Driven Lung Adenocarcinoma Research Articles

P2.03-26 Elucidating Mechanisms of Resistance to Targeted Therapies in Mutant EGFR or KRAS Driven Lung Adenocarcinoma Harboring Dual Loss of p53 and RB1

Inactivation of the two canonical tumor suppressors, p53 and RB1, is a genetic hallmark of small-cell lung cancer (SCLC). In contrast, lung adenocarcinomas (LUADs) preferentially harbor alterations in the p16 pathway over RB1. Nonetheless, despite being rare, concurrent loss of p53 and RB1 occurs in a subset of LUADs and this is hypothesized to be necessary for the histological transformation of LUAD to SCLC, observed during treatment with tyrosine kinase inhibitors (TKIs). However, whether the dual loss of p53 and RB1 is sufficient for this histological transformation remains unknown. Furthermore, loss of RB1 in LUADs with EGFR mutations is associated with poor response to TKIs in the absence of SCLC transformation. Here, we aimed to explore how loss of p53/RB1 affects the biology of p16 pathway-altered LUAD, particularly in the context of acquired resistance mechanisms to targeted therapies. Four TP53-mutated LUAD cell lines were used: two EGFR mutation-positive (PC9 and H1975) and two KRAS mutation-positive (H1792 and H358). All these cell lines possess p16 pathway alterations: p16 (CDKN2A) mutations in PC9 and H1975, CDK4 amplification in H1792, and silenced p16 in H358. Inactivation of RB1 was carried out using CRISPR-Cas9 and RB1 knockout monoclonal cells were established. Cell proliferative and clonogenic abilities were assessed. In addition, osimertinib-resistant (PC9 and H1975) and trametinib-resistant (H1792 and H358) cells were generated (initial high dose and/or stepwise dose escalation methods). Acquired resistance mechanisms were evaluated by MSK-IMPACT profiling. Two RB1 knockout clones were established for each cell line. No advantageous effects were observed for proliferative and clonogenic abilities after RB1 knockout. Although loss of p53 and RB1 has been reported to result in lineage shift in prostate cancer through the upregulation of SOX2, deregulation of SOX2 expression was not observed upon RB1 knockout in the TP53-mutant LUAD cells. In addition, although loss of RB1 caused a modest reduction in osimertinib and trametinib sensitivities of H1975 and H358 cells, respectively, no effect was observed in PC9 and H1792 cells. After becoming resistant to osimertinib or trametinib, transformation to SCLC was not observed. Individual resistance mechanisms are currently being assessed by MSK-IMPACT. Dual loss of p53 and RB1 caused no advantageous effects in TP53-mutated and p16 pathway-altered LUAD cells and did not initiate transformation to SCLC as a resistance mechanism to targeted therapies. TP53/RB1 deficiency-related acquired resistance mechanisms to osimertinib or trametinib will be further explored and presented.

P2.03-48 Tumor-Derived Granulocyte Chemotactic Protein 2 Cooperates with Proteases to Drive Lung Adenocarcinoma

Lung adenocarcinoma (LADC) commonly arises in the lungs of smokers that are heavily affected by chronic inflammation. Inflammatory signaling from tumor to host cells is critically involved in the pathogenesis of LADC, but the exact mechanisms by which the lung epithelium interacts with the immune system during carcinogenesis are obscure. Objectives: We discovered that murine and human LADC cell lines overexpress the inflammatory and angiogenic CXC chemokine granulocyte chemotactic protein 2 (GCP2, CXCL6) compared with normal epithelial cells. GCP2 is processed by immune proteases: neutrophil elastase (NE, ELANE), proteinase 3 (PR3, PRTN3) and matrix metalloproteinase 9 (MMP9, MMP9). We suppose that GCP2 interacts with these proteases to drive LADC and aimed at investigating its function(s). GCP2, MMP9 and PR3 expressions were determined by ELISA and immunohistochemistry. Mouse and human microarray data were analyzed using Affymetrix Transcriptome Analysis Console. GCP2 silencing using dedicated shRNA pools (SantaCruz Biotechnology) and NE/PR3 or MMP9 compound knockout mice were used to study GCP2 interaction with immune proteases in LADC progression. Murine and human LADC tumors and cell lines overexpress GCP2, MMP9, NE and PR3 at the mRNA and protein levels. GCP2 is sequestered to tumor cells, whereas proteases are produced by tumor-infiltrating immune cells. LADC-secreted GCP2 is incompletely processed and require MMP9, NE or PR3 for full activation and vasoactive effects. Moreover, GCP2-silenced LADC cells injected in mice show a lack in metastasis formation. Our results indicate that tumor-originated GCP2 cooperates with immune proteases to drive LADC, providing a paradigm of how the respiratory epithelium coopts the innate immune system during carcinogenesis.

