Structural, Biochemical, and Clinical Characterization of Epidermal Growth Factor Receptor (EGFR) Exon 20 Insertion Mutations in Lung Cancer

In the paper

“The Ki value for A763_Y764insFQEA (2.5 nM) was comparable to that we measured for the L858R mutant (6.4 nM).”

In the paper

“All other EGFR exon 20 insertions and T790M–bearing mutations had 50% inhibitory concentrations (IC50s) that exceeded 2 µM erlotinib (Fig. 1B, 1C and Sup. Table 2).”

• Also stated in: § DISCUSSION · boosted

  “our data confirm that most exon 20 insertions are not inhibited by submicromolar concentrations of reversible EGFR TKIs.”

In the paper

“All were able to proliferate in the absence of IL3 (Sup. Fig. 1), indicating transforming ability of these mutations.”

In the paper

“both insertion mutants exhibited higher catalytic activity than WT EGFR; although they were not as active as EGFR-L858R (Table 1).”

In the paper

“the inhibition of these downstream phosphorylation events and upregulation of BIM were seen with EGFR-A763_Y764insFQEA in a similar fashion as with inhibitor-sensitive L858R (Fig. 1D, 1E)”

In the paper

“In both BID007 and H3255, EGFR-specific siRNAs decreased expression of EGFR and suppressed cell growth (Fig. 3B).”

In the paper

“In vitro characterization of A763_Y764insFQEA demonstrated that it had an IC50 of < 0.05µM to erlotinib or gefitinib, which is more than 10-fold lower than the clinically achievable serum concentrations of these EGFR TKIs”

In the paper

“Together with D770, the three inserted residues form a tight turn, with a hydrogen bond formed between the main chain carbonyl of D770 and the amide of the inserted glycine (Fig. 4B, Sup. Fig. 10B, 10C).”

In the paper

“The D770_N771insNPG mutant adopts an active conformation with the C-helix in its inward position (Fig. 4A).”

In the paper

“submicromolar concentrations of erlotinib inhibited phosphorylation of EGFR in A763_Y764insFQEA mutant cells, led to deactivation of the PI3K/AKT and MAPK/ERK pathways, and subsequently induced apoptosis in our models.”

In the paper

“Differences, which likely arise from the divergent crystal packing in D770_N771insNPG, include a small change in the relative orientation of the N- and C-lobes, and the conformation of the activation loop, which is fully ordered in the D770_N771insNPG structure but not in the present L858R structure.”

In the paper

“The region around chromosome 7, corresponding to EGFR, demonstrated slight amplification (Sup. Fig. 9B).”

In the paper

“Our kinetic characterization of this mutant revealed that it is activated, relative to WT EGFR, and that it is much more potently inhibited by gefitinib than is WT EGFR.”

In the paper

“In both models, the insertion was modeled in a helical conformation, shifting the register of the C-helix toward its N-terminus, and resulting in a longer loop at its N-terminal end (the β3-αC loop).”

In the paper

“inspection of the model indicates that the FQEA insertion is compatible with formation of the asymmetric dimer and, consistent with this, analysis of the A763_Y764insFQEA mutant using size-exclusion chromatography revealed a monomer-dimer equilibrium in solution (Sup. Fig. 11).”

In the paper

“We found that E762Q_insFQEA, like A763_Y764insFQEA, was highly active in a ligand independent manner but A763_Y764insFQQA was inactive (Fig. 5B).”

In the paper

“BID007 had a focal area of deletion encompassing the CDKN2A tumor suppressor gene (Sup. Fig. 9C); an area commonly deleted in cancers 45.”

