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Osimertinib is a third-generation epidermal growth factor receptor and tyrosine kinase inhibitor (EGFR-TKI) approved for the treatment of lung adenocarcinoma patients harboring EGFR mutations. However, acquired resistance to this targeted therapy is inevitable, leading to disease relapse within a few years. Therefore, understanding the molecular mechanisms of osimertinib resistance and identifying novel targets to overcome such resistance are unmet needs of cancer patients. Here, we investigated the efficacy of two novel CDK12/13 inhibitors, AU-15506 and AU-16770, in osimertinib-resistant EGFR mutant lung adenocarcinoma cells in culture and xenograft models in vivo. We demonstrate that these drugs, either alone or in combination with osimertinib, are potent inhibitors of osimertinib-resistant as well as -sensitive lung adenocarcinoma cells in culture. Interestingly, only the CDK12/13 inhibitor in combination with osimertinib, although not as monotherapy, suppresses the growth of resistant tumors in xenograft models in vivo. Taken together, the results of this study suggest that inhibition of CDK12/13 in combination with osimertinib has the potential to overcome osimertinib resistance in EGFR mutant lung adenocarcinoma patients.

Supplementary Methods, Figure Legends

Supplementary Figure S1. Serial MRI imaging and lung histopathology analyses show rapid tumor progression of mutant EGFR-driven tumorigenesis in Mig6-/- background. Supplementary Figure S2. Representative photomicrographs of CC10rtTA/EGFRL858R/Mig6+/- mouse lungs at early stages of doxycycline treatment show variability of tumor progression. Supplementary Figure S3. Rapid progression of tumorigenesis 7 days after doxycycline induction of mutant EGFR in Mig6-/- mice. Supplementary Figure S4. No loss of heterozygosity (LOH) in lung tumors of Mig6+/- mice. Supplementary Figure S5. QPCR analysis of mRNA expression in lung tumor samples from Mig6+/+, Mig6+/-, and Mig6-/- mice. Supplementary Figure S6. QPCR analysis of mRNA expression in lung samples from Mig6+/+, Mig6+/-, and Mig6-/- mice at early stages of doxycycline treatment. Supplementary Figure S7. MS and MS/MS spectra of Y394/395 containing tryptic peptide identified in MIG6 (gene symbol ERRFI1). Supplementary Figure S8. Specificity of monoclonal antibodies made against wild type and mutant EGFRs. Supplementary Figure S9. Co-localization of (A-B) MIG6 and EEA1, a marker of early endosomes with both WT EGFR and EGFRL858R and (C-D) LAMP1, a late endosomal marker with WT EGFR, but not EGFRL858R.

Supplementary Table 1. Identification of mice used is Western blots and immunoprecipitation experiments (M1-M20) and the mice used for the early time periods upon doxycycline induction.

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.

Table S1

Supplementary Fig.S7

Affected pathways

PDF file - 76K, Examples of negative results with the NanoString assay for kinase fusions.

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.

Effects of XPO1 knockdown on NF-κB and cell proliferation

PDF file - 308K, Complete list of target sequences for NanoString probes

Background: The mutually exclusive pattern of the major driver oncogenes in lung cancer suggests that other mutually exclusive oncogenes exist. We conducted a systematic search for tyrosine kinase fusions by screening all tyrosine kinases for aberrantly high RNA expression levels of the 3′ kinase domain (KD) exons relative to more 5′ exons.Methods: We studied 69 patients (including five never smokers and 64 current or former smokers) with lung adenocarcinoma negative for all major mutations in KRAS, EGFR, BRAF, MEK1, HER2, and for ALK fusions (termed “pan-negative”). A NanoString-based assay was designed to query the transcripts of 90 tyrosine kinases at two points: 5′ to the KD and within the KD or 3′ to it. Tumor RNAs were hybridized to the NanoString probes and analyzed for outlier 3′ to 5′ expression ratios. Presumed novel fusion events were studied by rapid amplification of cDNA ends (RACE) and confirmatory reverse transcriptase PCR (RT-PCR) and FISH.Results: We identified one case each of aberrant 3′ to 5′ ratios in ROS1 and RET. RACE isolated a GOPC-ROS1 (FIG-ROS1) fusion in the former and a KIF5B-RET fusion in the latter, both confirmed by RT-PCR. The RET rearrangement was also confirmed by FISH. The KIF5B-RET patient was one of only five never smokers in this cohort.Conclusion: The KIF5B-RET fusion defines an additional subset of lung cancer with a potentially targetable driver oncogene enriched in never smokers with “pan-negative” lung adenocarcinomas. We also report in lung cancer the GOPC-ROS1 fusion originally discovered and characterized in a glioma cell line. Clin Cancer Res; 18(24); 6599–608. ©2012 AACR.

