somatic alterations as the basis for resistance to targeted therapies

Journal of PathologyJ Pathol 2014; 232: 244–254Published online in Wiley Online Library(wileyonlinelibrary.com) DOI: 10.1002/path.4278

INVITED REVIEW

Somatic alterations as the basis for resistance to targetedtherapiesBrian G Blair,1 Alberto Bardelli2 and Ben Ho Park1*

1 Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD, USA2 Institute for Cancer Research and Treatment (IRCC), University of Torino Medical School, Candiolo, Torino, Italy

*Correspondence to: BH Park, Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, 1650 Orleans Street, Room 151, Baltimore, MD21287, USA. E-mail: [email protected]

AbstractRecent advances in genetics and genomics have revealed new genes and pathways that are somatically alteredin human malignancies. This wealth of knowledge has translated into molecularly defined targets for therapyover the past two decades, serving as key examples that translation of laboratory findings can have great impacton the ability to treat patients with cancer. However, given the genetic instability and heterogeneity that arecharacteristic of all human cancers, drug resistance to virtually all therapies has emerged, posing further andfuture challenges for clinical oncology. Here we review the history of targeted therapies, including examples ofgenetically defined cancer targets and their approved therapies. We also discuss resistance mechanisms that havebeen uncovered, with an emphasis on somatic genetic alterations that lead to these phenotypes.Copyright 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Keywords: targeted therapies; drug resistance; somatic alterations; cancer

Received 10 August 2013; Revised 23 August 2013; Accepted 21 September 2013

No conflicts of interest were declared.

First-generation targeted therapies

Multi-kinase inhibitorsThe first drug that revolutionized the concept ofsmall molecule kinase inhibitors was imatinib (STI571,Gleevec, Glivec). Imatinib was originally developedin the late 1990s as a targeted therapy against theBCR–ABL protein, present in virtually all chronicmyelogenous leukaemia (CML) patients (reviewed in[1]). CML develops from the reciprocal transloca-tion between chromosomes 9 and 22, leading to the‘Philadelphia chromosome’ (Ph+), which is a genetichallmark of this disease [2,3]. This translocation createsthe BCR–ABL fusion gene product, encoding a hyper-activated ABL tyrosine kinase. Thus, inhibition ofABL kinase activity would, in theory, specifically deterthe proliferation of the malignant Ph+ clonal cancercells, yet non-CML cells would be unaffected. High-throughput screening against protein kinase C (PKC)activity teased out imatinib as having high potential forpharmacological inhibition [1]. Prior to the use of ima-tinib, only 30% of CML patients survived 5 years afterdiagnosis. After FDA approval of imatinib in 2001, thispercentage has steadily risen to nearly 90% [4].

Imatinib occupies an area of the ATP-binding pocketof ABL, locking it in the inhibited or closed confirma-tion [5]. In this way, the normally constitutively activeBCR–ABL kinase is turned off and it can no longer

phosphorylate downstream targets. However, imatinibis also an inhibitor to other kinases, most notably c-Kitand PDGFR [4]. In hindsight, this promiscuity is in factadvantageous, as imatinib has proved to be an effectivetreatment for gastrointestinal stromal tumours (GISTs),which exhibit mutated c-KIT or activated PDGFR [6].

Although an undeniable success, at least 10%of patients will not demonstrate prolonged benefitfrom imatinib therapy [7]. This can be due to initialresistance or an acquired resistance after an initialresponse to the drug. Several mechanisms for innateand acquired resistance to imatinib have been pro-posed. Amplification or duplication of the BCR–ABLkinase, c-KIT or PDGFR may be one such modeof resistance, in which the increased tyrosine kinaseproduction outcompetes the action of the drug [7,8].Importantly, point mutations in the ABL gene havealso been linked to imatinib resistance, most frequentlya threonine-to-isoleucine substitution (T315I) in thekinase domain that prevents imatinib association withthe ATP-binding pocket [7–9]. Not only do thesemutations confer resistance to imatinib treatment butthey also exhibit increases in cancerous phenotypes[10]. Many other mutations of the ABL gene havebeen identified as contributing to resistant phenotypes,including mutations within the P-loop, SH2 domain,A-loop, C-helix and substrate-binding domain [7].Mutations in c-KIT and PGDFR are also associ-ated with imatinib resistance in GIST patients [8].