What the sources say

• Not checked yetcites [45] Weir BA Woo MS Getz G Perner S Ding L Beroukhim R Lin WM Province MA Kraja A Johnson LA Shah K Sato M Thomas RK Barletta JA Borecki IB Broderick S Chang AC Chiang DY Chirieac LR Cho J Fujii Y Gazdar AF Giordano T Greulich H Hanna M Johnson BE Kris MG Lash A Lin L Lindeman N Mardis ER McPherson JD Minna JD Morgan MB Nadel M Orringer MB Osborne JR Ozenberger B Ramos AH Robinson J Roth JA Rusch V Sasaki H Shepherd F Sougnez C Spitz MR Tsao MS Twomey D Verhaak RG Weinstock GM Wheeler DA Winckler W Yoshizawa A Yu S Zakowski MF Zhang Q Beer DG Wistuba II Watson MA Garraway LA Ladanyi M Travis WD Pao

In the paper

“Unlike EGFR-L858R and exon 19 deletion mutants, the D770_N771insNPG mutation lacks this “Achilles’ heel”, since it activates EGFR without markedly diminishing its affinity for ATP or increasing its affinity for EGFR TKIs.”

In the paper

“The most common EGFR mutations are in-frame deletions around the LREA motif (amino-acid residues 747 to 750) of exon 19 (45% of EGFR mutations) and the exon 21 L858R point mutation (40% of EGFR mutations) 5,6,8.”

What the sources say

• Not checked yetcites [5] Shigematsu H Lin L Takahashi T Nomura M Suzuki M Wistuba II Fong KM Lee H Toyooka S Shimizu N Fujisawa T Feng Z Roth JA Herz J Minna JD Gazdar AF Clinical and biological features associated with epidermal growth factor receptor gene mutations in lung cancers J Natl. Cancer Inst 2005 97 339 346 15741570
• Not checked yetcites [6] Sequist LV Bell DW Lynch TJ Haber DA Molecular predictors of response to epidermal growth factor receptor antagonists in non-small-cell lung cancer J Clin. Oncol 2007 25 587 595 17290067
• Not checked yetcites [8] Tokumo M Toyooka S Kiura K Shigematsu H Tomii K Aoe M Ichimura K Tsuda T Yano M Tsukuda K Tabata M Ueoka H Tanimoto M Date H Gazdar AF Shimizu N The relationship between epidermal growth factor receptor mutations and clinicopathologic features in non-small cell lung cancers Clin. Cancer Res 2005 11 1167 1173 15709185

In the paper

“Exon 20 insertion mutations comprise 4–10% of all EGFR mutations 27,29–32.”

What the sources say

• Not checked yetcites [27] Mitsudomi T Yatabe Y Mutations of the epidermal growth factor receptor gene and related genes as determinants of epidermal growth factor receptor tyrosine kinase inhibitors sensitivity in lung cancer Cancer Sci 2007 98 1817 1824 17888036 PMC11159145
• Not checked yetcites [29] Choi YL Soda M Yamashita Y Ueno T Takashima J Nakajima T Yatabe Y Takeuchi K Hamada T Haruta H Ishikawa Y Kimura H Mitsudomi T Tanio Y Mano H EML4-ALK mutations in lung cancer that confer resistance to ALK inhibitors N. Engl. J Med 2010 363 1734 1739 20979473
• Not checked yetcites [30] Pao W Chmielecki J Rational, biologically based treatment of EGFR-mutant non-small-cell lung cancer Nat. Rev. Cancer 2010 10 760 774 20966921 PMC3072803 source
• Not checked yetcites [31] Oxnard GR Lo PC Nishino M Dahlberg SE Lindeman NI Butaney M Jackman DM Johnson BE Janne PA Natural history and molecular characteristics of lung cancers harboring EGFR exon 20 insertions J Thorac. Oncol 2013 8 179 184 23328547 PMC3549533
• Not checked yetcites [32] Arcila ME Nafa K Chaft JE Rekhtman N Lau C Reva BA Zakowski MF Kris MG Ladanyi M EGFR exon 20 insertion mutations in lung adenocarcinomas: prevalence, molecular heterogeneity, and clinicopathologic characteristics Mol. Cancer Ther 2013 12 220 229 23371856 PMC3714231

In the paper

“Other EGFR mutations have also been associated with some sensitivity to gefitinib and erlotinib. These include exon 18 point mutations in position G719 (G719A, C or S - ∼3% of EGFR mutations), rare inframe exon 19 insertions 25, and the exon 21 L861Q mutant (∼2% of EGFR mutations) 26–28.”

