◥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.
Phenotype-based screening can identify small molecules that elicit a desired cellular response, but additional approaches are required to characterize their targets and mechanisms of action. Here, we show that a compound termed LCS3, which selectively impairs the growth of human lung adenocarcinoma (LUAD) cells, induces oxidative stress. To identify the target that mediates this effect, we use thermal proteome profiling (TPP) and uncover the disulfide reductases GSR and TXNRD1 as targets. We confirm through enzymatic assays that LCS3 inhibits disulfide reductase activity through a reversible, uncompetitive mechanism. Further, we demonstrate that LCS3-sensitive LUAD cells are sensitive to the synergistic inhibition of glutathione and thioredoxin pathways. Lastly, a genome-wide CRISPR knockout screen identifies NQO1 loss as a mechanism of LCS3 resistance. This work highlights the ability of TPP to uncover targets of small molecules identified by high-throughput screens and demonstrates the potential therapeutic utility of inhibiting disulfide reductases in LUAD.
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...
Small-cell lung cancer (SCLC) is neuroendocrine in origin and rarely contains mutations in the MAPK pathway. Likewise, non-SCLC (NSCLC) that transform to SCLC concomitantly with development of therapy resistance downregulate MAPK signaling, suggesting an inverse relationship between pathway activation and lineage state. To test this, we activated MAPK in SCLC through expression of mutant KRAS or EGFR, which revealed suppression of the neuroendocrine differentiation via ERK. We found that ERK induces expression of ETS factors that mediate transformation into a NSCLC-like state. ATAC-seq demonstrated ERK-driven changes in chromatin accessibility at putative regulatory regions and global chromatin rewiring at neuroendocrine and ETS transcriptional targets. Further, induction of ETS factors and suppression of neuroendocrine differentiation were dependent on histone acetyltransferases CBP/p300. Overall, we describe how the ERK-CBP/p300-ETS axis promotes a lineage shift between neuroendocrine and non-neuroendocrine phenotypes and provide rationale for the disruption of this program during transformation-driven resistance to targeted therapy.
MEK inhibitors (MEKi) have limited efficacy in KRAS mutant lung adenocarcinoma (LUAD) patients, and this is attributed to both intrinsic and adaptive mechanisms of drug resistance. While many studies have focused on the former, there remains a dearth of data regarding acquired resistance to MEKi in LUAD. We established trametinib-resistant KRAS mutant LUAD cells through dose escalation and performed targeted MSK-IMPACT sequencing to identify drivers of MEKi resistance. Comparing resistant cells to their sensitive counterparts revealed alteration of genes associated with trametinib response. We describe a state of “drug addiction” in resistant cases where cells are dependent on continuous culture in trametinib for survival. We show that dependence on ERK2 suppression underlies this phenomenon and that trametinib removal hyperactivates ERK, resulting in ER stress and apoptosis. Amplification of KRASG12C occurs in drug-addicted cells and blocking mutant-specific activity with AMG 510 rescues the lethality associated with trametinib withdrawal. Furthermore, we show that increased KRASG12C expression is lethal to other KRAS mutant LUAD cells, consequential to ERK hyperactivation. Our study determines the drug-addicted phenotype in lung cancer is associated with KRAS amplification and demonstrates that toxic acquired genetic changes can develop de novo in the background of MAPK suppression with MEK inhibitors. We suggest that the presence of mutant KRAS amplification in patients may identify those that may benefit from a “drug holiday” to circumvent drug resistance. These findings demonstrate the toxic potential of hyperactive ERK signaling and highlight potential therapeutic opportunities in patients bearing KRAS mutations.
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