clinical testing (reviewed in REF. 17). Gefitinib

*Massachusetts General Hospital Cancer Center and Harvard Medical School, 149 13th Street, Charlestown, Massachusetts 02129, USA. ‡Present address: National Human Genome Research Institute, National Institutes of Health, Bethesda, Maryland 20892, USA. Correspondence to D.A.H. e-mail: [email protected]. harvard.edu doi:10.1038/nrc2088

Neural crest A pluripotent, ectodermally derived ridge-like cluster of cells found on either side of the neural tube in vertebrate embryos.

Cytotoxic chemotherapy Chemicals or drugs that kill proliferating cells, especially cancer cells. Their side effects are typically related to the inhibition of normal cell proliferation, with a narrow window of selectivity for cancer cells.

Epidermal growth factor receptor mutations in lung cancer Sreenath V. Sharma*, Daphne W. Bell*‡, Jeffrey Settleman* and Daniel A. Haber*

Abstract | The development and clinical application of inhibitors that target the epidermal growth factor receptor (EGFR) provide important insights for new lung cancer therapies, as well as for the broader field of targeted cancer therapies. We review the results of genetic, biochemical and clinical studies focused on somatic mutations of EGFR that are associated with the phenomenon of oncogene addiction, describing ‘oncogenic shock’ as a mechanistic explanation for the apoptosis that follows the acute treatment of susceptible cells with kinase inhibitors. Understanding the genetic heterogeneity of epithelial tumours and devising strategies to circumvent their rapid acquisition of resistance to targeted kinase inhibitors are essential to the successful use of targeted therapies in common epithelial cancers.

NATURE REVIEWS | C A N C E R VO LU M E 7 | M A R C H 2 0 0 7 | 169

REVIEWS

Molecular-targeted therapeutics Chemicals or drugs that target known proteins that are important in cancer cell proliferation or survival at the same time as being dispensable to normal cells. Although side effects are typically less severe than with cytotoxic agents, the effective inhibition of the target protein might not translate into generally effective therapies, hence the importance of reliable biomarkers.

Autocrine loop A mode of cell signalling in which soluble ligands released by cells stimulate receptors on their own cell surfaces.

Reversible inhibitors Inhibitors that bind non- covalently with biological molecules and interfere with their activity.

(Iressa; AstraZeneca) and erlotinib (Tarceva; OSI Pharmaceuticals, Genentech), two small-molecule drugs that specifically target the tyrosine kinase activity of EGFR (EGFR-tyrosine kinase inhibitors (EGFR-TKIs)), received fast-track approval from the US Food and Drug Administration (FDA) in 2003 and 2004, respectively, for patients with advanced NSCLC who had failed to respond to conventional chemotherapy5. Both drugs are reversible inhibitors of the EGFR kinase, designed to act as competitive inhibitors of ATP-binding at the active site of the EGFR kinase18,19. The observation that sensitivity to gefitinib and erlotinib correlated very strongly with a newly discovered class of somatic activating mutations in the EGFR kinase domain20–22 explained the unique subset of drug-responsive cases, notably those aris- ing in non-smokers and more frequently in women, individuals of Asian ethnic background and those with adenocarcinoma and bronchioloalveolar histology (for a review of the recent clinical literature see Sequist et al.23). In addition to providing a genetic marker for a highly EGFR-TKI-responsive subset of NSCLCs, this correlation has also highlighted the crucial importance of mutationally activated kinases as anticancer drug targets (reviewed in REF. 24) (FIG. 1).

In unselected NSCLC samples, EGFR mutations are present in ~10% of cases in North America and Western Europe, but ~30–50% of cases in individuals of East Asian descent, and are associated with most (over 50%) adeno-

carcinomas with bronchioloalveolar features that arise in non-smokers25–34. EGFR kinase domain mutations target four exons (18–21), which encode part of the tyrosine kinase domain (the entire kinase domain is encoded by exons 18–24) and are clustered around the ATP-binding pocket of the enzyme25,35–39. Consistent with their pur- ported role in the aetiology of NSCLC, recent studies have shown that exon 19 deletions that involve the LREA motif, L858R, G719S and ins 770(NPG)-mutated EGFR proteins are oncogenic in both cell culture and transgenic mouse studies40–42. These mutations also increase the kinase activity of EGFR, leading to the hyperactivation of downstream pro-survival pathways, and consequently confer oncogenic properties on EGFR43–45.

Kinase domain mutations in EGFR are generally referred to as activating mutations, as they seem to result in the increased kinase activity of the receptor. However, this does not imply that these mutated EGFRs are necessarily constitutively or fully active, as their degree of ligand independence might be a function of experimental context40,43,44,46,47. These partially activated mutant EGFRs can be rendered fully ligand-independent, and therefore constitutively active, by second site substitutions in EGFR, such as the T790M mutation in exon 20 (REF. 46). In vitro biochemical studies using purified recombinant wild-type and mutant (L858R and ∆E746–A750) EGFR cytoplasmic domains have shown that mutants have increased kcat values and an increased Km for ATP48,49. Moreover, as has been observed in cell-based studies, the mutants show an increased sensitivity to inhibition by erlotinib (reduced Ki) in these in vitro kinase assays. The reduced ATP affinity seen with mutant kinases most probably accounts for their increased sensitivity to the selective EGFR-TKIs, which compete with ATP for binding to the catalytic site. Another study, in which the phage-display method was used to exam- ine the interaction of a large panel of kinases with selective inhibitors, concluded that EGFR mutations, includ- ing ∆E746-A750, do not themselves affect the affinity for gefitinib and erlotinib50.

EGFR-targeted therapy of NSCLC For unknown reasons, EGFR kinase domain mutations seem to be restricted to a subset of NSCLC, although very rare mutations have also been reported in SCLC, cholangiocarcinoma, ovarian, colorectal, head and neck, oesophageal and pancreatic cancers51–56. This Review discusses the genetic and biochemical determinants of erlotinib and gefitinib sensitivity in NSCLC. In light of the rapid acquisition of resistance to these EGFR-TKIs, we discuss the mechanisms by which resistance might occur and the possibilities for alternative therapeutics.

Genetic determinants of sensitivity to gefitinib and erlo- tinib. Early NSCLC clinical trials with gefitinib and erlo- tinib were modestly encouraging, with partial responses observed in approximately 10% of treated patients with NSCLC57–60. Most responses were seen in East Asians, females or non-smoking patients with NSCLC. These patients had a high frequency of adenocarcinoma with bronchioloalveolar features, and many showed a dramatic and lasting response to second- or third-line

At a glance

• Advanced non-small-cell lung cancer (NSCLC) is the leading cause of cancer- related deaths in the world.

• Epidermal growth factor receptor (EGFR) is expressed in 50% of NSCLCs, and its expression is correlated with poor prognosis. These two factors make EGFR and its family members prime candidates for the development of targeted therapeutics.

• Two EGFR-targeting small-molecule inhibitors, gefitinib (Iressa: AstraZeneca, approved in May 2003) and erlotinib (Tarceva: OSI-Genentech, approved in November 2004) received fast-track approval from the US Food and Drug Administration as treatment for patients with advanced NSCLC who had failed to respond to conventional chemotherapy.

• Early clinical data showed that 10% of patients with NSCLC responded to gefitinib or erlotinib. Although infrequent, the speed and magnitude of clinical responses were unique, as was the fact that they were seen in specific subsets of cases (non-smokers, women, East Asians and patients with adenocarcinomas with bronchioloalveolar histology).

• Molecular analysis showed that in most instances, responders harboured specific mutations in the gene that encodes EGFR. Exon 19 mutations characterized by in-frame deletions of amino-acids 747–750 account for 45% of mutations, exon 21 mutations resulting in L858R substitutions account for 40–45% of mutations, and the remaining 10% of mutations involve exon 18 and 20.

• EGFR kinase domain mutations hyperactivate the kinase and confer a dependence on the mutated kinase for the survival of the NSCLC tumour cells.

• The treatment of sensitive cells with targeted therapeutics such as gefitinib and erlotinib seems to trigger a form of ‘oncogenic shock’, which is postulated to result from the differential decay of downstream signals leading to a temporary predominance of apoptotic signals.

• Acquired resistance to gefitinib and erlotinib might involve the recurrent mutation T790M which affects the gatekeeper residue in the catalytic domain of the kinase that weakens the interaction of the inhibitor with its target. Resistance can be overcome in vitro by irreversible inhibitors of EGFR

R E V I E W S

∆E746-A750 ∆E746-T751 ∆E746-A750 (ins RP) ∆E746-T751 (ins A/I) ∆E746-T751 (ins VA) ∆E746-S752 (ins A/V) ∆L747-E749 (A750P) ∆L747-A750 (ins P) ∆L747-T751 ∆L747-T751 (ins P/S) ∆L747-S752 ∆L747-752 (E746V) ∆L747-752 (P753S) ∆L747-S752 (ins Q) ∆L747-P753 ∆L747-P753 (ins S) ∆S752-I759

L858R (40–45%) N826S A839T K846R L861Q G863D

V765A T783A

G719C G719S G719A V689M N700D E709K/Q S720P

68 8

72 8

72 9

76 1

76 2

82 3

82 4

87 5

Autophosphorylation

Exon 2 5 1613 17 18–21 28

EGF binding EGF binding Tyrosine kinase

7 22–24

Exon 18 (nucleotide-binding loop)

Exon 19 Exon 21 (activation loop)

Exon 20

D761Y

T790M (50%)* D770_N771 (ins NPG) D770_N771 (ins SVQ) D770_N771 (ins G), N771T V769L S768I

(5%)

(45%)

(<1%)

(40–45%)

(5%)(<1%)

Mutations associated with drug sensitivity

Mutations associated with drug resistance

Unselected patients A cohort of patients identified on the basis of tissue diagnosis but not correlated with biomarkers (that is, sequencing of the EGFR gene was not used as a selection criterion).

Ligand independence The activation of a receptor in the absence of interaction with its cognate ligand.

Kcat The overall catalytic rate of an enzyme (that is, the number of substrate molecules converted to product by each catalytic site per unit of time.

Km The Michaelis–Menten constant. Km is a measure of the affinity of a substrate for an enzyme, and is the substrate concentration at half the maximal velocity of an enzyme.

Ki The dissociation constant for the binding of an inhibitor to an enzyme.

Phage-display method A method in which proteins or peptides are displayed on the surface of filamentous bacteriophages, which can then be used to study the interaction of the peptide with other proteins or chemicals.

gefitinib or erlotinib monotherapy. The sequencing of the EGFR gene in tumour samples from these responders showed somatic gain-of-function mutations20–22 (FIG. 1). Overall, the incidence of EGFR mutations in NSCLC among clinical responders to gefitinib or erlotinib is 77%, compared with 7% in NSCLC cases that are refractory to gefitinib or erlotinib20–22,28,30,33,61–73. Additional studies have shown some differences in the clinical outcomes that are associated with different mutations27,30,74,75. For example, NSCLCs that harbour exon 19 deletion muta- tions seem to respond better to gefitinib and erlotinib than tumours with point mutations in exon 21, such as L858R30,74,75. So far, insertion mutations in exon 20 have never been found to confer gefitinib or erlotinib

sensitivity in vitro, nor have they ever been reported to occur in responsive cases, despite the fact that, at least in some instances (for example, ins 770 (NPG)), they seem to activate EGFR to a similar degree as sensitizing mutations in exons 19 or 21 (REF. 40).

