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European Biopharmaceutical Review

Resisting Mutation, Restricting Resistance


Biomarker-driven targeted therapies for non-small cell lung cancer have come on in leaps and bounds over the last decade. Now drug developers are tackling the challenge of acquired resistance.

Lung cancer is the leading cause of cancer-related mortality in both men and women, with more than 1.6 million new cases of lung cancer estimated to have occurred globally in 2008. In Europe, lung cancer accounts for 20 per cent of all cancer deaths (28 per cent in men and 10 per cent in women). Of these, approximately 80 per cent are non-small cell lung cancers (NSCLC), which can be further subdivided based on histology, including squamous cell carcinomas, large cell lung carcinomas, and adenocarcinomas.

Throughout the past decade, further sub-categorisation has led to the realisation that NSCLCs are also very heterogeneous at the molecular level, harbouring ‘driver’ mutations in distinct genes that disrupt normal signalling and lead to the uncontrolled growth and proliferation of tumour cells (see Figure 1). The molecular understanding and subsequent targeting of these subsets has completely revolutionised disease treatment, further supporting the ‘oncogene addiction’ hypothesis – that many tumours have an ‘Achilles' heel’, and hence can be uniquely targeted with specific small molecule inhibitors and antibodies (1).

Of the many defined subsets, activating mutations in the epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) genes may be the most well-defined, and in both cases, small molecule inhibitors targeting each have been approved by the FDA. This article presents a review of both targets, including approved agents, mechanisms of resistance, and next-generation agents in development, many designed specifically to target disease-resistant mutations.

Mutant EGFR and NSCLC

EGFR is a member of the ERB-family of receptor tyrosine kinases whose ligand-induced homo- and heterodimerisation leads to intracellular signal transduction controlling key cellular functions, including growth and cell survival. Early targeting of wild-type EGFR for cancer treatment was based on observations that gene amplification leading to receptor over-expression was observed in multiple human tumours. This led to a body of preclinical data demonstrating small molecule efficacy in multiple mouse tumour models, including NSCLC (2). Based on these early experiments, gefitinib and erlotinib, two small molecule tyrosine kinase inhibitors designed to target wild type EGFR, were advanced into clinical trials in unselected NSCLC patients who had failed prior therapies. Although overall response rates in these early trials were underwhelming, confirmatory trials identified unique clinical characteristics in those responders, including tumour histology (adenocarcinoma), gender (female), ethnicity (east Asian), and ‘never’ or ‘light’ smokers (3,4).

In 2004, several landmark papers identified the existence of somatic mutations in the EGFR gene, whose presence correlated well with patient sensitivity to both erlotinib and gefitinib, and with those aforementioned clinical characteristics (5-7). In 2009, results from the IPASS trial unequivocally validated the predictive nature of EGFR mutations to gefitinib (8). In a randomised subgroup analysis of 261 previously untreated NSCLC patients harbouring activating mutations in EGFR, versus carboplatin-paclitaxel, significantly longer progression-free survival (PFS) (9.5 versus 6.3 months) and response rates (71.2 per cent versus 47.3 per cent) were attained. Moreover, the IPASS trial demonstrated that the patients without EGFR mutations were insensitive to gefitinib and fared better with standard platinum-based chemotherapy.

Despite what are now the obvious clinical successes in treating patients harbouring activating EGFR mutations with small molecule inhibitors, responses are not durable, and all patients ultimately develop resistance to both drugs. The most common mechanism of resistance is a mutation at the gatekeeper position (T790M), which is observed in roughly 50 per cent of all gefitinib/erlotinib-resistant patients (9,10). The presence of T790M has almost no effect on gefitinib binding, but rather restores ATP affinity, increasing binding site competition and rendering both compounds clinically ineffective against this drug-resistant mutant (9).

Although few reversible EGFR-T790M inhibitors have been reported, multiple compounds targeting the active site cysteine (C797) have been described (2). These ‘irreversible’ inhibitors form a covalent bond to the protein via C797, thus permanently inactivating the enzyme. Notable amongst the reported inhibitors are neratinib, canertinib, pelitinib, PF0029904 (all from Pfizer), and afatinib (BIB W-2992, Boehringer Ingelheim). Although many of these inhibitors are active pre-clinically in EGFR-T790M-driven models, both in vitro and in vivo, to date, no second-generation irreversible inhibitors have demonstrated clinically significant, single agent improvements in patients harbouring the T790M-resistant mutation. All designed to target the T790M mutation, most second-generation irreversible inhibitors simultaneously inhibit native EGFR at lower concentrations. This potentially promotes toxicity at circulating plasma levels well below what may be required for clinical efficacy in this patient population (2). Native EGFR is the ‘natural’ receptor; it is expressed throughout the body, but at particularly high levels in both the skin and gastrointestinal (GI) tract. Inhibition, therefore, can lead to clinically observed skin and GI toxicity with non-selective agents. More recently, an irreversible inhibitor with T790M selectivity relative to WT EGFR was reported, but its development status remains unclear (11). In short, there remains a real unmet medical need for compounds selectively targeting the EGFR-T790M gatekeeper mutation in resistant NSCLC patients.

