Circumventing Cancer Drug Resistance in the Era of Personalized Medicine

Secondary Genetic Alterations in the Target (Onco)Protein

One of the most common drug resistance mechanisms involves additional genetic alterations within the target oncogene itself. This mechanism was first described in patients with CML treated with imatinib; however, so-called secondary somatic alterations have subsequently been detected in a wide variety of tumors from patients treated with different kinase inhibitors (26–30). Several lines of evidence have shown that these mutations exist at low levels before drug treatment and undergo positive selection during exposure to a clinically effective targeted agent (24, 31).

Secondary mutations can impede the effects of a kinase inhibitor by altering contact points for drug binding or by perturbing the conformational state of the kinase (32). Among contact point mutations, the most therapeutically challenging is the so-called gatekeeper mutation (32). The gatekeeper is a conserved amino acid residue situated within the catalytic cleft of tyrosine kinases that determines the accessibility of a hydrophobic pocket critical to binding of many small-molecule tyrosine kinase inhibitors [TKI (33)]. Gatekeeper mutations have been detected from patients with a variety of TKI-resistant cancers after treatment with selective inhibitors. Examples include imatinib-, nilotinib-, and dasatinib-resistant CML (T315I); gefitinib- and erlotinib-resistant epidermal growth factor receptor (EGFR)–mutant non–small cell lung cancer [NSCLC (T790M)]; imatinib-resistant GIST (KIT^T670I); and crizotinib-resistant, ALK-rearranged NSCLC [ALK^L1196M (26–28, 30, 34)]. Despite this evolutionary convergence in target-oriented resistance to TKIs, the specific mechanism by which the gatekeeper mutation leads to drug resistance may vary. For example, the ABL^T315I gatekeeper mutation produces steric hindrance that disrupts imatinib binding (35), whereas the analogous EGFR^T790M mutation increases the affinity of EGFR for adenosine-5′-triphosphate (ATP), thus rendering the EGFR kinase inhibitors gefitinib and erlotinib less effective in displacing this molecule (36).

Beyond the gatekeeper mechanism, many other secondary mutations have been reported that alter the conformation state of kinase drug targets. In general, these mutations either promote adoption of an active conformation by the kinase or alter the flexibility of the P-loop that prevents conformational changes required for drug binding. This mechanism has been most extensively described and studied for imatinib, which binds the inactive conformation of ABL (32). Mutations that alter the conformational state of ABL are predicted to disfavor imatinib binding and, consequently, its efficacy. Secondary mutations in KIT that occur in the kinase activation loop (which likely promote the active conformation) are associated with resistance to the KIT inhibitors imatinib and sunitinib in vitro and a worse clinical outcome in patients treated with sunitinib (37–39). In contrast, mutations in the drug/ATP binding pocket of KIT, although common in patients with gastrointestinal stromal cell tumors (GIST) who develop imatinib resistance, are less often detected from in vitro studies or from patients with GISTs that develop sunitinib resistance (37–39). These differences may be related to the differential potencies of KIT inhibition between imatinib and sunitinib. More recently, 3 mutations in ALK (F1174L, C1156Y, and L1152R) were detected in patients with ALK-rearranged cancers that developed clinical resistance to the ALK inhibitor crizotinib (29, 30). All of these mutations are located outside the drug-binding region. Biochemical studies of the F1174L mutation reveal that it results in an increase in ATP affinity analogous to the EGFR^T790M drug resistance ­mutation (40).

Most small-molecule kinase inhibitors in clinical use fall into 1 of 2 categories: type I inhibitors (which occupy the ATP-binding pocket when the kinase assumes its active conformation) or type IIa inhibitors (which bind both the ATP-binding site and adjacent motifs that are revealed when the kinase resides in an inactive conformation). The EGFR inhibitor erlotinib represents a well-known type I kinase inhibitor, whereas the ABL/KIT/platelet-derived growth factor receptor (PDGFR) inhibitor imatinib is a classic type IIa inhibitor. On the other hand, inhibitors of the MEK serine–threonine kinase exemplify “type IIb” inhibitors: small molecules that occupy an allosteric pocket adjacent to the ATP-binding motif and are thus non-ATP competitive agents (33). In vitro mutagenesis studies have indicated that secondary MEK resistance mutations may involve hydrophobic-to-hydrophilic amino acid substitutions along the C-helix or at various other key positions within the allosteric pocket (41). In addition, a clinically observed MEK1 mutation (MEK1 P124L) may simultaneously modulate the negative regulatory affects of the recently described A-helix based on its predicted proximity to this N-terminal motif in 3-dimensional models (41).