What the sources say

• Not checked yetcites [25] He M Capelletti M Nafa K Yun CH Arcila ME Miller VA Ginsberg MS Zhao B Kris MG Eck MJ Janne PA Ladanyi M Oxnard GR EGFR exon 19 insertions: a new family of sensitizing EGFR mutations in lung adenocarcinoma Clin Cancer Res 2012 18 1790 1797 22190593 PMC3306520
• Not checked yetcites [26] Mitsudomi T Yatabe Y Epidermal growth factor receptor in relation to tumor development: EGFR gene and cancer FEBS J 2010 277 301 308 19922469
• Not checked yetcites [27] Mitsudomi T Yatabe Y Mutations of the epidermal growth factor receptor gene and related genes as determinants of epidermal growth factor receptor tyrosine kinase inhibitors sensitivity in lung cancer Cancer Sci 2007 98 1817 1824 17888036 PMC11159145
• Not checked yetcites [28] Yeh P Chen H Andrews J Naser R Pao W Horn L DNA-Mutation Inventory to Refine and Enhance Cancer Treatment (DIRECT): A Catalog of Clinically Relevant Cancer Mutations to Enable Genome-Directed Anticancer Therapy Clin Cancer Res 2013 19 1894 1901 23344264 PMC4121886

In the paper

“Based on this measure, A763_Y764insFQEA is predicted to be almost as sensitive to gefitinib as the L858R mutant, whereas D770_N771insNPG is expected to be many fold less sensitive (Table 1).”

In the paper

“Most of these mutations lie near the end of the C-helix within the N-lobe of the kinase, after residue M766, but a small subset map to the middle of the C-helix (affecting amino-acids E762 to Y764) 5,33,34.”

What the sources say

• Not checked yetcites [5] Shigematsu H Lin L Takahashi T Nomura M Suzuki M Wistuba II Fong KM Lee H Toyooka S Shimizu N Fujisawa T Feng Z Roth JA Herz J Minna JD Gazdar AF Clinical and biological features associated with epidermal growth factor receptor gene mutations in lung cancers J Natl. Cancer Inst 2005 97 339 346 15741570
• Not checked yetcites [33] Murray S Dahabreh IJ Linardou H Manoloukos M Bafaloukos D Kosmidis P Somatic mutations of the tyrosine kinase domain of epidermal growth factor receptor and tyrosine kinase inhibitor response to TKIs in non-small cell lung cancer: an analytical database J Thorac. Oncol 2008 3 832 839 18670300
• Not checked yetcites [34] Linardou H Dahabreh IJ Bafaloukos D Kosmidis P Murray S Somatic EGFR mutations and efficacy of tyrosine kinase inhibitors in NSCLC Nat. Rev. Clin Oncol 2009 6 352 366 19483740

In the paper

“Unlike EGFR exon 19 deletions and L858R–bearing tumors, most NSCLCs with EGFR exon 20 insertion mutations don’t respond radiographically or clinically to gefitinib or erlotinib. The reported response rate (RR) is below 5% and most patients have short intervals of disease control 35.”

What the sources say

• Not checked yetcites [35] Yasuda H Kobayashi S Costa DB EGFR exon 20 insertion mutations in non-small-cell lung cancer: preclinical data and clinical implications Lancet Oncol 2012 13 e23 e31 21764376 source

In the paper

“Our study was limited by a number of factors, including a paucity of available NSCLC cell lines with EGFR exon 20 insertion mutations, a lack of in vivo models and the small number of exon 20 insertion mutated NSCLC cases that were treated with reversible EGFR TKIs in our centers.”

In the paper

“our structure and kinetic data show that D770_N771insNPG promotes the active conformation, as reflected by its enhanced catalytic activity (Table 1) and a propensity to form the asymmetric dimer as seen in the crystal structure (Sup. Fig. 10A) and in solution (Sup. Fig. 11).”

In the paper

“Alternatively, a shift toward the N-terminus would displace this residue by one turn, but effectively replace it with a glutamic acid residue introduced by the insertion (FQEA, Fig. 5A).”

In the paper

“mutation of L747 in this cluster to proline or serine is believed to underlie the activity of EGFR-exon 19 insertions 25.”