Although EGFR mutations were present in most cases of NSCLC that were identified by virtue of their dramatic clinical response to TKIs, controversy has surrounded the predictive value of EGFR muta- tions in unselected patients31,32,61,69. Approximately 10–20% of patients who do show a partial response to gefitinib do not have identifiable EGFR muta- tions, indicating that EGFR mutations are not the sole determinants of TKI response20,22,28,30,31,33,61–64,68–70,72,73,76.

Figure 1 | Gefitinib- and erlotinib-sensitizing mutations of EGFR in NSCLC. A cartoon representation of epidermal growth factor receptor (EGFR) showing the distribution of exons in the extracellular domain (EGF binding), transmembrane domain (TM) and intracellular domain (comprising the tyrosine kinase and autophosphorylation regions). The cysteine-rich regions in the extracellular domain (EGF binding; purple shaded region) and the tyrosine kinase region in the intracellular domain (cyan shaded region) are also represented. Exons 18–21 in the tyrosine kinase region where the relevant mutations are located are expanded (represented by the cyan bar), and a detailed list of EGFR mutations in these exons that are associated with sensitivity (magenta boxes) or resistance (yellow boxes) to gefitinib or erlotinib is shown. The most prevalent of EGFR kinase domain mutations, accounting for 45% of EGFR mutations in non- small-cell lung cancer (NSCLC), are in-frame deletions of exon 19, nested around the LREA string of amino-acids located between residues 747–750 of the EGFR polypeptide175. Another recurrent mutation is the L858R substitution in exon 21, within the activation loop of EGFR, which comprises approximately 40–45% of EGFR mutations. Nucleotide substitutions in exon 18 (for example, G719C or G719S) account for another 5% of EGFR mutations, as do in-frame insertions in exon 20. The most noteworthy, clinically relevant mutation in exon 20 is T790M, which is detected in 50% of the cases (denoted by *) as a second site mutation associated with acquired gefitinib and erlotinib resistance25,35–39. Recently, D761Y, a T790M-like secondary mutation in exon 19 of EGFR (at the border of exon 19 and exon 20), was also reported to be associated with resistance to gefitinib and erlotinib in NSCLC cells that contain the L858R-EGFR mutation71,176. Although the inclusion of most of these sensitizing mutations are based on their occurrence in drug responders, increased biochemical and cellular activity of these mutations has been documented in some cases. The main mutations in each class are shown in bold type. Data compiled from20–22,28,30,31,33,71,177.

R E V I E W S

a Drug dosing

b Patient selection and inclusion criteria

Gefitinib (ISEL)

Erlotinib (BR21)

Drug (study)

Response to chemotherapy preceding entry into trial

Criteria for inclusion in ISEL and BR21 clinical trials

150

600 250

150

MTD (mg day–1) Trial dose (mg day–1)

0% 10% 20% 30% 40% 50% 60% 70% 80% 90%

100%

ISEL BR21

ISEL: development of progressive disease within 90 days of the preceding round of chemotherapy (early relapse)

BR21: no selection for early relapse

Progressive disease Stable disease Partial response

Other molecular abnormalities, including the amplifica- tion of wild-type EGFR or alterations in other ErbB family members have been detected, although it is unclear whether they account for most gefitinib-responsive cases that lack EGFR mutations31,61,69,77–79. In particular, the amplification of EGFR has been difficult to interpret by itself, because gene copy number alterations that affect both mutant and wild-type EGFR alleles have not been distinguished in most studies. In addition, inter-study variability stemming from the different techniques used to measure copy number, including quantitative PCR (qPCR), which provides a ‘global’ copy number assess- ment, and fluorescence in situ hybridization (FISH), which evaluates copy number at the single cell level, have yielded divergent results, possibly owing to the use of different threshold measurements and the distinction between specific amplification of the EGFR locus versus more general alterations in gene copy numbers linked to aneuploidy. Significantly, EGFR kinase mutations seem to be highly correlated with clinical characteristics that are predictors of TKI-responsive disease, whereas EGFR gene amplification, as measured by qPCR, seems to be more common in smoking-associated cancers, and does not show the same predilection towards distinct ethnic background and tumour histology61.

Most retrospective studies to date have reported that 50–80% of EGFR-mutant NSCLCs respond to gefitinib or erlotinib, and more recent studies from Asia, where EGFR mutant NSCLC is 2–3 times more prevalent, have reported responses in more than 75% of NSCLC cases with mutant EGFR67,80,81. Most significantly, although improvement in overall survival for the small fraction of EGFR-mutant cases treated with gefitinib or erlotinib has not reached statistical significance in US and European studies, this has been readily appar- ent in Asian studies with larger fractions of mutant cases28,31,33,62,69,73. Taken together, the data suggest that a subset of cases, marked primarily by EGFR mutations and in some cases EGFR amplification, show dramatic responses to TKIs. These responses might occasionally be durable (that is, >3 years), but in most cases they only last for ~6–12 months before resistant disease recurs. Given the low frequency of EGFR-mutant NSCLC, a modest (2–3 months) improvement in overall survival has been observed in US and European retrospective trials, driven primarily by the stabilization of disease rather than tumour shrinkage, which is not tightly linked to the presence of EGFR mutations31. In these studies, increased EGFR gene copy number and high levels of aneuploidy, as measured by FISH, seemed to be more predictive of disease stabilization after treatment with TKIs59,82. The effect on overall survival in genotypi- cally uncharacterized cases was observed with erlotinib (BR21 trial), but not gefitinib (ISEL trial), contributing to the withdrawal of gefitinib from the US and European market and the approval of erlotinib as third-line therapy in NSCLC irrespective of tumour genotype59,82. A closer examination reveals differences in the dose of the two agents, together with differences in the composition of the patient population that might account for the observed differences in outcome between gefitinib and erlotinib83 (FIG. 2). Nonetheless, gefitinib (which is still in use in Asia) and erlotinib are comparable in virtually all laboratory analyses, and the appropriate clinical role of EGFR mutation analysis in the treatment of NSCLC remains an evolving question, awaiting prospective studies with adequate tumour analysis.

Biochemical determinants of sensitivity to gefitinib and erlotinib. Unpublished results from our laboratory suggest that sensitivity to EGFR-TKIs is not simply recapitulated by expressing the mutant constructs in transfected cells, pointing to the importance of cell- ular context in conferring dependency on the EGFR pathway. Furthermore, caution should be exercised in interpreting in vitro data using NSCLC cell lines as surrogates for clinical responses (FIG. 3). However, in vitro studies with NSCLC cell lines have highlighted the fact that gefitinib- and erlotinib-sensitizing muta- tions invariably hyperactivate the EGFR signalling pathway and promote EGFR-mediated anti-apoptotic and pro-survival signals through the Ras–Raf–MEK (mitogen-activated and extracellular-signal regulated kinase kinase)–ERK1 and ERK2 (extracellular-signal- regulated kinase 1 and 2), PI3K–Akt (phosphatidyli- nositol-3 kinase–Akt) and STAT3 and STAT5 (signal

Figure 2 | Why gefitinib failed in the United States. a | The maximum tolerated dose (MTD) for gefitinib and erlotinib, and the dose of the two drugs used in the ISEL and BR21 trials. Although erlotinib was used at its MTD in the BR21 trial, gefitinib was used at the sub-MTD level of 250 mg a day. b | The composition of the patients and their response to chemotherapy at the time of recruitment for the ISEL and BR21 trials. The histogram represents patients with progressive disease (orange), stable disease (green) and partial response (yellow). Note that the patient pool recruited to the ISEL trial had a significantly larger percentage of individuals with progressive disease as compared to patients recruited to the BR21 trial (45% versus 28%), and conversely had a lower percentage of patients that had a partial response to chemotherapy (18% versus 38%). Also shown are the inclusion criteria for patient enrollment in both of the trials. In the ISEL trial, only patients who had progressive disease within 90 days of cessation of chemotherapy were included, but no similar time-limited exclusion criterion was implemented for the inclusion of patients in the BR21 trial83. The differences in dosage used and the patient selection criteria might have contributed to the differences in outcomes between the two trials.

R E V I E W S

Hypersensitive SensitiveSensitivity

Clinical dose of gefitinib (G) or erlotinib (E)

250 mg day–1 150 mg day–1

Insensitive

In vitro equivalent (µM) 0.0001 0.001 0.01 0.1 1 10

NSCLC cell lines

Genetic signatures

NCI H2170 (200 nM)

NCI H2073 (250 nM)

NCI-H3255 (1 nM) PC9 (4 nM)

NCI-H1975 (12 µM)

NCI-H1650 (9 µM)

NCI-H460 (20 µM)

EGFR: wild typeEGFR: L858R EGFR: ∆(E746-A750)

EGFR: T790M PTEN loss KRAS

G E

Oncogenic shock A mechanism to explain oncogene addiction, in which the acute inactivation of an oncoprotein is associated with differential attenuation rates of pro-survival and pro-apoptotic signals emanating from the oncoprotein, such that apoptotic signals become predominant and kill the cancer cell.

Differential signal decay A signalling imbalance created by the rapid decay of pro- survival signals and persistence of the relatively long-lived pro-apoptotic signals after acute oncogene inactivation.

transducer and activator of transcription proteins 3 and 5) pathways such that cancer cells might become dependent on a functional EGFR for their survival43,84–86. Interestingly, these are the same pathways that are activated after ligand engagement and are inhibited by gefitinib, including the ERK pathway involved in cell proliferation and the pro-survival Akt pathway87–89. The obvious implication is that shutting off EGFR with specific kinase inhibitors, antibodies or RNA interference would extinguish these proliferative and survival signals on which the tumour cell is dependent, therefore resulting in tumour cell death. Normal cells (or non-EGFR-dependent tumour cells that do not respond to gefitinib or erlotinib) remain unaffected, as their pro-survival signals are either driven by other genes or can be compensated for by other RTKs in the event of EGFR inhibition. This is consistent with the observation that gefitinib and erlotinib response in sen- sitive cells results in the downregulation of ERK, Akt and STAT3 and STAT5, whereas a similar downregulation is not evident in insensitive or resistant cells43,87–90.

Although these pro-survival signalling pathways are probably controlled by many RTK outputs in normal cells, their dependency on mutated and/or activated EGFR in some NSCLC tumours and cell lines bears the hallmark of oncogene addiction (BOX 1).

The molecular mechanisms that underpin onco- gene addiction remain to be elucidated. As commonly understood, alterations of the signal-transduction pathways in cancer cells are thought to underlie drug hypersensitivity91. Based on modelling studies in vitro, we have recently proposed that unbalanced pro-apop- totic and pro-survival signals lead to a phenomenon that we refer to as oncogenic shock, and might account for the observed apoptotic outcome following the acute inacti- vation of a crucial oncogene in an addicted cancer cell92 (FIG. 4). According to this model, an addicting oncogene gives rise to both pro-apoptotic and pro-survival signal outputs. While the oncogene is active, the pro-survival signals pre-dominate and keep the pro-apoptotic signals in check, enabling the survival and proliferation of the cancer cell. After acute oncogene inactivation, the relatively short-lived pro-survival signals decay first, whereas the longer-lasting pro-apoptotic outputs are maintained during a crucial window of time. Therefore, differential signal decay leading to a signal imbalance and a temporary predominance in pro-apoptotic outputs sets in motion the apoptotic cascade and commits the cell irrevocably to apoptosis, even if the signalling imbal- ance is subsequently redressed. In support of the onco- genic shock model, the apoptotic response to oncogene inactivation in oncogene-addicted cells is abrogated if the disruption of oncogene-derived signals is extended over a period of time, rather than being acute, or if pro- survival signals are transiently applied during the crucial window of time following acute withdrawal92. Therefore, the cell is not hard-wired to depend on a given oncogene, but rather it requires time to adapt to the loss of such a signal, and is highly susceptible to apoptosis during that window of time. The implications of this model for clinical practice, if confirmed, are considerable, as it would argue against the co-administration of TKIs with chemotherapy drugs that, by virtue of their own effects on DNA-damage checkpoints, might attenuate the acute effect of growth factor signal withdrawal. For RTKs like EGFR, it is also possible that the acute effect of EGFR-TKIs in abrogating kinase activity might be quali- tatively different from that of anti-receptor antibodies, which might enable a more gradual signal attenuation, therefore explaining the differential effect of these two classes of agents on EGFR-mutant NSCLC86.

