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Lung cancer is the leading cause of cancer mortality worldwide. The treatment of patients with lung cancer harboring mutant EGFR with orally administered EGFR tyrosine kinase inhibitors (TKI) has been a paradigm shift. Osimertinib and rociletinib are third-generation irreversible EGFR TKIs targeting the EGFR T790M mutation. Osimertinib is the current standard of care for patients with EGFR mutations due to increased efficacy, lower side effects, and enhanced brain penetrance. Unfortunately, all patients develop resistance. Genomic approaches have primarily been used to interrogate resistance mechanisms. Here we characterized the proteome and phosphoproteome of a series of isogenic EGFR-mutant lung adenocarcinoma cell lines that are either sensitive or resistant to these drugs, comprising the most comprehensive proteomic dataset resource to date to investigate third generation EGFR TKI resistance in lung adenocarcinoma. Unbiased global quantitative mass spectrometry uncovered alterations in signaling pathways, revealed a proteomic signature of epithelial–mesenchymal transition, and identified kinases and phosphatases with altered expression and phosphorylation in TKI-resistant cells. Decreased tyrosine phosphorylation of key sites in the phosphatase SHP2 suggests its inhibition, resulting in subsequent inhibition of RAS/MAPK and activation of PI3K/AKT pathways. Anticorrelation analyses of this phosphoproteomic dataset with published drug-induced P100 phosphoproteomic datasets from the Library of Integrated Network-Based Cellular Signatures program predicted drugs with the potential to overcome EGFR TKI resistance. The PI3K/MTOR inhibitor dactolisib in combination with osimertinib overcame resistance both in vitro and in vivo. Taken together, this study reveals global proteomic alterations upon third generation EGFR TKI resistance and highlights potential novel approaches to overcome resistance. Significance: Global quantitative proteomics reveals changes in the proteome and phosphoproteome in lung cancer cells resistant to third generation EGFR TKIs, identifying the PI3K/mTOR inhibitor dactolisib as a potential approach to overcome resistance.

Exportin 1 (XPO1) mediates nuclear export of many cellular factors known to play critical roles in malignant processes, and selinexor (KPT-330) is the first XPO1-selective inhibitor of nuclear export compound in advanced clinical development phase for cancer treatment. We demonstrated here that inhibition of XPO1 drives nuclear accumulation of important cargo tumor suppressor proteins, including transcription factor FOXO3a and p53 in thymic epithelial tumor (TET) cells, and induces p53-dependent and -independent antitumor activity in vitro Selinexor suppressed the growth of TET xenograft tumors in athymic nude mice via inhibition of cell proliferation and induction of apoptosis. Loss of p53 activity or amplification of XPO1 may contribute to resistance to XPO1 inhibitor in TET. Using mass spectrometry-based proteomics analysis, we identified a number of proteins whose abundances in the nucleus and cytoplasm shifted significantly following selinexor treatment in the TET cells. Furthermore, we found that XPO1 was highly expressed in aggressive histotypes and advanced stages of human TET, and high XPO1 expression was associated with poorer patient survival. These results underscore an important role of XPO1 in the pathogenesis of TET and support clinical development of the XPO1 inhibitor for the treatment of patients with this type of tumors. Cancer Res; 77(20); 5614-27. ©2017 AACR.