ALK Translocations and NSCLC

Anaplastic lymphoma kinase (ALK) – a receptor tyrosine kinase in the insulin receptor super-family – was first identified as a nucleophosmin (NPM) chromosomal rearrangement (NPMALK fusion gene) in anaplastic large cell lymphoma (ALCL). Subsequently it has also been identified in other tumour types, including diffuse large-cell lymphoma (DLCL) and inflammatory myofibroblastic tumours (IMT) (12). Additionally, echinoderm microtubule-associated protein-like-ALK (EML4-ALK) has more recently been identified in three to seven per cent of NSCLCs (13). In all cases, the ALK fusion partner (for example NPM or EML4) induces a ligand-independent conformational change resulting in constitutive kinase activation and aberrant and continuous downstream signalling. Preclinical studies with small molecule inhibitors demonstrate that ALK inhibition induces apoptosis and tumour regression in multiple ALK-driven models, highlighting a discrete Achilles heel – similar to activating mutations EGFR – and identifying ALK translocations as ‘driver’ mutations further underscoring their potential as viable therapeutic targets (12).

Crizotinib (PF-02341066), a small molecule TKI, was the first compound evaluated clinically for activity in ALK-positive NSCLC patients (14). After screening approximately 1,500 NSCLC patients, 82 (5.5 per cent) were identified as ALK-positive by fluorescence in situ hybridisation (FISH) and enrolled in the trial (15). Encouragingly, at the mean treatment duration of 6.4 months, the overall response rate was 57 per cent, and the estimated probability of six-month PFS was 72 per cent. In a companion publication to the Phase 1 study, crizotinib also demonstrated activity in an IMT patient driven by a separate ALK translocation (16). Together, these data provide clinical validation for ALK as a target and further highlight the value of early genotyping in effective clinical trial design and in streamlining the drug discovery process. On the basis of a larger Phase 3 registration trial comparing crizotinib to single-agent chemotherapy in advanced NSCLC patients harbouring the ALK gene, crizotinib was recently granted accelerated approval by the FDA. What makes this story so remarkable is the relatively short time from target gene identification (EML4-ALK, 2007) to US regulatory approval (2011) – a span of approximately four years. This is a likely increasing paradigm for many targeted agents now in clinical development.

Unfortunately, as with other targeted therapies involving ‘driver’ mutations such as BCR-ABL and EGFR, acquired resistance in crizotinib-treated patients has now been reported, leading to eventual relapse (17). To date, three single-point kinase domain mutations in ALK have been reported. Two were reported in tumour cells identified from a single NSCLC patient, including L1 196M at the ‘gatekeeper’ position, and C1 156Y just preceding the C-alpha helix (17). Similar to the gatekeeper mutations in BCR-ABL (T315I) and EGFR (T790M) – the site most frequently mutated in acquired resistance to TKI treatment – the EML4- ALK L1 196M mutation precludes effective compound-binding, which substantially reduces potency and leads to patient relapse at clinically achievable crizotinib plasma levels. Although C1 156Y does not contact crizotinib directly, presumably it destabilises the crizotinib-ALK binding conformation, which leads to reduced potency. More recently, mutation at F1 174L in an IMT patient harbouring the RANBP2-ALK translocation was reported (18). Coincidentally, mutation at this position (in full length ALK) has been detected in neuroblastomas and is reported to be transforming (19). Similar to the C1156Y mutation, L1174 does not make direct inhibitor contact, but is more likely to shift the ALK conformational equilibrium away from that required for optimal crizotinib binding.

Next on the Horizon: Overcoming Resistance

Several compounds have recently been reported to have activity against crizotinib-resistant mutations, including TAE684 (Novartis), X-396 (Xcovery), and CH5424802 (Chugai). TAE684, an ALK inhibitor based on the pyrimidine template, was shown in two independent mutagenesis studies to maintain substantial activity against a wide range of crizotinib mutations, including L1196M (20-22). Similarly, X-396, an aminopyridizane-based ALK inhibitor that shares common structural features with crizotinib was reported to be active against both L1196M and C1156Y (23). Lastly, CH5424802, a structurally unique ALK inhibitor derived from a screening lead, was also reported to have activity in multiple preclinical models, including oral in vivo efficacy in EML4-ALK 1196M-driven tumours (24). Of the three inhibitors, only CH5424802 has advanced into clinical trials, in Japan, but no data highlighting clinical activity has been reported.

