CHIR-124 kinase domain mutants against ERBB2 inhibitors. Lapatinib is a dual inhibitor of EGFR and ERBB2 kinases. In the present study the efficacy of lapatinib against ERBB2 variants was studied. Moreover, a cell based screening strategy was employed to identify lapatinib resistant ERBB2 kinase domain mutations. The effect of another reversible dual EGFR/ERBB2 inhibitor AEE 788, was tested against ERBB2 mutants. Together, comprehensive drug sensitivity profiles for various ERBB2 mutations that were reported in several cancers were established along with the identification of lapatinib resistant mutations. Furthermore, irreversible ERBB2 inhibitors were identified which potentially can overcome lapatinib resistance.
Results and Discussion Identification of lapatinib resistant ERBB2 kinase domain mutations It has been demonstrated that the drug sensitivity of different mutations varies against selective inhibitors. Thus, we aimed to PLoS ONE | www.plosone.org 1 October 2011 | Volume 6 | Issue BIX 02189 10 | e26760 8 test the efficacy of reversible ERBB2 inhibitors lapatinib and AEE788 against a panel of ERBB2 kinase domain mutations that were reported in various solid cancers. Analogous mutations in EGFR were reported for most of the ERBB2 mutations analyzed in this study, suggesting that these mutations are not passenger mutations but functionally important. Additionally, a gatekeeper mutation T798M was cloned for analysis. ERBB2 T798M is analogous to EGFR T790M that was shown to cause resistance towards EGFR inhibitors.
The locations of the kinase domain mutants investigated in this study are depicted in Figure 1. Four mutations are located in the N lobe of the kinase. L755 is located at a loop adjacent to helix C, V773 and V777 are at or near the C terminal portion of helix C, and T798 is at the gatekeeper position in the ATP binding site. Of the remainder, N857 is located in helix D, T862A forms the base of the ATP binding site, and H878 is in the activation loop. All the mutations analyzed retained autokinase activity and activated downstream signaling pathways when expressed in HEK293 cells. Moreover mutations L755S, L755P, V777L, T798M and T862A displayed enhanced activation of JNK/SAPK and to a lesser extent of ERK1/2 compared to wt ERBB2.
Enhanced autophosphorylation as well as activation of downstream signaling molecules was also observed upon stimulation with either EGF or heregulin of serum starved HEK293 cells expressing ERBB2 in combination with EGFR or ERBB3 indicating that the mutations did not interfere with ligand induced heterodimerization of the ERBB2 mutants Table 1. Summary of ERBB2 mutants analyzed along with the IC50 values against reversible inhibitors lapatinib and AEE788. ERBB2 mutation Exon Functional region Cancer type Lapatinib AEE788 Reference WT NA NA Breast cancer 30 257 NA L755S 19 ATP binding region Breast and gastric cancer.2000 897 4 L755P 19 ATP binding region NSCLC 1545 1216 2,3 V773A 20 ATP binding region SCCHN 146 200 6 V777L 20 ATP binding region Gastric, colon and lung 27 215 3,4 T798M 20 Gate keeper residue NA 1433.2000 NA N857S 21 Activation loop Ovarian cancer 75 246 2 T862A 21 Activation loop Primary gastric cancer 125 191 7 H878Y 21 Activation loop Hepatocellular carcinoma 14 168 5 ERBB2 kinase domain mutations that were repor