Tumor-specific mutated proteins can create non-self, mutation-containing ‘neoepitopes' that are immunogenic and are attractive targets for adoptive T-cell therapies. To avoid the complexity of patient-specific, private neoepitopes and their T-cell responses, there has been major interest in targeting common shared mutations in driver genes using off-the-shelf T-cell receptors engineered into autologous lymphocytes. The diversity of MHC alleles which could present these select neoantigens is an obstacle to this strategy. Most neoepitopes will not be successfully presented by most HLA alleles, and algorithms predicting epitope-HLA binding will either miss valid epitopes or identify too many candidate alleles to investigate thoroughly. One method to definitively demonstrate that an MHC allele can successfully present an epitope is to elute peptides bound to that MHC allele on the cell surface and analyze them by mass spectrometry (MS). It has the additional advantages of defining the precise epitope processed from an antigen and estimating its abundance. We undertook a comprehensive analysis by MS of several common shared mutations in driver oncogenes and their presentation by common Class I HLA alleles. To overcome the low sensitivity of MS (compared to T-cells), we created mono-allelic cell lines expressing one Class I HLA allele and one common mutated oncogene. Peptides bound to MHC on the surface of these cell lines were immunoprecipitated, eluted, and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). As expected, most HLA alleles could not present a detectable neoepitope from the candidate antigens. Our system was able to identify precise neoepitopes derived from four commonly mutated oncogenes (KRAS, EGFR, BRAF, and PIK3CA) presented by the HLA-A*03 HLA superfamily. The majority of mutated peptides were consistently found in biological replicates, demonstrating the reproducibility of our system. By evaluating intensities and fragmentation data for mutated peptides, we were able to improve the reliability of detection of neoepitopes at the cell surface. Although most of the identified epitopes were predicted to bind using the canonical NetMHCpan algorithm, many epitopes predicted to bind with high affinity were not observed using our pipeline. HLA-A*0201 has been intensely studied because of its high frequency in the U.S. population but we have not found it to consistently present mutated peptides from our candidate oncogenes. Our data show that we could utilize a MS approach to demonstrate which shared oncogene-derived neoepitopes are unequivocally processed and presented by common HLA alleles, which will allow greater focused efforts on developing TCRs against these promising HLA-neoantigen pairs. Citation Format: Catherine M. Ade, Yue A. Qi, Sudipto Das, Ken-ichi Hanada, Tapan Maity, Xu Zhang, Thorkell Andresson, Udayan Guha, James C. Yang. A mass spectrometry survey of frequent HLA alleles successfully presenting common tumor specific mutations for immune recognition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 273.

Epidermal growth factor receptor (EGFR) signaling is frequently dysregulated in a variety of cancers. The ubiquitin ligase Cbl ubiquitinates the activated EGFR, targeting it for degradation, and acts as an adaptor, recruiting proteins required for EGFR trafficking. To study Cbl adaptor function, we used Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) mass spectrometry (MS) to compare Cbl complexes in 3 groups: without or with EGF stimulation, and with EGF stimulation in the presence of an endosomal trafficking inhibitor. This approach identified over a hundred novel Cbl interactors, whose abundance in the complex with Cbl changed with the treatments. Secondary screening with an in cell Elisa assay demonstrated that siRNA mediated knockdown of Flotillin-2 (FLOT2) resulted in increased phosphorylation of FLOT2. Further evaluation of knockdown of FLOT2 in HeLa cells by immunoblotting showed that loss of FLOT2 led to increased phosphorylation, increased Cbl recruitment to the EGFR, increased ubiquitination of the EGFR, and degradation of EGFR upon EGF stimulation compared to negative control siRNA in HeLa cells. CRISPR knockout of FLOT2 in HeLa cells reduced steady-state levels of EGFR, but increased EGF-stimulated phosphorylation and signaling downstream of EGFR. In lung adenocarcinoma H441 cells, knockdown of FLOT2 increased EGF-stimulated EGFR phosphorylation, ubiquitination, and downstream signaling. This effect was reversed by the EGFR inhibitor erlotinib. Conversely, stable overexpression of FLOT2 in HeLa cells decreased EGF-stimulated EGFR phosphorylation and ubiquitination. Overexpression of wild type (WT) FLOT2 inhibited EGFR phosphorylation