PDF file - 32K, Clinicopathological characteristics of study cohort

Supplementary Methods

Supplementary Figure S6. Efficient knock-down of ERBB2 and ERBB3 in PC9 and H3255 cells.

Supplementary Methods. Description of additional methods and procedures used in the study.

Supplementary Table S1. List of mice treated with erlotinib.

Legends for Supplementary Figures S1-S7.

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.

Background Small cell lung cancer (SCLC) has the strongest association with smoking among lung cancers. The characteristics of never smokers with SCLC is not known. Research Question Are the clinical characteristics, prognostic factors, survival, genomic alterations, and tumor mutational burdens of SCLC in patients who have never smoked different from those who have smoked? Study Design and Methods A retrospective multicenter cohort study of patients with clinician-confirmed SCLC was performed with the use of a longitudinal and nationally representative electronic medical records database. Smoking history was assessed through technology-enabled abstraction and confirmed for never smokers via chart review. Genomic characteristics of never smoker patients with SCLC were examined with the use of a next-generation sequencing-based gene panel and whole exome sequencing. Results One hundred of 5,632 patients (1.8%) with SCLC were never smokers. Relative to smokers, never smokers were more likely to be female (66.0% vs 52.4%; P = .009) and present with extensive stage (70.0% vs 62.2%; P = .028). Never smokers had a higher proportion of patients in age groups 35 to 49 years (7.0% vs 3.0%; P = .006) and ≥80 years (17.0% vs 8.2%; P = .006). Known risk factors for lung cancer were found in Interpretation The sex- and age-specific distribution of SCLC among never smokers, along with differences that were identified by genomic analyses, suggests a distinct biology of SCLC in never smokers compared with smokers.

Epidermal growth factor receptor (EGFR) signaling is frequently dysregulated in various cancers. The ubiquitin ligase Casitas B-lineage lymphoma proto-oncogene (Cbl) regulates degradation of activated EGFR through ubiquitination and acts as an adaptor to recruit proteins required for trafficking. Here, we used stable isotope labeling with amino acids in cell culture mass spectrometry to compare Cbl complexes with or without epidermal growth factor (EGF) stimulation. We identified over a hundred novel Cbl interactors, and a secondary siRNA screen found that knockdown of Flotillin-2 (FLOT2) led to increased phosphorylation and degradation of EGFR upon EGF stimulation in HeLa cells. In PC9 and H441 cells, FLOT2 knockdown increased EGF-stimulated EGFR phosphorylation, ubiquitination, and downstream signaling, reversible by EGFR inhibitor erlotinib. CRISPR knockout (KO) of FLOT2 in HeLa cells confirmed EGFR downregulation, increased signaling, and increased dimerization and endosomal trafficking. Furthermore, we determined that FLOT2 interacted with both Cbl and EGFR. EGFR downregulation upon FLOT2 loss was Cbl dependent, as coknockdown of Cbl and Cbl-b restored EGFR levels. In addition, FLOT2 overexpression decreased EGFR signaling and growth. Overexpression of wildtype (WT) FLOT2, but not the soluble G2A FLOT2 mutant, inhibited EGFR phosphorylation upon EGF stimulation in HEK293T cells. FLOT2 loss induced EGFR-dependent proliferation and anchorage-independent growth. Lastly, FLOT2 KO increased tumor formation and tumor volume in nude mice and NSG mice, respectively. Together, these data demonstrated that FLOT2 negatively regulated EGFR activation and dimerization, as well as its subsequent ubiquitination, endosomal trafficking, and degradation, leading to reduced proliferation in vitro and in vivo.