Copyright 2013 Pathological Society of Great Britain and Ireland. J Pathol 2014; 232: 244–254Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk www.thejournalofpathology.com

Somatic alterations as the basis for resistance to targeted therapies 245

Alternative mechanisms of imatinib resistance havealso been described and include changes in druginflux/efflux, epigenetic modification and activation ofalternative growth pathways independent of tyrosinekinase receptors [7,8].

Following the success of imatinib in treating CMLand GIST, the development of kinase inhibitors to treatcancers began in earnest. Sorafenib and sunitinib aretwo compounds that are multi-kinase inhibitors. Thesedrugs have shown to inhibit various growth signallingpathways mediated by receptor tyrosine kinases [11].Sorafenib and sunitinib interact with the ATP-bindingpocket within the kinase domain of their respective tar-gets, competitively deactivating the growth signallingof the various kinases [12,13]. Originally designed asa RAF kinase inhibitor, sorafenib also targets VEGFR,PDGFR and the RAF kinases CRAF and BRAF,and has been utilized as a successful treatment forrenal and liver cancers [12,14]. Sunitinib is approvedfor use in renal cell carcinoma and GIST, and hasbeen shown to target VEGFR, PDGFR, KIT, RET,FLT3 and other receptor tyrosine kinases [11,15–19].Sunitinib inhibits c-KIT and PDGF in a manner dis-tinctly different than imatinib, and therefore it is clini-cally used for the treatment of imatinib-resistant GIST[20]. The multi-kinase inhibitory properties also makethese drugs less selective for cancer cells, resultingin numerous side-effects for most patients. Resistance

can arise to either or both of the anti-proliferative andanti-angiogenic properties of these kinase inhibitors.Activation of alternative signalling pathways (AKT,PI3K, etc.), drug sequestration, up-regulation of non-VEGF-dependent angiogenesis factors (FGF, IL8, etc.)and pro-angiogenic pericyte and monocyte recruitmentare some of the documented mechanisms of resis-tance to PDGFR and VEGFR inhibitors, includingsorafenib and sunitinib [8,21,22]. In addition, hepato-cyte growth factor/MET signalling has also been pro-posed as an alternative pro-angiogenic pathway con-tributing to VEGF inhibitor resistance [23]. Althoughthese drugs are effective for certain indications, theirlack of specificity and toxicity profiles dictated furtherrefinements in targeted therapies for cancer.

mTOR inhibitorsThe mammalian target of rapamycin (mTOR) isa regulator of cell signalling found downstreamof several growth pathways that are mutated incancers, including the PI3K–AKT signalling cas-cade (Figure 1). mTOR was discovered as a tar-get for the anti-fungal rapamycin in yeast cells[24–26]. Rapamycin interacts with the protein FKBP12(Rbp1), forming a complex that then binds the FRB(FKB) domain of mTOR, thereby inhibiting mTORkinase activity [27,28]. mTOR associates with two

ActivatingKRAS mutations

EGFR RTK

p

p

p

IRS-

1

p85

KRAS

p110α

PIP2 PIP3 PIP2

PTEN

AKT

Cell cycleprogression

Increased cellsurvival

Proteinsynthesis andcell growth

Apoptosis

AlternativeAKT pathway

activation

*

*

*

*

RAF

MEK

ERK

EGFR Antibodies

BRAF V600Emutation

Alternative ERKpathway activation

Understanding Disease

Figure 1. The MAP kinase and PI3 kinase pathways, targeted therapies and somatic alterations leading to resistance. Epidermal growthfactor receptor (EGFR), a receptor tyrosine kinase (RTK), is a target for antibody therapies. The PI3 kinase and MAP kinase pathways thatare activated by aberrant EGFR signalling in cancers are shown. *Mechanisms of resistance at key nodal points.


246 BG Blair et al

proteins; RAPTOR and RICTOR, forming two com-plexes, mTORC1 and mTORC2, respectively [25,28].Rapamycin predominantly interacts with mTORC1-RAPTOR and inhibits the phosphorylation of the tar-gets S6 kinase 1 (S6K1, p70) and eukaryotic initiationfactor 4E binding protein (4EBP1) [29,30].