Implicit in the oncogenic shock model is the paradoxical requirement that activated oncogenes gen- erate pro-survival and pro-apoptotic signals simultane- ously93. Such a coupling of antagonistic signals is well documented for Ras94,95, Src96,97, BCR-ABL98, EGFR43,99, MYC100,101 and even viral oncogenes such as adenoviral E1A102. Taken together in the context of NSCLC, mutated EGFR might represent the genetic lesion to which the tumour is addicted, and the acute withdrawal of these signals by EGFR-TKIs might trigger oncogenic shock and tumour cell apoptosis.

Figure 3 | NSCLC cell lines: in vitro surrogates of in vivo drug sensitivity. Understanding the biochemical basis of sensitivity to gefitinib (G) and erlotinib (E) has been aided by the generation and use of human tumour-derived non-small-cell lung cancer (NSCLC) cell lines that show varying degrees of sensitivity to these inhibitors, ranging from hypersensitive (IC

50 in the low nM; graded magenta box), to sensitive (IC

in the high nM; graded orange box) to extremely insensitive (IC 50

in the high µM; graded yellow box). Representative examples of NSCLC cell lines from each category, including their distinguishing genetic features, are also shown. The hypersensitive cell lines NCI- H3255 and PC9 harbour the EGFR tyrosine kinase domain mutations L858R and ∆E746- A750, respectively. Insensitive cell lines such as NCI-H1975 and NCI-H1650, although harbouring the same kinase domain mutations (L858R and ∆E746-A750), have additional changes such as T790M (NCI-H1975), phosphatase and tensin homologue (PTEN) loss (NCI-H1650) or KRAS mutations in NCI-H460 cells. Although these cell lines have been used extensively, conclusions derived from such in vitro systems should be interpreted with caution in view of the off-target effects seen with these inhibitors50, especially at supra-physiological concentrations, in excess of 1 and 2.5µM for gefitinib and erlotinib, respectively. The in vitro concentrations used in tissue culture roughly correlate to the plasma concentrations of these drugs in patients treated with the standard doses of these agents (250 mg a day of gefitinib and 150 mg a day of erlotinib), and have been used by researchers as a useful threshold to distinguish sensitive from insensitive and/or resistant cell lines90,112,178–180.

R E V I E W S

Gatekeeper residue Amino acids with small side chains found at the catalytic site of enzymes that, when mutated to amino acids with bulkier side chains, can sterically impede the binding of a drug at the active site of the enzyme at the same time as retaining substrate binding.

Resistance to EGFR-targeted therapy Not all EGFR kinase mutations are associated with hyper- sensitivity to gefitinib and erlotinib. An overarching conclusion that has emerged from studies of primary insensitivity to EGFR-TKIs is that most cells that express EGFR will show effective attenuation of EGFR activity, but only in EGFR-addicted cancers will this be accompanied by tumour shrinkage. Tumours that fail to respond to gefitinib or erlotinib despite the presence of an EGFR mutation might have sustained additional genetic lesions that relieve this addiction, a mechanism that could also mediate acquired resistance in previously sensitive tumours.

Primary resistance. Recent studies suggest that inser- tion mutations in exon 20 of the EGFR gene might render the receptor about 100-fold less sensitive to EGFR-TKIs compared with other sensitizing EGFR kinase mutations40. It is unclear whether these muta- tions differ from classical activating mutations in their downstream signals (therefore attenuating oncogene addiction), or whether they do not share the dif- ferential binding affinity to ATP and the inhibitors. However, these mutations are relatively rare, and in most cases of NSCLC that fail to respond to EGFR- TKIs it is likely that genetic lesions other than EGFR are driving tumorigenesis. In most instances the T790M mutation is associated with acquired resistance. However, it has also been linked to primary resistance, occurring together with a sensitizing mutation in four unresponsive cases of NSCLC36,39.

About 15–30% of NSCLCs harbour activating muta- tions in codons 12 and 13 of the KRAS gene103,104. By and large, KRAS and EGFR mutations seem to be mutually exclusive in NSCLC, and define distinct subsets of tumours, with EGFR mutations being characteristic of tumours that arise in non-smokers 25, whereas KRAS mutations are more common in smoking-associated cancers105,106. Mutant EGFR and KRAS might also

have overlapping and/or redundant signalling roles in NSCLC aetiology25,27,29,107, which might explain the conspicuous absence of KRAS mutations in EGFR- TKI-responsive tumours108. Mutations in KRAS have been proposed as a mechanism of primary resistance to gefitinib and erlotinib108, although KRAS mutations are almost always found in NSCLCs with wild-type EGFR. Therefore, it is difficult to unequivocally determine whether insensitivity is due to the presence of mutated KRAS or the absence of mutated EGFR.

Tumour cells that are sensitive to gefitinib and erlotinib are characterized by a rapid decrease in Akt activity in response to drug treatment87,88, and failure to downregulate Akt is a hallmark of insensitivity to the drugs43,90. The activation of Akt is indirectly regulated by the tumour suppressor phosphatase and tensin homo- logue (PTEN), which is frequently lost in human can- cers109. Although genetic alterations in PTEN are found in <10% of cases of NSCLC110, the absence of PTEN expression is evident in as many as 70%, and might be mediated by epigenetic mechanisms such as PTEN promoter methylation111. In some cell types, restoring PTEN expression is asso-ciated with increased sensi- tivity to gefitinib or erlotinib, suggesting that it might modulate sensitivity in vivo89,112. Insulin-like growth factor receptor 1 (IGFR1), ERBB3 or activated ERBB2 expression have also been proposed to have a role in mediating resistance to gefinitib113–115. However, recent studies of a large cohort of gefitinib-treated NSCLC cases failed to uncover a correlation between PTEN or IGFR1 status and response to gefitinib treatment, and have questioned the role of these proteins in mediating primary insensitivity to gefitinib116.

Acquired resistance. Despite dramatic responses in EGFR- mutant cases of NSCLC treated with gefitinib or erlotinib, the development of drug resistance within 6–12 months of the initiation of therapy greatly limits the ability of these drugs to significantly prolong patient survival. A deeper understanding of the molecular and cellular basis of this phenomenon is crucial to the future development of alter- native therapies to overcome this resistance.

A single secondary mutation in EGFR exon 20, T790M, is present in a subset of EGFR-mutant tumours that recur after an initial response to gefitinib or erlotinib35–37. Using allele-specific PCR, the T790M mutation is detect- able in approximately 50% of patients with NSCLC who relapse after an initial response to TKIs, although in some cases the mutation seems to be underrepresented in the tumour cell genome relative to the total number of EGFR alleles37,117. This suggests that T790M might either be present in only a subset of resistant cancer cells, or might be present only in a minority of copies of the EGFR gene in each tumour cell39,118. Some studies have also shown that T790M mutations are present before the patient is exposed to the drug25,38,56,119,120, thereby sug- gesting that this mutation might confer some selective advantage to tumour outgrowth and might be further selected after the exposure of the tumour to TKIs39. The T790M mutation in EGFR is structurally analogous to the mutated gatekeeper residue T315I in BCR-ABL,

Box 1 | Oncogene addiction

The term oncogene addiction was first coined in 2000 by Bernard Weinstein91,168,169 to describe the phenomenon by which a tumour cell, despite many other genetic alterations, can become completely dependent on a single oncogenic pathway for its proliferation and/or survival. Implicit in this dependency is the fact that the tumour cell should be exquisitely sensitive to the targeted inhibition of the addicting oncogene. Beyond gefitinib- and erlotinib-responsive NSCLC, oncogene addiction is thought to explain responses of chronic myeloid leukaemia, gastrointestinal stromal tumours and chronic myelomonocytic leukaemia to imatinib, which targets the BCR-ABL, c-Kit, and platelet derived growth factor receptor-β (PDGFRβ) kinases170. Transgenic mouse tumour models have shown a similar addiction phenomenon, although they are somewhat biased in that the inducible expression of an oncogene is used to trigger the genesis of a tumour that is then shown to be dependent on the continued expression of the transgene for its survival (for example, HRAS in melanoma, KRAS in lung carcinoma and MYC in lymphoma and leukaemia171–173 (reviewed in REF. 174)). An interesting parallel is the observation that, in some cases, the continued expression of the transgene does lead to the emergence of cells that have sustained additional somatic genetic lesions and have consequently acquired independence from the triggering oncogene173. A similar mechanism might occur in human cancers that show resistance to TKIs, despite the presence of the mutated kinase.

R E V I E W S

Signal intensity

Time

Pro-survival signals

Adaptation and drug resistance

Vulnerable window of drug sensitivity

Baseline/cancer cell survival

Oncogenic shock Period during which pro-apoptotic signals exceed pro-survival signals

Pro-apoptotic signals

Oncoprotein disruption byTKI

Irreversible inhibitors Inhibitors that bind covalently with biological molecules and interfere with their activity.

T670I in c-KIT and T674I in platelet-derived growth factor receptor-α (PDGFRα) that weaken the interac- tion of inhibitors with the kinase and that have previ- ously been shown to confer resistance to targeted agents such as imatinib and other ATP-mimicking kinase inhibitors121–123. Besides T790M, the only other study of acquired resistance in clinical samples suggests that an A disintegrin and metalloproteinase 17 (ADAM17)- mediated heregulin-dependent autocrine loop activates both ERBB2 and ERBB3 signalling pathways in NSCLC and mediates resistance to EGFR-TKIs124.

The phenomenon of acquired resistance to gefit- inib has been modelled in vitro using highly sensitive NSCLC cell lines with EGFR mutations125. Mechanisms of acquired drug resistance have been defined in vitro, including the acquisition of (or selection for) the T790M mutation118 and altered EGFR trafficking37. Other possible mechanisms that confer resistance include amplification of the mutant EGFR or the hyperactivation of downstream signalling components that circumvent EGFR inhibition, causing the increased expression of signal-attenuating molecules or cellular changes that alter the bioavailability of the drug126. Some studies have raised the possibility that the multi- drug resistance protein ATP-binding cassette G2 (ABCG2) might actively pump gefitinib from cells and

therefore confer resistance to the drug127,128, although others have suggested that gefitinib itself inactivates the multi-drug transporters ABCG2 and the ABC transporter P-glycoprotein129–133. These alternative mechanisms of gefitinib and erlotinib resistance still await validation in vivo — an issue confounded by the limited amounts of clinical specimens from recurrent tumours and the absence of defined genetic lesions that can be detected in tissue sections.