upon EGF stimulation in HEK293T cells. Conversely, overexpression of a soluble G2A FLOT2 mutant (which does not get recruited to the plasma membrane) did not inhibit EGF induced EGFR phosphorylation. In order to determine the effect of FLOT2 on EGFR-dependent cancer cell growth, both HeLa and H441 cells were treated in the presence or absence of both EGF and erlotinib in FLOT2 knockdown or knockout cells, and growth was monitored by cell counts over 7 days. HeLa cells exhibited increased growth and soft-agar colony formation in response to FLOT2 knockout, however this growth was not reduced by erlotinib treatment suggesting this was EGFR independent. However, EGF treatment greatly increased HeLa growth in the FLOT2 knockout group as compared to the control CRISPR and this increase was abrogated by erlotinib, indicating that FLOT2 knockout sensitizes HeLa cells to increased EGF-induced HeLa cell growth. H441 cells had an increase in growth over time in response to FLOT2 knockdown, and this growth was erlotinib sensitive, showing that the increase in growth was EGFR-dependent. In summary, these data demonstrate that FLOT2 negatively regulates EGFR activation and its subsequent ubiquitination and degradation. Further, we present evidence that FLOT2 negatively regulates cancer cell growth in an EGFR-dependent manner in H441 cells. Citation Format: David John Wisniewski, Mariya S. Liyasova, Xu Zhang, Samuel F. Gilbert, Alexis Catalano, Donna Voeller, Udayan Guha, Stanley Lipkowitz. Elucidating the role of Flotillin-2 in EGFR signaling and cancer growth [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 117.

Introduction: The epidermal growth factor receptor (EGFR) is one of the most frequently mutated oncogenes in human lung cancer. Although immune checkpoint inhibitors have been therapeutically effective in multiple cancers, patients harboring EGFR mutations have not responded well to this immunotherapy. Adoptive T cell therapy (ACT) has resulted in complete and durable regression of metastatic cancers. ACT requires that neoantigens are presented by major histocompatibility complex (MHC) class I molecules on tumor but not normal cells. The MHC binding algorithms, such as IEDB, most commonly used for antigen prediction on MHC molecules is far from perfect; thus, experimental proof of MHC-bound peptides is needed to provide direct evidence of antigen presentation. Method: We developed a mass spectrometry (MS)-based platform integrated with a whole exome sequencing (WES)-based protein database to identify tumor-specific mutant peptides presented by MHC class I. Experiments were conducted using EGFR mutant PC9 lung adenocarcinoma cell line that harbors the EGFRDel746-750mutant. MHC class I proteins and their associated peptides were immunoprecipitated, and the peptides separated from MHC proteins. The MHC proteins were subjected to trypsin digestion and HLA typing using MS. Class I-associated peptides underwent C-18 separation and tandem MS analysis. A PC9-specific database was built adding all SNVs and INDELs identified by WES to the normal human database and used to search MS data. Peptides identified by MS were subjected to IEDB algorithm search using PC9 specific HLA types. Results: The MS HLA typing identified A*02, A*24, and B*39 in PC9 cells. We identified 13,765 MHC bound peptides. 10,711 peptides were 6~15 amino acid residues in length (6~15mer), which are generally considered as class I epitopes. Using the PC9-specific protein database, 11 variant peptides (neo-peptides) were identified. These include plectin isoform 1 (PLEC_H1459R, 9mer), proteasome subunit beta type-4 (PSMB4_I234T, 9mer), prostaglandin reductase 1 (PTGR1_A27S, 8mer), cytochrome b-c1 complex subunit Rieske mitochondrial (UQCRFS1_S6A, 7mer) and obscurin-like protein 1 (OBSL1_E1365D, 10mer). The IEDB results predicted the binding of PLEC_H1459R with HLA-B*39, OBSL1_E1365D with HLA-A*02 and PSMB4_I234T with HLA-A*24; however, 8 identified variant peptides were not predicted by IEDB. Conclusion: Our MS data provided direct experimental evidence for MHC class I presentation of 11 neopeptides in PC9 cells. Neopeptides discovered by this proteogenomic platform could potentially trigger T cell response, which needs to be validated by immunological assays. Experiments are underway to utilize this workflow to identify variant neopeptides from patient tumors and patient-derived xenografts (PDXs). Citation Format: Yue Qi, Tapan K. Maity, Xu Zhang, Shaojian Gao, Nitin Roper, Meriam Bahta, Khoadang Nguyen, Constance M. Cultraro, Udayan Guha. Integrated proteogenomics utilizing mass spectrometry to identify MHC-associated neopeptides in EGFR mutant lung adenocarcinoma [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 1438.