The pursuit of mTOR inhibitors as an anti-cancertherapy first gained traction with the discovery ofthe rapamycin analogues (rapalogues) temsirolimus,everolimus and ridaforolimus (deforolimus). Likerapamycin, the rapalogues target FKBP-12 rapamycin-binding domain (FRB, FKB) of the mTORC1 complex[29,30]. Unlike rapamycin, these rapalogues do nothave the same degree of immunosuppressive proper-ties. Temsirolimus and everolimus are prodrugs, suchthat their metabolites are the active rapamycin-likeforms. These compounds inhibit several downstreamtargets of mTOR, such as HIF1α, cyclin D and VEGF,thereby affecting angiogenesis, cell proliferation,cellular metabolism and survival [31]. Temsirolimus,the first rapalogue to be approved for clinical use,is primarily administered for the treatment of renalcancer. In the past few years, everolimus has beenapproved for the treatment of kidney, pancreatic andER positive, HER2-negative breast cancers.

Mutations in FKBP12 or the FKBP12-bindingdomain of mTORC1 can result in rapalogue resistance,due to a drop in binding affinity [32–34]. Additionally,up-regulation of PI3K, AKT, MAPK, PIM kinaseand PDK activity has been observed in response torapamycin and rapalogue treatment [35–37]. Thismechanism of resistance is common among targetedtherapies, that is, disruption of a signalling pathwayleads to activation of alternative pathways by negativefeedback loops. This mechanism of resistance isespecially vexing with rapalogues because mTORC2phosphorylates AKT at serine 473 in response to nega-tive feedback from mTORC1 inhibition [38]. Decreasesin 4E-BP1 protein levels can also confer resistance torapalogues, and increased expression of eIF4E can alsolead to resistance [39,40]. Increases in anti-apoptoticsignals, through up-regulation of Bcl-2 or the IAPs,has also been indicated to contribute to rapalogueresistance [41,42]. It has also been proposed that inhi-bition of HIF-1α by rapalogues can trigger decreases inangiogenesis and limit tumour growth, and alternativeangiogenesis signals may bypass this component ofthe anti-tumour effect of rapalogues [42,43].

Second-generation mTOR inhibitors (mTOR kinaseinhibitors) are being developed that competitivelyinhibit the serine/threonine kinase activity of mTOR[44]. The advantage of this mode of mTOR inhibi-tion is that it is independent of FKBP12 and tar-gets both mTORC1 and mTORC2. Another potentialmethod to overcome the common causes of rapalogueresistance is treatment with rapalogues together withcompounds that target alternative proliferative path-ways, such as the PI3 kinase and/or MAP kinase path-ways. Furthermore, single compounds that block both

PI3K and mTOR are being explored for improvedefficacy [44,45].

Current and future cancer-targeted therapies

CD20 and rituximabOne of the first and most successful antibodies incancer therapy is rituximab, a monoclonal antibody(mAb) against the B cell antigen CD20. CD20, initiallydescribed in 1980, is a member of the membrane-spanning 4-A (MS4A) family of proteins [46–49]. Theclinical significance of this protein was quickly realizedupon the observation that CD20 is highly expressed inB cell lymphomas, hairy cell leukaemias and B cellchronic lymphocytic leukaemias [47,48].

Rituximab is a chimeric mouse/human antibodyagainst CD20 [50]. It was approved to treat non-Hodgkin’s lymphoma and is now approved for useagainst other lymphomas and leukaemias, as wellas autoimmune diseases such as rheumatoid arthri-tis. Rituximab marks CD20-positive cells for destruc-tion by natural killer (NK) cells, neutrophils andmacrophages, thereby selectively targeting malignantB cells for antibody-dependent cellular cytotoxicity(ADCC) [51,52]. The CD20-bound rituximab recruitsthe innate effector cells responsible for ADCC throughan interaction of the Fc region of rituximab and theFcγ receptor (FcγR, CD16) on NK cells [53,54].

Rituximab, while highly effective in many patients,is subject to therapeutic resistance. It is estimated that30–60% of patients may show resistance to rituximab[55]. As there are several mechanisms of rituximabcytotoxicity, there are likewise many means by whichresistance may develop. For example, the destructionof rituximab-bound B cells by immune effector cells isdependent on two variables: the binding of rituximabto FcγR and the congregation of the bound CD20into lipid rafts [54–56]. Thus, decreases in the affinityof FcγR to rituximab or low numbers of NK cellswith FcγR can diminish the efficacy of the treatment[55,56]. Similarly, a lack of rituximab–CD20 polar-ization can inhibit ADCC [51]. Over-expression of theMAP kinase or PI3 kinase pathways, as well as NF-κBhyperphosphorylation, can all contribute to an attenu-ation of the pro-apoptotic nature of rituximab [56,57].While it would seem that the most likely mechanismfor resistance would be changes to CD20 expression oraffinity to rituximab, evidence for this is lacking [55].

