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Supplemental Table 1.

Figure S1. Fifty percent (50%) inhibitory concentration of neratinib, afatinib and dacomitinib in Ba/F3 cells expressing different EGFR or HER2 exon 20 insertion mutations and the EGFR exon 20 insertion patient derived cell lines. to second generation EGFR-TKIs; Figure S2. Efficacy of neratinib, afatinib or dacomitinib against HER2 exon 20 insertion mutants measured in a soft agar assay; Figure S3. Patient derived EGFR exon 20 insertion cell lines; Figure S4. Comparison of amino acid sequences of exon 20 of EGFR to HER2.

Insertion mutations in EGFR and HER2 both occur at analogous positions in exon 20. Non–small cell lung cancer (NSCLC) patients with tumors harboring these mutations seldom achieve clinical responses to dacomitinib and afatinib, two covalent quinazoline–based inhibitors of EGFR or HER2, respectively. In this study, we investigated the effects of specific EGFR and HER2 exon 20 insertion mutations from NSCLC patients that had clinically achieved a partial response after dacomitinib treatment. We identified Gly770 as a common feature among the drug-sensitive mutations. Structural modeling suggested that this mutation may facilitate inhibitor binding to EGFR. Introduction of Gly770 into two dacomitinib-resistant EGFR exon 20 insertion mutants restored sensitivity to dacomitinib. Based on these findings, we used afatinib to treat an NSCLC patient whose tumor harbored the HER2 V777_G778insGSP mutation and achieved a durable partial response. We further identified secondary mutations in EGFR (T790M or C797S) and HER2 (C805S) that mediated acquired drug resistance in drug-sensitive EGFR or HER2 exon 20 insertion models. Overall, our findings identified a subset of EGFR and HER2 exon 20 insertion mutations that are sensitive to existing covalent quinazoline–based EGFR/HER2 inhibitors, with implications for current clinical treatment and next-generation small-molecule inhibitors. Cancer Res; 77(10); 2712–21. ©2017 AACR.

Pancreatic Ductal Adenocarcinoma (PDAC) is a lethal disease with a five-year survival rate of 11%. PDAC develops from pancreatic precursor lesions, including intraductal papillary mucinous neoplasm (IPMN); studying these lesions is important to understand the multi-step process of pancreatic tumorigenesis. Development of model systems and associated tools is a critical step that is 1) scientifically necessary to understand IPMN biology and 2) clinically important for improving patient outcome. To date, only one patient-derived IPMN cell line that grows in a monolayer has been reported, illustrating the challenges of growing precancerous cells in this format. While protocols have previously been established to generate patient-derived IPMN models, the majority of these cell-based models utilize 3D organoids. To date, no study has successfully converted patient-derived IPMN organoids into 2D culture. Converting these cellular models to propagate and divide in a monolayer allows us to expand our in vitro toolbox to perform experimental methods that are less efficient or not practical in 3D culture. In this study, we optimized the conversion of a high-grade IPMN patient-derived organoid model into a monolayer culture. We considered conditions that could recapitulate an extracellular matrix as well as growth factors and/or inhibitors that could promote IPMN cell growth. In total, we tested 15 different conditions and were able to successfully culture the human high-grade IPMN cells in a monolayer culture for up to 5 passages (~1 month). Additionally, while human IPMN cellular models exists, genetic modification of these in vitro models has not been reported. Such approaches allow direct interrogation of the role of specific genes in premalignant pancreatic tumorigenesis. In this study, we report successful transduction of IPMN cells via lentivirus. By first converting the organoid into a monolayer culture, transducing them in 2D, and then converting them back into 3D culture as spheroids, we were able to successfully express EGFP into our IPMN cellular model. In summary, we optimized culture conditions to temporarily grow a patient derived 3D IPMN model in a 2D monolayer form, genetically modified this IPMN cell line model, and successfully converted it back into a 3D spheroid. These protocols have potential to be replicated in other IPMN models and can provide further insight into overall IPMN biology and pancreatic tumorigenesis. Citation Format: Raymond M. Paranal, Julie Schlanz, Maria A. Trujillo, Nicholas J. Roberts, Laura D. Wood. Optimizing an in vitro toolbox to interrogate pancreatic tumorigenesis from a patient-derived intraductal papillary mucinous neoplasm sample [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6511.