Abstract 3138: SMARCA4 mutations in KRAS-mutant lung adenocarcinoma: A multi-cohort analysis

Abstract KRAS is a key oncogenic driver in lung adenocarcinoma (LUAD). Chromatin-remodeling gene SMARCA4 was co-mutated with KRAS in LUAD; however, the impact of SMARCA4 mutations on clinical outcome has not been adequately established. This study sought to shed light on the clinical significance of SMARCA4mutations in LUAD. The association of SMARCA4 mutations with survival outcomes was interrogated in 4 independent cohorts totaling 564 patients: KRAS-mutant patients with LUAD who received non-immunotherapy treatment from 1) The Cancer Genome Atlas (TCGA) and 2) the MSK-IMPACT Clinical Sequencing (MSK-CT) cohorts; and KRAS-mutant patients with LUAD who received immune checkpoint inhibitor-based immunotherapy treatment from 3) the MSK-IMPACT (MSK-IO) and 4) the Wake Forest Baptist Comprehensive Cancer Center (WFBCCC) immunotherapy cohorts. Of the patients receiving non-immunotherapy treatment, in the TCGA cohort (n=155), KRAS-mutant patients harboring SMARCA4 mutations (KS) showed poorer clinical outcome (P=6e-04 for disease-free survival (DFS) and .031 for overall survival (OS), respectively), compared to KRAS-TP53 co-mutant (KP) and KRAS-only mutant (K) patients; in the MSK-CT cohort (n=314), KS patients also exhibited shorter OS than KP (P=.03) or K (P=.022) patients. Of patients receiving immunotherapy, KS patients consistently exhibited the shortest progression-free survival (PFS; P=.0091) in the MSK-IO (n=77), and the shortest PFS (P=.0026) and OS (P=0.0014) in the WFBCCC (n=18) cohorts, respectively. Therefore, mutations of SMARCA4 represent a genetic factor that lead to adverse clinical outcome in lung adenocarcinoma treated by either non-immunotherapy or immunotherapy. Citation Format: LIANG LIU, Tamjeed Ahmed, Willam Jeffrey Petty, Stefan Grant, Jimmy Ruiz, Thomas W. Lycan, Umit Topaloglu, Ping-Chieh Chou, Lance D. Miller, Gregory A. Hawkins, Martha A. Alexander-Miller, Stacey S. O’Neill, Bayard L. Powell, Ralph B. D’Agostino, Reginald F. Munden, Boris Pasche, Wei Zhang. SMARCA4 mutations in KRAS-mutant lung adenocarcinoma: A multi-cohort analysis [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 3138.

Abstract 931: The T790M mutation is acquired through 5-methylcytosine deamination after EGFR TKI treatment in lung cancer

Abstract Background: Epidermal growth factor receptor (EGFR) activation mutations occur in 10-50% of lung adenocarcinomas. EGFR tyrosine kinase inhibitors (TKIs) are the mainstay of treatment for stage IV non-small cell lung cancer (NSCLC) patients with EGFR mutations. However, acquired EGFR mutations are the main mechanism of on-target resistance to TKIs. T790M mutation that occurs after first generation TKI treatment, is a cytosine to thymine (C>T) single nucleotide transition leading to a threonine to methionine amino acid change at position 790 (i.e. T790M). Our data suggest that resistant mutations are acquired events secondary to cytosine deamination through Activation Induced Cytosine Deamination enzyme (AICDA). Interestingly, treatment with Osimertinib, that overcomes the T790M mutation, leads to other acquired resistant mutations (C797S, G796S/R and L792F/H) that are mostly cytosine based. Results: Sub clones of the lung adenocarcinoma cell line PC9 with no evidence of T790M mutation by digital droplet PCR (ddPCR) at baseline, were treated with EGFR TKI. After serially increasing the treatment dose, T790M mutation was detected by ddPCR associated with a significant increase in AICDA expression. Similarly, when the resistant T790M PC9 clones were treated with Osimertinib, the expression of AICDA was also induced. Knocking down AICDA by shRNA or CRISPR-Cas9, decreases the development of T790M in PC9 cell lines after TKI exposure. Using mass spectrometry, we established that cytosine at codon 790 is methylated; thus, deamination of 5-methylcytosine leads to thymine directly, explaining the T790M C>T mutation. In addition, using ChIP assay and pharmacological inhibition we confirm that upon TKI exposure, NFκB binds AICDA promoter and induces its expression. In a mouse xenograft model, the induction of NFκB and AICDA after EGFR TKI exposure is abrogated by concurrent use of an NFκB inhibitor. Finally, patients treated with EFR TKI had an increased expression of AICDA upon progression. Conclusion: In EGFR driven lung adenocarcinoma, NFκB pathway is activated upon exposure to EGFR TKIs which induces AICDA expression. AICDA deaminates cytosine into other nucleotides leading to treatment resistance. Citation Format: Najwa El Kadi, Luo Wang, April Davis, Alexander Cooke, Varun Vadnala, Hasan Korkaya, Gregory P. Kalemkerian, Khaled A. Hassan. The T790M mutation is acquired through 5-methylcytosine deamination after EGFR TKI treatment in 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 931.