Increased gene dosage has long been recognized as a potential means to engender resistance to enzymatic inhibitors, dating back to early “dominant genetics” studies in yeast and parasites (42, 43). Toward this end, amplification of the drug target has been identified as an additional somatic resistance mechanism in relapsing tumor cells. BCR-ABL amplification has been detected both in vitro and in imatinib-resistant CML specimens (26). In addition, amplification of the EGFR^T790M allele was detected in an in vitro model of resistance to the irreversible EGFR inhibitor PF299804 [dacomitinib (44)], and amplification of BRAF has been observed in an in vitro study of resistance to MEK inhibition (45, 46).

A novel drug resistance mechanism resulting from alternatively spliced form of an oncoprotein has been recently described. A 61-kDA variant of BRAF^V600E (p61BRAF^V600E), resulting from a splicing isoform and lacking the RAS binding domain, was detected in both vemurafenib in vitro-resistant cells and from resistant patient tumor biopsies (47). The p61BRAF^V600E produces enhanced dimerization with other RAF family members and resistance to vemurafenib but not MEK inhibitors (47).

Activation of Bypass Mechanisms

An increasingly recognized drug resistance mechanism occurs through engagement of a so-called bypass signaling module. This results in the activation of a critical downstream signaling effector—normally activated by the kinase and extinguished by a kinase inhibitor—through a parallel mechanism that is indifferent to the kinase-directed therapy. An illustrative example of bypass-mediated resistance has been described in EGFR-mutant NSCLC. Here, reactivation of PI3K/AKT signaling (a critical pathway augmented as a result of oncogenic RTK activation) results in drug resistance (48). Aberrant activation of PI3K/AKT signaling in the presence of the EGFR kinase inhibitor gefitinib can occur as a result of activation of MET (through either MET amplification or by its ligand hepatocyte growth factor [HGF]) or insulin growth factor-1 receptor (IGF-IR) signaling (49–53). In each of these examples, gefitinib is no longer able to turn off PI3K/AKT signaling as a means to inhibit tumor growth. Analogous bypass mechanisms may confer resistance to vemurafenib in melanomas harboring the BRAF^V600E mutation. In vitro and in vivo studies have provided evidence that upregulation of the COT kinase promotes sustained ERK activation by circumventing BRAF inhibition (54). Receptor tyrosine kinase dysregulation may constitute an additional RAF inhibitor bypass mechanism. Toward this end, PDGFRA and IGF-IR overexpression have been associated with resistance to vemurafenib (or related compounds) in vitro and in vivo (55, 56). Vemurafenib-resistant BRAF^V600E melanoma cells with IGF-IR overexpression exhibited sustained ERK phosphorylation that was dependent on both A-RAF and C-RAF overexpression, suggesting a target ortholog-dependent bypass mechanism (55, 56).

Another avenue though which bypass resistance mechanisms may operate involves modulation of feedback loops. Cell signaling cascades are commonly regulated by feedback inhibition of various network components. In particular, pharmacologic inhibition of an individual node within an oncogenic signaling pathway may result in relief of feedback inhibition at multiple upstream nodes. Several recent studies have documented this phenomenon and raise the possibility that such feedback modulation may contribute to de novo or acquired resistance. Examples include HER3 pathway upregulation in the setting of AKT inhibition (57), PI3K/AKT activation by TOR inhibitors (58), and augmentation of AKT signaling by MEK inhibitors (59).