BRCA and PARP inhibitorsCarriers of BRCA1 and BRCA2 mutations have upto an 80% lifetime risk of developing breast cancer[58]. These patients also have up to a 55% chance ofdeveloping ovarian cancer [58]. BRCA1 and BRCA2act as part of a complex of proteins, most notablyinteracting with the recombinase RAD1, to repairdouble-strand breaks (DSBs) through homologous



recombination (HR) and stabilization of stalled DNAsynthesis [59–62]. Cells defective in BRCA1 orBRCA2 demonstrate increased mutational rates due tothe breakdown of DNA repair, and thus are likely tobecome tumourigenic.

Poly(ADP-ribose) polymerases (PARPs), are afamily of proteins whose primary function is tolocate and repair single-strand DNA breaks (SSBs).PARPs, and in particular PARP1, bind to both single-and double-strand breaks and initiate DNA repair[63–65]. In cases where BRCA1 or BRCA2 functionis compromised, PARPs play an important role in thecellular response to this loss of function. In additionto repairing SSBs and restarting stalled forks, in theabsence of BRCA1/BRCA2 it is believed that PARPscan serve a further role in DSB repair [66,67]. There-fore, in patients with defective BRCA homologousrecombination-mediated DNA repair, PARPs can bekey proteins in regulating the amount of DNA damageaccumulated within cancer cells.

The interplay of BRCA1/2 and PARPs in DNArepair offers a unique target for clinical treatment.Cells lacking HR, due to defective BRCA1, BRCA2or other repair proteins such as PALB2, have provedto be excellent targets for PARP inhibition. In thesecells, loss of PARP activity will increase the numberof SSBs, eventually leading to DSBs when replicationforks become stalled [68]. The vast increase in dam-aged DNA in cells lacking both HR and PARP activityinitiates a synthetic lethal effect [66]. Additionally, itis thought that PARP inhibitors ‘trap’ PARP proteins atthe site of DNA damage and that this PARP ‘trapping’is also toxic to the cell [69]. Both modes of actionfor PARP inhibitors are selective only for cancerouscells that lack HR. Many PARP inhibitors have beendeveloped, including olaparib, veliparib, rucaparib andMK4827.

Mechanisms of PARP inhibitor resistance are justbeginning to be understood. It has been found thatisoforms of BRCA2 can arise in culture that restoresome HR function to cancer cells harbouring BRCA2mutations, allowing these cells to bypass the syn-thetic lethality of PARP inhibition [70,71]. Also, manyBRCA1-defective cancers have low or no PARP1expression, and resistant or refractory cancers oftenshow decreased PARP activity relative to cancersthat show high PARP inhibitor sensitivity [72]. PARPinhibitor resistance can also arise from differentialexpression of other redundant DNA repair pathways.For example, it has been shown that some BRCA1-deficient breast cancers lose 53BP1 expression, achange that can lead to restoration of HR repair andPARP inhibitor resistance [73,74].

HER2HER2 (ErbB2) expression is amplified and the pro-tein over-expressed in approximately 15–20% of breastcancers [75]. HER2 is a transmembrane receptor tyro-sine kinase belonging to the ErbB/HER family, which

includes EGFR (HER1/ErbB1) and HER 2, 3 and 4.While each member of the family shares common tyro-sine kinase domain motifs, they each contain uniqueligand-binding domains [76]. HER2 is activated byautophosphorylation upon homo- and heterodimeriza-tion, and can dimerize with other members of the ErbBfamily. Over-expression of HER2 alone is often ade-quate to trigger receptor phosphorylation and activa-tion, even in the absence of ligand. HER2 activatesseveral proliferative pathways upon phosphorylation,including the MAP kinase and PI3 kinase pathways.