Lung cancer is the leading cause of cancer mortality. Tumor heterogeneity is a major cause of treatment failure. Intra- and inter-metastatic tumor heterogeneity has been demonstrated by next generation sequencing (NGS) studies. However, heterogeneity in the proteome and phosphoproteome has been less studied. Integrated proteogenomics is essential to understanding the intricacies of tumor heterogeneity affecting treatment response. Here, we performed integrated mass spectrometry-based proteogenomics to characterize spatial and temporal heterogeneity of an exceptional responder lung adenocarcinoma patient who survived with metastatic disease for more than 7 years while on combination treatment with HER2-targeted and chemotherapy. We employed Super-SILAC and TMT labeling strategies to quantify the proteome and phosphoproteome of a lung metastatic site and ten different metastatic progressive lymph nodes from our patient collected across a span of seven years, including at autopsy. To further interrogate the mass spectrometry data, patient-specific database was built to incorporate all the somatic variants identified by NGS. An extensive validation pipeline was built for confirmation of variant peptides. CRISPR-Cas9-mediated gene knockout, cell viability assays, and confocal microscopy were used for further validation of novel variants. A total of 6214 and 4061 proteins were identified from Super-SILAC and TMT experiments, respectively. 3648 proteins were identified and quantified in both experiments. More than 2000 proteins had catalytic activity, including kinases, phosphatases and metabolic enzymes. We identified 78 and 23 mutant peptides from Super-SILAC and TMT experiments, respectively. Three somatic variants, CDK12-G879V, FASN-R1439Q and HNRNPF-A105T, were confirmed using our variant peptide detection pipeline. Multiple reaction monitoring in a triple quadrupole mass spectrometer successfully identified and relatively quantified two of the variant tryptic peptides harboring the mutations, CDK12-G879V and FASN-R1439Q from the lung and lymph node metastatic sites, respectively. We investigated the consequences of loss of CDK12 function, as predicted from the novel CDK12-G879V mutant, in chemotherapy sensitivity. A549 lung adenocarcinoma cells, upon knockdown of CDK12, exhibited greater chemotherapy sensitivity that was rescued by wild type CDK12, but not by CDK12-G879V mutant. We demonstrate the importance of integrated proteogenomic analyses to identify variant peptides in mass spectrometry data and studying proteomic heterogeneity affecting treatment response. CDK12-G879V mutation results in a nonfunctional CDK12 kinase and chemotherapy susceptibility in lung metastatic sites, likely explaining the “cure” of lung metastatic sites in this patient. Citation Format: Xu Xhang, Khoa Dang P. Nguyen, Paul Rudnick, Nitin Roper, Emily Kawaler, Tapan K. Maity, Shivangi Awasthi, Shaojian Gao, Romi Biswas, Abhilash Venugopalan, Constance Cultraro, David Fenyo, Udayan Guha. Quantitative mass spectrometry to interrogate proteomic heterogeneity in metastatic lung adenocarcinoma and validate a novel somatic mutation CDK12-G879V [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 4528.

