◥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...

Introduction: Targeted therapies for lung adenocarcinoma (LUAD) have improved patient outcomes; however, drug resistance remains a major problem. One strategy to achieve durable response is to develop combination-based therapies that target both mutated oncogenes and key modifiers of oncogene-driven tumorigenesis. This is based on the premise that mutated oncogenes, although necessary, are not sufficient for malignant transformation. We aimed to uncover genetic alterations that cooperate with mutant EGFR during LUAD development. Methods: We performed integrative genomic analyses, combining copy number, gene expression and mutational information for over 500 LUAD tumors. Co-immunoprecipitation and Western blot analysis were performed in LUAD cell lines to confirm candidate interactions while RNA interference and gene overexpression were used for in vitro and in vivo functional assessment. Results: We identified frequent amplifications/deletions of chromosomal regions affecting the activity of genes specifically in the context of EGFR mutation, including amplification of the mutant EGFR allele and deletion of dual specificity phosphatase 4 (DUSP4), which have both previously been reported. In addition, we identified the novel amplification of a segment of chromosome arm 16p in mutant-EGFR tumors corresponding to increased expression of Golgi Associated, Gamma Adaptin Ear Containing, ARF Binding Protein 2 (GGA2), which functions in protein trafficking and sorting. We found that GGA2 interacts with EGFR, increases EGFR protein levels and modifies EGFR degradation after ligand stimulation. Furthermore, we show that overexpression of GGA2 enhances EGFR mediated transformation while GGA2 knockdown reduces the colony and tumor forming ability of EGFR mutant LUAD. Conclusions: These data suggest that overexpression of GGA2 in LUAD tumors results in the accumulation of EGFR protein and increased EGFR signaling, which

Targeting the epigenome to modulate gene expression programs driving cancer development has emerged as an exciting avenue for therapeutic intervention. Pharmacological inhibition of the bromodomain and extraterminal (BET) family of chromatin adapter proteins has proven effective in this regard, suppressing growth of diverse cancer types mainly through downregulation of the c-MYC oncogene, and its downstream transcriptional program. While initially effective, resistance to BET inhibitors (BETi) typically occurs through mechanisms that reactivate MYC expression. We have previously shown that lung adenocarcinoma (LAC) is inhibited by JQ1 through suppression of FOSL1, suggesting that the epigenetic landscape of tumor cells from different origins and differentiation states influences BETi response. Here, we assessed how these differences affect mechanisms of BETi resistance through the establishment of isogenic pairs of JQ1 sensitive and resistant LAC cell lines. We found that resistance to JQ1 in LAC occurs independent of FOSL1 while MYC levels remain unchanged between resistant cells and their JQ1-treated parental counterparts. Furthermore, while epithelial–mesenchymal transition (EMT) is observed upon resistance, TGF-β induced EMT did not confer resistance in JQ1 sensitive LAC lines, suggesting this is a consequence, rather than a driver of BETi resistance in our model systems. Importantly, siRNA knockdown demonstrated that JQ1 resistant cell lines are still dependent on BRD4 expression for survival and we found that phosphorylation of BRD4 is elevated in resistant LACs, identifying casein kinase 2 (CK2) as a candidate protein mediating this effect. Inhibition of CK2, as well as downstream transcriptional targets of phosphorylated BRD4—including AXL and activators of the PI3K pathway—synergize with JQ1 to inhibit BETi resistant LAC. Overall, this demonstrates that the mechanism of resistance to BETi varies depending on cancer type, with LAC cells developing JQ1 resistance independent of MYC regulation, and identifying CK2 phosphorylation of BRD4 as a potential target to overcome resistance in this cancer.