What the sources say

• Not checked yetcites [25] He M Capelletti M Nafa K Yun CH Arcila ME Miller VA Ginsberg MS Zhao B Kris MG Eck MJ Janne PA Ladanyi M Oxnard GR EGFR exon 19 insertions: a new family of sensitizing EGFR mutations in lung adenocarcinoma Clin Cancer Res 2012 18 1790 1797 22190593 PMC3306520

In the paper

“Likewise, we expect that binding of gefitinib and erlotinib would be unperturbed in this mutant, consistent with the closely similar Ki values for gefitinib we measure for D770_N771insNPG and WT EGFR (Table 1).”

In the paper

“Thus we hypothesize that the de facto I759A replacement caused by the FQEA insertion may activate EGFR via a similar mechanism (destabilization of the inactive conformation).”

In the paper

“This mode of interaction mediates EGFR activation by ligand-induced dimerization 48, and is typically recapitulated in crystals of the EGFR kinase in its active conformation.”

What the sources say

• Not checked yetcites [48] Zhang X Gureasko J Shen K Cole PA Kuriyan J An allosteric mechanism for activation of the kinase domain of epidermal growth factor receptor Cell 2006 125 1137 1149 16777603

In the paper

“the four inserted residues are expected to form approximately one turn of helix and shift the register of adjacent residues in the helix, either toward the N-terminus or toward the C-terminus (Fig. 5A).”

In the paper

“PD168393 is a pre-clinical irreversible anilinoquinazoline inhibitor that is structurally related to afatinib and dacomitinib 46,47.”

What the sources say

• Not checked yetcites [46] Engelman JA Zejnullahu K Gale CM Lifshits E Gonzales AJ Shimamura T Zhao F Vincent PW Naumov GN Bradner JE Althaus IW Gandhi L Shapiro GI Nelson JM Heymach JV Meyerson M Wong KK Janne PA PF00299804, an irreversible pan-ERBB inhibitor, is effective in lung cancer models with EGFR and ERBB2 mutations that are resistant to gefitinib Cancer Res 2007 67 11924 11932 18089823 source
• Not checked yetcites [47] Li D Ambrogio L Shimamura T Kubo S Takahashi M Chirieac LR Padera RF Shapiro GI Baum A Himmelsbach F Rettig WJ Meyerson M Solca F Greulich H Wong KK BIBW2992, an irreversible EGFR/HER2 inhibitor highly effective in preclinical lung cancer models Oncogene 2008 27 4702 4711 18408761 PMC2748240 source

In the paper

“The L858R and L861Q mutations within this cluster are well-characterized activating mutations 28”

What the sources say

• Not checked yetcites [28] Yeh P Chen H Andrews J Naser R Pao W Horn L DNA-Mutation Inventory to Refine and Enhance Cancer Treatment (DIRECT): A Catalog of Clinically Relevant Cancer Mutations to Enable Genome-Directed Anticancer Therapy Clin Cancer Res 2013 19 1894 1901 23344264 PMC4121886

In the paper

“Thus, we conclude that the insertion shifts the register of the helix in the N-terminal direction and that the glutamic acid residue in the FQEA insertion assumes the active site role of E762 in the WT kinase.”

In the paper

“L858R and exon 19 deletion mutants exhibit increased Km[ATP] and higher affinity (5–10 fold) for gefitinib or erlotinib than the WT receptor 39,50,56.”

What the sources say

• Not checked yetcites [39] Carey KD Garton AJ Romero MS Kahler J Thomson S Ross S Park F Haley JD Gibson N Sliwkowski MX Kinetic analysis of epidermal growth factor receptor somatic mutant proteins shows increased sensitivity to the epidermal growth factor receptor tyrosine kinase inhibitor, erlotinib Cancer Res 2006 66 8163 8171 16912195
• Not checked yetcites [50] Eck MJ Yun CH Structural and mechanistic underpinnings of the differential drug sensitivity of EGFR mutations in non-small cell lung cancer Biochim. Biophys. Acta 2010 1804 559 566 20026433 PMC2859716
• Not checked yetcites [56] Mulloy R Ferrand A Kim Y Sordella R Bell DW Haber DA Anderson KS Settleman J Epidermal growth factor receptor mutants from human lung cancers exhibit enhanced catalytic activity and increased sensitivity to gefitinib Cancer Res 2007 67 2325 2330 17332364

In the paper

“Structurally, EGFR-A763_Y764insFQEA is very different from other exon 20 insertions.”