The clinical efficacy of epidermal growth factor receptor (EGFR)–targeted therapy in EGFR-mutant non–small cell lung cancer is limited by the development of drug resistance. One mechanism of EGFR inhibitor resistance occurs through amplification of the human growth factor receptor (MET) proto-oncogene, which bypasses EGFR to reactivate downstream signaling. Tumors exhibiting concurrent EGFR mutation and MET amplification are historically thought to be codependent on the activation of both oncogenes. Hence, patients whose tumors harbor both alterations are commonly treated with a combination of EGFR and MET tyrosine kinase inhibitors (TKIs). Here, we identify and characterize six patient-derived models of EGFR-mutant, MET-amplified lung cancer that have switched oncogene dependence to rely exclusively on MET activation for survival. We demonstrate in this MET-driven subset of EGFR TKI-refractory cancers that canonical EGFR downstream signaling was governed by MET, even in the presence of sustained mutant EGFR expression and activation. In these models, combined EGFR and MET inhibition did not result in greater efficacy in vitro or in vivo compared to single-agent MET inhibition. We further identified a reduced EGFR:MET mRNA expression stoichiometry as associated with MET oncogene dependence and single-agent MET TKI sensitivity. Tumors from 10 of 11 EGFR inhibitor–resistant EGFR-mutant, MET-amplified patients also exhibited a reduced EGFR:MET mRNA ratio. Our findings reveal that a subset of EGFR-mutant, MET-amplified lung cancers develop dependence on MET activation alone, suggesting that such patients could be treated with a single-agent MET TKI rather than the current standard-of-care EGFR and MET inhibitor combination regimens.

A subset of drug-resistant EGFR -mutant MET -amplified lung cancer switches oncogenic dependence to MET and is treatable with MET inhibitor monotherapy.

Insertion mutations in EGFR and HER2 both occur at analogous positions in exon 20. Non–small cell lung cancer (NSCLC) patients with tumors harboring these mutations seldom achieve clinical responses to dacomitinib and afatinib, two covalent quinazoline–based inhibitors of EGFR or HER2, respectively. In this study, we investigated the effects of specific EGFR and HER2 exon 20 insertion mutations from NSCLC patients that had clinically achieved a partial response after dacomitinib treatment. We identified Gly770 as a common feature among the drug-sensitive mutations. Structural modeling suggested that this mutation may facilitate inhibitor binding to EGFR. Introduction of Gly770 into two dacomitinib-resistant EGFR exon 20 insertion mutants restored sensitivity to dacomitinib. Based on these findings, we used afatinib to treat an NSCLC patient whose tumor harbored the HER2 V777_G778insGSP mutation and achieved a durable partial response. We further identified secondary mutations in EGFR (T790M or C797S) and HER2 (C805S) that mediated acquired drug resistance in drug-sensitive EGFR or HER2 exon 20 insertion models. Overall, our findings identified a subset of EGFR and HER2 exon 20 insertion mutations that are sensitive to existing covalent quinazoline–based EGFR/HER2 inhibitors, with implications for current clinical treatment and next-generation small-molecule inhibitors. Cancer Res; 77(10); 2712–21. ©2017 AACR.

The mechanism by which the wild-type KRAS allele imparts a growth inhibitory effect to oncogenic KRAS in various cancers, including lung adenocarcinoma (LUAD), is poorly understood. Here, using a genetically inducible model of KRAS loss of heterozygosity (LOH), we show that KRAS dimerization mediates wild-type KRAS-dependent fitness of human and murine KRAS mutant LUAD tumor cells and underlies resistance to MEK inhibition. These effects are abrogated when wild-type KRAS is replaced by KRASD¹⁵⁴Q, a mutant that disrupts dimerization at the α4-α5 KRAS dimer interface without changing other fundamental biochemical properties of KRAS, both in vitro and in vivo. Moreover, dimerization has a critical role in the oncogenic activity of mutant KRAS. Our studies provide mechanistic and biological insights into the role of KRAS dimerization and highlight a role for disruption of dimerization as a therapeutic strategy for KRAS mutant cancers. The tumor-suppressive function of wild-type KRAS depends on its dimerization capacity with mutant KRAS.

