In responses to activation of receptor tyrosine kinases (RTKs), crucial cell fate decisions depend on the duration and dynamics of ERK signaling. In PC12 cells, epidermal growth factor (EGF) induces transient ERK activation that leads to cell proliferation, whereas nerve growth factor (NGF) promotes sustained ERK activation and cell differentiation. These differences have typically been assumed to reflect distinct feedback mechanisms in the Raf-MEK-ERK signaling network, with the receptors themselves acting as simple upstream inputs. We failed to confirm the expected differences in feedback type when investigating transient versus sustained signaling downstream of the EGF receptor (EGFR) and NGF receptor (TrkA). Instead, we found that ERK signaling faithfully followed RTK dynamics when receptor signaling was modulated in different ways. EGFR activation kinetics, and consequently ERK signaling dynamics, were switched from transient to sustained when receptor internalization was inhibited with drugs or mutations, or when cells expressed a chimeric receptor likely to have impaired dimerization. In addition, EGFR and ERK signaling both became more sustained when substoichiometric levels of erlotinib were added to reduce duration of EGFR kinase activation. Our results argue that RTK activation kinetics play a crucial role in determining MAP kinase cascade signaling dynamics and cell fate decisions, and that signaling outcome can be modified by activating a given RTK in different ways.

◥Osimertinib, a mutant-specific third-generation EGFR tyrosine kinase inhibitor, is emerging as the preferred first-line therapy for EGFR-mutant lung cancer, yet resistance inevitably develops in patients. We modeled acquired resistance to osimertinib in transgenic mouse models of EGFR L858R -induced lung adenocarcinoma and found that it is mediated largely through secondary mutations in EGFR-either C797S or L718V/Q. Analysis of circulating free DNA data from patients revealed that L718Q/V mutations almost always occur in the context of an L858R driver mutation. Therapeutic testing in mice revealed that both erlotinib and afatinib caused regression of osimertinib-resistant C797S-containing tumors, whereas only afatinib was effective on L718Q mutant tumors. Combination first-line osimertinib plus erlotinib treatment prevented the emergence of secondary mutations in EGFR. These findings highlight how knowledge of the specific characteristics of resistance mutations is important for determining potential subsequent treatment approaches and suggest strategies to overcome or prevent osimertinib resistance in vivo.Significance: This study provides insight into the biological and molecular properties of osimertinib resistance EGFR mutations and evaluates therapeutic strategies to overcome resistance.