Genomic Alterations Affecting Upstream or Downstream Effectors

A third category of acquired drug resistance involves genomic alterations that dysregulate signaling proteins acting either upstream or downstream of the target (onco)protein. To date, this type of drug resistance mechanism has been most extensively described for MEK and RAF inhibitors. In particular, activating mutations in MEK1 or NRAS have been observed in tumors that progressed in the setting of vemurafenib treatment and were also shown to be sufficient to confer resistance to RAF inhibition in vitro (55, 60). Both mutations also lead to constitutive ERK signaling, even in the presence of RAF inhibition. In the case of MEK1 mutations (which exemplify downstream effector dysregulation), MEK signaling becomes uncoupled from the inhibited BRAF oncoprotein (60). In contrast, oncogenic NRAS (illustrative of upstream effector dysregulation) activates ERK signaling through CRAF, thus bypassing BRAF inhibition in an ortholog-dependent manner (55).

In a recent study of erlotinib resistance in lung cancer, dysregulation of NF-κB signaling was implicated through a short hairpin RNA (shRNA) suppressor screen and supported indirectly by clinical data showing that such dysregulation was associated with decreased patient survival (61). NF-κB signaling has been postulated as a gating mechanism downstream of both MAP kinase and PI3 kinase signaling in EGFR-mutant lung cancer. Amplification of BRAF has been described as an alternative “upstream” mechanism of resistance to the MEK inhibitor AZD6244 (45, 46). This mechanism leads to increased MEK phosphorylation and consequently ERK activation, which no longer can be inhibited by AZD6244 (45, 46). In addition, an oncogenic PIK3CA mutation was sufficient to engender gefitinib resistance in EGFR-mutant cancer cells and has also been observed in an erlotinib-resistant EGFR-mutant tumor, thus suggesting that downstream effectors may ultimately become relevant in this context as well (62, 63).

Pathway-Independent Resistance Mechanisms

As noted, the most common and best understood mechanisms of resistance to kinase oncoprotein inhibitors involve sustained activation of the salient downstream signaling pathway. However, several studies have indicated that resistance may also arise even in the setting of sustained downstream pathway inhibition. For example, several in vitro studies, and evaluation of tumor specimens, of resistance to EGFR inhibitors in lung cancer showed that the epithelial–mesenchymal transition (EMT) may underpin an EGFR pathway-independent resistance process (63–65). The basis for EMT induction in this setting remains incompletely characterized; however, several recent reports have raised the possibility of a connection between EMT and the acquisition of stem/progenitor cell characteristics within heterogeneous tumor subpopulations [for a review, see Singh and Settleman (66)]. Given that drug resistance is often considered a hallmark feature of such stem-like cancer cell populations, a functional link between EMT and cancer stem cell biology presents an intriguing model worthy of additional investigation.

Studies of EGFR-mutant lung cancer cells that persist in the setting of erlotinib exposure have implicated a distinct chromatin state mediated by the histone demethylase KDM5A that results in resistance in a pathway-independent manner (67). On the clinical side, anecdotal reports of BRAF-mutant melanomas exhibiting resistance to RAF inhibition despite sustained MAP kinase inhibition have recently been reported, although the mechanisms of resistance in this setting remain to be determined. One possibility for these observations includes cancer-cell nonautonomous mechanisms of drug resistance mediated by the tumor microenvironment. Recent studies demonstrate that the tumor microenvironment can influence the efficacy of polo-like kinase and Janus-activated kinase 2 inhibitors (68, 69). These can be mediated by several different stromal secreted cytokines (69). HGF can be produced by tumor-derived stromal fibroblasts and cause resistance to EGFR kinase inhibitors (70). Altered tumor angiogenesis has also been demonstrated to lead to resistance to EGFR kinase inhibitors and to the anti-EGFR therapeutic antibody in preclinical models (71, 72). Continued studies elucidating the molecular basis for “pathway-independent” resistance to kinase inhibitors will have important implications for future therapeutic combinations in several genetically defined tumor subtypes. Furthermore, there is no reason to believe that “pathway-dependent” and “independent” mechanisms of drug resistance could not develop simultaneously. This underscores one of the challenges of developing effective clinical therapies to combat drug-resistant cancers.

