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EGFR -TkI resistance pathways
- 1. Third-generation EGFR TKIs Maria Jubran, M.sc. D.M.D Student Laboratory of Biophysics and Cancer Research The Faculty of Dental Medicine
- 2. WHY EGFR ? Rationale for developing EGFR inhibtors :EGFR is expressed on the majority of Lung Cancer . EGFR is associated with poor prognosis
- 3. EGFR Identity EGFR, also known as ErbB1 one of four members of the ErbB/HER family of transmembrane tyrosine kinase growth factor receptors EGFR forms dimers with itself and other members of the ErbB family Dimer formation promotes tyrosine autophosphorylation of the EGFR intracellular domain the resultant open configuration of the kinase domain enhances access by ATP and substrate and creates binding sites for signaling molecules.
- 5. First generation inhibitors Gefitinib and erlotinib are reversible small-molecule ATP analogues originally designed to inhibit the tyrosine kinase (TK) activity of wild-type EGFR. During their clinical development, these first-generation TK inhibitors (TKI) were found to be most effective in patients with advanced non–small cell lung cancer (NSCLC) recurrent somatic activating mutations (EGFRm+) occurring in the exons encoding the kinase domain of EGFR: small multinucleotide in-frame deletions in exon 19 (ex19del) a point mutation in exon 21 leading to substitution of leucine for arginine at position 858 WHY does inhibition of EGFR work? Biological: EGFR mutant tumors are addicted to EGFR signaling ,cell survival and proliferation pathway are solely controlled by EGFR Pharmacologic : Gefitinib binds 20 fold more tightly to mutant EGFR than WT EGFR
- 6. Somatic mutations in EGFR
- 7. Unfortunately, patients who respond to therapy ultimately develop disease progression after about 9 to 14 months of treatment First generation inhibitors
- 8. Mechanism of erlotinib and gefitinib resistance Mechanism Definietion Primary genetic and other signaling aberrations Secondary / acquired Tumor growth following complete /partial response while on drug the most common resistance mechanism and is detected in tumor cells from more than 50% of patients after disease progression: that acquisition of a second mutation in EGFR, resulting in substitution of threonine at the “gatekeeper” amino acid 790 to methionine (T790M). The T790M effects on both steric hindrance and increased ATP affinity of the reversible drugs
- 9. Mechanism of erlotinib and gefitinib resistance 3 categories of resistance to kinase inhibitors Bypass Mechanism Target oriented of EGFR itself Insurgence of secondary mutations in the EGFR gene EGFR amplification Alteration in down stream effectors (proteins that activated by the target oncoprotein) Secondary EGFR mutation/ EGFR T790M 50% Bypass signaling via ERBB3 /MET amplificat ion 20%/other
- 11. Second-generation However, as monotherapy, they have failed to overcome T790M- mediated resistance in patients ,because: concentrations are not achievable in humans due to dose-limiting toxicity related to nonselective inhibition of wild-type EGFR irreversible EGFR TKIs such as: afatinib and dacomitinib are effective in untreated EGFR-mutant lung cancer .
- 12. First generation + second generation Therefore, there remains a significant unmet need for an EGFR TKI agent that can more effectively target T790M tumors while sparing the activity of wild-type EGFR
- 13. Third -generation has led to the development of “third- generation” EGFR TKIs that are designed to target T790M and EGFR TKI– sensitizing mutations more selectively than wild-type EGFR. WZ4002 rociletinib (CO-1686) AZD9291: is a mono-anilino–pyrimidine compound that is structurally and pharmacologically distinct from all other TKIs, including CO-1686 and WZ4002.