Alternative EGFR-targeted therapeutics The development of resistance to EGFR-TKIs calls for alternative strategies that still target EGFR signalling but circumvent the insensitivity to kinase inhibitors. Mutations such as T790M might have far-reaching implications in the context of various receptor and non-receptor tyrosine kinases, and represent a general problem that needs to be overcome in TK-targeted therapy123. Therefore, one of the main challenges in the treatment of NSCLC is to design inhibitors that can overcome the steric interference to drug binding conferred by the T790M mutation. Irreversible inhibitors seem to show some promise in this regard (TABLE 1). In most cases, irreversible inhibitors form a covalent bond with crucial cysteine residues — Cys797 within EGFR or Cys805 within ERBB2 — in the active site of the respective enzymes134,135. Given the fact that only EGFR and ERBB2 (as opposed to ERBB4) have cysteines at these corresponding positions, irreversible ErbB inhibi- tors show very high specificity for EGFR and ERBB2. Previous studies from our laboratory have shown that the irreversible dual EGFR and ERBB2 inhibitors, HKI-272 (REF. 136) and HKI-357 (REF. 37), as well as the irreversible EGFR inhibitor EKB-569 (REF. 137) were all able to overcome gefitinib resistance owing to T790M in cis with an L858R mutation in EGFR37,138.

Interestingly, resistance to irreversible dual inhibitors is not achieved as rapidly as resistance to gefitinib and erlotinib in the laboratory37. Similarly, other studies have shown that the irreversible EGFR inhibitor CL-387,785 (REF. 139), and the irreversible pan-ErbB inhibitor CI-1033 (also known as canertinib)140 can overcome resist- ance to L858R-mutated EGFR harbouring the T790M resistance-conferring mutation, whereas the reversible EGFR and ERBB2 inhibitor GW-572016 (also known as lapatinib) was ineffective in this regard35,141. CL-387,785 is also able to overcome gefitinib and erlotinib resistance mediated by in-frame insertions in exon 20 of EGFR40. A small subset of NSCLCs harbour mutations in ERBB2 (but not EGFR), and tumour cells that harbour the G776 insVG/C in ERBB2, although insensitive to erlotinib, are sensitive to the EGFR and ERBB2 dual irreversible inhibitor, HKI-272 (REF. 142). Similarly, a small subset of NSCLCs that express the EGFR mutant variant III (EGFRvIII) are also insensitive to gefitinib and erlotinib but show sensitivity to HKI-272 (REF. 143). HKI-272 is currently being evaluated in multi-center clinical trials in NSCLC patients. Therefore, several independent lines of evidence underscore the use of irreversible erbB inhibitors, especially for situations in which reversible inhibitors of EGFR lose efficacy.

Figure 4 | The role of differential signal attenuation in inducing oncogenic shock. The oncogenic shock model proposes that pro-survival (orange curve) and pro- apoptotic (red curve) signals emanating from an active oncoprotein in a tumour cell are normally balanced so that the survival output predominates and results in the survival of the cancer cell. After the acute disruption of oncogene function by targeted kinase inhibitors (TKIs), pro-survival signals dissipate very rapidly, whereas pro- apoptotic signals are relatively longer lived. During this vulnerable window of drug sensitivity, the longer-lived pro-apoptotic signals gain the upper hand and cause the cells to irrevocably undergo apoptosis. One possible mechanism by which tumour cells acquire resistance to a therapeutic target is that they are able to adapt to and overcome oncogenic shock.

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Although the effectiveness of the irreversible inhibi- tor HKI-272 against EGFR is assumed to be responsible for its ability to suppress the proliferation of cells with acquired resistance to gefitinib and erlotinib, its ability to target ERBB2 is also of potential interest, given the role of other ErbB family members in EGFR signalling. ErbB family members undergo ligand-induced homo- and hetero-dimerization as a prelude to signal transduction after receptor activation, with each receptor dimer show- ing distinct ligand specificity (reviewed in REF. 7). The complex functional interactions among members of the ErbB family, combined with the ability to target several family members, might open new avenues for overcoming resistance to EGFR inhibitors.

Another strategy to overcome acquired resistance resulting from the T790M mutation is based on the obser- vation that various EGFR mutants, including the double mutant L858R/T790M, associate with the molecular chaperone heat shock protein 90 (HSP90) (REF. 144). This interaction can be very specifically disrupted by the ben- zoquinone ansamysin, geldanamycin, which results in the degradation of gefitinib- and erlotinib-resistant mutant EGFR and leads to the apoptosis of EGFR-dependent tumour cells that harbour the mutated receptors144,145. The selective HSP90 inhibitor 17-(allylamino)-17-demeth- oxygeldanamycin (17-AAG) and its derivative IPI-504

(REF. 146) are currently in clinical trials in patients with advanced solid tumours, and might be useful therapeutic alternatives for gefitinib- and erlotinib-resistant NSCLC. By overcoming the gatekeeper-residue mutations, such a strategy, if successful, might be of value for the treatment of a broad range of mutant receptor-driven cancers.

Rational combinations: is there a rationale? At present, gefitinib and erlotinib, either alone or in com- bination with other regimens, are being evaluated in 157 clinical trials for various cancers (see the NCI clinical trials website). Despite some caveats147, EGFR-targeted therapy has not been shown to have any beneficial effects in combination with standard chemotherapeutic regi- mens, which begs the question of whether or not there is a rationale for the combination of therapeutics in the treatment of NSCLC.

Insights into EGFR-dependent signalling might provide a first clue to the rational design of combination therapies. Research over the past 40 years has uncovered some of the crucial players in the EGFR signal-transduction pathway, which can be roughly divided into two categories: the pro- survival arm of the pathway comprising the PI3K–mTOR (mammalian target of rapamycin)–Akt cascade, and the proliferative arm consisting of the Ras–Raf–MEK–ERK pathway148 (FIG. 5). This simplistic framework would

Table 1 | Targeted therapeutics currently approved or being evaluated for the treatment of NSCLC

Class Therapeutic Target Company Stage of development (tumour type)

EGFR TKI (single reversible)

Gefitinib (Iressa; ZD-1839)

EGFR AstraZeneca Approved (NSCLC)

Erlotinib (Tarceva; OSI-774)

EGFR OSI, Genentech and Roche

Approved (NSCLC)

EGFR TKI (single irreversible)

EKB-569 EGFR Wyeth Phase II * (colorectal)

CL-387,785 EGFR Wyeth Preclinical *

ErbB family TKI (multiple reversible)

Lapatinib (GW572016; Tykerb)

EGFR, ERBB2 GlaxoSmithKline Phase III (breast)

ErbB family TKI (multiple irreversible)

Canertinib (CI- 1033; PD183805)

EGFR, ERBB2, ERBB4

Pfizer Phase II * (NSCLC, breast)

HKI-272 EGFR, ERBB2 Wyeth Phase I/II * (NSCLC, breast)

BIBW 2992 EGFR, ERBB2 Boehringer Ingelheim

Phase I/II (breast, prostate, ovarian)

HKI-357 EGFR, ERBB2 Wyeth Preclinical *

RTK family TKI (multiple reversible)

ZD-6474 EGFR, ERBB2, FLT1, KDR

AstraZeneca Phase III * (NSCLC, thyroid)

AEE 788 EGFR, ERBB2, KDR

Novartis Phase I/II (glioblastoma)

XL647 EGFR, ERBB2, KDR, EPHB4

Exelexis Phase II (NSCLC)

ErbB family heterodimerization

BMS-599626 EGFR, ERBB2 Bristol-Myers Squibb Phase I (metastatic solid tumours)

HSP90 IPI-504 Mutant EGFR Infinity Pharmaceuticals

Phase I/II * (multiple myeloma, GIST)

17-AAG Mutant EGFR Kosan Phase I/II * (solid tumours) *Ability to overcome resistance to gefitinib or erlotinib. EGFR, epidermal growth factor receptor; FLT1, fms-like tyrosine kinase 1; GIST, gastrointestinal stromal tumour; KDR, kinase domain region; NSCLC, non-small-cell lung cancer; TKI, tyrosine kinase inhibitor; RTKI, receptor TKI.

R E V I E W S

GAP RAS

SOS

RAF

MEK

ERK mTOR

PTEN

SHIP

AKT

p110

AKT

PDK1

TSC2

PHLPP

GTP

GDP PIP2 PIP3

Cell growth Protein synthesis Cell survival

ErbB family Extracellular

Intracellular

P P

p85GRB2

suggest that inhibitors that target different key compo- nents of this network (in combination with EGFR-TKIs) might provide greater therapeutic efficacy, particularly in a setting where EGFR-TKI monotherapy follows a consistent pattern of diminishing returns and eventually becomes ineffective. Preclinical studies in an NSCLC cell line xenograft model have suggested that a PI3K inhibi- tor, PX-866, sensitizes otherwise insensitive tumours to gefitinib149 (TABLE 2). Efforts are currently underway to develop PI3K inhibitors with greater specificity150. The serine-threonine kinase mTOR lies downstream of PI3K, and is inhibited by rapamycin and rapamycin analogues (TABLE 2). Preclinical studies suggest that mTOR inhibitors might also have synergistic effects when combined with targeted EGFR inhibitors151,152. At present, combinations of gefitinib or erlotinib with sirolimus, temsirolimus or everolimus are undergoing phase I and II evaluation in patients with advanced NSCLC, recurrent malignant glioma, prostate cancer and metastatic breast cancer153. However, despite their dramatic effects in some preclinical studies (reviewed in REF. 154), monotherapy with mTOR inhibitors have so far yielded disappointing results in clini- cal trials155. The selective inhibition of mTOR might in fact lead to the activation of the PI3K pathway and result in feedback activation of the pro-survival mediator Akt156,157. Therefore, recent studies have tried to overcome this

problem through the use of dual inhibitors of PI3K and mTOR, which seem effective in preclinical studies of glioma cell lines150. The evaluation of these dual PI3K and mTOR inhibitors, either as monotherapy or in combina- tion with targeted ErbB family inhibitors, in clinical studies might therefore hold considerable promise.

The Ras–MAPK (mitogen-activated protein kinase) pathway is another important cell-proliferation pathway downstream of EGFR that is frequently activated in cancer. Although mutations in Ras oncogenes do not seem to coexist with EGFR mutations, the pathway might be important in mediating EGFR-mutant signals, and therefore the inhibition of Ras or Raf in combination with gefitinib or erlotinib might have some benefit. The MEK inhibitor PD-325901 is currently being evaluated as a single agent in phase II clinical trials in patients with advanced NSCLC. However, activation of the Ras–MAPK pathway has not been as well correlated with response to EGFR inhibitors as the PI3K–Akt pathway158.

In addition to manipulating components of EGFR sig- nalling pathways, complementary molecular therapeutic approaches that involve simultaneously targeting distinct pathways have potential benefit. Although most of these approaches are empirical by nature, a rationale does exist for targeting both the tumour and stromal components of

Figure 5 | Cell-survival pathways downstream of activated erbB receptor tyrosine kinases. Two important cell- survival pathways that operate downstream of activated ErbB transmembrane receptor tyrosine kinases (represented by pairs of yellow, and yellow and blue receptors to represent homo- and hetero-dimers, respectively), along with some of the key constituent signalling molecules are shown. The Ras–Raf–MEK–ERK pathway is shown on the left, and the phosphatidylinositol 3-kinase (PI3K)–Akt pathway is shown on the right. Key points along the pathway where targeted inhibition seems to exert a blockade are indicated by red circles, showing the relevant proteins they target (specific examples cited in TABLE 2). ERK, extracellular signal-regulated kinase; GRB2, growth factor receptor-bound protein 2; mTOR, mammalian target of rapamycin; SOS, son of sevenless.