Studies of Isogenic Tumor Cells Generated In Vitro

One of the most common experimental approaches to study cancer drug resistance involves culturing of drug-sensitive cancer cell lines in the presence of the query drug until subpopulations emerge that proliferate avidly at high drug concentrations. This has been a very successful approach because many cancer cell lines that harbor a genomic alteration (mutation, amplification of genomic rearrangement) in the drug target are representative model systems and undergo apoptosis, an in vitro equivalent of tumor shrinkage observed in patients with cancer, after treatment with a kinase inhibitor. These inogenic, drug-resistant subpopulations are then compared with the drug-sensitive parental line to identify genetic, molecular, or biochemical differences that might account for the resistance phenotype. Such analyses usually begin with an interrogation of the cellular target and pathway inhibited by the drug and include sequencing of known drug targets, biochemical studies of target protein function (e.g., measurement of substrate phosphorylation if the target is a protein kinase), and determination of whether the resistance phenotype is reversible on drug discontinuation. It is also typical to extend these studies using unbiased omic approaches such as gene expression profiling (using microarrays or transcriptome sequencing), genomic studies (e.g., array comparative genomic hybridization or whole exome sequencing), or systematic protein-based studies (with phospho-antibody or reverse phase protein arrays).

When such studies are focused in scope, their interpretation often becomes straightforward. For example, characterization of EGFR, MEK1/2, and ALK genes in cell lines rendered resistant by stepwise selection to EGFR, MEK, or ALK inhibitors identified mutations in each gene that confer resistance through reduced drug binding (41, 73, 74). Similarly, interpretation of unbiased studies becomes clear when a clear target or pathway based alteration is revealed. For example, analysis of high-density single-nucleotide polymorphism arrays generated using isogenic EGFR-mutant NSCLC lines selected for resistance to gefitinib revealed focal MET amplification in several cases (49), thus pointing to an obvious bypass effector hypothesis. This resistance mechanism was first identified in drug-resistant cell lines and then validated in tumors from patients that had developed gefitinib or erlotinib resistance (49). However, characterization of resistant isogenic lines may become more complicated when no single dominant candidate effector mechanism is uncovered. In such cases, it is tempting to use enrichment-based analytical approaches to nominate candidate pathways or networks associated with resistance. Although often helpful, these approaches typically identify multiple candidate gene sets whose strength of statistical association can be influenced by the size of the gene set or confounded by adaptive or feedback-related changes that may not drive resistance per se. Choosing the best hypothesis for experimental follow-up is often a subjective process that may be further biased by prior knowledge (e.g., gravitation toward recognizable candidates) in a manner that hampers novel discovery.

In the absence of a clear resistance hypothesis (e.g., a new genetic alteration or “outlier” differential gene expression), follow-up studies in isogenic cell lines must rely on rigorous interrogation of the necessity and sufficiency of candidate effectors. When candidate effectors are ectopically expressed (or silenced) in the drug-sensitive parental lines, the extent to which the resulting pharmacologic IC₅₀ shift (if any) recapitulates the IC₅₀ of the resistant isogenic strains must be carefully considered. Conversely, the magnitude of IC₅₀ reversal should be stringently assessed when putative gain-of-function effectors are silenced genetically (e.g., by RNA interference) or pharmacologically (e.g., using small-molecule inhibitors if available) in drug-resistant cell populations. In some isogenic studies, no single effector mechanism may be sufficient to fully recapitulate the magnitude of resistance present in the drug-resistant “daughter” populations; thus, multiple cellular perturbations may be necessary to resensitize resistant cells. Detailed attention to these issues should avoid the risk of over-interpreting effects that are significantly but not causally associated with the acquisition of drug resistance in vitro.