Epidermal growth factor receptor (EGFR) signaling is frequently dysregulated in various cancers. The ubiquitin ligase Cbl (Casitas B-lineage lymphoma proto-oncogene) regulates degradation of activated EGFR through ubiquitination and acts as an adaptor to recruit proteins required for trafficking. We used Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) mass spectrometry (MS) to compare Cbl complexes with or without epidermal growth factor (EGF) stimulation. We identified over a hundred novel Cbl interactors, and a secondary siRNA screen found that knockdown of Flotillin-2 (FLOT2) led to increased phosphorylation and degradation of EGFR upon EGF stimulation in HeLa cells. In PC9 and H441 cells, FLOT2 knockdown increased EGF-stimulated EGFR phosphorylation, ubiquitination, and downstream signaling, reversible by the EGFR inhibitor erlotinib. CRISPR knockout (KO) of FLOT2 in HeLa cells confirmed EGFR downregulation, increased signaling, and increased dimerization and trafficking to the early endosome. FLOT2 interacted with both Cbl and EGFR. EGFR downregulation upon FLOT2 loss was Cbl-dependent, as co-knockdown of Cbl and Cbl-b restored EGFR levels. Overexpression of FLOT2 decreased EGFR sjgnaling and growth. Overexpression of wild type (WT) FLOT2, but not the soluble G2A FLOT2 mutant, inhibited EGFR phosphorylation upon EGF stimulation in HEK293T cells. FLOT2 loss induced EGFR-dependent proliferation and anchorage-independent growth. Lastly, FLOT2 KO increased tumor formation and tumor volume in nude mice and NSG mice, respectively. These data demonstrated that FLOT2 negatively regulated EGFR activation and dimerization, as well as its subsequent ubiquitination, endosomal trafficking, and degradation, leading to reduced proliferationin vitroandin vivo.

Diabetes mellitus is the leading cause of cardiovascular and renal disease in the United States. In spite of the beneficial interventions available for patients with diabetes, there remains a need for additional therapeutic targets and therapies in diabetic kidney disease (DKD). Inflammation and oxidative stress are increasingly recognized as important causes of renal diseases. Inflammation is closely associated with mitochondrial damage. The molecular connection between inflammation and mitochondrial metabolism remains to be elucidated. Recently, nicotinamide adenine nucleotide (NAD+) metabolism has been found to regulate immune function and inflammation. In the present studies we tested the hypothesis that enhancing NAD metabolism could prevent inflammation in and progression of DKD. We found that treatment ofdb/dbmice with type 2 diabetes with nicotinamide riboside (NR) prevented several manifestations of kidney dysfunction (i.e., albuminuria, increased urinary kidney injury marker-1 (KIM1) excretion and pathologic changes). These effects were associated with decreased inflammation, at least in part via inhibiting the activation of the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) signaling pathway. An antagonist of the serum stimulator of interferon genes (STING) and whole-body STING deletion in diabetic mice showed similar renoprotection. Further analysis found that NR increased SIRT3 activity and improved mitochondrial function, which led to decreased mitochondrial DNA damage, a trigger for mitochondrial DNA leakage which activates the cGAS-STING pathway. Overall, these data show that NR supplementation boosted NAD metabolism to augment mitochondrial function, reducing inflammation and thereby preventing progression of diabetic kidney disease.

SUMMARY We perform an immunogenomics analysis utilizing whole-transcriptome sequencing of 657 pediatric extra-cranial solid cancer samples representing 14 diagnoses, and additionally utilize transcriptomes of 131 pediatric cancer cell lines and 147 normal tissue samples for comparison. We describe patterns of infiltrating immune cells, T cell receptor (TCR) clonal expansion, and translationally relevant immune checkpoints. We find that tumor-infiltrating lymphocytes and TCR counts vary widely across cancer types and within each diagnosis, and notably are significantly predictive of survival in osteosarcoma patients. We identify potential cancer-specific immunotherapeutic targets for adoptive cell therapies including cell-surface proteins, tumor germline antigens, and lineage-specific transcription factors. Using an orthogonal immunopeptidomics approach, we find several potential immunotherapeutic targets in osteosarcoma and Ewing sarcoma and validated PRAME as a bona fide multi-pediatric cancer target. Importantly, this work provides a critical framework for immune targeting of extracranial solid tumors using parallel immuno-transcriptomic and -peptidomic approaches., Graphical Abstract, In brief Brohl et al. perform immunogenomics analysis of a cohort of 788 pediatric extracranial solid tumors and cell lines, representing 14 diagnoses, utilizing RNA sequencing. They broadly describe prognostically relevant immunophenotypes and provide proof of concept of a transcriptomics-informed adoptive cellular therapy approach, validating PRAME as a multi-pediatric cancer immunotherapy target.