R E V I E W S

a tumour. Tumour vasculature is a particularly important target for therapeutic intervention, and it has been the basis for the development of dual inhibitors of EGFR and ERBB2 and the vascular endothelial growth factor (VEGF) receptors FLT1 (fms-like tyrosine kinase 1) and KDR (kinase domain region). Consistent with this con- cept, the dual EGFR and VEGFR inhibitor ZD6474159,160 has shown efficacy in tumour xenografts that are resistant to cetuximab or gefitinib161,162. The use of the EGFR and VEGFR dual inhibitors ZD6464, AEE788163 and XL647 is currently being evaluated in clinical trials163,164.

The way forward Experience with gefitinib and erlotinib has taught us the following important lessons: first, RTKs can be very useful targets for therapeutic intervention in epithelial cancers. Second, the targeted inhibition of RTKs might only be efficacious in a small subset of patients, and mutations in RTKs might be one of the useful predic- tors of response. Third, acquired TKI resistance substan- tially limits the therapeutic efficacy of these agents. The first lesson validates the usefulness of drug-discovery programmes focused on screening for RTK inhibitors, and reinforces the ongoing efforts of organizations engaged in this endeavor. However, the second lesson might point to the need for a reorientation of our tradi- tional approach to drug discovery. The low frequency of genetically-defined responsive patient subsets calls for the consideration of a far broader sampling of individual cancer types, so as to achieve a representation of genetic diversity at all levels of analysis, from mutation detection in RTK genes to identifying new drug targets through functional assays and screening for efficacy in preclinical experiments. For example, to detect the 10% response to gefitinib or erlotinib typically seen in NSCLC patients, a cell-based drug screen would require a minimum of 100, and ideally 1,000, different NSCLC-derived cell lines, far beyond traditional cell-based screens. In fact, given the current cell-based screening strategies that generally involve a few cell lines representative of each

tumour type, it is likely that gefitinib or erlotinib would never have been picked up as a ‘hit’. A similar rationale for increased sampling size can be applied to genetic analyses of tumour samples in order to detect mutations in EGFR in an unselected cohort of NSCLC patients.

Recent genetic studies underscore the affect of large- scale genomic analyses in highlighting the complexity of the ‘cancer landscape’, and more importantly in pin- pointing the specific genetic alterations that are key to the genesis of these tumours. For example, by analysing 120 primary lung tumours, the Sanger Center cancer kinome sequencing project showed that ERBB2 muta- tions occur in 4% of lung tumours165. In a recent whole- cancer-genome sequencing project at Johns Hopkins University in the United States, the number of distinct genetic lesions in an individual tumour were fewer than might have been predicted, but few of these were found to be recurrent, even among different cases of the same type of cancer166, pointing to potentially small subsets of genetically-defined tumours across various histolo- gies (different needles in different haystacks). Achieving the ambitious goals of the US National Institutes of Health (NIH) Cancer Genome Atlas (TCGA), to com- prehensively annotate all cancer-associated mutations, might therefore require the analysis of many individual tumours within each histological type. Complementary functional approaches, including the use of short hairpin RNA libraries to identify genes that are essential to cancer cell viability167, would also need to be applied across a broad spectrum of different cancer cell lines, each rep- resenting a different genetic context and potential addic- tion to a different oncogenic pathway. In summary, as the lessons learned from EGFR inhibition and cancer therapy continue to evolve, they have already provided a powerful example of clinical therapeutic affect achieved through an understanding of molecular abnormalities in cellular signalling, at the same time as warning of the genetic complexity in cancer that will require the coupling of different therapeutic strategies to individual genetic variation.

Table 2 | Targeted therapeutics used alone or in combination with EGFR-TKIs

Mode of action Therapeutic Target Company Stage of development (tumour type)

p110α-specific inhibition

PX-866 (combination with gefitinib) PI3K ProlX Pharmaceuticals

Preclinical (NSCLC)

Rapamycin analogues

Sirolimus (combination with gefitinib)

mTOR Wyeth Phase I/II (NSCLC, glioblastoma)

Temsirolimus (CCI-779; combination with erlotinib)

mTOR Wyeth Phase I/II (glioblastoma)

Everolimus (RAD001; combination with gefitinib or erlotinib)

FKBP12, mTOR

Novartis Phase I/II (NSCLC, glioblastoma, breast)

AP23573 mTOR Ariad Phase I/II (endometrial)

MAPK pathway Sorafenib (BAY49-9006; alone or in combination with erlotinib)

Raf, (KDR, p38α?)

Bayer Phase I/II (NSCLC, glioblastoma)

PD-325901 (single agent) MEK Pfizer Phase II (NSCLC) Representative examples of different classes of targeted inhibitors that are undergoing evaluation either alone or in combination with EGFR-TKIs are indicated. KDR, kinase domain region; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated and extracellular-signal regulated kinase kinase; mTOR, mammalian target of rapamycin; NSCLC; non-small-cell lung cancer; PI3K, phosphatidylinositol 3-kinase.

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1. Brambilla, E., Travis, W. D., Colby, T. V., Corrin, B. & Shimosato, Y. The new World Health Organization classification of lung tumours. Eur. Respir. J. 18, 1059–1068 (2001).

2. Rapp, E. et al. Chemotherapy can prolong survival in patients with advanced non-small-cell lung cancer — report of a Canadian multicenter randomized trial. J. Clin. Oncol. 6, 633–641 (1988).

3. Breathnach, O. S. et al. Twenty-two years of phase III trials for patients with advanced non-small-cell lung cancer: sobering results. J. Clin. Oncol. 19, 1734–1742 (2001).

4. Blackhall, F. H., Shepherd, F. A. & Albain, K. S. Improving survival and reducing toxicity with chemotherapy in advanced non-small cell lung cancer: a realistic goal? Treat Respir. Med. 4, 71–84 (2005).

5. Blackhall, F., Ranson, M. & Thatcher, N. Where next for gefitinib in patients with lung cancer? Lancet Oncol. 7, 499–507 (2006).

6. Krause, D. S. & Van Etten, R. A. Tyrosine kinases as targets for cancer therapy. N. Engl. J. Med. 353, 172–187 (2005).

7. Hynes, N. E. & Lane, H. A. ERBB receptors and cancer: the complexity of targeted inhibitors. Nature Rev. Cancer 5, 341–354 (2005).

8. Nicholson, R. I., Gee, J. M. & Harper, M. E. EGFR and cancer prognosis. Eur. J. Cancer 37 (Suppl. 4), S9–S15 (2001).

9. Veale, D., Ashcroft, T., Marsh, C., Gibson, G. J. & Harris, A. L. Epidermal growth factor receptors in non- small cell lung cancer. Br. J. Cancer 55, 513–516 (1987).

10. Haeder, M. et al. Epidermal growth factor receptor expression in human lung cancer cell lines. Cancer Res. 48, 1132–1136 (1988).

11. Ohsaki, Y. et al. Epidermal growth factor receptor expression correlates with poor prognosis in non-small cell lung cancer patients with p53 overexpression. Oncol. Rep. 7, 603–607 (2000).

12. Hirsch, F. R. et al. Epidermal growth factor receptor in non-small-cell lung carcinomas: correlation between gene copy number and protein expression and impact on prognosis. J. Clin. Oncol. 21, 3798–3807 (2003).

13. Testa, J. R. & Siegfried, J. M. Chromosome abnormalities in human non-small cell lung cancer. Cancer Res. 52, 2702s–2706s (1992).

14. Putnam, E. A. et al. Autocrine growth stimulation by transforming growth factor-α in human non-small cell lung cancer. Surg. Oncol. 1, 49–60 (1992).

15. Rusch, V. et al. Differential expression of the epidermal growth factor receptor and its ligands in primary non- small cell lung cancers and adjacent benign lung. Cancer Res. 53, 2379–2385 (1993).

16. Mendelsohn, J. Epidermal growth factor receptor as a target for therapy with antireceptor monoclonal antibodies. J. Natl Cancer Inst. Monogr. 125–131 (1992).

17. Giaccone, G. Epidermal growth factor receptor inhibitors in the treatment of non-small-cell lung cancer. J. Clin. Oncol. 23, 3235–3242 (2005).

18. Barker, A. J. et al. Studies leading to the identification of ZD1839 (IRESSA): an orally active, selective epidermal growth factor receptor tyrosine kinase inhibitor targeted to the treatment of cancer. Bioorg. Med. Chem. Lett. 11, 1911–1914 (2001).

19. Wakeling, A. E. et al. ZD1839 (Iressa): an orally active inhibitor of epidermal growth factor signaling with potential for cancer therapy. Cancer Res. 62, 5749–5754 (2002).

20. Lynch, T. J. et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N. Engl. J. Med. 350, 2129–2139 (2004).

Identified mutations in the ATP-binding pocket of EGFR that confer hypersensitivity to gefitinib and translates to a gefitinib response accompanied by tumour shrinkage in patients with NSCLC in a clinical setting.

21. Paez, J. G. et al. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 304, 1497–1500 (2004).

Identified mutations in the kinase domain of EGFR that predict gefitinib response.

22. Pao, W. et al. EGF receptor gene mutations are common in lung cancers from ‘never smokers’ and are associated with sensitivity of tumors to gefitinib and erlotinib. Proc. Natl Acad. Sci. USA 101, 13306–13311 (2004).

23. Sequist, L. V. et al. Epidermal growth factor receptor mutation testing in the care of lung cancer patients. Clin. Cancer Res. 12, 4403s–4408s (2006).

24. Settleman, J. Mutant kinases as targets for cancer drugs. Drug Discov. Today Dis. Mech. 2, 139–144 (2005).

25. Kosaka, T. et al. Mutations of the epidermal growth factor receptor gene in lung cancer: biological and clinical implications. Cancer Res. 64, 8919–8923 (2004).

26. Janne, P. A., Engelman, J. A. & Johnson, B. E. Epidermal growth factor receptor mutations in non- small-cell lung cancer: implications for treatment and tumor biology. J. Clin. Oncol. 23, 3227–3234 (2005).

27. Shigematsu, H. et al. Clinical and biological features associated with epidermal growth factor receptor gene mutations in lung cancers. J. Natl Cancer Inst. 97, 339–346 (2005).

28. Han, S. W. et al. Predictive and prognostic impact of epidermal growth factor receptor mutation in non- small-cell lung cancer patients treated with gefitinib. J. Clin. Oncol. 23, 2493–2501 (2005).

29. Marchetti, A. et al. EGFR mutations in non-small-cell lung cancer: analysis of a large series of cases and development of a rapid and sensitive method for diagnostic screening with potential implications on pharmacologic treatment. J. Clin. Oncol. 23, 857–865 (2005).

30. Mitsudomi, T. et al. Mutations of the epidermal growth factor receptor gene predict prolonged survival after gefitinib treatment in patients with non-small-cell lung cancer with postoperative recurrence. J. Clin. Oncol. 23, 2513–2520 (2005).

31. Cappuzzo, F. et al. Epidermal growth factor receptor gene and protein and gefitinib sensitivity in non-small- cell lung cancer. J. Natl Cancer Inst. 97, 643–655 (2005).

32. Tsao, M. S. et al. Erlotinib in lung cancer- molecular and clinical predictors of outcome. N. Engl J. Med. 353, 133–144 (2005).

