Poziotinib for EGFR exon 20-mutant NSCLC: Clinical efficacy, resistance mechanisms, and impact of insertion location on drug sensitivity

Abstract

We report a phase II study of 50 advanced non-small cell lung cancer (NSCLC) patients with point mutations or insertions in EGFR exon 20 treated with poziotinib (NCT03066206). The study achieved its primary endpoint, with confirmed objective response rates (ORRs) of 32% and 31% by investigator and blinded independent review, respectively, with a median progression-free survival of 5.5 months. Using preclinical studies, in silico modeling, and molecular dynamics simulations, we found that poziotinib sensitivity was highly dependent on the insertion location, with near-loop insertions (amino acids A767 to P772) being more sensitive than far-loop insertions, an observation confirmed clinically with ORRs of 46% and 0% observed in near versus far-loop, respectively (p = 0.0015). Putative mechanisms of acquired resistance included EGFR T790M, MET amplifications, and epithelial-to-mesenchymal transition (EMT). Our data demonstrate that poziotinib is active in EGFR exon 20-mutant NSCLC, although this activity is influenced by insertion location.

Keywords: epidermal growth factor receptor; exon 20 insertion; non-small cell lung carcinoma.

Conflict of interest statement

Declaration of interests The research being reported in this publication is research in which The University of Texas M.D. Anderson Cancer Center has an institutional financial conflict of interest. Because M.D. Anderson is committed to the protection of human subjects and the effective management of its financial conflicts of interest in relation to its research activities, M.D. Anderson has implemented an Institutional Conflict of Interest Management and Monitoring Plan to manage and monitor the conflict of interest with respect to M.D. Anderson’s conduct of this research. M.D. Anderson, including J.P.R., M.B.N., and J.V.H., have filed a patent for the use of poziotinib for treating EGFR and HER2 mutant cancers and licensed the technology to Spectrum Pharmaceuticals (SP). J.P.R. and J.V.H. receive research support from SP and Takeda. M.D. Anderson, including M.B.N., J.P.R., and J.V.H., have a pending patent submitted for treatment of EGFR TKI-resistant NSCLC and another, including J.P.R., S.H., and J.V.H., for the classification of EGFR mutations. J.P.R. and M.B.N. have no nonfinancial competing interests. J.P.R. is now a full-time employee of AstraZeneca. J.V.H. also receives grant or research support from AstraZeneca (AZ) and GlaxoSmithKline (GSK) and has served on advisory committees for AZ, Boehringer Ingelheim (BI), Bristol Myers Squibb, Catalyst, EMD Serono, Foundation Medicine (FMI), Hengrui Therapeutics, Genentech, GSK, Guardant Health (GH), Eli Lilly, Merck, Novartis, Pfizer, Roche, Sanofi, and Seattle Genetics. As nonfinancial competing interests, J.V.H. serves as a scientific advisor for Rexanna’s Foundation and the EGFR resisters. Y.Y.E. discloses research support from Spectrum, AstraZeneca, Takeda, Eli Lilly, Xcovery, and Tuning Point Therapeutics; and advisory role for AstraZeneca, Eli Lilly, and Turning Point; and accommodation expenses from Eli Lilly. X.L. receives consultant and advisory fees from Eli Lilly, AstraZeneca, and EMD Serono and research funding from Eli Lilly, Boehringer Ingelheim, and Spectrum Pharmaceuticals. M.A. reports research funding to the M.D. Anderson Cancer Center from Genentech, Nektar Therapeutics, Merck, GlaxoSmithKline, Novartis, Jounce Therapeutics, Bristol Myers Squibb, Eli Lilly, and Adaptimmune and receives advisory fees from GlaxoSmithKline and Shattuck Labs. V.K.L. reports advisory role fees from Takeda, Seattle Genetics, Bristol Myers Squibb, and AstraZeneca and research funding from Bristol Myers Squibb, AstraZeneca, and Merck. D.L.G. reports honoraria for scientific advisory boards of AstraZeneca, Sanofi, Alethia Biotherapeutics, Lilly, and Janssen and research support from Janssen, Takeda, Ribon Therapeutics, Astellas, and AstraZeneca. G.B. receives personal fees and research funding from Amgen, Bayer, Bristol Myers Squibb, Celgene, Daiichi Sankyo, Genentech, MedImmune, Merck, Roche, and Xcovery; research funding from Adaptimmune, Exelixis, GlaxoSmithKline, Immatics, Immunocore, Incyte, Kite Pharma, Macrogenics, Torque, AstraZeneca, Tmunity, Regeneron, Beigene, Novartis, and Repertoire Immune Medicines; and personal fees from Abbvie, Adicet, Amgen, Araid, Clovis Oncology, AstraZeneca, Bristol Myer Squibb, Celgene, Genentech, Gilead, Merck, Novartis, Roche, Virogin Biotech, Johnson & Johnson/Janssen, and Maverick Therapeutics. A.S.T. reports advisory board/consultant fees from Bristol Myers Squibb, Eli Lilly, Genentech, Roche, Novartis, Ariad, EMD Serono, Merck, Seattle Genetics, AstraZeneca, Boehringer Ingelheim, Sellas Life Science, Takeda, Epizyme, and Huron and receives research grants from Eli Lilly, Millennium, Polaris, Genentech, Merck, Boehringer Ingelheim, Bristol Myers Squibb, Ariad, Epizyme, Seattle Genetics, Takeda, and EMD Serono.

Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1.. Tumor response to poziotinib in EGFR exon 20 mutant NSCLCA

A. Waterfall plot of maximum percent decrease from baseline in the sum of diameters of target tumors based on investigator assessment in patients with evaluable disease. The dashed line at −30% represents the cutoff for RECIST response. Patients harboring point mutation(s) are indicated. SLD, sum of the longest diameters. B. Kaplan-Meier curve for estimated duration of response. Duration of response was defined as time from first objective response per RECIST 1.1 to objective disease progression or death from any cause. Median duration of response was 8.6 months (95% CI: 3.7 to 19.3) C. Kaplan-Meier curve for progression-free survival of patients treated with poziotinib (N=50). Progression free survival was defined as the time from the administration of the first dose of poziotinib to objective disease progression or death from any cause. Median progression-free survival was 5.5 months (95% CI: 5.4 to 10.4). See also Figures S1-S3 and Tables S1-S3.

Figure 2.. Duration of poziotinib treatment

Duration of poziotinib treatment in all treated patients (n=50). Each bar each bar denotes a single patient. See also Figures S4 and S5.

Figure 3.. Far loop mutants are lessensitive to poziotinib.

A. Schematic representation of the first 15 amino acids of exon 20 of EGFR divided by structural features corresponding to amino acids. Mutations are listed with the frequency observed in this study. Bars are representative of overall frequency of variants at the indicated amino acid. B-C. (B) Waterfall plot and (C) bar graph of evaluable patient confirmed response to poziotinib divided by mutation location. Objective response rate (ORR) and disease control rate (DCR) are shown for the intent to treat population for near and far. Statistical differences were determined by Chi-square test. D. Bar graph of RECIST responses for patients’ best objective response divided by amino acid location. Bars are representative of average RECIST response + SEM, and dots are representative of individual evaluable patients (n=44). Statistical differences were determined by two-tailed students’ t-test. E. Dot plot of patients’ best RECIST response after poziotinib treatment plotted against amino acid location of mutation (n=44). F. Kaplan-Meier plot of progression free survival of patients with near loop and far loop mutants in the intent to treat population. Long-Rank Mantel-Cox approach was used to determine p-value. B-F. Near loop: A767-P772, n=32 patients and far loop: H773-C775, n=12 patients.

Figure 4.. Exon 20 insertion location effects the orientation of the P-loop and drug-receptor interactions.