- 14. AZD9291vs other generations The data represent the geomean IC50 nmol/L value dose-limiting toxicity First generation second generation third generation
- 16. Purpose To identify novel mechanisms of resistance to third- generation EGFR inhibitors in patients. Experimental Design seven patients with lung adenocarci- noma before, during, and/or after treatment with: AZD9291 or rociletinib (CO- 1686) *Targeted sequencing * FISH analyses and the relevance of candidate genes in vitro models. analyzed tumor biopsies from
- 17. Primary and acquired Resistance to third generation inhibitors
- 18. Tumor heterogeneity complicates the assignment of the correct anti- cancer therapy, and underlies drug resistance
- 19. Fish analysis and targeted sequenced genes 7.3 gene copies 3.7 gene copies probe for chromosomal loci containing ERBB2 centromere for reference 1 2
- 20. 6 days treatment ERBB2 amplification has been shown to confer resistance to first-generation EGFR TKIs Hypotheses: the amplified ERBB2 might substitute for EGFR signaling in this context and explain the lack of response to of AZD9291 and rociletinibin P1andP2 Test the hypothesis: evaluated the impact of ERBB2 overexpression on the sensitivity to AZD9291 and rociletinib in PC9GR cells (EGFRT790M) Despite a relatively low overexpression of ERBB2 in PC9GR cells, we observed decreased sensitivity to both drugs at nanomolar concentrations our results indicate that ERBB2 activation may contribute to resistance against rociletinib and AZD9291.
- 21. Activation of MET confers resistance to third-generation EGFR inhibitors 4.42 MET copiesMET/CEN7 ratios probe for chromosomal loci containing ERBB2 centromere for reference 3 4 high-level MET amplification (MET/ CEN7 ratios 1.88, 21.92 MET copies ERBB2 amplification (4.25 gene copies) 5
- 22. Activation of MET confers resistance to third-generation EGFR inhibitors Does MET amplification reduce the sensitivity to AZD9291 or rociletinib? ü overexpressed EGFRT790M-mutant constructs in: q HCC827 cells (MET wild-type) q HCC827GR cells (MET amplified)gefitinib-resistant derivate. the presence of MET amplification decreased the sensitivity to both third-generation EGFR inhibitors A-AZD9291 R-ROCILETINIB C-CRIZOTINIB =MET INHIBITOR
- 23. The presence of MET amplification decreased the sensitivity to both third-generation EGFR inhibitors q HCC827 cells (MET wild-type) q HCC827GR cells (MET amplified). ERBB2 and MET amplification has been found to be associate with loss of efficacy of third-generation EGFR inhibitors in EGFR-mutant tumors .
- 24. 3 categories of resistance to kinase inhibtors Bypass Mechanism Target oriented Secondary mutation Alteration in down stream effectors (proteins that activated by the target oncoprotein)
- 25. The liver biopsy the retention of the initial EGFR exon19 deletion intermediate-level MET amplification (MET/CEN7 ratios 1.14, 5.67 MET copies 6 Presence of a secondary EGFR C797S mutation that was not present in the pretreatment biopsy confirm resistance
- 26. 3 categories of resistance to kinase inhibtors Bypass Mechanism Target oriented Secondary mutation Alteration in down stream effectors (proteins that activated by the target oncoprotein)
- 27. Acquired resistance to AZD9291 by acquired kRAS 7
- 28. In-vitro
- 29. 3 categories of resistance to kinase inhibtors Bypass Mechanism Target oriented Secondary mutation Alteration in down stream effectors (proteins that activated by the target oncoprotein)
- 30. Summary ERBB2and MET activation can confer resistance to third-generation EGFR inhibitors , in recurrent amplification of either MET orERBB2 in tumors KRASG12S mutation in a patient with acquired resistance to AZD9291 can be potential driver of acquired resistance dual inhibition of EGFR/MEK might be a viable strategy to over- come resistance in EGFR- mutant cells expressing mutant KRAS.