Osimertinib, a mutant‐specific third generation EGFR TKI, is emerging as the preferred first‐line therapy for EGFR mutant lung cancer. Despite initial responses in patients, however, resistance inevitably develops over time. In order to investigate mechanisms of resistance to first‐line osimertinib, we modeled acquired resistance to this drug in transgenic mouse models of EGFRL858R‐induced lung adenocarcinoma and found that it is mediated largely through secondary mutations in EGFR – either C797S or L718V/Q (Figure 1A and 1B). Analysis of circulating free DNA data from patients with EGFR mutant lung cancer revealed that L718Q/V mutations almost always arise in the context of an L858R driver mutation. Therapeutic testing in mice revealed that both erlotinib and afatinib caused regression of osimertinib‐resistant C797S‐containing tumors, whereas only afatinib was effective in L718Q mutant tumors (Figure 1C and 1D). Combination first‐line osimertinib plus erlotinib treatment prevented the emergence of secondary mutations in EGFR. Our data identify specific secondary EGFR mutations as a major mechanism of acquired resistance to first‐line osimertinib treatment and highlight potential strategies to overcome or prevent osimertinib resistance in vivo. Furthermore, these findings emphasize how knowledge of the specific characteristics of resistance mutations are important for determining potential subsequent treatment approaches. Support or Funding Information This work was supported by ‐‐‐‐‐Yale’s Specialized Program of Research Excellence in Lung Cancer grant (to K. Politi, S.B. Goldberg and M.A. Lemmon) and funding from AstraZeneca (to K. Politi). Additional support came from the NIH/NCI‐funded Yale Cancer Biology Training Program T32 CA193200‐01A1 and F31 CA228268‐01A1 (to J.H. Starrett), R01 CA198164 (M.A. Lemmon), the Ginny and Kenneth Grunley Fund for Lung Cancer Research, and the Canadian Institutes of Health Research Project Grant PJT‐148725 (to W.W. Lockwood). W.W. Lockwood is supported by a Michael Smith Foundation for Health Research Scholar and NIHR New Investigator Awards, A. Guernet is a fellow funded by the IMED AstraZeneca postdoc program, A. Nagelberg is supported by a scholarship from the CIHR, and K.D. Ashtekar is an Arnold and Mabel Beckman Foundation Postdoctoral Fellow. Yale Cancer Center Shared Resources used for this work were in part supported by NIH/NCI Cancer Center Support Grant P30 CA016359. Acquired resistance to first‐line osimertinib arises partially due to the emergence of secondary mutations in EGFR, which are differentially sensitive to other EGFR TKIs. A. Schema of the experiment. CCSP‐rtTA;TetO‐EGFRL858R mice were administered doxycycline (dox) for the duration of the experiment and developed tumors after ~6 weeks on dox. When tumors were detected by MRI (see pre‐treatment image), osimertinib treatment was initiated (25 mg/kg QD M‐F) which elicited a response (see representative response MRI) and treated until the emergence of resistant tumors by MRI. Coronal MR images are shown...

Tyrosine kinase inhibitors (TKIs) are used to treat non-small cell lung cancers (NSCLC) driven by epidermal growth factor receptor (EGFR) mutations in the tyrosine kinase domain (TKD). TKI responses vary across tumors driven by the heterogeneous group of exon 19 deletions and mutations, but the molecular basis for these differences is not understood. Using purified TKDs, we compared kinetic properties of several exon 19 variants. Although unaltered for the second generation TKI afatinib, sensitivity varied significantly for both the first and third generation TKIs erlotinib and osimertinib. The most sensitive variants showed reduced ATP-binding affinity, whereas those associated with primary resistance retained wild type ATP-binding characteristics (and low KM, ATP). Through crystallographic and hydrogen-deuterium exchange mass spectrometry (HDX-MS) studies, we identify possible origins for the altered ATP-binding affinity underlying TKI sensitivity and resistance, and propose a basis for classifying uncommon exon 19 variants that may have predictive clinical value.

Purpose: The uncommon EGFR exon 19 deletion (ex19del), L747_A750>P, demonstrates reduced sensitivity to osimertinib compared to the common ex19del, E746_A750del in preclinical models. The clinical efficacy of osimertinib in patients with non-small cell lung cancer (NSCLC) harboring L747_A750>P and other uncommon ex19dels is not known. Design: The AACR GENIE database was interrogated to characterize the frequency of individual ex19dels relative to other variants, and a multi-center retrospective cohort was used to compare clinical outcomes for patients with tumors harboring E746_A750del, L747_A750>P, and other uncommon ex19dels who received osimertinib in the first line (1L) or in second or later lines of therapy and were T790M+ (≥2L). Results: Ex19dels comprised 45% of EGFR mutations, with 72 distinct variants ranging in frequency from 28.1% (E746_A750del) to 0.03%, with L747_A750>P representing 1.8% of the EGFR mutant cohort. In our multi-institution cohort (N=200), E746_A750del was associated with significantly prolonged progression free survival (PFS) with 1L osimertinib vs. L747_A750>P (median 21.3 months [95% CI 17.0-31.7] vs. 11.7 months [10.8-29.4], adjusted hazard ratio [HR] 0.52 [0.28-0.98] p=0.043). Osimertinib efficacy in patients with other uncommon ex19dels varied based on the specific mutation present. Conclusions: The ex19del L747_A750>P is associated with inferior PFS compared to the common E746_A750del mutation in patients treated with 1L osimertinib. Understanding differences in osimertinib efficacy among EGFR ex19del