A. Box plot of binding pocket volumes (Å3) of poziotinib bound to S768dupSVD and H773insNPH over the duration of the simulation. B. In silico model of the most conformations of S768dupSVD (blue) and H773insNPH (purple) in poziotinib bound simulations. Dashed line shows differences in orientation of the loop connecting the α-c-helix and the ß3 strand. Red arrow shows differences in orientation of P-loop highlighting F723 that interacts with most TKIs in the binding cleft. The dark blue arrow indicates the location of the SVD insertion (light blue) at S768, and the purple arrow indicates the location of the NPH insertion (light blue) at H773. C. Table of binding energy calculations in kCal/mol for poziotinib for indicated mutations. D. In silico representation of poziotinib (purple) bound to S768dupSVD (light blue) demonstrate a small distance (black dashed line) between reactive acrylamide group (purple arrow) and reactive cysteine (dark blue arrow). E. In silico representation of poziotinib (purple) bound to H773insNPH (teal) demonstrate a larger distance (black dashed line) between reactive acrylamide group (purple arrow) and reactive cysteine (dark grey arrow). F. Distance between the reactive acrylamide (electrophile) and reactive cysteine (nucleophile) during MDS with poziotinib and S768dupSVD (black) and H773insNPH (red) over time. G. Bar graph of average IC50 values of Ba/F3 cell lines expressing EGFR exon 20 point mutation or insertion mutations treated with poziotinib for 72 hours. Dots are representative of average IC50 value of each cell line determined in triplicate, and line is representative of average IC50 value of all point mutations or insertion mutations + SEM. Differences between groups was determined by two-sided students’ t-test. H. Dot plots of average IC50 values of Ba/F3 cell lines expressing various EGFR exon 20 insertion mutations (n = 24 cell lines) treated with poziotinib compared to amino acid residue of EGFR exon 20 insertion mutation. Red dashed line indicates the average IC50 value of Ba/F3 cells expressing WT EGFR + 10ng/ml EGF. Dots are representative of the average IC50 value determined in biological triplicate. Data was fit to a linear regression model, and two-tailed Pearson correlation was determined. See also Figure S6.

Figure 5:. Poziotinib resistance may be driven by EMT but retains sensitivity to SAC inhibitors in preclinical models.

A. IC50 values of Ba/F3 cells expressing the indicated EGFR exon 20 mutations. Bars are representative of the average IC50 value ± SEM. Dotted red line indicates the average IC50 value of Ba/F3 cells expressing WT EGFR. All IC50 values were determined in at least three independent replicates. B.In silico modelling of EGFR D770insNPG (purple) with T790M (orange) and poziotinib (blue). The methionine at 790 (orange) displaces the terminal halogenated ring of poziotinib (blue) away from the hydrophobic cleft, increasing the distance between the quinazoline core of poziotinib and methionine 793 (yellow, dashed lines). C. Heatmap of protein expression from YUL-0019 (YUL) and YUL-0019 poziotinib resistant (PR) cell lines as determined by RPPA analysis of EMT associated proteins. D. Dot plots of relative fold change in protein expression determined by RPPA for indicated EMT associated markers in YUL-0019 parental (YUL) and YUL-0019 PR cell lines (PR), normalized to YUL parental controls. Dots are representative of fold change in protein expressing for biological replicates for YUL-0019 parental cells and the average of n=4 replicates for each poziotinib-resistant cell line (PR1-PR-8). Bars are representative of average ± standard deviation (SD), and p-values were determined by two-sided, unpaired students’ t-test. E. Average IC50 values of indicated inhibitors as determined by the colony formation assay after 14 days. Bars are representative of average ± SEM (n=2). See also Figure S7.

Figure 6:. Resistance to poziotinib is driven by both EGFR-dependent and –independent mechanisms

A. Potential poziotinib-acquired resistance mechanisms identified in 14 out of 23 patients with matched pre-poziotinib and on disease progression samples. Each column represents a patient. The letters on top of each column denotes the primary exon 20 mutation as follows: A: H773_V774VdupHV, B: D770_N771insG, C: S768_D770dupSVD, D: H773_V774insAH, E: A767_V769dupASV, F: D770_N771dupDN, G: H773dupH, H: P772_H773dupPH, I: P772_H773insPNP. The left-side column lists the alterations acquired at resistance. Red box: mutation, blue box: amplification. Objective response to poziotinib is shown in the bottom row. Green box: partial response, orange box: stable disease. B. Relative mRNA expression of epithelial and mesenchymal markers determined by RT-PCR from indicated cell lines. Bars are representative of average ± standard deviation (SD) fold change in mRNA expression normalized to GAPDH and YUL-0019 cell line expression, and p-values were determined by two-sided unpaired t-tests. C. Western blot of epithelial and mesenchymal markers from YUL-0019 and MDA-004K cell lines. See also Figure S8 and Table S4.