Immune checkpoint inhibitor (ICI) therapy has been a paradigm shift in the treatment of cancer. ICI therapy results in durable responses and survival benefit for a large number of tumor types. Osimertinib, a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) has shown great efficacy treating EGFR mutant lung cancers, however, all patients eventually develop resistance. ICI therapy has not benefitted EGFR mutant lung cancer. Herein, we employed stable isotope labeling by amino acids in cell culture (SILAC) quantitative mass spectrometry-based proteomics to investigate potential immune escape molecular mechanisms in osimertinib resistant EGFR mutant lung adenocarcinoma by interrogating the alterations in the human leukocyte antigen (HLA) Class I-presented immunopeptidome, Class I-interactome, and the whole cell proteome between isogenic osimertinib-sensitive and -resistant human lung adenocarcinoma cells. Our study demonstrates an overall reduction in HLA class I-presented immunopeptidome and downregulation of antigen presentation core complex (e.g., TAP1 and ERAP1/2) and immunoproteasome in osimertinib resistant lung adenocarcinoma cells. Several key components in autophagy pathway are differentially altered. S100 proteins and SLC3A2 may play critical roles in reduced antigen presentation. Our dataset also includes ~1000 novel HLA class I interaction partners and hundreds of Class I-presented immunopeptides in EGFR mutant lung adenocarcinoma. This large-scale unbiased proteomics study provides novel insights and potential mechanisms of immune evasion of EGFR mutant lung adenocarcinoma.

The clinical success of EGFR inhibitors in EGFR-mutant lung cancer is limited by the eventual development of acquired resistance. We hypothesize that enhancing apoptosis through combination therapies can eradicate cancer cells and reduce the emergence of drug-tolerant persisters. Through high-throughput screening of a custom library of ∼1,000 compounds, we discover Aurora B kinase inhibitors as potent enhancers of osimertinib-induced apoptosis. Mechanistically, Aurora B inhibition stabilizes BIM through reduced Ser87 phosphorylation, and transactivates PUMA through FOXO1/3. Importantly, osimertinib resistance caused by epithelial-mesenchymal transition (EMT) activates the ATR-CHK1-Aurora B signaling cascade and thereby engenders hypersensitivity to respective kinase inhibitors by activating BIM-mediated mitotic catastrophe. Combined inhibition of EGFR and Aurora B not only efficiently eliminates cancer cells but also overcomes resistance beyond EMT.

Simple Summary ctDNA assay is a promising non-invasive method to detect genomic alterations associated with lung cancer. In this prospective study of 25 patients with EGFR-mutant lung adenocarcinoma receiving osimertinib, ctDNA progression predated radiographic progression by 118 days in 11 of 20 patients with disease progression. Saliva-based ctDNA analysis and plasma NGS detected additional patients with ctDNA progression preceding clinical progression, suggesting the potential complementary roles of different ctDNA detection methodologies. Baseline mutant ctDNA level predicted progression-free survival while tumor volume measurements by volumetric CT did not. Serial ctDNA analysis of plasma and saliva is a clinically useful tool to monitor response and resistance to osimertinib. Abstract Background: We assessed whether serial ctDNA monitoring of plasma and saliva predicts response and resistance to osimertinib in EGFR-mutant lung adenocarcinoma. Three ctDNA technologies—blood-based droplet-digital PCR (ddPCR), next-generation sequencing (NGS), and saliva-based EFIRM liquid biopsy (eLB)—were employed to investigate their complementary roles. Methods: Plasma and saliva samples were collected from patients enrolled in a prospective clinical trial of osimertinib and local ablative therapy upon progression (NCT02759835). Plasma was analyzed by ddPCR and NGS. Saliva was analyzed by eLB. Results: A total of 25 patients were included. We analyzed 534 samples by ddPCR (n = 25), 256 samples by NGS (n = 24) and 371 samples by eLB (n = 22). Among 20 patients who progressed, ctDNA progression predated RECIST 1.1 progression by a median of 118 days (range: 61–272 days) in 11 (55%) patients. Of nine patients without ctDNA progression by ddPCR, two patients had an increase in mutant EGFR by eLB and two patients were found to have ctDNA progression by NGS. Levels of ctDNA measured by ddPCR and NGS at early time points, but not volumetric tumor burden, were associated with PFS. EGFR/ERBB2/MET/KRAS amplifications, EGFR C797S, PIK3CA E545K, PTEN V9del, and CTNNB1 S45P were key resistance mechanisms identified by NGS. Conclusion: Serial assessment of ctDNA in plasma and saliva predicts response and resistance to osimertinib, with each assay having supplementary roles.