Target-Based Mutagenesis Approaches

The gain of secondary mutations within a target protein comprises a common mechanism of resistance to targeted agents, as described previously. Thus, several groups have used random mutagenesis to define a spectrum of mutations within the drug target that may confer resistance. Mutagenesis libraries (using an expression vector harboring the target cDNA) are generated using error-prone polymerase chain reaction techniques or by culturing the expression construct in “mutator” strains of Escherichia coli (75). These libraries are packaged into viral delivery systems and introduced into drug-sensitive cancer cell lines. The resulting populations are cultured in the presence of the targeted agent at concentrations that inhibit the parental cell line. Individual drug-resistant clones emerge as isolated colonies; after recovery of these clones, the target cDNA is characterized for mutations that may confer resistance. The random mutagenesis screening approach was pioneered through studies of imatinib resistance in CML (75). It has since been used to study target-based resistance to several other agents, including MEK and FLT3 inhibitors (41). A complementary approach exposes sensitive cell lines to mutagens such as N-ethyl N-nitrosourea (ENU) followed by drug selection and targeted sequencing. ENU mutagenesis screens have been used to identify resistance mutations to several kinase inhibitors, including imatinib, sunitinib, and nilotinib (38, 76). The advantages of these approaches are a relatively unbiased view of mutations within the target protein that may confer drug resistance. Such studies may anticipate future clinical findings, validate the cellular targets of small molecules, and facilitate the development of next-generation inhibitors whose effects are not blunted by the same mutations. The major disadvantage is the limited scope—usually restricted to the cDNA encoding the primary drug target.

Historically, individual resistant clones arising after mutagenesis screens required expansion and cDNA sequencing in parallel, which was tedious and labor-intensive. However, the advent of massively parallel sequencing has circumvented this limitation by enabling pooling and expansion of hundreds or thousands of clones followed by deep sequencing of the target cDNA (41). Indeed, massively parallel sequencing technology could eventually obviate the need for plasmid-based mutagenesis libraries altogether.

Systematic Gain- and Loss-of-Function Resistance Screens

The emergence of reagents that enable near genome-scale functional screens in mammalian systems has opened up powerful new genetic avenues. Application of systematic RNAi knockdown or open reading frame (ORF) expression-based studies to the question of cancer drug resistance has considerable appeal because these approaches are categorical in scope, unbiased, and functional as opposed to descriptive in nature. Furthermore, the use of selective small-molecule inhibitors and cell growth phenotypes (as opposed to loss of viability) allows robust signal-to-noise readouts for both screening and validation studies. Together, global functional screens could in principle define the universe of individual genes whose overexpression or silencing is sufficient to confer cellular resistance to many types of targeted agents.

Several proof-of-principle studies of pooled RNAi suppressor screens that identify genes whose knockdown confers resistance have been performed. Notable examples include the demonstration that CDK10 and PTEN silencing may cause resistance to tamoxifen and trastuzumab, respectively, in breast cancer (77, 78). As analytical tools improve and the scope of such studies expands, many additional validated effectors should emerge from genome-scale loss-of-function resistance efforts.

On the gain-of-function side, overexpression-based studies (or those that engender increased gene dosage) have long been used to query drug resistance in model organisms (so-called dominant genetics) as described previously. Recently, a related approach was applied to the study of resistance to selective RAF inhibition in melanoma. An arrayed, kinome-wide ORF expression library was introduced into BRAF^V600E melanoma cells that were highly sensitive to the RAF inhibitor PLX4720 [an analogue of vemurafenib (54)]. This effort identified COT as a novel kinase that directs robust resistance to RAF inhibition. Of note, several other kinases also scored as “hits” in this study, including C-RAF and 3 receptor tyrosine kinases (54). C-RAF is the key resistance effector downstream of mutant NRAS, which has been observed in some vemurafenib-resistant clinical specimens; receptor tyrosine kinases as a class have also been implicated in clinical resistance to RAF inhibition, as noted previously (55). Importantly, most of these kinases conferred resistance through sustained extracellular signal-regulated kinase/mitogen-activated protein kinase activation, thus affirming the paramount importance of this hallmark melanoma tumor dependency in the resistance phenotype. Overall, systematic functional screens hold considerable promise to define a spectrum of resistance mechanisms, many of which may have immediate clinical relevance.