Recently, a Phase 1/2 clinical trial was initiated for AP26113, an investigational dual ALK/EGFR inhibitor (NSCLC, NCT01449461). AP26113 is unique in that it targets both ALK and EGFR, and so has the potential to be two drugs in one. Similar to the crizotinib clinical trial structure, the Phase 1 component will probe initial safety, tolerability, pharmacokinetic profile and the recommended dose, while the Phase 2 component will probe preliminary antitumour activity through four genetically defined cohorts, including:

  • ALK+ NSCLC patients with no prior ALK inhibitor therapy
  • ALK+ NSCLC patients resistant to one ALK inhibitor
  • EGFR+ NSCLC patients resistant to at least one prior EGFR inhibitor
  • Patients with other ALK+ expressing cancers or other known targets of AP26113

In preclinical models, in addition to being at least 10 times more potent against the native form of ALK, AP26113 is active against crizotinib-resistant mutations both in vitro and in vivo (25). Additionally, AP26113 is active against both activating and resistant mutants of EGFR while sparing the native form of the protein (26). This is in contrast to the aforementioned irreversible inhibitors, which while potent against T790M, simultaneously inhibit the native form of the protein, which may contribute to their observed clinical toxicity. Importantly, oral doses that are efficacious in mice against activated and T790M EGFR are similar to those active against native and crizoitinib-resistant ALK mutants, suggesting that AP26113 has the potential to address both clinical needs. Polypharmacology through multitargeted kinase inhibition is not unique to AP26113, but simultaneously targeting two well-defined and important subsets of NSCLC make this investigational agent appealing, and it is believed that expedited development based on this profile should be feasible.

Conclusion

The US approval of crizotinib, and its companion diagnostic (Abbott), for the identification and treatment of NSCLC patients harbouring activating ALK mutations is the most recent example of an evolving paradigm of biomarker-driven, targeted therapy for cancer treatment. Together with the identification of activating EGFR mutations, almost 15 per cent of all NSCLC patients are thus candidates for treatment with targeted agents based on the identification of one of these two molecular abnormalities. Unfortunately, treatment of patients with these ‘driver’ mutations can lead to acquired resistance and so the development of newer, more potent agents will remain an important goal.

References

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  3. Kris MG, Natale RB, Herbst RS 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 randomised trial, JAMA 290: pp2,149-2,158, 2003
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  7. Pao W, Miller V, Zakowski M et al, EGF receptor gene mutations are common in lung cancers from ‘never smokers’ and are associated with sensitivity of tumours to gefitinib and erlotinib, Proc Natl Acad Sci: 101: pp13,306-13,311, 2004
  8. Mok TS, Wu Y-L, Thongprasert S et al, Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinomas, NEJM 361: pp947-957, 2009
  9. Yun C-H, Mengwasser KE, Toms AV et al, The T790M mutation in EGFR kinases causes drug resistance by increasing the affinity for ATP, Proc Natl Acad Sci 105: pp2,070-2,075, 2008
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  12. Webb TR, Slavish J, George RE et al, Anaplastic lymphoma kinase: role in cancer pathogenesis and small-molecule inhibitor development for therapy, Expert Rev Anticancer Ther 9: pp331-356 and references cited therein, 2009
  13. Soda M, Choi YL, Enomto M et al, Identification of the transforming EML4- ALK fusion gene in non-small-cell lung cancer, Nature 448: pp561-566, 2007
  14. Christensen JG, Zou HY, Arango ME et al, Cytoreductive antitumour activity of PF¬2341066, a novel inhibitor of anaplastic lymphoma kinase and c-Met, in experimental models of anaplastic large-cell lymphoma, Mol Cancer Ther 6: pp3,314-3,322, 2007
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  16. Butrynski JE, D’Adamo DR, Hornick JL et al, Crizotinib in ALK-rearranged inflammatory myofibroblastic tumour, NEJM 363: pp1,727-1,733, 2010
  17. Choi YL, Soda M, Yamashita Y et al, EML4-ALK mutations in lung cancer that confer resistance to ALK inhibitors, NEJM 363: pp1,734-1,739, 2010
  18. Sasaki T, Okuda K, Zeng W et al, The neuroblastoma associated F1 174L ALK mutation causes resistance to an ALK kinase inhibitor in ALK translocated cancers, Cancer Res 17: pp6,051-6,060, 2010
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  21. Heuckmann JM, Hölzel M, Sos ML et al, ALK mutations conferring differential resistance to structurally diverse ALK inhibitors, Clin Cancer Res 10(1): pp1,078-0432, 2011
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  23. Lovly CM, Heuckmann JM, de Stanchina E et al, Insights into ALKdriven cancers revealed through development of novel ALK tyrosine kinase inhibitors, Cancer Res 71(14): pp4,920-4,931, 2011
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  26. Miret JJ, Wang F, Anjum R, Zhang S et al, AP261 13, a potent ALK inhibitor, is also active against EGFR T790M in mouse models of NSCLC. In: Proceedings and related abstract of the International Association for the Study of Lung Cancer (IASLC) 14th World Conference on Lung Cancer, Amsterdam, The Netherlands, July 3-11, 2011. (MO11.12 Abstract)

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William C Shakespeare has served as ARIAD’s Vice President, Drug Discovery since April 2009. Previously, he served as Senior Director, Chemistry from January 2007 to March 2009, Research Director from September 2004 to December 2006 and Principal Scientist from August 2000 to August 2004. Prior to joining ARIAD in July 1996, William was a Senior Research Scientist at Astra AB where he focused on CNS research. William received his BSc in Chemistry from Gettysburg College and his PhD in Organic Chemistry from the University of New Hampshire. He received his postdoctoral training in organic chemistry at the University of Pennsylvania.
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