Abstract 1601: Genetic and epigenetic intratumor heterogeneity of lung adenocarcinoma

Abstract Genomic studies have revealed remarkable intratumoral heterogeneity (ITH) in lung adenocarcinoma (LUAD) and increasing evidence suggests its impact in cancer treatment. However, to what extent epigenomic abnormalities and somatic copy number alterations (SCNAs) are involved in shaping genetic clones in lung tumors and are associated with clinical outcome is under-appreciated. Here we applied Infinium HumanMethylation450K BeadChips, Infinium Global Screening Arrays and ultra-deep (>500X) targeted sequencing of 35 LUAD driver genes to 369 samples (including 77 normal tissues) from 84 LUAD patients to investigate the genetic and epigenetic architecture of subclonal diversification. SCNAs were mostly clonal events. For patients with more than three tumor regions assayed, 66% SCNAs were located on the trunk of the evolutionary trees. Similar evolutionary trajectories were inferred from SCNAs and DNA methylation. The driver genes’ sequencing depth allowed to identify subclonality even in TP53, EGFR, and BRAF mutations, which were largely reported as clonal events in previous studies. Elevated copy number ITH was associated with higher risk of death, while no significant association was found between clinical outcome and DNA methylation ITH or heterogeneous CpG island methylator phenotype (CIMP), in contrast with the observed prognostic capability of DNA methylation ITH in hematological malignancies. These findings expand our understanding of the coevolution of genetic and epigenetic aberrations in tumorigenesis, the subclonal complexity for therapeutic targets and the implication of ITH in cancer prognosis. Citation Format: Wei Zhao, Xing Hua, Tongwu Zhang, Dario Consonni, Angela Cecilia Pesatori, Jianxin Shi, Maria Teresa Landi. Genetic and epigenetic intratumor heterogeneity of 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 1601.

ADAM17 selectively activates the IL‐6 trans‐signaling/ERK MAPK axis in KRAS‐addicted lung cancer

Oncogenic KRAS mutations are major drivers of lung adenocarcinoma (LAC), yet the direct therapeutic targeting of KRAS has been problematic. Here, we reveal an obligate requirement by oncogenic KRAS for the ADAM17 protease in LAC. In genetically engineered and xenograft (human cell line and patient‐derived) KrasG12D‐driven LAC models, the specific blockade of ADAM17, including with a non‐toxic prodomain inhibitor, suppressed tumor burden by reducing cellular proliferation. The pro‐tumorigenic activity of ADAM17 was dependent upon its threonine phosphorylation by p38 MAPK, along with the preferential shedding of the ADAM17 substrate, IL‐6R, to release soluble IL‐6R that drives IL‐6 trans‐signaling via the ERK1/2 MAPK pathway. The requirement for ADAM17 in KrasG12D‐driven LAC was independent of bone marrow‐derived immune cells. Furthermore, in KRAS mutant human LAC, there was a significant positive correlation between augmented phospho‐ADAM17 levels, observed primarily in epithelial rather than immune cells, and activation of ERK and p38 MAPK pathways. Collectively, these findings identify ADAM17 as a druggable target for oncogenic KRAS‐driven LAC and provide the rationale to employ ADAM17‐based therapeutic strategies for targeting KRAS mutant cancers.

Adenylate kinase 4 modulates oxidative stress and stabilizes HIF-1\u03b1 to drive lung adenocarcinoma metastasis

BackgroundAdenylate kinase 4 (AK4) has been identified as a biomarker of metastasis in lung cancer. However, the impacts of AK4 on metabolic genes and its translational value for drug repositioning remain unclear.MethodsIngenuity upstream analyses were used to identify potential transcription factors that regulate the AK4 metabolic gene signature. The expression of AK4 and its upstream regulators in lung cancer patients was examined via immunohistochemistry. Pharmacological and gene knockdown/overexpression approaches were used to investigate the interplay between AK4 and its upstream regulators during epithelial-to-mesenchymal transition (EMT). Drug candidates that reversed AK4-induced gene expression were identified by querying a connectivity map. Orthotopic xenograft mouse models were established to evaluate the therapeutic efficacy of drug candidates for metastatic lung cancer.ResultsWe found that HIF-1α is activated in the AK4 metabolic gene signature. IHC analysis confirmed this positive correlation, and the combination of both predicts worse survival in lung cancer patients. Overexpression of AK4 exaggerates HIF-1α protein expression by increasing intracellular ROS levels and subsequently induces EMT under hypoxia. Attenuation of ROS production with N-acetylcysteine abolishes AK4-induced invasion potential under hypoxia. Pharmacogenomics analysis of the AK4 gene signature revealed that withaferin-A could suppress the AK4-HIF-1α signaling axis and serve as a potent anti-metastatic agent in lung cancer.ConclusionsOverexpression of AK4 promotes lung cancer metastasis by enhancing HIF-1α stability and EMT under hypoxia. Reversing the AK4 gene signature with withaferin-A may serve as a novel therapeutic strategy to treat metastatic lung cancer.