High-throughput phenotype-based screening of large libraries of novel compounds without known targets can identify small molecules that elicit a desired cellular response, but additional approaches are required to find and characterize their targets and mechanisms of action. Here we show that a compound termed lung cancer screen 3 (LCS3), previously selected for its ability to impair the growth of human lung adenocarcinoma (LUAD) cell lines, but not normal lung cells, induces oxidative stress and activates the NRF2 signaling pathway by generating reactive oxygen species (ROS) in sensitive LUAD cell lines. To identify the target that mediates this effect, we applied thermal proteome profiling (TPP) and uncovered the disulfide reductases GSR and TXNRD1 as LCS3 targets. Through enzymatic assays using purified protein, we confirmed that LCS3 inhibits disulfide reductase activity through a reversible, uncompetitive mechanism. Further, we demonstrate that LCS3-sensitive LUAD cells are correspondingly sensitive to the synergistic inhibition of glutathione and thioredoxin pathways. Lastly, a genome-wide CRISPR knockout screen identified the loss of NQO1 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 utility of inhibiting disulfide reductases as a therapeutic strategy for LUAD.

Molecular subtypes of small cell lung cancer (SCLC) defined by the expression of key transcription regulators have recently been proposed in cell lines and limited number of primary tumors. The clinical and biological implications of neuroendocrine (NE) subtypes in metastatic SCLC, and the extent to which they vary within and between patient tumors and in patient-derived models is not known. We integrate histology, transcriptome, exome, and treatment outcomes of SCLC from a range of metastatic sites, revealing complex intra- and intertumoral heterogeneity of NE differentiation. Transcriptomic analysis confirms previously described subtypes based on ASCL1, NEUROD1, POU2F3, YAP1, and ATOH1 expression, and reveal a clinical subtype with hybrid NE and non-NE phenotypes, marked by chemotherapy-resistance and exceedingly poor outcomes. NE tumors are more likely to have RB1, NOTCH, and chromatin modifier gene mutations, upregulation of DNA damage response genes, and are more likely to respond to replication stress targeted therapies. In contrast, patients preferentially benefited from immunotherapy if their tumors were non-NE. Transcriptional phenotypes strongly skew towards the NE state in patient-derived model systems, an observation that was confirmed in paired patient-matched tumors and xenografts. We provide a framework that unifies transcriptomic and genomic dimensions of metastatic SCLC. The marked differences in transcriptional diversity between patient tumors and model systems are likely to have implications in development of novel therapeutic agents.

Immune checkpoint inhibitor and adoptive lymphocyte transfer-based therapies have shown great therapeutic potential for cancers with high tumor mutation burden (TMB). Here, we employed mass spectrometry (MS)-based proteogenomic large-scale profiling to identify potential immunogenic human leukocyte antigen (HLA) Class I-presented peptides in both melanoma, a “hot tumor” with high TMB, and EGFR mutant lung adenocarcinoma, a “cold tumor” with low TMB. We identified several classes of neopeptides, including mutated neoantigens and more than 1000 post-translationally modified peptides representing 58 different PTMs. We constructed a cancer germline (CG) antigen database with 285 antigens and identified 42 Class I-presented CG antigens. Finally, we developed a non-canonical peptide discovery pipeline to identify 44 lncRNA-derived peptides and validated Class I binding for select neopeptides. We provide direct evidence of HLA Class I presentation of a large number of neopeptides for potential vaccine or adoptive cell therapy in melanoma and mutant EGFR lung cancer.