Editor's Notes
- EGFR and cancer The 170 kD Epidermal Growth Factor Receptor (EGFR, also known as ErbB1), is one of four members of the ErbB/HER family of transmembrane tyrosine kinase growth factor receptors (Figure 1). EGFR forms dimers and higher order oligomers with itself and other members of the ErbB family (ErbB2/HER2/neu, Erbb3/HER3, and ErbB4/HER4) via a primary dimerization domain and several secondary receptor–receptor contact points [1-3]. Binding of naturally ocurring extracellular ligands (e.g., amphiregulin, epiregulin, HB-EGF) to the extracellular ligand-binding domain (domain III) of EGFR induces conformational shifts that permit homo- and hetero-dimerization events between EGFR molecules and its family members. Dimer and multimer formation promotes tyrosine autophosphorylation of the EGFR intracellular domain; the resultant open configuration of the kinase domain enhances access by ATP and substrate and creates binding sites for signaling molecules. The EGFR kinase is also active at a low level when the protein is in the unliganded state, with the degree of activity varying by cell type, and influenced by both cell-intrinsic and environmental factors
- Monoclonal antibodies (mAbs) directed to the extracellular domain of EGFR Small molecular inhibitors of EGFR kinase intracellular domain
- theseTumors account for approximately 10% to 15% and 40% of NSCLC in Western and Asian populations, respectively . Unfortunately, although patients with EGFRm+ tumors typically show good initial responses to first-generation TKIs, most patients who respond to therapy ultimately develop disease progression after about 9 to 14 months of treatment (7–11). Furthermore, these first-generation TKIs are associated with side effects that include skin rash and diarrhea that are due to the inhibition of wild-type EGFR in skin and gastrointestinal organs .
- Preclinical modeling and analysis of tumor tissue obtained from patients after the development of disease progression have led to the identification of a number of mechanisms that mediate EGFR TKI resistance. The T790M mutation is believed to render the receptor refractory to inhibition by these reversible EGFR TKIs through exerting effects on both steric hindrance and increased ATP affinity
- Preclinical modeling and analysis of tumor tissue obtained from patients after the development of disease progression have led to the identification of a number of mechanisms that mediate EGFR TKI resistance.
- http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X2014001100929
- theseTumors account for approximately 10% to 15% and 40% of NSCLC in Western and Asian populations, respectively . Unfortunately, although patients with EGFRm+ tumors typically show good initial responses to first-generation TKIs, most patients who respond to therapy ultimately develop disease progression after about 9 to 14 months of treatment (7–11). Furthermore, these first-generation TKIs are associated with side effects that include skin rash and diarrhea that are due to the inhibition of wild-type EGFR in skin and gastrointestinal organs .
- https://www.sciencedirect.com/science/article/pii/S0223523417303884 However, this combination has substantial skin toxicity, with 20% of patients reporting Common Terminology Criteria for Adverse Events (CTCAE) grade 3 or higher rash (33). Therefore, there remains a significant unmet need for an EGFR TKI agent that can more effectively target T790M tumors while sparing the activity of wild-type EGFR. This has led to the development of “third-generation” EGFR TKIs that are designed to target T790M and EGFR TKI–sensitizing mutations more selectively than wild-type EGFR. WZ4002 was the first such agent to be published (34), although it has not progressed to clinical trials. A second agent closely related to the WZ4002 series, CO-1686, has been recently reported (35) and is currently in early phase II clinical trials. HM61713 is another “third-generation” agent that is currently in early phase I trials. Here, we describe identification, characterization, and early clinical development of AZD9291, a novel, irreversible, EGFR TKI with selectivity against mutant versus wild-type forms of EGFR. AZD9291 is a mono-anilino–pyrimidine compound that is structurally and pharmacologically distinct from all other TKIs, including CO-1686 and WZ4002.
- theseTumors account for approximately 10% to 15% and 40% of NSCLC in Western and Asian populations, respectively . Unfortunately, although patients with EGFRm+ tumors typically show good initial responses to first-generation TKIs, most patients who respond to therapy ultimately develop disease progression after about 9 to 14 months of treatment (7–11). Furthermore, these first-generation TKIs are associated with side effects that include skin rash and diarrhea that are due to the inhibition of wild-type EGFR in skin and gastrointestinal organs .
- AZD9291 demonstrates greater inhibition of viability against mutant EGFR cell lines compared with wild-type, as assessed using a Sytox Green live/dead assay measured after 3 days of treatment. The data represent the geomean IC50 nmol/L value from at least two separate experiments (expressed with 95% confidence intervals where n > 3).
