Using targeted, massively parallel sequencing, we identified an activating MEK1 mutation in a patient who developed resistance to a selective RAF inhibitor. Although the tumor was initially highly responsive to PLX4032 treatment, rapid disease progression ensued after 4 months of treatment. The MEK1C121S mutation results in increased kinase activity and confers resistance to both RAF and MEK inhibition in vitro. Taken together, these results indicate one likely mechanism by which this patient's tumor became resistant to RAF inhibition.
allele was not detected in the pretreatment biopsy sample by mass spectrometric genotyping; we speculate that it was present at low levels and underwent selection during the course of treatment. Indeed, studies of BCR-ABL
, and MET
suggest that resistance mutations are commonly present at low levels in treatment-naive tumors and undergo clonal selection during treatment.19,51,67–71
Conceivably, this phenomenon might also help explain the development of simultaneous pan-resistance seen in this patient. Metastatic lesions have been shown to cross seed via circulating tumor cells, which may in principle distribute small numbers of resistant cells across many metastatic foci.72
In such a case, treatment-refractory clones could emerge at multiple sites simultaneously, resulting in a widespread resistance phenotype. Alternatively, multiple resistance mechanisms (genetic, epigenetic, and/or others) may have arisen independently in this patient.
Emerging Mechanisms of Resistance to Kinase Oncogene Inhibition
As noted earlier, genetic mechanisms of acquired resistance to targeted kinase inhibitors typically fall within one of the following two categories: mutations affecting the target kinase, or alterations of other genes within the target signaling pathway that may compensate for or bypass target oncoprotein inhibition ( and ). Resistance mutations in the target kinase commonly populate the catalytic domain, where they sterically impede drug binding while maintaining (or in some cases increasing) catalytic activity. This mechanism has been well described for several tyrosine kinases, including ABL, KIT, PDGFRA (imatinib), EGFR (erlotinib or gefitinib), FLT3 (PKC412), and ALK (crizotinib).18–22,38,40–47
Of particular importance are the gatekeeper mutations that occur in a conserved threonine residue near the kinase active site.19–21,45
These mutations substitute a larger hydrophobic residue for the conserved threonine, thereby increasing the kinase affinity for adenosine triphosphate (ATP). Because most kinase inhibitors in clinical use are ATP-competitive agents, the increased ATP affinity that results from gatekeeper substitutions provides a kinetic means of drug resistance.
Exemplary Mechanisms of Acquired Resistance to Kinase Inhibitors
In addition to gatekeeper mutations, other types of second mutations in the target oncogene have also been reported, albeit less commonly.38–40,42,46
Some of these mutations destabilize the autoinhibitory (inactive) protein conformation bound by certain targeted drugs. Alternatively, gene amplification of the target kinase oncogene may override the ability of the drug to fully extinguish oncoprotein activity. Amplification as a means for resistance has been described for BCR-ABL and KIT in patients with chronic myeloid leukemia and GI stromal tumor (GIST), respectively, as well as for EGFR in lung cancer cell lines.18,47,48
Genetic alterations upstream of the target oncogene may provide an additional mechanism; these events cause resistance through upregulation or activation of the target oncoproteins. Toward this end, some BRAF
-mutant melanoma tumors and cell lines that are resistant to RAF inhibition have been found to harbor NRAS
Furthermore, melanoma cell lines cultured in vitro in the presence of MEK inhibitors may exhibit BRAF
In the PLX4032-resistant melanoma examined here, we did not observe additional mutations or amplifications involving the BRAF
Bypass resistance mechanisms may involve genomic alterations that dysregulate a cellular effector acting in parallel to the drug target. As described in the introduction, the MET
oncogene, which is amplified in approximately 20% of EGFR-mutant lung cancers after treatment with erlotinib or gefitinib, can activate PI3K/AKT and ERK signaling even in the presence of EGFR inhibition.51–53,73
Other bypass resistance mechanisms, including activation of IGF-1Rβ/IRS-1 signaling and signaling via the MET ligand HGF, have also been described in cell lines with acquired resistance to inhibition by erlotinib, gefitinib, and/or trastuzumab.51,73–77
In melanoma, several bypass mechanisms resulting in resistance to PLX4032 have been recently described. Elevated expression of the kinase COT (MAP3K8) drives resistance to PLX4032 in melanoma cell lines and, apparently, in some tumor samples.60
CRAF activation also results in resistance to PLX4032 in cell lines.60,78
Both COT and CRAF dysregulation reactivate the MAPK pathway. Another recently described bypass mechanism—upregulation of PDGFRβ—may activate a MAPK-independent pathway.37
Receptor tyrosine kinases such as AXL, ERBB2, and IGF1R may also confer resistance to RAF inhibition in a MAPK-independent manner, at least in vitro.60,60a
In the current study, we observed mutations in the receptor tyrosine kinases ERBB4
. Although ERBB4
mutations have been implicated in the pathogenesis of melanoma,79
the presence of the ERBB4
mutation in both pretreatment and postrelapse tumor DNA argues against a specific role in acquired resistance in this patient. Similarly, our in vitro analysis suggests that the RET
mutation observed here is unlikely to contribute a major mechanism of resistance, although an additive role in vivo cannot be excluded.