<div>AbstractPurpose:<p>The uncommon <i>EGFR</i> exon 19 deletion (ex19del), L747_A750>P, demonstrates reduced sensitivity to osimertinib compared with the common ex19del, E746_A750del in preclinical models. The clinical efficacy of osimertinib in patients with non–small cell lung cancer harboring L747_A750>P and other uncommon ex19dels is not known.</p>Design:<p>The AACR GENIE database was interrogated to characterize the frequency of individual ex19dels relative to other variants, and a multicenter retrospective cohort was used to compare clinical outcomes for patients with tumors harboring E746_A750del, L747_A750>P, and other uncommon ex19dels who received osimertinib in the first line (1L) or in second or later lines of therapy and were T790M+ (≥2L).</p>Results:<p>ex19dels comprised 45% of <i>EGFR</i> mutations, with 72 distinct variants ranging in frequency from 28.1% (E746_A750del) to 0.03%, with L747_A750>P representing 1.8% of the <i>EGFR</i> mutant cohort. In our multi-institutional cohort (<i>N</i> = 200), E746_A750del was associated with significantly prolonged progression-free survival (PFS) with 1L osimertinib versus L747_A750>P [median 21.3 months (95% confidence interval, 17.0–31.7) vs. 11.7 months (10.8–29.4); adjusted HR 0.52 (0.28–0.98); <i>P</i> = 0.043]. Osimertinib efficacy in patients with other uncommon ex19dels varied on the basis of the specific mutation present.</p>Conclusions:<p>The ex19del L747_A750>P is associated with inferior PFS compared with the common E746_A750del mutation in patients treated with 1L osimertinib. Understanding differences in osimertinib efficacy among EGFR ex19del subtypes could alter management of these patients in the future.</p></div>

<div>AbstractPurpose:<p>The uncommon <i>EGFR</i> exon 19 deletion (ex19del), L747_A750>P, demonstrates reduced sensitivity to osimertinib compared with the common ex19del, E746_A750del in preclinical models. The clinical efficacy of osimertinib in patients with non–small cell lung cancer harboring L747_A750>P and other uncommon ex19dels is not known.</p>Experimental Design:<p>The AACR GENIE database was interrogated to characterize the frequency of individual ex19dels relative to other variants, and a multicenter retrospective cohort was used to compare clinical outcomes for patients with tumors harboring E746_A750del, L747_A750>P, and other uncommon ex19dels who received osimertinib in the first line (1L) or in second or later lines of therapy and were T790M+ (≥2L).</p>Results:<p>ex19dels comprised 45% of <i>EGFR</i> mutations, with 72 distinct variants ranging in frequency from 28.1% (E746_A750del) to 0.03%, with L747_A750>P representing 1.8% of the <i>EGFR</i> mutant cohort. In our multi-institutional cohort (<i>N</i> = 200), E746_A750del was associated with significantly prolonged progression-free survival (PFS) with 1L osimertinib versus L747_A750>P [median 21.3 months (95% confidence interval, 17.0–31.7) vs. 11.7 months (10.8–29.4); adjusted HR 0.52 (0.28–0.98); <i>P</i> = 0.043]. Osimertinib efficacy in patients with other uncommon ex19dels varied on the basis of the specific mutation present.</p>Conclusions:<p>The ex19del L747_A750>P is associated with inferior PFS compared with the common E746_A750del mutation in patients treated with 1L osimertinib. Understanding differences in osimertinib efficacy among EGFR ex19del subtypes could alter management of these patients in the future.</p></div>

<p>Supplementary Figure S3. Progression Free Survival for patients with tumors harboring select exon 19 deletions treated with second or later line osimertinib (T790M+). The point estimate for median progression free survival associated with each mutation is listed to the right of the figure legend.</p>