Identification of a highly lethal V3+ TP53+ subset in ALK+ lung adenocarcinoma.

Tyrosine kinase inhibitors (TKI) have improved prognosis in metastatic anaplastic lymphoma kinase (ALK)-driven lung adenocarcinoma, but patient outcomes vary widely. We retrospectively analyzed the clinical course of all cases with assessable baseline TP53 status and/or ALK fusion variant treated at our institutions (n = 102). TP53 mutations were present in 17/87 (20%) and the echinoderm microtubule-associated protein-like 4 (EML4)-ALK variant 3 (V3) in 41/92 (45%) patients. The number of metastatic sites at diagnosis was affected more by the presence of V3 than by TP53 mutations, and highest with both factors (mean 5.3, p < 0.001). Under treatment with ALK TKI, progression-free survival (PFS) was shorter with either TP53 mutations or V3, while double positive cases appeared to have an even higher risk (hazard ratio [HR] = 2.9, p = 0.015). The negative effect of V3 on PFS of TKI-treated patients was strong already in the first line (HR = 2.5, p = 0.037) and decreased subsequently, whereas a trend for PFS impairment under first-line TKI by TP53 mutations became stronger and statistically significant only when considering all treatment lines together. Overall survival was impaired more by TP53 mutations (HR = 4.9, p = 0.003) than by V3 (HR = 2.4, p = 0.018), while patients with TP53 mutated V3-driven tumors carried the highest risk of death (HR = 9.1, p = 0.02). Thus, TP53 mutations and V3 are independently associated with enhanced metastatic spread, shorter TKI responses and inferior overall survival in ALK+ lung adenocarcinoma. Both markers could assist selection of cases for more aggressive management and guide development of novel therapeutic strategies. In combination, they define a patient subset with very poor outcome.

P3.12-12 Genomic Profiling of Pulmonary Large-Cell Neuroendocrine Carcinoma (LCNEC) Reveals Distinct Mutational Landscape

The controversial classification of lung neuroendocrine tumor has been amended a few times since recognised as a separate entity. LCNEC shares clinical features with small cell lung carcinoma (SCLC) and they were both classified as lung neuroendocrine carcinoma according to the 2015 WHO lung primary pathology classification, numerous studies have revealed barely satisfactory outcomes when it was treated as SCLC. However the underlying molecular basis for such commonalities and discrepancies are poorly understood. In this study, we interrogated the genomic landscape of LCNEC and SCLC along with their histologically related subtypes: carcinoids and atypical carcinoids to define the molecular pattern of LCNEC. We performed targeted sequencing in 35 tissue samples using a panel covering 520 cancer related genes, spanning 1.6MB of human genome, with an average sequencing depth of 1,418x. Among them, 15 were diagnosed with SCLC, 9 with LCNEC, 6 with carcinoid and 5 with atypical carcinoid. On average, LCNEC exhibited 13.5 mutations per million base pairs (Mb) and a C:G>A:T transversion rate of 34%, which is indicative of tobacco exposure. LCNEC had SCLC (16.7 Mb) had comparable TMB (p=0.18), which is significantly higher than carcinoids (1.2/Mb, p<0.001) and atypical carcinoids (2.4/Mb, p<0.001). The most frequently mutated gene in LCNEC is TP53 (89%, 8/9), followed by NOTCH1 (33%), KEAP1 (22%), RB1 (22%) and a few chromatin modifiers, including KMT2D (33%), KMT2C (33%). Co-mutation in TP53 and RB1, a hallmark of SCLC, was found in 22% (2/9) of LCNEC patients; in contrast, 80% of SCLC patients harbored concurrent mutation. 67% carcinoid (4/6) and 20% (1/5) atypical carcinoid patients had no mutation identified from this panel. No classic lung adenocarcinoma driver mutations were found in any subtype. Copy number analyses revealed significantly higher copy number variation (CNV) in SCLC and LCNEC comparing with carcinoids and atypical carcinoids, which yield virtually no CNV. Our analysis revealed a comparable CNV status of SCLC and LCNEC (p=0.158), with an enrichment in amplification of chromatin modifiers. Our study, comprehensively characterized 4 subtypes of neuroendocrine tumors, revealed a high TMB and CG:AT transversion rate in LCNEC patients as well as a distinctive mutation landscape, with an enrichment of mutations occurring at chromatin remodelers. Furthermore, LCNEC has comparable TMB and CNV status as SCLC, which are significantly higher than carcinoid and atypical carcinoids.