Background: Osimertinib is effective in EGFR-mutant NSCLC in both first- and second-line settings. However, the efficacy of osimertinib is limited by the universal development of resistance. Retrospective studies have shown a survival benefit to local ablative therapy (LAT) in those who develop oligoprogressive disease (progression at a limited number of anatomic sites) on EGFR-tyrosine kinase inhibitor (EGFR-TKI) therapy. Methods: We initiated a prospective study of LAT in patients with EGFR-mutant NSCLC and oligoprogression during treatment with osimertinib (NCT02759835). Patients with no prior EGFR-TKI therapy (cohort 1) or progression after 1st/2nd generation EGFR-TKI therapy and acquired T790M mutation (cohort 2) start osimertinib. Upon progression, eligible patients with Results: Between 04/2016 and 01/2018, 24 patients were enrolled (cohort 1: 16, cohort 2: 5, cohort 3: 3). Median age was 59.8 (range 36-77). The most common adverse events (AEs) on osimertinib treatment were rash, diarrhea, thrombocytopenia, and QTc prolongation with most of the AEs being grade 1 or 2. Among evaluable patients, confirmed objective response rates prior to LAT in cohorts 1 and 2 were 66.7 % (8/12) and 75% (3/4), respectively, with 11.2 months median PFS (95% CI: 3.6 months-undefined) in cohort 1 and 10.9 months in cohort 2 (95% CI: 6.7-12.8 months). To date, 12 patients had progressive disease, 8 of which had oligoprogression and subsequently underwent LAT (cohort 1: 3; cohort 2: 2; cohort 3: 3). Two patients were treated with combination of surgery and radiotherapy (RT), 4 patients with surgery, and 2 patients with RT. Median PFS2 was 2.3 months (95% CI: 1.0-5.9 months). Whole exome sequencing (WES) was performed on tumor tissues obtained pre-treatment and upon progression on osimertinib. MET amplification, transformation to small cell lung cancer, and EGFR C797S mutation were identified as mechanisms of resistance to osimertinib. Results of molecular analyses of tumor tissue and blood ctDNA will be presented. Conclusions: Patients with EGFR-mutant NSCLC and oligoprogression after EGFR-TKI therapy can be safely treated with LAT. In select patients, this approach could potentially maximize duration of EGFR-TKI treatment and prevent premature switching to other systemic therapies. Citation Format: Chul Kim, Nitin Roper, Chuong Hoang, Laura Wisch, Maureen Connolly, Hsien-Chao Chou, Jun Wei, Manoj Tyagi, Constance M. Cultraro, Liqiang Xi, Maryam Waris, Khoa Dang Nguyen, Eva Szabo, Emerson Padiernos, Aparna Kesarwala, Shaojian Gao, Seth M. Steinberg, Mark Raffeld, Arun Rajan, Javed Khan, Udayan Guha. Local ablative therapy for oligoprogressive, EGFR-mutant, non-small cell lung cancer (NSCLC) after treatment with osimertinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT106.

EGF receptor (EGFR) signaling is dysregulated by gene amplification or activating mutations of the EGFR in a variety of human malignancies. Activation of the EGFR by EGF leads to recruitment of the ubiquitin ligase Cbl, which ubiquitinates EGFR targeting it for degradation and acts as an adaptor, recruiting additional proteins required for EGFR internalization and trafficking to the lysosome. However, the detailed molecular mechanisms remain elusive. Complete characterization of proteins involved in EGFR trafficking and degradation may uncover additional mechanisms of EGFR dysregulation in cancer. To characterize the EGFR degradative pathway, we investigated protein complexes formed on Cbl with Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) mass spectrometry. By labeling proteins with stable isotopes in culture, we compared complexes in 3 groups: 1) complexes formed on Cbl without EGF stimulation (control), 2) complexes formed on Cbl with EGF stimulation for 30 min and 3) complexes formed on Cbl with EGF stimulation in the presence of YM201636, a trafficking inhibitor, which was added to prevent degradation of the Cbl/EGFR complex and to enrich it for other proteins important for EGFR trafficking. This approach identified over a hundred novel Cbl interactors, whose abundance in the complex with Cbl changed significantly with the treatments. Fifty of these proteins were selected for RNAi knockdown to