Targeted covalent small molecules have shown promise for cancers driven by KRAS G12C. Allosteric compounds that access an inducible pocket formed by movement of a dynamic structural element in KRAS, switch II, have been reported, but these compounds require further optimization to enable their advancement into clinical development. We demonstrate that covalent quinazoline-based switch II pocket (SIIP) compounds effectively suppress GTP loading of KRAS G12C, MAPK phosphorylation, and the growth of cancer cells harboring G12C. Notably we find that adding an amide substituent to the quinazoline scaffold allows additional interactions with KRAS G12C, and remarkably increases the labeling efficiency, potency, and selectivity of KRAS G12C inhibitors. Structural studies using X-ray crystallography reveal a new conformation of SIIP and key interactions made by substituents located at the quinazoline 2-, 4-, and 7-positions. Optimized lead compounds in the quinazoline series selectively inhibit KRAS G12C-dependent signaling and cancer cell growth at sub-micromolar concentrations.

Introduction: Non-small cell lung cancers harboring sensitizing gain-of-function mutations in the epidermal growth factor receptor (EGFR) are treated with small molecule EGFR kinase inhibitors including erlotinib and osimertinib. Unfortunately, the clinical efficacy of these inhibitors is limited by progression to drug resistance. Genomic amplification of the hepatocyte growth factor receptor (MET) is a prevalent mechanism of resistance to clinical EGFR inhibitors. EGFR mutant lung cancers with MET amplification are co-dependent on signaling through EGFR and MET kinases, whereby either oncogene can phosphorylate the EGFR family receptor ERBB3, activating downstream signaling and promoting survival. These EGFR/MET co-dependent cancers are characteristically resistant to treatment with individual drugs, but retain sensitivity to combination treatment with EGFR and MET inhibitors. Here, we describe three patient-derived EGFR mutant, MET amplified lung cancer models that exhibit a switch away from EGFR, to MET oncogene dependence. Methods: We established and characterized patient-derived lung cancer cell lines and xenograft models harboring concurrent EGFR activating mutations (EGFR L858R or EGFR Del19) and genomic MET copy number gain. Three patient-derived models—DFCI81, DFCI161, and DFCI307—were developed from pleural effusion and core biopsy specimens of patients whose tumors progressed on erlotinib. Clinical samples were immediately sorted and stably cultured in vitro or engrafted into immunodeficient mice. Results: DFCI81, DFCI161, and DFCI307 cell lines and xenograft models retained mutant EGFR expression, but exhibited resistance to clinical EGFR inhibitors and sensitivity to single-agent c-MET inhibitors, including crizotinib and savolitinib. Comparing our EGFR-mutant and MET-dependent cell lines to EGFR/MET-codependent models, we observed that EGFR expression was significantly reduced in our models compared to controls. Ectopic overexpression of EGFR Del19 and EGFR L858R in DFCI81 and DFCI161 cell lines, respectively, was sufficient to confer crizotinib resistance and induce EGFR/MET co-dependency. We demonstrated that MET-mediated ERBB3 phosphorylation drives downstream PI3K activation to promote cell proliferation and survival in DFCI81 and DFCI161 cells. In these contexts, ERBB3 reactivation by recombinant ligand treatment was sufficient to induce EGFR-mediated ERBB3 activation, conferring resistance to single-agent crizotinib treatment. Conclusions: We have identified and characterized three patient-derived models of treatment refractory EGFR mutant lung cancer that exhibit a switch to MET oncogene dependency. Clinically, we predict a subset of EGFR mutant, MET-dependent tumors exists, and can be identified de novo by a reduced EGFR to MET expression ratio: a potential biomarker predictive of sensitivity to single-agent MET inhibition. Citation Format: Pinar Ö. Eser, Raymond M. Paranal, Michael J. Poitras, Man Xu, Stephen Wang, Atsuko Ogino, Jihyun Choi, Pavlos Missios, Heidi M. Haikala, Jieun Son, Mika Lin, Masahiko Yanagita, Prafulla C. Gokhale, George Q. Daley, Pasi A. Jänne. Switch to MET oncogene dependence as a novel mechanism of drug resistance in EGFR-mutant lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1732.