- Mechanisms of primary and acquired resistance tothirdgenerationEGFR inhibitors : We obtained specimens from seven patients treated with thirdgenerationEGFRinhibitorsAZD9291(n¼5)orrociletinib(n¼2), within two clinical trials (clinicaltrials.gov, NCT01802632 and NCT02147990) after acquired EGFRT790M-positive resistance to first- or second-generation EGFR kinase inhibitors. The PFS for third-generationEGFRTKItherapyrangedfrom1.2to19.4months (median 4.2 months; 95% CI, 3.7–4.7 months, Fig. 1A). The best reduction of the sum of the largest tumor diameters under treatment, according to RECIST V1.1 (22), ranged between 0.1% and 79.2%,with 3 patients presenting growth of non-target lesions(P1, P3, and P5, Fig. 1B). The pattern of response varied across these patients: two patients(P1andP3)experiencedprimaryresistancetorociletinib(defined as progression on the first restaging scan), whereas two patients had stable disease (P2 and P4) under treatment with AZD9291. Finally, three cases (P5, P6, and P7) presented with partial responses to treatment with AZD9291, with reductions in diameters of more than 50% (Fig. 1A and B). For these patients, unfortunately, disease recurred after 16.5, 19.4, and 7 months, respectively. AddwhatsPD SD ….and do it aspargraph
- To understand the mechanism thatoislinked to heterogenous response
- To characterize the molecular mechanisms that may be linked with such heterogeneous response patterns, we performed FISH for known EGFR resistance markers (ERBB2 and MET) and targeted next-generation sequencing on tumor tissue from these patients (see Patients and Methods). FISH analyses of a massively progressive pleural effusion (nontarget lesion) collectedafter3weeksoftreatmentinitiationfromP1(PD,625 mg rociletinib twice daily) and a lung biopsy sample collected before treatment from P2 (SD, 180 mg AZD9291 daily), revealed ERBB2 amplification (7.3 and 3.7 gene copies, respectively; Fig. 1C).
- Because ERBB2 amplification has been shown to confer resistance to first-generation EGFR TKIs (23), we hypothesized that amplified ERBB2 might substitute for EGFR signaling in this context and explain the lack of response to of AZD9291 and rociletinibinP1andP2.Totestthishypothesis,weevaluatedthe impact of ERBB2 overexpression on the sensitivity to AZD9291 and rociletinib in PC9GR cells (EGFRT790M) . Despite a relatively low overexpression of ERBB2 in PC9GR cells, we observed decreased sensitivity to both drugs at nanomolar concentrations (Fig. 1D and Supplementary Fig. S1A). Thus, our results indicate that ERBB2 activation may contribute to resistance against rociletinib and AZD9291. Our functional in vitro results suggest that the activity of both AZD9291 and rociletinib may decrease through specific activation of ERBB2 signaling.These observations go in line with the studies reporting ERBB2 as a mechanism of resis- tance to first-generation EGFR inhibitors
- ThetumorbiopsycollectedbeforetreatmentofP3(PD,625mg rociletinib twice daily) showed high-level amplification of MET (MET/CEN7 ratios 2.10, 4.42 MET copies; Fig. 2A). Unfortunately, no baseline comparative sample (before erlotinib treatment) was available for this patient. In the case of P4 (SD, 80 mg AZD9291 daily), an initial tumor reduction (20.8%) was followed by an increase in the tumor volume after 4.1 months of treatment (Fig. 2B). In this patient too, the biopsy taken after the initial response exhibited high-level amplification of MET (MET/ CEN7ratio5.35,11.42METcopies)thatwasnotobservedbefore therapy withAZD9291(Fig.2C).