examine their effects on EGFR degradation. We found that knockdown of Flotillin 2 (FLOT2) protein, a major scaffold protein within caveolar lipid rafts, led to increased phosphorylation and accelerated degradation of EGFR upon EGF stimulation compared to negative control siRNA in HeLa cells. Conversely, overexpression of FLOT2 in HEK293T inhibited EGFR phosphorylation and formation of the EGFR/Cbl complex upon EGF stimulation and led to decreased EGFR ubiquitination and degradation. CRISPR knockout of FLOT2 in HeLa cells led to decreased steady-state levels of EGFR, increased phosphorylation of EGFR and ERK1/2 compared to non-targeting CRISPR control. Stable overexpression of FLOT2 in HeLa cells increased steady-state levels of EGFR, but decreased EGFR phosphorylation and ubiquitination upon EGF stimulation. FLOT2 is frequently amplified and overexpressed in breast, endometrial and lung cancers. In a lung adenocarcinoma cell line H441, in which FLOT2 gene is amplified, knockdown of FLOT2 increased EGFR phosphorylation and ubiquitination, as well as EGFR signaling, as detected by the phosphorylation of ERK1/2. Our data indicate that FLOT2 negatively regulates EGFR phosphorylation, ubiquitination and degradation. Although it is not clear how FLOT2 amplification could contribute to cancer pathogenesis, we speculate that studying how FLOT2 regulates EGFR signaling could lead to a better understanding of its role in normal physiology and cancer. Citation Format: Mariya Liyasova, Xu Zhang, Udayan Guha, Stanley Lipkowitz. Flotillin 2 negatively regulates EGFR phosphorylation and Cbl-mediated ubiquitination and degradation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2506.

Lung cancer remains the leading cause of cancer-related deaths worldwide. Epidermal growth factor receptor(EGFR) is the primary driver oncogene in 10-30% lung adenocarcinoma. Kinase domain mutations in EGFR (L858R and E746-A750 deletion) provide sensitivity to EGFR-specific tyrosine kinase inhibitors (TKIs). Despite good tumor inhibitory effects, after approximately a year, patients acquire resistance to EGFR TKIs. We have carried out a stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative mass spectrometry (MS) on a Thermo Orbitrap Elite to identify and characterize the dynamics of tyrosine phosphorylation upon TKI treatment of human lung adenocarcinoma cell lines. The degree of inhibition of phosphorylation of several phospho-sites correlated with the extent of TKI-sensitivity. Several kinases and adaptor proteins that were significantly differentially phosphorylated include AHNAK-Y160, -Y715, STAT5A-Y694, DAPP1-Y139, ERRFI1-Y394, NEDD9-Y164, NF1-Y2579, INNPL1-Y1135, suggesting that these are a subset of potential biomarkers of TKI response. To corroborate our results, the next logical step is to carry out clinical validation of these phospho-sites in human lung adenocarcinoma patients, however no absolute quantification assay is currently available for these tyrosine phosphorylated peptides in human samples. The MS method most often used to complement shotgun methods is selected/multiple reaction monitoring (SRM/MRM) because of its high sensitivity, speed and dynamic range. We have performed SRM/MRM-MS using a nano-flow high performance liquid chromatography instrument coupled to a nano-electrospray ionization source attached to a Triple Quadrupole MS (Agilent 6495). In this method, digested proteins are chromatographically separated and ionized. The resulting precursor ions are m/z filtered in the first quadrupole(Q1) and collision induced dissociation is carried out in Q2 and the resultant product ions are m/z filtered in Q3. Each precursor-product pair is termed as a transition. For absolute quantification, heavy isotope labelled versions of the target peptides are spiked in biological samples to build a calibration curve. For the robust assay development, we have carried out the initial empirical refinement process using the heavy labelled versions of the target peptides, including selection of optimal transitions, normalizing retention times of peptides, running peptide stability assays and digestion time course experiments. We are currently validating these assays in human lung adenocarcinoma cell lines with known EGFR mutations with or without treatment with 1st and 3rd generation EGFR TKIs. The overall goal of these targeted studies is to develop absolute quantitation assays to precisely and accurately quantify these potential biomarkers in clinical samples. Citation Format: Shivangi Awasthi, Xu Zhang, Tapan Maity, Benjamin L. Oyler, David R. Goodlett, Udayan Guha. A SRM/MRM based targeted proteomics strategy for absolute quantification of potential biomarkers of TKI sensitivity in EGFR mutated lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2203. doi:10.1158/1538-7445.AM2017-2203