In the paper

“Together with the oncogene-addicted state of tumors bearing these mutations 39,50,56, these factors explain their sensitivity to EGFR TKIs 50.”

What the sources say

• Not checked yetcites [50] Eck MJ Yun CH Structural and mechanistic underpinnings of the differential drug sensitivity of EGFR mutations in non-small cell lung cancer Biochim. Biophys. Acta 2010 1804 559 566 20026433 PMC2859716

In the paper

“we hypothesize that it leads to catalytic activation in a related manner.”

In the paper

“Together with the oncogene-addicted state of tumors bearing these mutations 39,50,56, these factors explain their sensitivity to EGFR TKIs 50.”

What the sources say

• Not checked yetcites [39] Carey KD Garton AJ Romero MS Kahler J Thomson S Ross S Park F Haley JD Gibson N Sliwkowski MX Kinetic analysis of epidermal growth factor receptor somatic mutant proteins shows increased sensitivity to the epidermal growth factor receptor tyrosine kinase inhibitor, erlotinib Cancer Res 2006 66 8163 8171 16912195
• Not checked yetcites [50] Eck MJ Yun CH Structural and mechanistic underpinnings of the differential drug sensitivity of EGFR mutations in non-small cell lung cancer Biochim. Biophys. Acta 2010 1804 559 566 20026433 PMC2859716
• Not checked yetcites [56] Mulloy R Ferrand A Kim Y Sordella R Bell DW Haber DA Anderson KS Settleman J Epidermal growth factor receptor mutants from human lung cancers exhibit enhanced catalytic activity and increased sensitivity to gefitinib Cancer Res 2007 67 2325 2330 17332364

In the paper

“The EGFR-T790M mutation confers resistance by reversing the sensitization conferred by primary activating mutations, but it does so by altering the gatekeeper residue in the ATP-site that is known to be an important determinant of inhibitor specificity 50,57.”

What the sources say

• Not checked yetcites [50] Eck MJ Yun CH Structural and mechanistic underpinnings of the differential drug sensitivity of EGFR mutations in non-small cell lung cancer Biochim. Biophys. Acta 2010 1804 559 566 20026433 PMC2859716
• Not checked yetcites [57] Yun CH Mengwasser KE Toms AV Woo MS Greulich H Wong KK Meyerson M Eck MJ The T790M mutation in EGFR kinase causes drug resistance by increasing the affinity for ATP Proc. Natl. Acad. Sci. U. S. A 2008 105 2070 2075 18227510 PMC2538882

In the paper

“Indeed, it seems that EGFR monoclonal antibodies 60 and EGFR WT kinase sparing covalent T790M selective TKIs 61 are ineffective in typical EGFR exon 20 insertion mutants.”

What the sources say

• Not checked yetcites [60] Cho J Chen L Sangji N Okabe T Yonesaka K Francis JM Flavin RJ Johnson W Kwon J Yu S Greulich HE Johnson BE Eck MJ Janne PA Wong KK Meyerson M Cetuximab response of lung cancer-derived EGF receptor mutants is associated with asymmetric dimerization Cancer Res 2013 PMC3903789 24063894
• Not checked yetcites [61] Walter AO Tjin Tham SR Haringsma HJ Ohashi K Sun J Lee K Dubrovskiy A Labenski M Zhu Z Wang Z Sheets M St Martin T Karp R van Kalken D Chaturvedi P Niu D Nacht M Petter RC Westlin W Lin K Jaw-Tsai S Raponi M Van Dyke T Etter J Weaver Z Pao W Singh J Simmons AD Harding TC Allen A Discovery of a mutant-selective covalent inhibitor of EGFR that overcomes T790M–mediated resistance in NSCLC Cancer Discov 2013 PMC4048995 24065731 source

In the paper

“our structural analysis and supporting mutagenesis indicate that the inserted FQEA sequence shifts the register of the C-helix toward its N-terminus, altering the length of the β3-αC loop leading into the helix and effectively leading to an I759A replacement.”