Lysyl hydroxylases are transcription targets for GATA3 driving lung cancer cell metastasis

Metastasis associates with late stages of lung cancer progression and remains the main cause of patient death due to the lack of clinically effective therapeutics. Here we report that the transcription factor GATA3 and its co-factor FOG2 commonly promote the expression of the lysyl hydroxylase (LH) family members, including LH2 and LH3, which in turn drive lung adenocarcinoma cell migration, invasion, and metastasis. We show evidence that both LH2 and LH3 are direct transcription targets for GATA3. Knockdown of either LH2 or LH3 suppresses migration and invasion; on the contrary, forced expression of LH2 or LH3 promotes growth and migration, suggesting that the two LHs exert redundant oncogenic functions. Importantly, re-expression of LH2 is sufficient to restore the metastatic capacity of GATA3-depleted cells, suggesting a role for LHs as the downstream mediators of GATA3. Collectively, our data reveal a pro-metastatic GATA3-LHs axis for lung cancer, supporting the notion that targeting LHs may be useful for treating lung cancer.

HP1γ Promotes Lung Adenocarcinoma by Downregulating the Transcription-Repressive Regulators NCOR2 and ZBTB7A.

Lung adenocarcinoma is a major form of lung cancer, which is the leading cause of cancer death. Histone methylation reader proteins mediate the effect of histone methylation, a hallmark of epigenetic and transcriptional regulation of gene expression. However, their roles in lung adenocarcinoma are poorly understood. Here, our bioinformatic screening and analysis in search of a lung adenocarcinoma-promoting histone methylation reader protein show that heterochromatin protein 1γ (HP1γ; also called CBX3) is among the most frequently overexpressed and amplified histone reader proteins in human lung adenocarcinoma, and that high HP1γ mRNA levels are associated with poor prognosis in patients with lung adenocarcinoma. In vivo depletion of HP1γ reduced K-RasG12D-driven lung adenocarcinoma and lengthened survival of mice bearing K-RasG12D-induced lung adenocarcinoma. HP1γ and its binding activity to methylated histone H3 lysine 9 were required for the proliferation, colony formation, and migration of lung adenocarcinoma cells. HP1γ directly repressed expression of the transcription-repressive regulators NCOR2 and ZBTB7A. Knockdown of NCOR2 or ZBTB7A significantly restored defects in proliferation, colony formation, and migration in HP1γ-depleted lung adenocarcinoma cells. Low NCOR2 or ZBTB7A mRNA levels were associated with poor prognosis in patients with lung adenocarcinoma and correlated with high HP1γ mRNA levels in lung adenocarcinoma samples. NCOR2 and ZBTB7A downregulated expression of tumor-promoting factors such as ELK1 and AXL, respectively. These findings highlight the importance of HP1γ and its reader activity in lung adenocarcinoma tumorigenesis and reveal a unique lung adenocarcinoma-promoting mechanism in which HP1γ downregulates NCOR2 and ZBTB7A to enhance expression of protumorigenic genes.Significance: Direct epigenetic repression of the transcription-repressive regulators NCOR2 and ZBTB7A by the histone reader protein HP1γ leads to activation of protumorigenic genes in lung adenocarcinoma. Cancer Res; 78(14); 3834-48. ©2018 AACR.

Abstract 5509: Early suppression of the TBX2 subfamily of transcription factors during in vivo tobacco carcinogen-mediated lung adenocarcinoma development

Abstract Targets for early treatment of lung adenocarcinoma (LUAD), the leading cause of cancer mortality among smokers, are far and few largely due to the paucity in our knowledge of the molecular pathogenesis of the malignancy. In our efforts to address this glaring gap in knowledge, we previously surveyed the expression of members of the T-box 2 subfamily of transcription factors (TBXs 2, 3, 4 and 5) in normal and malignant lung tissues owing to their evolutionarily conserved expression and function in normal lung organogenesis,. We found that all four members were profoundly suppressed in human premalignant lesions and malignant LUADs. While these findings showed that the TBX2 subfamily of transcription factors is suppressed in overt LUADs, their role in the molecular and temporal oncogenesis of the malignancy in smokers is still elusive. To fill this void, we first surveyed the expression of these genes in a mouse model of LUAD we had developed comprised of knockout of the airway-lineage specific gene Gprc5a (Gprc5a-/-) and exposure to the tobacco-specific carcinogen nicotine-derived nitrosamine ketone (NNK). We had previously found that NNK-exposed Gprc5a-/- mice developed adenomas and LUADs that harbored somatic activating mutations (p.G12D and p.Q61R) in the Kras oncogene – the same variants posited to function as drivers of LUAD in human smokers. In ongoing RNA-Seq efforts cataloging expression in frozen-fixed temporally collected (up to seven months post-NNK) Gprc5a-/- normal tissues, adenomas and LUADs, we found that all four TBX genes were markedly down-regulated (all P &amp;lt; 0.05) in all lesions and across all time points following exposure, relative to normal lung tissues. Concurrent analysis of canonical genes directly activated or repressed by the TBX2 subfamily were found to be consistently down-regulated or elevated, respectively, in the same NNK-exposed lung lesions relative to normal lung. We employed a mouse LUAD cell line (mF471 cells) we had previously derived from an NNK-exposed Gprc5a-/- mouse tumor to further functionally probe the role of TBX2 subfamily in LUAD oncogenesis. Transient over-expression of each of the four TBX2 subfamily members in the mF471 LUAD cells significantly inhibited cell proliferation and growth evidenced by trypan blue dye exclusion, MTT and cell cycle analyses. Over-expression of each of the four TBX2 subfamily members yielded similar anti-growth effects with significant increases in sub-G0 phase of the cell cycle indicating cell cycle arrest (up to 35%) 48 hours following transfection. These findings suggest that the TBX2 subfamily genes are suppressed at early stages in the development of smoking-associated Kras-mutant LUAD and play key tumor suppressor roles in the molecular pathogenesis of this common and fatal malignancy. Citation Format: Athar Khalil, Junya Fujimoto, Tina McDowell, Paul Scheet, Georges Nemer, Humam Kadara. Early suppression of the TBX2 subfamily of transcription factors during in vivo tobacco carcinogen-mediated lung adenocarcinoma development [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 5509.