Recent large-scale cancer genome sequencing studies have uncovered extensive diverse mutational landscapes in lung cancer patients. Furthermore, tumor heterogeneity has been widely recognized and it has significant clinical implications in selection of targeted treatment strategies as well as treatment response. Using whole genome sequencing, we demonstrate an unprecedented genomic heterogeneity between sequentially acquired lung and lymph node metastatic sites from an African American never-smoker lung adenocarcinoma patient who has survived with metastatic disease for over seven years while being treated with single or combination HER2-directed therapies. We determined that less than 1% of somatic variants were common between the two tumor sites. Copy number variations were more intense in the lung tumor than in the metastatic lymph node. We identified several novel somatic mutations in key cancer genes in both sites. Interestingly, one novel translocation, PLAG1-ACTA2 was highly expressed in both the lung and lymph node metastases resulting in overexpression of ACTA2, which has been suggested to increase the metastatic potential in lung adenocarcinoma. Using ultra deep targeted re-sequencing, we validated all non-synonymous variants, and approximately 80% of those identified in the metastatic lymph node were also present in a second lymph node biopsied two years after the first one. Although this degree of tumor heterogeneity was surprising, somatic variants affected key hallmarks of tumorigenesis in both sites. These findings suggest a model of early metastatic spread and parallel clonal evolution in disparate metastatic sites. Citation Format: Shaojian Gao, Constance Cultraro, Romi BIswas, Corey A. Carter, Tapan K. Maity, Anish Thomas, Arun Rajan, Paul Meltzer, David Schrump, Giuseppe Giaccone, Javed Khan, Udayan Guha. Whole genome sequencing of sequentially acquired lung and lymph node metastatic sites from a never smoker lung adenocarcinoma patient revealed extensive genomic heterogeneity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4759. doi:10.1158/1538-7445.AM2015-4759

Lung cancer is the leading cause of cancer related mortality in the United States and is the most common cancer worldwide. Somatic mutations in the kinase domain of EGFR such as in-frame deletion of LREA amino acids in exon 19 and L858R point mutation in exon 21 largely account for 15-30% of lung adenocarcinoma. Although lung cancer patients carrying either of these mutations respond dramatically to EGFR-directed tyrosine kinase inhibitors (TKIs) such as erlotinib and gefitinib, acquired resistance to these drugs has been the main obstacle in treating these patients for the long term. Secondary mutation at the gatekeeper residue of the kinase domain (T790M) of EGFR accounts for drug resistance in about 50% of patients. We have used doxycycline inducible mutant EGFR transgenic mice to model human lung adenocarcinoma that is sensitive and resistant to TKIs. Our overall goal is to examine the treatment response to EGFR-directed TKIs in lung adenocarcinoma harboring various EGFR mutants. Our approach is aimed to identify imaging surrogates and proteomics profile of treatment response in mouse models of mutant EGFR-driven lung tumorigenesis. In this study, we have used transgenic mice that express exon 19 deletion mutant, L858R mutant, and L858R/T790M double mutant in type II pneumocytes upon doxycycline induction. Tumor burden in these mice was monitored by serial MRI imaging. Each group of mice with significant tumor burden was treated with erlotinib, a reversible EGFR