Studies of Resistance in Genetically Engineered Mouse Models

Although the aforementioned in vitro approaches offer powerful and scalable avenues for resistance characterization, they are limited to tumor cell autonomous mechanisms and cannot interrogate effects of the microenvironment in the context of an intact organism. Accordingly, several groups have endeavored to model resistance using genetically engineered mouse models that form autochthonous cancers. For example, RNAi-mediated silencing of TOP2A, which encodes the topoisomerase 2 enzyme, induced resistance to doxorubicin (a topoisomerase inhibitor) in a murine lymphoma model (79). On the other hand, knockdown of TOP1 (which encodes topoisomerase 1) conferred resistance to camptothecin [a topoisomerase 1 inhibitor (79)].

More recently, resistance to PI3K inhibitors was investigated in a mammary tumor model in which PIK3CA H1047R, which encodes an oncogenic PI3K variant commonly found in many epithelial tumors, was rendered under the control of a doxycycline-inducible promoter (80). Induction of mutant PIK3CA caused mammary tumor formation, as expected; however, many of these tumors failed to regress on removal of doxycycline or after treatment with PI3K inhibitors. Thus, mutant PI3K was required for the genesis but not the maintenance of some tumors; in other words, they exhibited de novo resistance to PI3K inhibition. Detailed genetic and molecular studies revealed that some of these tumors had acquired MYC amplification, whereas others had acquired MET upregulation. These studies may inform ongoing clinical trials of PI3K inhibitors. Chronic treatment studies of genetically engineered mouse model of EGFR-mutant NSCLC have also been performed with erlotinib (81). The drug-resistant tumors develop both EGFR^T790M and MET amplification, drug resistance mechanisms also found in patients with cancer (27, 28, 49). These examples illustrate the power of endogenous tumor models to uncover pivotal insights into drug resistance mechanisms. In the future, the use of patient-derived xenografts for preclinical studies of drug resistance may emerge as a powerful murine counterpart to the genetically engineered models described previously.

Genomic and Molecular Studies of Drug-Resistant Clinical Tumors

As noted previously, the gold standard for any resistance study is confirmation that a given mechanism is relevant to patients with cancer. Thus, the characterization of human tumors that relapse after exposure to anticancer agents has become an area of intense research activity. Toward this end, 2 overarching avenues may be pursued: candidate driven studies, in which a specific gene or mechanism identified in vitro is queried in human tumors, or unbiased studies, in which omic or related large-scale technologies are applied to these samples. Ideally, these investigations should use tumor specimens that are both patient-matched and lesion-matched, because rather subtle changes in gene or protein expression may need to be measured. Regarding the latter, frozen material is preferable to archival tissue, particularly when phospho-protein studies are interrogated. Indeed, measurement of protein phosphorylation often represents a crucial component of clinical resistance studies; such assays are usually required to determine if the relevant effector pathway has become constitutively activated despite drug treatment.

Thus far, relatively few whole genome/exome or transcriptome studies of pre- and postrelapse tumors have been published. A targeted massively parallel sequencing study focusing on approximately 140 known cancer genes identified a somatic MEK1 mutation in a BRAF-mutant melanoma patient who relapsed after a dramatic response to vemurafenib treatment (60). This mutation was evident in the postrelapse sample but not in the pretreatment counterpart. Going forward, it will undoubtedly be of great interest to perform both whole exome and transcriptome sequencing using pretreatment, postrelapse, and matched normal DNA from many patients treated with targeted anticancer therapeutics. However, these discovery-oriented efforts must be coupled with detailed functional follow-up studies and/or parallel preclinical efforts to validate the resistance mechanisms operant in each case—paying special attention to issues of necessity, sufficiency, and effects of such perturbations on the index tumor dependency and downstream pathway. This aspect could pose a formidable challenge if multiple resistance associated mutations and differentially regulated genes are identified in a given patient—an outcome that seems likely when genome-scale technologies are applied. In the future, an intersection of omic results from clinical specimens with validated resistance genes identified through systematic functional studies performed in vitro (as described previously) may prove fruitful in designating high-priority resistance effectors operant in patients with cancer.