Abstract 5836: Activation Induced Cytosine Deamination, AICDA, is induced after EGFR TKI exposure leading to secondary resistant mutations in lung adenocarcinoma

Abstract Background: Epidermal growth factor receptor (EGFR) activation mutations occur in 10-50% of lung adenocarcinomas. This leads to constitutive activation of EGFR, which triggers multiple downstream survival and proliferation pathways. EGFR tyrosine kinase inhibitors (TKIs) are the mainstay of treatment for stage IV non-small cell lung cancer (NSCLC) patients with EGFR mutations. Acquired EGFR mutations are the main mechanism of on-target resistance to TKIs. T790M mutation that occurs after first line TKI treatment, is a cytosine to thymine (C&amp;gt;T) single nucleotide transition leading to a threonine to methionine amino acid change at position 790 (i.e. T790M). Interestingly, treatment with Osimertinib, that overcomes the T790M mutation, leads to other acquired resistant mutations, C797S, G796S/R and L792F/H. Our data suggest that resistant mutations are acquired events secondary to cytosine deamination through Activation Induced Cytosine Deamination enzyme (AICDA). Results: Sub clones of the lung adenocarcinoma cell line PC9 with no evidence of T790M mutation by droplet digital PCR (ddPCR) at baseline, were treated with EGFR TKI. After serially increasing the treatment dose, T790M mutation was detected by ddPCR associated with a significant increase in AICDA expression. Knocking down AICDA by shRNA, decreases the development of T790M in PC9 cell lines after TKI exposure. Similarly, when the resistant T790M PC9 clones were treated with Osimertinib, the expression of AICDA was also induced. Using mass spectrometry, we established that cytosine at codon 790 is methylated; thus, deamination of 5-methylcytosine leads to thymine directly, explaining the T790M C&amp;gt;T mutation. In addition, using ChIP assay and pharmacological inhibition we confirm that upon TKI exposure, NFĸB binds AICDA promoter and induces its expression. In a mouse xenograft model, the induction of NFĸB and AICDA after EGFR TKI exposure is abrogated by concurrent use of an NFĸB inhibitor. Finally, patients treated with EFR TKI had an increased expression of AICDA upon progression. Conclusion: In EGFR driven lung adenocarcinoma, NFĸB pathway is activated upon exposure to EGFR TKIs which induces AICDA expression. AICDA deaminates cytosine into other nucleotides leading to treatment resistance. Citation Format: Najwa El Kadi, April Davis, Alexander Cooke, Luo Wang, Hasan Korkaya, Gregory Kalemkerian, Khaled Hassan. Activation Induced Cytosine Deamination, AICDA, is induced after EGFR TKI exposure leading to secondary resistant mutations in lung adenocarcinoma [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 5836.

Abstract B33: CRISPR/Cas9 generation of Ret and Ntrk1 fusion oncogenes and novel in vitro sgRNA screening method