Background: The longitudinal monitoring of actionable oncogenes in circulating tumor DNA (ctDNA) of non-small cell lung cancer (NSCLC) is crucial for clinicians to evaluate current therapeutic response and adjust therapeutic strategies. Saliva-based electric field-induced release and measurement (EFIRM) is liquid biopsy platform to that can directly detect mutation genes with a small volume of samples. Herein, we compared the effectiveness of longitudinal monitoring for the combination of epidermal growth factor receptor (EGFR) and phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) mutations between saliva-based EFIRM and plasma-based platforms (ddPCR and NGS) in two advanced NSCLC patients undergoing the treatment with osimertinib before and after local ablative therapy (LAT). Patients and Methods: Two patients with unresectable advanced NSCLC were enrolled into the National Institutes of Health Clinical Center (NIHCC) Study (ClinicalTrials.gov: 16-C-0092; local ablative therapy for the treatment of oligoprogressive, EGFR-mutated, non-small cell lung cancer after treatment with osimertinib). Serial collections of saliva, plasma, and metastatic tumor volume measurement by computed tomography (CT) were performed. Longitudinal paired saliva and plasma samples were analyzed for p.L858R EGFR, exon19 del EGFR, and p.E545K PIK3CA ctDNA using EFIRM (saliva) and ddPCR and NGS (plasma). Results: In Case 1, the saliva ctDNA curve of exon19 del EGFR by EFIRM demonstrated a strong similarity to those of tumor volume (R = 0.78, P = 0.00) and exon19 del EGFR in ddPCR (R = 0.53, P = 0.01). Moreover, the curve of p.E545K PIK3CA in EFIRM showed similarity to those of tumor volume (R = 0.70, P = 0.00) and p.E545K PIK3CA in NGS (R = 0.72, P = 0.00). In Case 2, the curve of p.E545K PIK3CA in EFIRM revealed a reverse relationship to that of tumor volume (R = -0.65, P = 0.01). Conclusion: In these two case reports, saliva-based EFIRM platform demonstrates a high level of concordance to plasma-based platforms (ddPCR and NGS) for longitudinally monitoring the combination of EGFR and PIK3CA ctDNA and can be a useful platform to monitor tumor progression and response to targeted therapy in NSCLC patients.

Because tobacco is a potent carcinogen, secondary causes of lung cancer are often diminished in perceived importance. To assess the extent of inherited susceptibility to small cell lung cancer (SCLC), the most lethal type of lung cancer, we sequenced germline exomes of 87 patients (77 SCLC and 10 extrapulmonary small cell) and considered 607 genes, discovering 42 deleterious variants in 35 cancer-predisposition genes among 43.7% of patients. These findings were validated in an independent cohort of 79 patients with SCLC. Loss of heterozygosity was observed in 3 of 14 (21.4%) tumors. Identification of variants influenced medical management and family member testing in nine (10.3%) patients. Unselected patients with SCLC were more likely to carry germline RAD51 paralog D (RAD51D), checkpoint kinase 1 (CHEK1), breast cancer 2 (BRCA2), and mutY DNA glycosylase (MUTYH) pathogenic variants than healthy controls. Germline genotype was significantly associated with the likelihood of a first-degree relative with cancer or lung cancer (odds ratio: 1.82, P = 0.008; and 2.60, P = 0.028), and longer recurrence-free survival after platinum-based chemotherapy (P = 0.002), independent of known prognostic factors. Treatment of a patient with relapsed SCLC and germline pathogenic mutation of BRCA1 interacting protein C-terminal helicase 1 (BRIP1), a homologous recombination–related gene, using agents synthetically lethal with homologous recombination deficiency, resulted in a notable disease response. This work demonstrates that SCLC, currently thought to result almost exclusively from tobacco exposure, may have an inherited predisposition and lays the groundwork for targeted therapies based on the genes involved.

Mutations in the epidermal growth factor receptor (EGFR) tyrosine kinase domain constitutively activate EGFR resulting in lung tumorigenesis. Activated EGFR modulates downstream signaling by altering phosphorylation-driven interactions that promote growth and survival. Secretory carrier membrane proteins (SCAMPs) are a family of transmembrane proteins that regulate recycling of receptor proteins, including EGFR. The potential role of SCAMPs in mutant EGFR function and tumorigenesis has not been elucidated. Using quantitative mass-spectrometry-based phosphoproteomics, we identified SCAMP3 as a target of mutant EGFRs in lung adenocarcinoma and sought to further investigate the role of SCAMP3 in the regulation of lung tumorigenesis. Here we show that activated EGFR, either directly or indirectly phosphorylates SCAMP3 at Y86 and this phosphorylation increases the interaction of SCAMP3 with both wild-type and mutant EGFRs. SCAMP3 knockdown increases lung adenocarcinoma cell survival and increases xenograft tumor growth in vivo, demonstrating a tumor suppressor role of SCAMP3 in lung tumorigenesis. The tumor suppressor function is a result of SCAMP3 promoting EGFR degradation and attenuating MAP kinase signaling pathways. SCAMP3 knockdown also increases multinucleated cells in culture, suggesting that SCAMP3 is required for efficient cytokinesis. The enhanced growth, increased colony formation, reduced EGFR degradation and multinucleation phenotype of SCAMP3-depleted cells were reversed by re-expression of wild-type SCAMP3, but not SCAMP3 Y86F, suggesting that Y86 phosphorylation is critical for SCAMP3 function. Taken together, the results of this study demonstrate that SCAMP3 functions as a novel tumor suppressor in lung cancer by modulating EGFR signaling and cytokinesis that is partly Y86 phosphorylation-dependent.