The emergence of a somatic MEK1
mutation in the setting of clinical RAF inhibition represents the first reported example, to our knowledge, of an acquired resistance mechanism in which the tumor develops an activating mutation downstream of the target kinase. We previously described a MEK1
mutation involving codon 124 (MEK1P124L
) arising in a patient with metastatic melanoma that developed resistance to the MEK inhibitor AZD6244.59
, this mutation was proximal to the C helix and conferred resistance to AZD6244 as well as a modest cross resistance to PLX4720 (B). The key difference here is that the MEK1C121S
mutation arose in the setting of RAF inhibition rather than MEK inhibition. Moreover, the pharmacologic and biochemical effect of MEK1C121S
was substantially greater than that of MEK1P124L
. Although downstream resistance mutations have also been described in clinical and preclinical studies of de novo resistance to trastuzumab and lapatinib through activation of the PI3K pathway80–83
and to anti-EGFR therapy via KRAS activation,84–86
a role for such mechanisms in acquired resistance has not previously been demonstrated.
Therapeutic Implications of Emerging Resistance Mechanisms
Mechanisms of acquired resistance often have important therapeutic implications. Overcoming resistance mutations in kinase oncogenes may sometimes be achieved simply by increasing the dose of the targeted agent. Toward this end, several studies have demonstrated increased efficacy of elevated imatinib doses in patients with chronic myeloid leukemia who experience relapse at standard dose levels.87–92
Similarly, one phase III study of imatinib in GIST showed that 33% of patients who experienced progression on 400 mg of imatinib experienced a second response and/or disease stabilization when the dose was increased to 800 mg.93
Dose escalation might also be an option in the setting of resistance as a result of gene amplifications or the setting of altered pharmacokinetics resulting from certain types of resistance mutations.
However, dose escalation may be limited by adverse drug effects and has proved largely ineffective in the presence of gatekeeper mutations. Thus, ongoing discovery efforts are geared toward the development of new drugs to circumvent on-target kinase resistance mechanisms. One promising avenue involves agents engineered to maintain efficacious kinase inhibition despite the presence of resistance mutations. Dasatinib and nilotinib are examples of newer agents that exhibit clinical activity in the setting of various resistance mutations in ABL1.89,94–106
Similarly, sunitinib, an inhibitor of KIT and PDGF, is active against imatinib-resistant KIT and has been approved for use in imatinib-refractory GIST.26,107
Although the aforementioned agents suppress many resistance mutations, they typically fail to inhibit the gatekeeper mutations. In particular, the T315I gatekeeper mutation in ABL1 is refractory to dasatinib and nilotinib as well as imatinib. The development of agents that effectively inhibit gatekeeper mutations remains an area of intense study.97,102
New, irreversible inhibitors of EGFR undergoing evaluation in preclinical and clinical studies include neratinib (HKI-272), BIBW-2992, and PF-00299804.108–116
These agents covalently bind Cys-797 of EGFR, allowing them to inhibit erlotinib- and gefitinib-resistant mutant EGFR. Results from phase I and phase II trials suggest that these agents are reasonably well tolerated and may be efficacious in patients whose tumors have developed resistance to gefitinib or erlotinib.40,109,111,114–119
As with the ABL1 gatekeeper mutation described earlier, some of these experimental drugs may have limited efficacy against the EGFRT790M
resistance mutation. For example, in vitro studies demonstrated that EGFRT790M
conferred acquired resistance to PF-0099804.47,120
A recent phase II study of neratinib also found no benefit in patients with T790M mutations, although dose-limiting toxicity (diarrhea) may have precluded sufficient target inhibition in this case.114
However, neratinib did show evidence of benefit in the setting of a rare G719X EGFR mutation, emphasizing the potential importance of detailed characterization of resistance mechanisms in the clinical setting.