The over-expression of HER2 in breast cancers(and now other cancers, such as stomach cancers)has made it a strong candidate for targeted therapies.In 1998, the FDA approved trastuzumab (Herceptin)as the first targeted treatment against HER2-positivebreast cancers. Trastuzumab is a monoclonal antibodyspecific to the juxtamembrane domain of HER2 andresults in a down-regulation of the protein [77–79].Trastuzumab also has other proposed anti-proliferativeeffects, such as inhibiting transcription and decreasingthe levels of a constitutively active truncated formof HER2 (p95-HER2) [78,80]. Also, similar to themethod of action for rituximab, trastuzumab can triggerantibody-dependent cellular toxicity (ADCC) throughan interaction between the antibody and Fc receptorson immune cells [54].

The successes of trastuzumab are balanced byoccurrences of innate or acquired resistance. Thesemechanisms of resistance include HER2-independentactivation of the PI3 kinase pathway, up-regulation ofthe other HER family member receptors and increasedproduction of p95-HER2 [81,82]. One of the primarysignalling pathways activated by ErbB receptors is thePI3 kinase pathway. PI3K is composed of two subunits,the p85 regulatory subunit and the p110 catalytic com-ponent. These subunits interact with activated HER2,thus beginning a signalling cascade, which ultimatelyresults in cancerous phenotypes. In many cancers, PI3Kor downstream targets, such as AKT and MAPK, canbecome constitutively activated independent of the sig-nalling dependence from HER2 activation [83–85].For example, mutations that lead to loss of PTENfunction, or activation of the PIK3CA gene (PIK3CAencodes for p110α) are frequent in breast cancer [83].In these cases, targeting HER2 over-expression withtrastuzumab potentially lessens its anti-proliferativeeffects, as these cancers can grow without HER2 acti-vation and have been associated with resistance inpreclinical models [86,87].

Redundancy in the downstream signalling pathwaysof receptor tyrosine kinases indicates that loss of sig-nal from one of these receptors may not be enough toimpede MAPK and PI3K activation [88]. As example,the HER2–HER3 heterodimer has been shown to bean essential interaction for HER2-dependent tumouri-genesis [89]. HER2-independent activation of HER3or EGFR may lead to HER2-positive cancers thatlack sensitivity to trastuzumab [89–91]. In addition toredundancy within the members of the ErbB family of


248 BG Blair et al

membrane receptors, IGF-1R has been demonstratedto activate PI3K through crosstalk with HER2 [92]. Ithas been proposed that IGF-1R stimulation in certaincancer cells may be a source of trastuzumab resis-tance, and inhibition of IGF-1R can restore trastuzumabsensitivity [93].

The truncated p95-HER2 protein lacks the amino-terminus of HER2 and therefore lacks the binding sitefor trastuzumab. Moreover, p95-HER2 is constitutivelyactive, and downstream targets become phosphorylatedindependent of ligand interaction. While trastuzumabtreatment has been shown to decrease p95-HER2expression, 30% of HER2-positive tumours over-express p95-HER2 [78,94]. Thus it has been proposedthat cancers with increased levels of p95-HER2 arerelatively resistant to trastuzumab’s anti-neoplasticeffects.

Additional treatments against HER2 have also beendeveloped. Pertuzumab is another monoclonal antibodydirected against HER2, but interacts with the dimer-ization domain of HER2, blocking the homo- and het-erodimerization of HER2 [82]. This can inhibit not onlyHER2-stimulated growth but also the dimerization-dependent activation of the other members of theErbB family, specifically HER3 [95–97]. Also, unliketrastuzumab, pertuzumab may be able to inhibit p95-HER2-mediated growth [98]. Clinical data indicate thattrastuzumab and pertuzumab are complementary andcan be highly effective when used in combination [99].

There have also been studies to increase the effec-tiveness of trastuzumab, leading to the developmentof TDM-1. TDM-1 is a conjugate of trastuzumabwith DM1 (derivative of maytansine 1), a micro-tubule inhibitor. This antibody–drug conjugate (ADC)is designed to combine the anti-proliferative effectsof trastuzumab with targeted delivery of the cytotoxiccompound DM-1 directly to HER2 over-expressingcells. TDM-1 shows promise in attacking cells that maybe resistant to trastuzumab, and demonstrates reten-tion of trastuzumab’s other anti-neoplastic functions,such as PI3K signal inhibition, decreases in p95-HER2-mediated signalling and ADCC [100,101].