33. Chou, T. Y. et al. Mutation in the tyrosine kinase domain of epidermal growth factor receptor is a predictive and prognostic factor for gefitinib treatment in patients with non-small cell lung cancer. Clin. Cancer Res. 11, 3750–3757 (2005).

34. Haneda, H. et al. A correlation between EGFR gene mutation status and bronchioloalveolar carcinoma features in Japanese patients with adenocarcinoma. Jpn J. Clin. Oncol. 36, 69–75 (2006).

35. Kobayashi, S. et al. EGFR mutation and resistance of non-small-cell lung cancer to gefitinib. N. Engl. J. Med. 352, 786–792 (2005).

Identified a second site intragenic mutation in EGFR (T790M) that confers acquired resistance to gefitinib in patients with NSCLC that stop responding to gefitinib therapy.

36. Pao, W. et al. Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain. PLoS Med. 2, e73 (2005). Identified the T790M mutation in exon 20 of EGFR that confers acquired resistance to EGFR-TKIs.

37. Kwak, E. L. et al. Irreversible inhibitors of the EGF receptor may circumvent acquired resistance to gefitinib. Proc. Natl Acad. Sci. USA 102, 7665–7670 (2005).

In vitro data showing that T790M-mediated acquired resistance to gefitinib can be overcome by irreversible EGFR-TKIs.

38. Gow, C. H., Shih, J. Y., Chang, Y. L. & Yu, C. J. Acquired gefitinib-resistant mutation of EGFR in a chemonaive lung adenocarcinoma harboring gefitinib-sensitive mutation L858R. PLoS Med. 2, e269 (2005).

39. Inukai, M. et al. Presence of epidermal growth factor receptor gene T790M mutation as a minor clone in non-small cell lung cancer. Cancer Res. 66, 7854–7858 (2006).

40. Greulich, H. et al. Oncogenic transformation by inhibitor-sensitive and-resistant EGFR mutants. PLoS Med. 2, e313 (2005).

41. Ji, H. et al. The impact of human EGFR kinase domain mutations on lung tumorigenesis and in vivo sensitivity to EGFR-targeted therapies. Cancer Cell 9, 485–495 (2006).

Showed the relevance of EGFR mutations to the development of NSCLC and response to EGFR-TKIs in a mouse tumour model.

42. Politi, K. et al. Lung adenocarcinomas induced in mice by mutant EGF receptors found in human lung cancers respond to a tyrosine kinase inhibitor or to down-regulation of the receptors. Genes Dev. 20, 1496–1510 (2006).

Modelled NSCLC in mice using transgenic kinase domain mutant EGFR and showed their response to EGFR-TKIs.

43. Sordella, R., Bell, D. W., Haber, D. A. & Settleman, J. Gefitinib-sensitizing EGFR mutations in lung cancer activate anti-apoptotic pathways. Science 305, 1163–1167 (2004).

EGFR kinase domain mutations hyperactivate survival pathways such as Akt and STAT 3, and gefitinib attenuates them in sensitive cell lines.

44. Jiang, J. et al. Epidermal growth factor-independent transformation of Ba/F3 cells with cancer-derived epidermal growth factor receptor mutants induces gefitinib-sensitive cell cycle progression. Cancer Res. 65, 8968–8974 (2005).

45. Prudkin, L. & Wistuba, II. Epidermal growth factor receptor abnormalities in lung cancer. Pathogenetic and clinical implications. Ann. Diagn. Pathol. 10, 306–315 (2006).

46. Kobayashi, S. et al. An alternative inhibitor overcomes resistance caused by a mutation of the epidermal growth factor receptor. Cancer Res. 65, 7096–7101 (2005).

47. Nagatomo, I. et al. The gefitinib-sensitizing mutant epidermal growth factor receptor enables transformation of a mouse fibroblast cell line. DNA Cell Biol. 25, 246–251 (2006).

48. Carey, K. D. et al. Kinetic analysis of epidermal growth factor receptor somatic mutant proteins shows increased sensitivity to the epidermal growth factor receptor tyrosine kinase inhibitor, erlotinib. Cancer Res. 66, 8163–8171 (2006).

49. Mulloy, R. et al. EGF receptor mutants from human lung cancers exhibit enhanced catalytic activity and increased sensitivity to gefitinib. Cancer Res. (in the press).

50. Fabian, M. A. et al. A small molecule-kinase interaction map for clinical kinase inhibitors. Nature Biotechnol. 23, 329–336 (2005).

51. Okamoto, I. et al. EGFR mutation in gefitinib- responsive small-cell lung cancer. Ann. Oncol. 17, 1028–1029 (2006).

52. Gwak, G. Y. et al. Detection of response-predicting mutations in the kinase domain of the epidermal growth factor receptor gene in cholangiocarcinomas. J. Cancer Res. Clin. Oncol. 131, 649–652 (2005).

53. Schilder, R. J. et al. Phase II study of gefitinib in patients with relapsed or persistent ovarian or primary peritoneal carcinoma and evaluation of epidermal growth factor receptor mutations and immunohistochemical expression: a Gynecologic Oncology Group Study. Clin. Cancer Res. 11, 5539–5548 (2005).

54. Barber, T. D., Vogelstein, B., Kinzler, K. W. & Velculescu, V. E. Somatic mutations of EGFR in colorectal cancers and glioblastomas. N. Engl. J. Med. 351, 2883 (2004).

55. Lee, J. W. et al. Somatic mutations of EGFR gene in squamous cell carcinoma of the head and neck. Clin. Cancer Res. 11, 2879–2882 (2005).

56. Kwak, E. L. et al. Epidermal growth factor receptor kinase domain mutations in esophageal and pancreatic adenocarcinomas. Clin. Cancer Res. 12, 4283–4287 (2006).

57. Fukuoka, M. et al. Multi-institutional randomized phase II trial of gefitinib for previously treated patients with advanced non-small-cell lung cancer (The IDEAL 1 Trial). J. Clin. Oncol. 21, 2237–2246 (2003).

58. Kris, M. G. et al. Efficacy of gefitinib, an inhibitor of the epidermal growth factor receptor tyrosine kinase, in symptomatic patients with non-small cell lung cancer: a randomized trial. JAMA 290, 2149–2158 (2003).

59. Shepherd, F. A. et al. Erlotinib in previously treated non-small-cell lung cancer. N. Engl. J. Med. 353, 123–132 (2005).

Reports on a phase III clinical trial that showed a significant survival benefit for individuals with advanced NSCLC who were treated with erlotinib.

60. Perez-Soler, R. et al. Determinants of tumor response and survival with erlotinib in patients with non-small- cell lung cancer. J. Clin. Oncol. 22, 3238–3247 (2004).

61. Bell, D. W. et al. Epidermal growth factor receptor mutations and gene amplification in non-small-cell lung cancer: molecular analysis of the IDEAL/INTACT gefitinib trials. J. Clin. Oncol. 23, 8081–8092 (2005).

62. Cortes-Funes, H. et al. Epidermal growth factor receptor activating mutations in Spanish gefitinib- treated non-small-cell lung cancer patients. Ann. Oncol. 16, 1081–1086 (2005).

63. Huang, S. F. et al. High frequency of epidermal growth factor receptor mutations with complex patterns in non- small cell lung cancers related to gefitinib responsiveness in Taiwan. Clin. Cancer Res. 10, 8195–8203 (2004).

R E V I E W S

64. Kim, K. S. et al. Predictors of the response to gefitinib in refractory non-small cell lung cancer. Clin. Cancer Res. 11, 2244–2251 (2005).

65. Kondo, M. et al. Mutations of epidermal growth factor receptor of non-small cell lung cancer were associated with sensitivity to gefitinib in recurrence after surgery. Lung Cancer 50, 385–391 (2005).

66. Mu, X. L. et al. Gefitinib-sensitive mutations of the epidermal growth factor receptor tyrosine kinase domain in chinese patients with non-small cell lung cancer. Clin. Cancer Res. 11, 4289–4294 (2005).

67. Niho, S. et al. First-line single agent treatment with gefitinib in patients with advanced non-small-cell lung cancer: a phase II study. J. Clin. Oncol. 24, 64–69 (2006).

68. Rosell, R. et al. Mutations in the tyrosine kinase domain of the EGFR gene associated with gefitinib response in non-small-cell lung cancer. Lung Cancer 50, 25–33 (2005).

69. Takano, T. et al. Epidermal growth factor receptor gene mutations and increased copy numbers predict gefitinib sensitivity in patients with recurrent non- small-cell lung cancer. J. Clin. Oncol. 23, 6829–6837 (2005).

70. Taron, M. et al. Activating mutations in the tyrosine kinase domain of the epidermal growth factor receptor are associated with improved survival in gefitinib- treated chemorefractory lung adenocarcinomas. Clin. Cancer Res. 11, 5878–5885 (2005).

71. Tokumo, M. et al. Double mutation and gene copy number of EGFR in gefitinib refractory non-small-cell lung cancer. Lung Cancer 53, 117–121 (2006).

72. Tomizawa, Y. et al. Clinicopathologic significance of the mutations of the epidermal growth factor receptor gene in patients with non-small cell lung cancer. Clin. Cancer Res. 11, 6816–6822 (2005).

73. Zhang, X. T. et al. The EGFR mutation and its correlation with response of gefitinib in previously treated Chinese patients with advanced non-small-cell lung cancer. Ann. Oncol. 16, 1334–1342 (2005).

74. Jackman, D. M. et al. Exon 19 deletion mutations of epidermal growth factor receptor are associated with prolonged survival in non-small cell lung cancer patients treated with gefitinib or erlotinib. Clin. Cancer Res. 12, 3908–3914 (2006).

75. Riely, G. J. et al. Clinical course of patients with non- small cell lung cancer and epidermal growth factor receptor exon 19 and exon 21 mutations treated with gefitinib or erlotinib. Clin. Cancer Res. 12, 839–844 (2006).

76. Tokumo, M. et al. The relationship between epidermal growth factor receptor mutations and clinicopathologic features in non-small cell lung cancers. Clin. Cancer Res. 11, 1167–1173 (2005).

77. Hirsch, F. R. et al. Increased epidermal growth factor receptor gene copy number detected by fluorescence in situ hybridization associates with increased sensitivity to gefitinib in patients with bronchioloalveolar carcinoma subtypes: a Southwest Oncology Group Study. J. Clin. Oncol. 23, 6838–6845 (2005).

78. Cappuzzo, F. et al. Increased HER2 gene copy number is associated with response to gefitinib therapy in epidermal growth factor receptor-positive non-small-cell lung cancer patients. J. Clin. Oncol. 23, 5007–5018 (2005).

79. Cappuzzo, F. et al. HER3 genomic gain and sensitivity to gefitinib in advanced non-small-cell lung cancer patients. Br. J. Cancer 93, 1334–1340 (2005).

80. Suzuki, R. et al. A phase II study of single-agent gefitinib as first-line therapy in patients with stage IV non-small-cell lung cancer. Br. J. Cancer 94, 1599–1603 (2006).

81. Inoue, A. et al. Prospective phase II study of gefitinib for chemotherapy-naive patients with advanced non-small-cell lung cancer with epidermal growth factor receptor gene mutations. J. Clin. Oncol. 24, 3340–3346 (2006).

82. Hirsch, F. R. et al. Molecular predictors of outcome with gefitinib in a phase III placebo-controlled study in advanced non-small-cell lung cancer. J. Clin. Oncol. 24, 5034–42 (2006).