Unlike the aforementioned on-target resistance mechanisms where newer inhibitors may be substituted for the index agents, overriding bypass resistance mechanisms may require combinatorial treatment strategies. Here, the goal is to intercept both the primary oncogene dependency and the bypass mechanism simultaneously. The progression of EGFR-mutant lung cancers that develop resistance to gefitinib or erlotinib through MET amplification may be inhibited by dual treatment with EGFR and MET kinase inhibitors.52
This combination is being investigated in clinical trials of patients with acquired resistance to gefitinib and erlotinib. Similarly, the combination of gefitinib and insulin-like growth factor 1 receptor inhibitors has been shown to inhibit the growth of gefitinib-resistant tumor cells73
and is also under clinical investigation. Concomitant inhibition of downstream pathway reactivation is also being examined in clinical trials. For example, PI3K pathway inhibitors are being tested in combination with EGFR inhibitors, HER2 inhibitors, and inhibitors of the MAPK pathway to overcome both primary and acquired resistance.52,73,82,121–123
The discovery of resistance-associated MEK1 mutations in the setting of BRAF-mutant melanoma predicts that salvage therapies intercepting the MAPK pathway at the level of MEK—or even further downstream—might prove beneficial. Accordingly, the addition of a second MAPK pathway inhibitor to a RAF inhibitor may be required to overcome the effect of the MEK1 resistance mutations and perhaps other bypass mechanisms that reactivate MAPK signaling. More generally, such downstream resistance mutations highlight the potential utility of combination therapy directed against multiple effectors within a driver pathway as opposed to across multiple parallel signaling pathways.
However, MEK1C121S adds an additional layer of complexity; this mutation confers resistance to both RAF inhibition and the allosteric MEK inhibitor chemotype currently in clinical development. Thus, MEK1C121S or similar mutations might confer resistance to both drugs, even when given in combination. This prospect is of particular concern given that clinical trials of combination PLX4032 and AZD6244 are currently under way. Overcoming MEK1C121S or related resistance mechanisms may therefore require inhibition downstream of MEK or alternative mechanisms of inhibiting MEK. Toward this end, ATP-competitive MEK inhibitors or ERK inhibitors represent intriguing possibilities for future therapeutic avenues in BRAF- mutant melanoma.
Challenges to Diagnosis and Management of Targeted Therapeutic Resistance
This study illustrates how knowledge of acquired resistance to kinase inhibitors may guide the conception and deployment of new agents and combination therapies. At the same time, several challenges limit the systematic characterization of resistance mechanisms in the clinical and translational settings. Timely acquisition of high-quality tumor material constitutes one such challenge. Although tissue procurement at the time of relapse would greatly aid the systematic understanding of acquired resistance, such material is only occasionally available in current clinical protocols. Circumventing this barrier will require dedicated multidisciplinary teams and patients to implement rigorous scholarly efforts in this area.
In addition, systematic genetic profiling of cancers remains underdeveloped, even in the research setting. Robust approaches applicable to routine clinical material are needed to procure the salient genetic information from each tumor both before treatment and after relapse. The routine implementation of new genomic technologies in the clinical diagnostic arena, such as the targeted, massively parallel sequencing approach used here, may greatly enable the study of acquired resistance to targeted agents.
Another challenge involves discerning which of the multiple somatic genetic changes that may be observed exerts pivotal resistance mechanisms in any given tumor. This challenge is additionally confounded by the possibility that multiple resistance mechanisms may conceivably occur at multiple tumor foci in the same patient. Here, preclinical models remain crucial as companion experimental avenues. Indeed, multiple resistance mechanisms to imatinib, gefitinib, erlotinib, and PCK412 were first predicted from studies in model systems before they were verified in the clinical setting.41,46,53,124
Our work affirms the importance of such systems in melanoma as well.59
In particular, the dual implementation of in vitro and patient-centered characterization (eg, using massively parallel sequencing) can be highly enabling by streamlining the identification and affirmation of clinically relevant resistance mutations. In addition, dedicated studies that query resistance mechanisms in multiple relapsing tumor sites within a given patient (to the extent possible in the clinical arena) may provide critical information pertaining to the clinical complexity of tumor drug resistance.
Finally, the field of oncology still remains limited in its ability to test rational drug combinations in the clinical trial setting once resistance has developed, even when the mechanisms of resistance are known. As we become increasingly able to genetically characterize individual patients' tumors, there will be a pressing need to develop more efficient ways to test combined therapeutic strategies in defined molecular contexts.
Understanding resistance to targeted anticancer agents has gained increasing importance in light of the success of multiple kinase inhibitors. The experience with RAF inhibition in melanoma offers an example of both the challenges and opportunities inherent in translational studies of cancer drug resistance. Our results illuminate a clinical mechanism of acquired resistance to RAF inhibition that may inform rational therapeutic approaches to target this salient tumor dependency. Profiling BRAF-mutant melanomas for genetic alterations affecting MEK and possibly other downstream effectors when resistance emerges may diagnose the mechanism of resistance and specify optimal salvage therapy.
This work also highlights the power of systematic tumor genomic profiling both before treatment and after relapse to stratify patients based on tumor genotype and to identify clinically relevant resistance mechanisms. In turn, characterizing resistance mechanisms should allow the development of novel therapeutic strategies that achieve more durable responses in cancers driven by a dominant oncogene. Together, these approaches may offer an unprecedented ability to identify druggable genetic changes associated with tumor progression and thereby speed the advent of personalized cancer medicine.