In addition to these antibody-based approaches fortargeting HER2, small molecules that inhibit the kinaseactivity of HER2 have been developed, most notablylapatinib. Lapatanib is a small molecule inhibitor ofEGFR and HER2, which competitively binds to theATP-binding domain, thus blocking autophosphoryla-tion. Due to the specificity for the ATP-binding pocket,lapatinib may have distinct mechanisms of resistancecompared to trastuzumab. Nonetheless, lapatinib resis-tance can still occur via several mechanisms. Acti-vating mutations of the PI3 kinase pathway may alsocontribute to lapatinib resistance, as can loss of PTENfunction [102]. A mutation in the ATP-binding pocketof HER2 (L755S) has also been reported to confer lapa-tinib resistance in vitro [103]. It has also been proposedthat activation of IGF-1R, ER or other receptor tyrosinekinases (such as AXL), may lead to tumours resistantto lapatinib treatment [104,105].

EGFR inhibitorsEGFR (HER1, ErbB1) is over-expressed and/ormutated in many cancers, including lung and coloncancers. EGFR is a receptor tyrosine kinase activatedby binding with epidermal growth factor (EGF), trans-forming growth factor-α (TGFα) and other ligands[88]. Activation of EGFR initiates several cell prolifer-ating signal transduction cascades, including the MAPkinase, PI3 kinase, JNK and STAT pathways [88].To date, several compounds have been developed fortreatment against EGFR-activated cancers, includingsmall molecule inhibitors such as gefitinib and erlotiniband antibody-based therapies such as cetuximab.

Gefitinib (Iressa) and erlotinib (Tarceva) are smallmolecule inhibitors of EGFR which were approvedfor the treatment of EGFR-positive lung cancer inthe past decade. Both compounds reversibly inhibitEGFR kinase activity by competitively binding theATP-binding site [106,107]. It was soon discoveredthat not only do these drugs block EGFR activation,but that the best predictors of response were specificEGFR-activating mutations [108]. These sensitizingmutations were found in the tyrosine kinase domain,either in deletions within exon 19 between codons 746and 759, or in exon 21 at L858R [108,109]. However,while these mutations can increase the sensitivityof tumour cells to gefitinib or erlotinib, subsequentanalyses have identified mutations that can conferdrug resistance. The most clinically relevant resistancemutation in EGFR is in exon 20 at T790M [110,111].This mutation is thought to block the binding ofinhibitors to the ATP-binding site, thus leading to theconstitutive activation of EGFR [112]. This mechanismof resistance may be overcome with the introductionof irreversible EGFR inhibitors [113]. Interestingly,another EGFR mutation, within exon 19 at D761Y,has been linked to gefitinib and erlotinib resistance intumours that contain the L858R mutation [114,115].As in the case with many kinase inhibitors, resistancecan also occur through activation of downstream path-ways, such as PI3K [116]. Likewise, activation of otherreceptor tyrosine kinases, such as MET, IGFR-1, PDGFand additional ErbB family members, has been linkedto acquired gefitinib and erlotinib resistance [97,116].

Cetuximab (Erbitux) and panitumumab (Vectibix)are monoclonal antibodies against EGFR that areclinically used for the treatment of colorectal andother cancers. These antibodies bind to the extra-cellular domain of EGFR and are thought to changethe conformation of the dimerization domain, therebysimultaneously blocking ligand binding and preventingreceptor dimerization [117]. The resultant inhibition ofEGFR phosphorylation combined with several othermechanisms of action contribute towards cetuximab’sand panitumumab’s efficacy against EGFR-positivetumours [118].

One well classified determinant of EGFR antibodytherapeutic sensitivity is the mutational status ofKRAS . Constitutive activation of the MAP kinase



signalling pathway downstream of EGFR bypassesthe effectiveness of cetuximab and panitumumab.In fact, patients with KRAS activating mutationshave a poorer predicted outcome than those withwild-type KRAS when treated with EGFR-directedtherapies [119–121]. As a result, KRAS mutationalstatus is screened for prior to the administrationof these therapeutic antibodies. More recently, twoindependent groups have demonstrated the appearanceof KRAS mutations within sites of progressive diseasein metastatic colon cancer patients treated withanti-EGFR antibody therapies [122,123]. Mutations inBRAF , a serine–threonine kinase in the MAP kinasepathway immediately downstream of KRAS , have alsobeen linked to EGFR antibody resistance. Specifically,the V600E activating mutation is strongly associatedto poor response to EGFR antibody therapy [124].Activating mutations of other downstream effectors ofEGFR have also been linked to antibody resistance.For example, constitutive activation of AKT or PI3K,or loss of PTEN, can diminish responses to EGFRantibody therapies [118,125]. Finally, MET amplifi-cation has also been recently shown to be associatedwith acquired resistance to EGFR antibody therapiesin colon cancers that are wild-type for KRAS [126].