inhibitor, BIBW-2992, an irreversible EGFR/ERBB2 dual inhibitor, or a combination of BIBW-2992 and cetuximab, an antibody directed against EGFR followed by 2[18F]Fluoro-2-deoxy-d-glucose (FDG)-PET imaging to assess any tumor regression. We observed a significant reduction of 18FDG uptake by tumors after the treatment with erlotinib in L858R mutant mice. However, in L858R/T790M mutant mice, no significant reduction of 18FDG uptake was observed upon treatment with erlotinib and BIBW-2992 had only mild effect. We are currently designing rational combination treatments based on the response in 18FDG-PET imaging. Finally, we are performing global phosphoproteomic analysis using quantitative mass spectrometry and immunohistochemistry with phospho-specific antibodies on tumor tissue before and after various treatments and correlating with the response in PET imaging. Results from these experiments will help us identify potential biomarkers (imaging and proteomic) of treatment response in lung adenocarcinoma driven by both TKI-sensitizing and resistant mutants of EGFR. Citation Format: Abhilash Venugopalan, Niu Gang, Jinxia Guo, Tapan Maity, Xu Zhang, Constance Cultraro, Xiaoyuan Chen, Udayan Guha. In vivo imaging and quantitative proteomics to study responsiveness to EGFR targeted therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 725. doi:10.1158/1538-7445.AM2013-725

Among various cancers, lung cancer is the leading cause of cancer-related mortality in both men and women in the US. The major driver mutations in lung cancer are in KRAS and EGFR. Mutations in EGFR account for 10-30% of lung adenocarcinoma. Somatic activating mutations in the kinase domain of EGFR occur in a subset of patients with lung adenocarcinoma and are associated with sensitivity to EGFR-directed tyrosine kinase inhibitors (TKIs). Unfortunately, all patients who respond dramatically to TKIs (gefitinib and erlotinib) eventually develop secondary resistance. A mutation in the gatekeeper residue in the ATP binding pocket of EGFR (T790M) occurs in around 50% of such patients. We hypothesized that mutant EGFR kinases have altered substrate specificities in vivo; these substrates play intermediary roles in transducing mutant EGFR signaling to downstream components to promote tumorigenesis. We have employed a screening method using an in vitro kinase assay on protein arrays containing around 17,000 proteins to compare the substrate specificity of the kinase domains of wild type EGFR, L858R EGFR (the most common activating mutant), and the EGFR harboring both the L858R and T790M mutations (TKI resistant mutant). We have identified several potential substrates such as Crk, Crk-like protein, Grb2, Stat3, Nck1, PIK3R3, etc., which have altered phosphorylation status in response to mutant EGFR kinase activity. We also performed a SILAC (Stable isotope labeling by amino acids in cell culture)-based global phosphoproteomic analysis in human lung adenocarcinoma cells using mass spectrometry and observed that phosphorylation of some of the potential substrates is modulated by EGF stimulation and TKI inhibition. We are currently validating promising substrates from the above assay by performing in vitro kinase assays in presence or absence of TKIs. Furthermore, to identify specific phosphorylation sites of the validated targets in vivo, we have adapted a SILAC-based mass spectrometry approach to immunoprecipitate specific substrates from isogenic human bronchial epithelial cells expressing various EGFR mutants followed by identification and quantification of phosphorylation. We have used siRNA-mediated gene silencing to interrogate the role of several of these substrates in survival of human lung adenocarcinoma cells. Finally, we are examining the phosphorylation status of identified substrates in tumor samples from patients of lung adenocarcinoma to correlate phosphorylation with EGFR mutation status. Citation Format: Tapan K. Maity, Hee-Sool Rho, Natalya Belinka, Zhi Xie, Xu Zhang, Abhilash Venugopalan, Romi Biswas, Constance Cultraro, Heng Zhu, Udayan Guha. Identification of substrates of lung cancer-specific mutant EGFR kinases. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5260. doi:10.1158/1538-7445.AM2013-5260