- P5 (PR,80mgAZD9291daily) hadaninitialtumorresponseof79%andpresentedanewlesion in the liver that harbored a high-level MET amplification (MET/ CEN7 ratios 1.88, 21.92 MET copies, Fig. 2D, left) and ERBB2 amplification (4.25 genecopies,Fig. 2D, right).Of note, targeted sequencing of these samples did not show additional clinically relevant mutations (Table 1). TofunctionallytestwhetherMETamplificationmightreduce the sensitivity to AZD9291 or rociletinib, we stably overexpressed EGFRT790M-mutant constructs in HCC827 cells (MET wild-type) and their gefitinib-resistant derivate, HCC827GR cells (MET amplified; Supplementary Fig. S1B). In line with previous reports (24), the presence of MET amplification decreased the sensitivity to both third-generation EGFR inhibitors (Fig. 2E) and led to sustained phosphorylation of ERK and AKT (Fig. 2F). Furthermore, concomitant inhibition with either AZD9291 or rociletinib and the MET inhibitor, crizotinib, resulted in pronounced cytotoxicity and reduced AKT and ERK activation in HCC827GREGFR-T790M cells (Fig. 2E and G). These results were confirmed in EGFRT790M-expressing cells (H1975 and PC9GR) where MET was activated through addition of exogenous HGF (Supplementary Fig. S1C–S1F). Thus,ourclinicalobservationsareinlinewithpreviousreports where both ERBB2 and MET amplification has been found to be associatewithlossofefficacyofthird-generationEGFRinhibitors in EGFR-mutant tumors (25). More importantly, we provide functional evidence that activation of ERBB2 and MET signaling may induce resistance to this new class of EGFR inhibitors
- P5 (PR,80mgAZD9291daily) hadaninitialtumorresponseof79%andpresentedanewlesion in the liver that harbored a high-level MET amplification (MET/ CEN7 ratios 1.88, 21.92 MET copies, Fig. 2D, left) and ERBB2 amplification (4.25 genecopies,Fig. 2D, right).Of note, targeted sequencing of these samples did not show additional clinically relevant mutations (Table 1). TofunctionallytestwhetherMETamplificationmightreduce the sensitivity to AZD9291 or rociletinib, we stably overexpressed EGFRT790M-mutant constructs in HCC827 cells (MET wild-type) and their gefitinib-resistant derivate, HCC827GR cells (MET amplified; Supplementary Fig. S1B). In line with previous reports (24), the presence of MET amplification decreased the sensitivity to both third-generation EGFR inhibitors (Fig. 2E) and led to sustained phosphorylation of ERK and AKT (Fig. 2F). Furthermore, concomitant inhibition with either AZD9291 or rociletinib and the MET inhibitor, crizotinib, resulted in pronounced cytotoxicity and reduced AKT and ERK activation in HCC827GREGFR-T790M cells (Fig. 2E and G). These results were confirmed in EGFRT790M-expressing cells (H1975 and PC9GR) where MET was activated through addition of exogenous HGF (Supplementary Fig. S1C–S1F). Thus,ourclinicalobservationsareinlinewithpreviousreports where both ERBB2 and MET amplification has been found to be associatewithlossofefficacyofthird-generationEGFRinhibitors in EGFR-mutant tumors (25). More importantly, we provide functional evidence that activation of ERBB2 and MET signaling may induce resistance to this new class of EGFR inhibitors *******The role of MET amplification in acquired resistance to first- generationEGFRinhibitors has been studied extensively (29–31). According to our results MET activation may similarly play a role in resistance to third-generation EGFR inhibitors as MET is not a relevant off-target for these drugs. Our observations also confirm previous reports on the efficacy of the combination of rociletinib with crizotinib in these tumors (32).
- . Finally, patient 6 (P6) presented with a tumor response of 72.6% followed by emergence of new liver metastases 19.4 months after start of therapy. The liver biopsy revealed the retention of the initial EGFR exon19 deletion (allelic fraction, AF:42.6%,SupplementaryFig.S2A)andtheEGFRT790Mmutation (AF:27.66%)togetherwiththepresenceofasecondaryEGFRC797S mutation (Fig. 2H) that was not present in the pretreatment biopsy (Supplementary Fig. S2B). Of note, the liver biopsy obtained at relapse also harbored an intermediate-level MET amplification (MET/CEN7 ratios 1.14, 5.67 MET copies, Fig. 2I). The EGFRC797S mutation abrogates the irreversible binding of third-generation EGFR inhibitors and has been described to confer resistance to AZD9291 thus providing a rational for the observed resistance phenotype in P6 (10, 26, 27).