Abstract Chromosomal rearrangements of ALK, RET, ROS1, and NTRK1 collectively represent approximately 10% of oncogenic drivers in lung adenocarcinoma. These rearrangements result in the aberrant expression and constitutive activation of a chimeric fusion kinase, which promotes growth, proliferation and survival of the cancer cell. CRISPR/Cas9 technology can now be employed to generate these chromosomal rearrangements both in vitro and in vivo. sgRNAs can be designed to target the Cas9 endonuclease to introns within the 5′ and 3′ gene partners of the rearrangement. A unique advantage of this approach is that it is compatible with immunocompetent mouse models. We have developed a novel in vitro screening strategy to identify sgRNAs that successfully generated the intended rearrangements. Ba/F3 cells are a murine B-cell cell line whose proliferation is normally dependent on the exogenous addition of the cytokine interleukin 3 (IL3), but can be rendered IL3 independent if an oncogenic alteration is introduced. We hypothesized that IL3-independence in Ba/F3 cells could be used as a method to select for cells that had successfully created chromosomal rearrangements leading to oncogenic fusions. This strategy eliminates the need for single cell cloning to confirm that successful CRISPR genome editing has occurred. We transfected Ba/F3 cells with pairs of sgRNAs within the PX330 Cas9 containing plasmid and then withdrew IL3. Cells that became IL3 independent were tested for rearrangements. Using this screening system, we successfully generated Kif5b-Ret, Trim24-Ret, and Tpm3-Ntrk1 rearrangements in Ba/F3 cells. These rearrangements were confirmed using PCR or RT-PCR and sequencing using fusion-specific primers. Additionally, the Ret rearrangements and Ntrk1 rearrangements were far more sensitive to RET and TRK inhibitors, respectively, than parental Ba/F3 cells, further confirming that the Ba/F3 cells' growth was now dependent on signaling from the fusion kinase. RET protein expression was also detected with Western blotting in the two Ret rearranged Ba/F3 cell lines. Our future plans are to introduce adenovirus containing validated sgRNAs into immune-competent mice, and confirm that they generate tumors with the intended rearrangements. Citation Format: Laura Schubert, Anh T. Le, Stephen P. Malkoski, Raphael Nemenoff, Robert C. Doebele. CRISPR/Cas9 generation of Ret and Ntrk1 fusion oncogenes and novel in vitro sgRNA screening method [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr B33.

Thy-1+ Cancer-associated Fibroblasts Adversely Impact Lung Cancer Prognosis

Cancer-associated fibroblasts (CAFs) regulate diverse intratumoral biological programs and can promote or inhibit tumorigenesis, but those CAF populations that negatively impact the clinical outcome of lung cancer patients have not been fully elucidated. Because Thy-1 (CD90) marks CAFs that promote tumor cell invasion in a murine model of KrasG12D–driven lung adenocarcinoma (KrasLA1), here we postulated that human lung adenocarcinomas containing Thy-1+ CAFs have a worse prognosis. We first examined the location of Thy-1+ CAFs within human lung adenocarcinomas. Cells that co-express Thy-1 and α-smooth muscle actin (αSMA), a CAF marker, were located on the tumor periphery surrounding collectively invading tumor cells and in perivascular regions. To interrogate a human lung cancer database for the presence of Thy-1+ CAFs, we isolated Thy-1+ CAFs and normal lung fibroblasts (LFs) from the lungs of KrasLA1 mice and wild-type littermates, respectively, and performed global proteomic analysis on the murine CAFs and LFs, which identified 425 proteins that were differentially expressed. Used as a probe to identify Thy-1+ CAF-enriched tumors in a compendium of 1,586 lung adenocarcinomas, the presence of the 425-gene signature predicted a significantly shorter survival. Thus, Thy-1 marks a CAF population that adversely impacts clinical outcome in human lung cancer.

Clinically significant sub-clonality for common drivers can be detected in 26% of KRAS/EGFR mutated lung adenocarcinomas.

Genetic sub-clonality has been described in multiple malignancies, however the presence of sub-clonality for major drivers in lung adenocarcinoma and its clinical significance is a subject under debate. Using molecular and morphometric approach, 347 lung adenocarcinoma samples were analyzed for KRAS and EGFR sub-clonality, which was further correlated with clinical and pathological variables.KRAS and EGFR mutations were identified in 100 (29%) and 82 (23%) cases, respectively. One hundred and forty four KRAS or EGFR positive cases were also available for morphometric analysis, among which 37 (26%) were defined as sub-clonal. The presence of sub-clonality was associated with shorter survival time (p=0.02). Interestingly, cases with sub-clonality were also associated with earlier disease stage (89% vs 66% stage I disease in sub-clonal vs clonal cases, respectively, p=0.01) and less lymph node involvement (8% vs 25% in sub-clonal vs clonal cases, respectively, p=0.02). Our findings demonstrate the presence of sub-clonality for mutations in common drivers in lung adenocarcinoma and link it both to earlier disease stage and to poor survival. These findings are in line with the different evolutionary models that can present with genetic sub-clonality.