BRAFThe cytoplasmic serine/threonine protein kinase BRAFis a critical member of the MAP kinase signal trans-duction pathway. Oncogenic mutations in BRAF havebeen thoroughly described, mutations reported in sev-eral cancer types including melanoma, colorectal, ovar-ian and papillary thyroid carcinomas [13]. Nearly 66%of melanomas harbour BRAF mutations and, of those,80% of the mutations are a single base pair substi-tution in the kinase domain, yielding V600E [127].Currently, there are FDA-approved therapies that targetthe V600E and other V600 mutations in clinical use:vemurafenib and dabrafenib. Vemurafenib, approvedfor clinical use in 2011, is a kinase inhibitor thatselectively targets cells harbouring V600E mutations,but has little effect on wild-type BRAF cells [128].When interacting with mutated BRAF, vemurafeniblocks onto the ATP-binding site and will down-regulateERK signalling, inducing cell cycle arrest and activat-ing apoptosis [128,129]. Treatment with vemurafenibcan be highly effective and many patients demonstratesignificant (up to 80%) clinical response [130]. Morerecently, dabrafenib in combination with the MEKinhibitor trametinib has also been shown as effectivetherapy for metastatic melanomas that harbour BRAFV600 mutations [131].

Up-regulation of PDGFRβ has been recognized as amode of acquired resistance in a subset of melanomas[132]. Alternatively, the up-regulation or mutation ofoncogenic NRAS can also contribute to loss of vemu-rafenib sensitivity [132], further substantiated by arecent clinical report demonstrating mutations in NRASand MEK being associated with vemurafenib-resistant

melanomas [133]. The tumour micro-environment canalso play a role in BRAF inhibitor resistance. Secre-tion of hepatocyte growth factor can stimulate MET,leading to activation of the PI3 kinase and MAP kinasepathways independent of RAF inhibition [134]. In addi-tion, early attempts using vemurafenib in BRAF V600Emutant colon cancers did not demonstrate any apprecia-ble response. Although mutant BRAF has been shownto predict for resistance against EGFR-mediated thera-pies, as mentioned above, recent work suggests an evenmore complex interplay. It appears that mutant BRAFsuppression leads to feedback activation of EGFR incolon cancer cells resulting in vemurafenib resistance,suggesting the need to simultaneously target both pro-teins [135]. Interestingly, it has been shown that vemu-rafenib treatment can paradoxically stimulate MAPkinase signalling and may promote tumourigenesis incells with wild-type RAF [129].

EML4–ALKNearly 5% of non-small cell lung cancer casesare estimated to harbour a somatic translocationbetween echinoderm microtubule-associated protein-like 4 (EML4) and anaplastic lymphoma kinase (ALK)[136]. The resultant EML4–ALK protein and othersproduced from ALK translocations are hyperactive ver-sions of the ALK receptor tyrosine kinase [137,138].Inspired by the success of BCR–ABL targeting withimatinib, a selective inhibitor of ALK was quicklydeveloped. Crizotinib is a competitive inhibitor of thekinase activity that interacts with the ATP-bindingdomain of ALK, ROS1 and MET [139]. Crizotinibinhibits cell proliferation and angiogenesis, initiatescell arrest and can induce apoptosis [140]. With aresponse rate of ∼60%, acquired and innate resistanceto crizotinib has also been documented [138].

Several mutations in ALK have been linked tocrizotinib resistance. The most well-documented ofthese is L1196M, which impedes inhibitor bindingto the active site [141]. Other resistance mutationshave been identified in the kinase domain, as well asmutations that increase ATP affinity, thus decreasingthe effectiveness of competitive inhibition [138].Copy number increases in ALK can also decrease theefficacy of competitive inhibition by crizotinib [142].Alternatively, activation of other proliferative mech-anisms can decrease tumour sensitivity to crizotinib.Specifically, up-regulation of EGFR, HER2 or HER3,constitutive activating mutations in EGFR (L858R andS7681), as well as KRAS G12C and G12V mutations,have been reported to be associated with a decrease inALK inhibitor sensitivity [138,143].