83. Thatcher, N. et al. Gefitinib plus best supportive care in previously treated patients with refractory advanced non-small-cell lung cancer: results from a randomised, placebo-controlled, multicentre study (Iressa Survival Evaluation in Lung Cancer). Lancet 366, 1527–1537 (2005).

Reports on a phase III clinical trial that showed a lack of survival benefit in individuals with advanced NSCLC who were treated with gefitinib.

84. Ono, M. et al. Sensitivity to gefitinib (Iressa, ZD1839) in non-small cell lung cancer cell lines correlates with dependence on the epidermal growth factor (EGF) receptor/extracellular signal-regulated kinase 1/2 and EGF receptor/Akt pathway for proliferation. Mol. Cancer Ther. 3, 465–472 (2004).

85. Han, S. W. et al. Epidermal growth factor receptor (EGFR) downstream molecules as response predictive markers for gefitinib (Iressa, ZD1839) in chemotherapy-resistant non-small cell lung cancer. Int. J. Cancer 113, 109–115 (2005).

86. Mukohara, T. et al. Differential effects of gefitinib and cetuximab on non-small-cell lung cancers bearing epidermal growth factor receptor mutations. J. Natl Cancer Inst. 97, 1185–1194 (2005).

87. Anderson, N. G., Ahmad, T., Chan, K., Dobson, R. & Bundred, N. J. ZD1839 (Iressa), a novel epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, potently inhibits the growth of EGFR-positive cancer cell lines with or without erbB2 overexpression. Int. J. Cancer 94, 774–782 (2001).

88. Moasser, M. M., Basso, A., Averbuch, S. D. & Rosen, N. The tyrosine kinase inhibitor ZD1839 (‘Iressa’) inhibits HER2-driven signaling and suppresses the growth of HER2-overexpressing tumor cells. Cancer Res. 61, 7184–7188 (2001).

89. She, Q. B., Solit, D., Basso, A. & Moasser, M. M. Resistance to gefitinib in PTEN-null HER- overexpressing tumor cells can be overcome through restoration of PTEN function or pharmacologic modulation of constitutive phosphatidylinositol 3’-kinase/Akt pathway signaling. Clin. Cancer Res. 9, 4340–4346 (2003).

90. Engelman, J. A. et al. ErbB-3 mediates phosphoinositide 3-kinase activity in gefitinib-sensitive non-small cell lung cancer cell lines. Proc. Natl Acad. Sci. USA 102, 3788–3793 (2005).

Gefitinib sensitivity is correlated with the heterodimerization of EGFR with ERBB2 and the attenuation of Akt activation in gefitinib-treated NSCLC cell lines.

91. Weinstein, I. B. Disorders in cell circuitry during multistage carcinogenesis: the role of homeostasis. Carcinogenesis 21, 857–864 (2000).

92. Sharma, S. V. et al. A common signaling cascade may underlie ‘addiction’ to the Src, BCR-ABL, and EGF receptor oncogenes. Cancer Cell 10, 425–435 (2006).

Details the molecular mechanistic basis for cancer cell killing by oncogene-targeted therapeutics.

93. Evan, G. & Littlewood, T. A matter of life and cell death. Science 281, 1317–1322 (1998).

94. Chen, C. Y. & Faller, D. V. Direction of p21ras- generated signals towards cell growth or apoptosis is determined by protein kinase C and Bcl-2. Oncogene 11, 1487–1498 (1995).

95. Mayo, M. W. et al. Requirement of NF-κB activation to suppress p53-independent apoptosis induced by oncogenic Ras. Science 278, 1812–1815 (1997).

96. Johnson, D. et al. Regulation of both apoptosis and cell survival by the v-Src oncoprotein. Cell Death Differ. 7, 685–696 (2000).

97. Webb, B. L., Jimenez, E. & Martin, G. S. v-Src generates a p53-independent apoptotic signal. Mol. Cell. Biol. 20, 9271–9280 (2000).

98. Wang, J. Y. Regulation of cell death by the Abl tyrosine kinase. Oncogene 19, 5643–5650 (2000).

99. Reginato, M. J. et al. Integrins and EGFR coordinately regulate the pro-apoptotic protein Bim to prevent anoikis. Nature Cell Biol. 5, 733–740 (2003).

100. Evan, G. I. et al. Induction of apoptosis in fibroblasts by c-myc protein. Cell 69, 119–128 (1992).

101. Shi, Y. et al. Role for c-myc in activation-induced apoptotic cell death in T cell hybridomas. Science 257, 212–214 (1992).

102. White, E. & Stillman, B. Expression of adenovirus E1B mutant phenotypes is dependent on the host cell and on synthesis of E1A proteins. J. Virol. 61, 426–435 (1987).

103. Rodenhuis, S. et al. Mutational activation of the K-ras oncogene. A possible pathogenetic factor in adenocarcinoma of the lung. N. Engl. J. Med. 317, 929–935 (1987).

104. Mitsudomi, T. et al. ras gene mutations in non-small cell lung cancers are associated with shortened survival irrespective of treatment intent. Cancer Res. 51, 4999–5002 (1991).

105. Husgafvel-Pursiainen, K. et al. K-ras mutations in human adenocarcinoma of the lung: association with smoking and occupational exposure to asbestos. Int. J. Cancer 53, 250–256 (1993).

106. Ahrendt, S. A. et al. Cigarette smoking is strongly associated with mutation of the K-ras gene in patients with primary adenocarcinoma of the lung. Cancer 92, 1525–1530 (2001).

107. Soung, Y. H. et al. Mutational analysis of EGFR and K-RAS genes in lung adenocarcinomas. Virchows Arch. 446, 483–488 (2005).

108. Pao, W. et al. KRAS mutations and primary resistance of lung adenocarcinomas to gefitinib or erlotinib. PLoS Med. 2, e17 (2005).

Shows the correlation between the presence of activated KRAS with NSCLC insensitivity to EGFR- TKIs.

109. Cully, M., You, H., Levine, A. J. & Mak, T. W. Beyond PTEN mutations: the PI3K pathway as an integrator of multiple inputs during tumorigenesis. Nature Rev. Cancer 6, 184–192 (2006).

110. Kohno, T., Takahashi, M., Manda, R. & Yokota, J. Inactivation of the PTEN/MMAC1/TEP1 gene in human lung cancers. Genes Chromosomes Cancer 22, 152–156 (1998).

111. Soria, J. C. et al. Lack of PTEN expression in non- small cell lung cancer could be related to promoter methylation. Clin. Cancer Res. 8, 1178–1184 (2002).

112. Bianco, R. et al. Loss of PTEN/MMAC1/TEP in EGF receptor-expressing tumor cells counteracts the antitumor action of EGFR tyrosine kinase inhibitors. Oncogene 22, 2812–2822 (2003).

113. Chakravarti, A., Loeffler, J. S. & Dyson, N. J. Insulin- like growth factor receptor I mediates resistance to anti-epidermal growth factor receptor therapy in primary human glioblastoma cells through continued activation of phosphoinositide 3-kinase signaling. Cancer Res. 62, 200–207 (2002).

114. Hurbin, A., Dubrez, L., Coll, J. L. & Favrot, M. C. Inhibition of apoptosis by amphiregulin via an insulin- like growth factor-1 receptor-dependent pathway in non-small cell lung cancer cell lines. J. Biol. Chem. 277, 49127–49133 (2002).

115. Erjala, K. et al. Signaling via ErbB2 and ErbB3 associates with resistance and epidermal growth factor receptor (EGFR) amplification with sensitivity to EGFR inhibitor gefitinib in head and neck squamous cell carcinoma cells. Clin. Cancer Res. 12, 4103–4111 (2006).

116. Cappuzzo, F. et al. Insulin-like growth factor receptor 1 (IGFR-1) is significantly associated with longer survival in non-small-cell lung cancer patients treated with gefitinib. Ann. Oncol. 17, 1120–1127 (2006).

117. Kosaka, T. et al. Analysis of epidermal growth factor receptor gene mutation in patients with non-small cell lung cancer and acquired resistance to gefitinib. Clin. Cancer Res. 12, 5764–5769 (2006).

118. Engelman, J. A. et al. Allelic dilution obscures detection of a biologically significant resistance mutation in EGFR-amplified lung cancer. J. Clin. Invest. 116, 2695–2706 (2006).

119. Bell, D. W. et al. Inherited susceptibility to lung cancer may be associated with the T790M drug resistance mutation in EGFR. Nature Genet. 37, 1315–1316 (2005).

Shows that the T790M mutation that confers acquired resistance to EGFR-TKIs might also predispose individuals to develop NSCLC.

120. Shih, J. Y., Gow, C. H. & Yang, P. C. EGFR mutation conferring primary resistance to gefitinib in non-small- cell lung cancer. N. Engl. J. Med. 353, 207–208 (2005).

121. Gorre, M. E. et al. Clinical resistance to STI-571 cancer therapy caused by BCR-ABL gene mutation or amplification. Science 293, 876–880 (2001).

122. Blencke, S. et al. Characterization of a conserved structural determinant controlling protein kinase sensitivity to selective inhibitors. Chem. Biol. 11, 691–701 (2004).

123. Daub, H., Specht, K. & Ullrich, A. Strategies to overcome resistance to targeted protein kinase inhibitors. Nature Rev. Drug Discov. 3, 1001–1010 (2004).

124. Zhou, B. B. et al. Targeting ADAM-mediated ligand cleavage to inhibit HER3 and EGFR pathways in non- small cell lung cancer. Cancer Cell 10, 39–50 (2006).

125. Koizumi, F., Shimoyama, T., Taguchi, F., Saijo, N. & Nishio, K. Establishment of a human non-small cell lung cancer cell line resistant to gefitinib. Int. J. Cancer 116, 36–44 (2005).

126. Haber, D. A. & Settleman, J. Overcoming acquired resistance to Iressa/Tarceva with inhibitors of a different class. Cell Cycle 4, 1057–1059 (2005).

R E V I E W S

127. Elkind, N. B. et al. Multidrug transporter ABCG2 prevents tumor cell death induced by the epidermal growth factor receptor inhibitor Iressa (ZD1839, Gefitinib). Cancer Res. 65, 1770–1777 (2005).

128. Ozvegy-Laczka, C., Cserepes, J., Elkind, N. B. & Sarkadi, B. Tyrosine kinase inhibitor resistance in cancer: role of ABC multidrug transporters. Drug Resist. Updat. 8, 15–26 (2005).

129. Yanase, K. et al. Gefitinib reverses breast cancer resistance protein-mediated drug resistance. Mol. Cancer Ther. 3, 1119–1125 (2004).

130. Nakamura, Y. et al. Gefitinib (‘Iressa’, ZD1839), an epidermal growth factor receptor tyrosine kinase inhibitor, reverses breast cancer resistance protein/ ABCG2-mediated drug resistance. Cancer Res. 65, 1541–1546 (2005).

131. Nagashima, S. et al. BCRP/ABCG2 levels account for the resistance to topoisomerase I inhibitors and reversal effects by gefitinib in non-small cell lung cancer. Cancer Chemother. Pharmacol. 58, 594–600 (2006).

132. Kitazaki, T. et al. Gefitinib, an EGFR tyrosine kinase inhibitor, directly inhibits the function of P-glycoprotein in multidrug resistant cancer cells. Lung Cancer 49, 337–343 (2005).

133. Yang, C. H. et al. Gefitinib reverses chemotherapy resistance in gefitinib-insensitive multidrug resistant cancer cells expressing ATP-binding cassette family protein. Cancer Res. 65, 6943–6949 (2005).