- KRAS mutation and resistance to third-generation EGFR inhibition: Inourcohort,aforthpatient(P7)relapsedafterhavinginitially responded to treatment with AZD9291. This patient received different lines of treatment before developing resistance through EGFRT790M under combination therapy with afatinib and cetuximab. After enrollment into the AURA trial (NCT01802632) the tumor responded to AZD9291 treatment (160 mg/d) with a PR (best response: 54.3%, Fig. 3A) for 8 months. However, the fifth follow-up revealed the appearance of a new lesion in a fast growing cervical lymph node. Targeted sequencing of the specimens obtained before and after treatment with AZD9291 (both from the cervical lymph nodes) showed a slight decrease in the fractionofreadscontainingtheoriginalactivatingEGFRmutation (43% before treatment with AZD9291 vs. 32% after treatment, Supplementary Fig. S2C) and the disappearance of EGFRT790M (25%beforetreatmentwithAZD9291,SupplementaryFig.S2D). Finally, in two independent sequencing runs we found a novel KRASG12S mutation (38% of the reads), which had not been detected before therapy with AZD9291 (Fig. 3B). In general, the disproportionate levels of C>T/G>A changes that occur as a consequence of formalin fixation are more apparent at low allelic fractions (1%–10%; ref. 28). In the relapse sample of P7, the C>T/G>A changes were comparable with other patient samplesorfreshlypreparedDNAfromcelllinessequencedinthe same batch, suggesting that the KRAS mutation detected is not a formalin-fixation artifact (Supplementary Fig. S2E). The EGFRC797S mutation had also been found in the plasma of this patient (10) but was not observed in the lymph node biopsy (SupplementaryFig.S2F).Moreover,theKRASG12S mutationwas not detected in the plasma of this patient. These observations suggest that heterogeneous mechanisms of acquired resistance might be operant in this patient. To investigate whether expression of an activated KRAS allele might contribute to resistance to third-generation EGFR inhibitors we stably transduced EGFR-mutant PC9 cells with the KRASG12S-mutant. PC9cells that expressed the KRASG12S-mutant wereselectedundertreatmentwithpuromycinforatleast2weeks. TheresultingPC9KRAS-G12S cellsexpressedboththeoriginalEGFR exon19deletionandtheKRASG12Smutation(SupplementaryFig. S3A).Concomitantexpressionofbothmutantswasalsoobserved in a PC9KRAS-G12S clone obtained by serial dilution (Supplementary Fig. S3B). When compared with the KRAS wild-type transduced cells (PC9KRAS-wt), PC9KRAS-G12S were less sensitive to AZD9291 (Fig. 3C and D). Furthermore, introduction of KRASG12S resulted in increased KRAS expression and sustained ERK phosphorylation under treatment with AZD9291 (Fig. 3E). Similar results were observed in HCC827KRAS-G12S cells (Supplementary Fig. S3C and S3D) where the introduction of the KRAS mutation also resulted in decreased sensitivity to rociletinib (Supplementary Fig. S3C). Confirming a role of MAPK signaling in inducing resistance in PC9KRAS-G12S cells, combined treatment with AZD9291 and the MEK inhibitor, trametinib, was highly synergistic [synergy score, 5.48 (ref. 21); P value of 4.14E08; Fig. 3F and Supplementary Fig. S4A]. Consistent with these results, AZD9291 failed to fully inhibit downstream MAPK signaling except in the presence of trametinib (Fig. 3G). Similar results were observed with the combination of AZD9291 and selumetinib (Fig. 3F and Supplementary Fig. S4B). Finally, concomitant EGFR and MEK inhibition was also synergistic in HCC827KRAS-G12S cells (Supplementary Fig. S4C and S4D). These findings demonstrate that mutant KRAS may induce acquired resistance to third-generation EGFR inhibitors.