Other targeted therapies and conclusions

The past success and future potential of targeted ther-apies for cancer are evident, and many new drugsare currently in development. An obvious candidate


250 BG Blair et al

Table 1. Summary of targeted therapies in cancer andmechanisms of resistance

Drug Target Resistance mechanism

Imatinib BCR–ABL BCR–ABL, c-KIT, PDGFRamplification/mutations

c-KIT Abl T315I mutationPDGFR

Sorafenib VEGFR Activation of alternative growthpathways

PDGFR SequestrationC-Raf/B-Raf

(V600E)Angiogenesis up-regulation

Sunitinib VEGFR Activation of alternative growthpathways

PDGFR SequestrationKIT Angiogenesis up-regulationRETFLT3

Rapamycin/rapalogues

mTOR Activation of alternative growthpathways

Mutations in FKBP12Changes in 4E-BP1 or eIF4EUp-regulation of Bcl-2 or IAPsAngiogenesis up-regulation

Rituximab CD20 Changes in mCRP expressionDecrease of C1qDecrease in FcγR affinity

PARPinhibitors

PARP PARP1 mutationsExpression of BCRA2 isoformsLoss of 53BP1

Trastuzumab HER2 Activation of PI3K pathwayHER family up-regulationIncreases in p95-HER2

Lapatanib EGFR Activation of PI3K pathwayHER2 Activation of mTORHER3 HER2 L755S mutation

Activation of IGF-1, ERGefitinib EGFR EGFR mutations: T790M, D761YErlotinib Activation of MET, IGFR-1, PDGF or

ErbB RTKsCetuximab EGFR KRAS mutationsPanitumumab BRAF V600E muation

Activation of alternative growthpathways

Angiogenesis up-regulationVemurafenib B-Raf

(V600E)PDGFR or N-RAS up-regulationHepatocyte growth factor secretionEGFR activation

Crizotinib EML4–ALK ALK mutations (L11196M)ALK copy number increasesEGFR, HER2, HER3 up-regulationEGFR mutations (L858R, S768I)KRAS mutation (G12C, G12V)

for targeted therapy is PI3K, or specifically the p110α

component encoded by the PIK3CA gene. PIK3CA isthe second most commonly mutated gene in the cancergenome, with a relatively high frequency found in lung,breast, brain, gastric, colon and other cancers [83,144].As such, there is currently much interest in devel-oping PI3K inhibitors for cancer therapy [145–147].AKT (protein kinase B) is a serine/threonine kinase inthe PI3K–AKT–mTOR pathway that is often mutatedand/or activated in human cancers [148]. Similar toPIK3CA, AKT is a heavily pursued target for targeted

therapies with numerous inhibitors at various stages ofclinical development. MEK is another kinase down-stream of RAS-RAF signalling within the MAP kinasepathway. Targeting of this kinase has resulted inthe development of several small molecule inhibitors,including trametinib, as mentioned above, and selume-tinib (AZD6244).

Over the past two decades, targeted therapies forcancer have made a significant impact on the morbidityand mortality inflicted by this group of diseases.These historic and recent examples demonstrate thatthe future of cancer medicine lies in the pursuit ofbetter patient and tumour-specific therapies, and thatgenetic knowledge of cancer genomes can help guidethese efforts. Despite the past successes, drug resistancecontinues to be a common problem among targetedtherapies, limiting their use (Table 1). Future researchwill therefore emphasize the use of multiple therapiesthat block cancer cell signalling across many pathways,as well as nodal points within each of these pathways.Clearly, great strides have been made in the assessmentand treatment of cancer, yet much work remainsin finding meaningful long-term therapies for thisdisease.

Acknowledgements

We apologize to our colleagues whose important workcould not be cited due to space constraints. BGBwas supported by a DOD BCRP Postdoctoral Fellow-ship (Award No. BC100972). BHP receives supportfrom the Flight Attendant Medical Research Insti-tute (FAMRI), the Breast Cancer Research Foun-dation, the Avon Foundation, the CommonwealthFoundation, JHU Singapore/Santa Fe Foundation, theSafeway Foundation and the Eddie and Sandy GarciaFoundation.

Author contributions

BGB, AB and BHP wrote and revised the manuscript.

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somatic alterations as the basis for resistance to targeted therapies

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