134. Singh, J. et al. Structure-based design of a potent, selective, and irreversible inhibitor of the catalytic domain of the erbB receptor subfamily of protein tyrosine kinases. J. Med. Chem. 40, 1130–1135 (1997).

135. Fry, D. W. et al. Specific, irreversible inactivation of the epidermal growth factor receptor and erbB2, by a new class of tyrosine kinase inhibitor. Proc. Natl Acad. Sci. USA 95, 12022–12027 (1998).

136. Rabindran, S. K. et al. Antitumor activity of HKI-272, an orally active, irreversible inhibitor of the HER-2 tyrosine kinase. Cancer Res. 64, 3958–3965 (2004).

137. Wissner, A. et al. Synthesis and structure-activity relationships of 6, 7-disubstituted 4-anilinoquinoline- 3-carbonitriles. The design of an orally active, irreversible inhibitor of the tyrosine kinase activity of the epidermal growth factor receptor (EGFR) and the human epidermal growth factor receptor-2 (HER-2). J. Med. Chem. 46, 49–63 (2003).

138. Haber, D. A. et al. Molecular targeted therapy of lung cancer: EGFR mutations and response to EGFR inhibitors. Cold Spring Harb. Symp. Quant. Biol. 70, 419–426 (2005).

139. Discafani, C. M. et al. Irreversible inhibition of epidermal growth factor receptor tyrosine kinase with in vivo activity by N-[4-[(3-bromophenyl)amino]-6- quinazolinyl]-2-butynamide (CL-387, 785). Biochem. Pharmacol. 57, 917–925 (1999).

140. Smaill, J. B. et al. Tyrosine kinase inhibitors. 17. Irreversible inhibitors of the epidermal growth factor receptor: 4-(phenylamino)quinazoline- and 4-(phenylamino)pyrido[3, 2-d]pyrimidine-6- acrylamides bearing additional solubilizing functions. J. Med. Chem. 43, 1380–1397 (2000).

141. Carter, T. A. et al. Inhibition of drug-resistant mutants of ABL, KIT, and EGF receptor kinases. Proc. Natl Acad. Sci. USA 102, 11011–11016 (2005).

Details the development of second generation TKIs that overcome resistance that arises from mutations of the gatekeeper residue in various tyrosine kinase oncogenes.

142. Shimamura, T. et al. Non-small-cell lung cancer and Ba/F3 transformed cells harboring the ERBB2 G776insV_G/C mutation are sensitive to the dual- specific epidermal growth factor receptor and ERBB2 inhibitor HKI-272. Cancer Res. 66, 6487–6491 (2006).

143. Ji, H. et al. Epidermal growth factor receptor variant III mutations in lung tumorigenesis and sensitivity to tyrosine kinase inhibitors. Proc. Natl Acad. Sci. USA 103, 7817–7822 (2006).

144. Shimamura, T., Lowell, A. M., Engelman, J. A. & Shapiro, G. I. Epidermal growth factor receptors harboring kinase domain mutations associate with the heat shock protein 90 chaperone and are destabilized following exposure to geldanamycins. Cancer Res. 65, 6401–6408 (2005).

145. Yang, S. et al. Association with HSP90 inhibits Cbl- mediated down-regulation of mutant epidermal growth factor receptors. Cancer Res. 66, 6990–6997 (2006).

146. Ge, J. et al. Design, synthesis, and biological evaluation of hydroquinone derivatives of 17-amino- 17-demethoxygeldanamycin as potent, water-soluble inhibitors of Hsp90. J. Med. Chem. 49, 4606–4615 (2006).

147. Herbst, R. S., Fukuoka, M. & Baselga, J. Gefitinib–a novel targeted approach to treating cancer. Nature Rev. Cancer 4, 956–965 (2004).

148. Shaw, R. J. & Cantley, L. C. Ras, PI(3)K and mTOR signalling controls tumour cell growth. Nature 441, 424–430 (2006).

149. Ihle, N. T. et al. The phosphatidylinositol-3-kinase inhibitor PX-866 overcomes resistance to the epidermal growth factor receptor inhibitor gefitinib in A-549 human non-small cell lung cancer xenografts. Mol. Cancer Ther. 4, 1349–1357 (2005).

150. Fan, Q. W. et al. A dual PI3 kinase/mTOR inhibitor reveals emergent efficacy in glioma. Cancer Cell 9, 341–349 (2006).

151. Goudar, R. K. et al. Combination therapy of inhibitors of epidermal growth factor receptor/ vascular endothelial growth factor receptor 2 (AEE788) and the mammalian target of rapamycin (RAD001) offers improved glioblastoma tumor growth inhibition. Mol. Cancer Ther. 4, 101–112 (2005).

152. Rao, R. D. et al. Disruption of parallel and converging signaling pathways contributes to the synergistic antitumor effects of simultaneous mTOR and EGFR inhibition in GBM cells. Neoplasia 7, 921–929 (2005).

153. Doherty, L. et al. Pilot study of the combination of EGFR and mTOR inhibitors in recurrent malignant gliomas. Neurology 67, 156–158 (2006).

154. Easton, J. B. & Houghton, P. J. Therapeutic potential of target of rapamycin inhibitors. Expert Opin. Ther. Targets 8, 551–564 (2004).

155. Margolin, K. et al. CCI-779 in metastatic melanoma: a phase II trial of the California Cancer Consortium. Cancer 104, 1045–1048 (2005).

156. Takeuchi, H. et al. Synergistic augmentation of rapamycin-induced autophagy in malignant glioma cells by phosphatidylinositol 3-kinase/protein kinase B inhibitors. Cancer Res. 65, 3336–3346 (2005).

157. Hay, N. The Akt-mTOR tango and its relevance to cancer. Cancer Cell 8, 179–183 (2005).

158. Adjei, A. A. Novel combinations based on epidermal growth factor receptor inhibition. Clin. Cancer Res. 12, 4446s-4450s (2006).

159. Hennequin, L. F. et al. Novel 4-anilinoquinazolines with C-7 basic side chains: design and structure activity relationship of a series of potent, orally active, VEGF receptor tyrosine kinase inhibitors. J. Med. Chem. 45, 1300–1312 (2002).

160. Wedge, S. R. et al. ZD6474 inhibits vascular endothelial growth factor signaling, angiogenesis, and tumor growth following oral administration. Cancer Res. 62, 4645–4655 (2002).

161. Ciardiello, F. et al. Antitumor activity of ZD6474, a vascular endothelial growth factor receptor tyrosine kinase inhibitor, in human cancer cells with acquired resistance to antiepidermal growth factor receptor therapy. Clin. Cancer Res. 10, 784–793 (2004).

162. Taguchi, F. et al. Anticancer effects of ZD6474, a VEGF receptor tyrosine kinase inhibitor, in gefitinib (‘Iressa’)- sensitive and resistant xenograft models. Cancer Sci. 95, 984–989 (2004).

163. Traxler, P. et al. AEE788: a dual family epidermal growth factor receptor/ErbB2 and vascular endothelial growth factor receptor tyrosine kinase inhibitor with antitumor and antiangiogenic activity. Cancer Res. 64, 4931–4941 (2004).

164. Lee, D. Phase II data with ZD6474, a small-molecule kinase inhibitor of epidermal growth factor receptor and vascular endothelial growth factor receptor, in previously treated advanced non-small-cell lung cancer. Clin. Lung Cancer 7, 89–91 (2005).

165. Stephens, P. et al. Lung cancer: intragenic ERBB2 kinase mutations in tumours. Nature 431, 525–526 (2004).

166. Sjoblom, T. et al. The consensus coding sequences of human breast and colorectal cancers. Science 314, 268–274 (2006). Highlights the genetic complexity and inter-tumour variability of human cancers.

167. Ngo, V. N. et al. A loss-of-function RNA interference screen for molecular targets in cancer. Nature 441, 106–110 (2006).

New approaches to anticancer target discovery. 168. Weinstein, I. B. Cancer. Addiction to oncogenes--the

Achilles heal of cancer. Science 297, 63–64 (2002). 169. Weinstein, I. B. & Joe, A. K. Mechanisms of disease:

Oncogene addiction-a rationale for molecular targeting in cancer therapy. Nature Clin. Pract. Oncol. 3, 448–457 (2006).

170. Druker, B. J. et al. Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells. Nature Med. 2, 561–566 (1996).

171. Chin, L. et al. Essential role for oncogenic Ras in tumour maintenance. Nature 400, 468–472 (1999).

172. Fisher, G. H. et al. Induction and apoptotic regression of lung adenocarcinomas by regulation of a K-Ras transgene in the presence and absence of tumor suppressor genes. Genes Dev. 15, 3249–3262 (2001).

173. Felsher, D. W. & Bishop, J. M. Reversible tumorigenesis by MYC in hematopoietic lineages. Mol. Cell 4, 199–207 (1999).

174. Felsher, D. W. Cancer revoked: oncogenes as therapeutic targets. Nature Rev. Cancer 3, 375–380 (2003).

175. Shigematsu, H. & Gazdar, A. F. Somatic mutations of epidermal growth factor receptor signaling pathway in lung cancers. Int. J. Cancer 118, 257–262 (2006).

176. Balak, M. N. et al. Novel D761Y and common secondary T790M mutations in epidermal growth factor receptor-mutant lung adenocarcinomas with acquired resistance to kinase inhibitors. Clin. Cancer Res. 12, 6494–6500 (2006).

177. Asahina, H. et al. Non-responsiveness to gefitinib in a patient with lung adenocarcinoma having rare EGFR mutations S768I and V769L. Lung Cancer 54, 419–422 (2006).

178. Baselga, J. et al. Phase I safety, pharmacokinetic, and pharmacodynamic trial of ZD1839, a selective oral epidermal growth factor receptor tyrosine kinase inhibitor, in patients with five selected solid tumor types. J. Clin. Oncol. 20, 4292–4302 (2002).

179. Janmaat, M. L., Kruyt, F. A., Rodriguez, J. A. & Giaccone, G. Response to epidermal growth factor receptor inhibitors in non-small cell lung cancer cells: limited antiproliferative effects and absence of apoptosis associated with persistent activity of extracellular signal-regulated kinase or Akt kinase pathways. Clin. Cancer Res. 9, 2316–2326 (2003).

180. Tracy, S. et al. Gefitinib induces apoptosis in the EGFRL858R non-small-cell lung cancer cell line H3255. Cancer Res. 64, 7241–7244 (2004).

Acknowledgements The authors would like to thank T.-M. Chin and L. Sequist Van Dam for their helpful comments. We apologize to our many colleagues whose work is not cited owing to space constraints. Another notable omission is the very important area of anti- body-based ErbB-targeted therapies, which is dealt with very well in other reviews.

Competing interests statement The authors declare no competing financial interests.

DATABASES The following terms in this article are linked online to: Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene ABCG2 | ABL | ADAM17 | BCR | EGF | EGFR | ERBB2| ERBB3 | ERBB4 | ERK1 | ERK2 | HSP90 | IGFR1 | KDR | KRAS | MEK | mTOR | MYC | PDGFRα | PI3K | STAT3 | STAT5 | TGFα | VEGF National Cancer Institute: http://www.cancer.gov lung cancer

FURTHER INFORMATION Massachusetts General Hospital Cancer Center: http://www.massgeneral.org/cancer/ NCI clinical trials website: http://www.cancer.gov/ clinicaltrials Access to this interactive links box is free online.

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