P2.03b-042 MET exon 14 Mutations Encode a Hyperactive Kinase and Therapeutic Target in Lung Adenocarcinoma: Topic: Biomarkers

Activation of the MET tyrosine kinase receptor can drive oncogenesis and metastasis in certain cancer types. Somatic mutations at or around the splice junctions for MET exon 14 (METΔ14) are a recurrent mechanism of MET activation. METΔ14 mutations lead to aberrant messenger RNA (mRNA) splicing resulting in a METΔ14 protein lacking the juxtamembrane domain. We analyzed RNAseq data across 12 cancer types to define the prevalence of METΔ14 in solid tumors. We explored the driver role of METΔ14 both in vitro and in vivo. Finally, we explored acquired resistance mechanisms in a patient treated with crizotinib. The METΔ14 mutation and aberrant mRNA transcript are most common in lung adenocarcinoma and also identified in other cancers. Endogenous levels of METΔ14 transcript transform human epithelial lung cells in an HGF-dependent manner. This allele also induces lung cancer in mice that is responsive to a clinically available MET inhibitor. Finally, we document clinical response to crizotinib in a patient with a METΔ14-driven NSCLC lung cancer. Upon clinical progression on crizotinib, indicating acquired resistance, we detected acquired MET mutations in cell-free DNA from the patient that converge on critical drug-binding residues in the MET activation loop. These findings qualify METΔ14 mutations as drivers of lung adenocarcinoma, demonstrate the utility of a new animal model of MET-driven disease and identify a subpopulation of patients who may benefit from further development of targeted MET/HGF therapies.

HER2 Transmembrane Domain (TMD) Mutations (V659/G660) That Stabilize Homo- and Heterodimerization Are Rare Oncogenic Drivers in Lung Adenocarcinoma That Respond to Afatinib

IntroductionErb-b2 receptor tyrosine kinase (HER2) transmembrane domain (TMD) mutations (HER2V659E, HER2G660D) have previously been identified in lung adenocarcinomas, but their frequency and clinical significance is unknown. MethodsWe prospectively analyzed 8551 consecutive lung adenocarcinomas using hybrid capture–based comprehensive genomic profiling (CGP) at the request of the individual treating physicians for the purpose of making therapy decisions. ResultsWe identified 15 cases (0.18%) of HER2 TMD mutations (HER2V659E/D, HER2G660D) through CGP of 8551 lung adenocarcinomas. HER2 TMD mutations were mutually exclusive from HER2 kinase domain mutations and other oncogenic drivers in lung adenocarcinoma. Only two cases with HER2 TMD mutations (13%) had concurrent Erb-b2 receptor tyrosine kinase 2 gene (HER2) amplification. Structural analysis of HER2 TMD association revealed that mutations at positions V659 and G660 to the highly polar residues glutamic acid, aspartic acid, or arginine should stabilize homodimerization and heterodimerization of HER2 in the active conformation. Treatment with afatinib, a pan-HER inhibitor, resulted in durable clinical response in three of four patients with lung adenocarcinoma, with two harboring HER2V659E and one with double HER2V659E/G660R mutations. HER2 TMD mutations (V659 and G660) are found in other non-NSCLC malignancies, and analogous TMD mutations are also found in EGFR, HER3, and HER4. ConclusionHER2 TMD mutations represent rare but distinct targetable driver mutations in lung adenocarcinoma. CGP capable of detecting diverse HER2 alterations, including HER2 TMD mutations, should be broadly adopted to identify all patients who may benefit from HER2-targeted therapies.

1267P - HER2 V659 and G660 transmembrane mutations that stabilize homo- and hetero-dimerization are rare oncogenic drivers in lung adenocarcinoma that are clinically responsive to afatinib

1267P - HER2 V659 and G660 transmembrane mutations that stabilize homo- and hetero-dimerization are rare oncogenic drivers in lung adenocarcinoma that are clinically responsive to afatinib

Comprehensive Characterization of Oncogenic Drivers in Asian Lung Adenocarcinoma

IntroductionThe incidence rate of lung adenocarcinoma (LUAD), the predominant histological subtype of lung cancer, is elevated in Asians, particularly in female nonsmokers. The mutation patterns in LUAD in Asians might be distinct from those in LUAD in whites. MethodsWe profiled 271 resected LUAD tumors (mainly stage I) to characterize the genomic landscape of LUAD in Asians with a focus on female nonsmokers. ResultsMutations in EGFR, KRAS, erb-b2 receptor tyrosine kinase 2 gene (ERBB2), and BRAF; gene fusions involving anaplastic lymphoma receptor tyrosine kinase gene (ALK), ROS1, and ret proto-oncogene (RET); and Met Proto-Oncogene Tyrosine Kinase (MET) exon 14 skipping were the major drivers in LUAD in Asians, exhibiting mutually exclusive and differing prevalence from those reported in studies of LUAD in non-Asians. In addition, we identified a novel mutational signature of XNX (the mutated base N in the middle flanked by two identical bases at the 5′ and 3′ positions) that was overrepresented in LUAD tumors in nonsmokers and negatively correlated with the overall mutational frequency. ConclusionsIn this cohort, approximately 85% of individuals have known driver mutations (EGFR 59.4%, KRAS 7.4%, ALK 7.4%, ERBB2 2.6%, ROS1 2.2%, RET 2.2%, MET 1.8%, BRAF 1.1%, and NRAS 0.4%). Seventy percent of smokers and 90% of nonsmokers had defined oncogenic drivers matching the U.S. Food and Drug Administration–approved targeted therapies.