Purpose Our preclinical work identified depletion of ATR as a top candidate for topoisomerase 1 (TOP1) inhibitor synthetic lethality and showed that ATR inhibition sensitizes tumors to TOP1 inhibitors. We hypothesized that a combination of selective ATR inhibitor M6620 (previously VX-970) and topotecan, a selective TOP1 inhibitor, would be tolerable and active, particularly in tumors with high replicative stress. Patients and Methods This phase I study tested the combination of M6620 and topotecan in 3-week cycles using 3 + 3 dose escalation. The primary end point was the identification of the maximum tolerated dose of the combination. Efficacy and pharmacodynamics were secondary end points. Results Between September 2016 and February 2017, 21 patients enrolled. The combination was well tolerated, which allowed for dose escalation to the highest planned dose level (topotecan 1.25 mg/m2, days 1 to 5; M6620 210 mg/m2, days 2 and 5). One of six patients at this dose level experienced grade 4 thrombocytopenia that required transfusion, a dose-limiting toxicity. Most common treatment-related grade 3 or 4 toxicities were anemia, leukopenia, and neutropenia (19% each); lymphopenia (14%); and thrombocytopenia (10%). Two partial responses (≥ 18 months, ≥ 7 months) and seven stable disease responses ≥ 3 months (median, 9 months; range, 3 to 12 months) were seen. Three of five patients with small-cell lung cancer, all of whom had platinum-refractory disease, had a partial response or prolonged stable disease (10, ≥ 6, and ≥ 7 months). Pharmacodynamic studies showed preliminary evidence of ATR inhibition and enhanced DNA double-stranded breaks in response to the combination. Conclusion To our knowledge, this report is the first of an ATR inhibitor-chemotherapy combination. The maximum dose of topotecan plus M6620 is tolerable. The combination seems particularly active in platinum-refractory small-cell lung cancer, which tends not to respond to topotecan alone. Phase II studies with biomarker evaluation are ongoing.

Candidate gene as well as genome-wide association studies identified several polymorphic variants to be associated with lung cancer worldwide including in India. However, contradictory results have failed to estimate the overall effect of the polymorphic variants on the disease. Textmining was conducted on PubMed following specific search strings to gather all the publications related to genetic association with lung cancer in India. Out of 211 PubMed hits only 30 studies were selected for meta-analysis following specific inclusion criteria. Heterogeneity between studies was calculated by Cochran's Q-test (P

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.

BACKGROUND: Small cell lung cancer (SCLC) has the strongest association with smoking among lung cancers. The characteristics of never smokers with SCLC is not known. RESEARCH QUESTION: Are the clinical characteristics, prognostic factors, survival, genomic alterations, and tumor mutational burdens of SCLC in patients who have never smoked different from those who have smoked? STUDY DESIGN AND METHODS: A retrospective multicenter cohort study of patients with clinician-confirmed SCLC was performed with the use of a longitudinal and nationally representative electronic medical records database. Smoking history was assessed through technology-enabled abstraction and confirmed for never smokers via chart review. Genomic characteristics of never smoker patients with SCLC were examined with the use of a next-generation sequencing-based gene panel and whole exome sequencing. RESULTS: One hundred of 5,632 patients (1.8%) with SCLC were never smokers. Relative to smokers, never smokers were more likely to be female (66.0% vs 52.4%; P = .009) and present with extensive stage (70.0% vs 62.2%; P = .028). Never smokers had a higher proportion of patients in age groups 35 to 49 years (7.0% vs 3.0%; P = .006) and ≥80 years (17.0% vs 8.2%; P = .006). Known risk factors for lung cancer were found in