- The EGFRC797S mutation had also been found in the plasma of this patient (10) but was not observed in the lymph node biopsy (SupplementaryFig.S2F).Moreover,theKRASG12Smutationwasnot detected in the plasma of this patient. These observations suggest that heterogeneous mechanisms of acquired resistance might be operant in this patient. To investigate whether expression of an activated KRAS allele might contribute to resistance to third-generation EGFR inhibitors we stably transduced EGFR-mutant PC9 cells with the KRASG12S-mutant. PC9cells that expressed the KRASG12S-mutant wereselectedundertreatmentwithpuromycinforatleast2weeks. TheresultingPC9KRAS-G12ScellsexpressedboththeoriginalEGFRexon19deletionandtheKRASG12Smutation(SupplementaryFig. S3A).Concomitantexpressionofbothmutantswasalsoobservedin a PC9KRAS-G12S clone obtained by serial dilution (Supplementary Fig. S3B). When compared with the KRAS wild-type transduced cells (PC9KRAS-wt), PC9KRAS-G12S were less sensitive to AZD9291 (Fig. 3C and D). Furthermore, introduction of KRASG12S resulted in increased KRAS expression and sustained ERK phosphorylation under treatment with AZD9291 (Fig. 3E). Similar results were observed in HCC827KRAS-G12S cells (Supplementary Fig. S3C and S3D) where the introduction of the KRAS mutation also resulted in decreased sensitivity torociletinib(Supplementary Fig. S3C). Confirming a role of MAPK signaling in inducing resistance in PC9KRAS-G12S cells, combined treatment with AZD9291 and the MEK inhibitor, trametinib, was highly synergistic [synergy score, 5.48 (ref. 21); P value of 4.14E08; Fig. 3F and Supplementary Fig. S4A]. Consistent with these results, AZD9291 failed to fully inhibit downstream MAPK signaling except in the presence of trametinib (Fig. 3G). Similar results were observed with the combination of AZD9291 andselumetinib(Fig. 3F and Supplementary Fig. S4B). Finally, concomitant EGFR and MEK inhibition was also synergistic in HCC827KRAS-G12S cells (Supplementary Fig. S4C and S4D). These findings demonstrate that mutant KRAS may induce acquired resistance to third-generation EGFR inhibitors. tumorrebiopsyobtained at the time of acquired resistance to AZD9291. This mutation is of particular interest because EGFR and KRAS mutations typically occur in a mutually exclusive fashion in lung cancer (16, 35). Indeed, a recent report evidenced that the expression of inducible KRASG12V constructs in EGFR- mutant PC9 cells results in decreased cell viability after 7 days of selection withdoxycyclin(16). Anecdotal reports show evidence of co-occurrence of KRAS and EGFR mutations in lung cancer patients (36, 37). More recentlyHataand colleagues (14) described two distinct evolutionary paths for the development of resistance to EGFR inhibition. In this study, cell lines that developed resistance at late stages were enriched for NRAS and KRAS mutations suggesting that under prolonged selection a distinct population of EGFR-mutant cells may well tolerate the presence of oncogenic MAPK pathway activation. In ourfunc-tionalexperiments, decreased cell viability was observed in cells expressing both EGFR and KRAS mutants; however, thispheno- type was overcome after about 10 passages under antibiotic selection. In line with our results, in the study performed byUnniand colleagues (16), the induction of mutant KRAS rescued PC9 cells from the lethal effects of erlotinib, thus implying that the toxicity ofcoexpressionof mutant KRAS and EGFR depend on the kinase activity of mutant EGFR. We speculate that EGFRinhibi-tionthroughAZD9291mayfunctionally depleteoncogenicEGFR (Continued.)
- initial EGFR exon19 deletion (allelic fraction, AF:42.6%,SupplementaryFig.S2A)andtheEGFRT790Mmutation (AF:27.66%)togetherwiththepresenceofasecondaryEGFRC797S mutation (Fig. 2H) that was not present in the pretreatment biopsy (Supplementary Fig. S2B). Of note, the liver biopsy obtained at relapse also harbored an intermediate-level MET amplification (MET/CEN7 ratios 1.14, 5.67 MET copies, Fig. 2I)
- According to our results MET activation may similarly play a role in resistance to third-generation EGFR inhibitors as MET is not a relevant off-target for these drugs. Our observations also confirm previous reports on the efficacy of the combination of rociletinib with crizotinib in these tumors (32). Combination therapies have proven successful in the setting of resistance to first-generation EGFR inhibitors (33) and early-phase clinical trials are evaluating the combination of rociletinib with crizotinib (32), or AZD9291 with the MET inhibitor, savolitinib, in EGFR-mutant lung cancer (34).