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Supplementary Data from Enhanced Vulnerability of LKB1-Deficient NSCLC to Disruption of ATP Pools and Redox Homeostasis by 8-Cl-Ado

Supplementary Methods. Supplementary Table 2. Details of the antibody used for TTF1 immunostaining.

Supplementary Figure S1 Details. Expression levels of 384 genes selected for the NMF algorithm in the three KRAS-mutant LUAC subsets.

Supplementary Table S1. Individual KRAS, STK11/LKB1, TP53, ATM and KEAP1 somatic mutations from the indicated clinical cohorts.

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Supplementary figure legend

PDF file - 167K, Different probes for the same gene vary within and across microarray platforms

Purpose: Epithelial–mesenchymal transition (EMT) has been associated with metastatic spread and EGF receptor (EGFR) inhibitor resistance. We developed and validated a robust 76-gene EMT signature using gene expression profiles from four platforms using non–small cell lung carcinoma (NSCLC) cell lines and patients treated in the Biomarker-Integrated Approaches of Targeted Therapy for Lung Cancer Elimination (BATTLE) study.Experimental Design: We conducted an integrated gene expression, proteomic, and drug response analysis using cell lines and tumors from patients with NSCLC. A 76-gene EMT signature was developed and validated using gene expression profiles from four microarray platforms of NSCLC cell lines and patients treated in the BATTLE study, and potential therapeutic targets associated with EMT were identified.Results: Compared with epithelial cells, mesenchymal cells showed significantly greater resistance to EGFR and PI3K/Akt pathway inhibitors, independent of EGFR mutation status, but more sensitivity to certain chemotherapies. Mesenchymal cells also expressed increased levels of the receptor tyrosine kinase Axl and showed a trend toward greater sensitivity to the Axl inhibitor SGI-7079, whereas the combination of SGI-7079 with erlotinib reversed erlotinib resistance in mesenchymal lines expressing Axl and in a xenograft model of mesenchymal NSCLC. In patients with NSCLC, the EMT signature predicted 8-week disease control in patients receiving erlotinib but not other therapies.Conclusion: We have developed a robust EMT signature that predicts resistance to EGFR and PI3K/Akt inhibitors, highlights different patterns of drug responsiveness for epithelial and mesenchymal cells, and identifies Axl as a potential therapeutic target for overcoming EGFR inhibitor resistance associated with the mesenchymal phenotype. Clin Cancer Res; 19(1); 279–90. ©2012 AACR.

Purpose: Although postoperative radiotherapy is often used to maintain local control after surgical resection and chemotherapy for locally advanced non–small cell lung cancer (NSCLC), both locoregional failure and distant metastasis remain problematic. The mechanisms of therapeutic resistance remain poorly understood.Experimental Design: We used reverse-phase protein arrays (RPPA) to profile the baseline expression of 170 total and phosphorylated proteins in 70 NSCLC cell lines to categorize pathways that may contribute to radiation resistance. Significant markers identified by RPPA were further analyzed in tissue microarrays (TMA) of specimens from 127 patients with NSCLC who had received surgery before receiving postoperative radiotherapy. Cox regression analysis and log-rank tests were used to identify potential predictive factors. We then validated the biological function of the markers in NSCLC cell lines in vitro.Results: Of the 170 proteins or phospho-proteins profiled, a subset of 12 proteins was found to correlate with radiation response parameters. TMA analysis of the 12 proteins showing the greatest differences in expression in the RPPA analysis demonstrated that RAD50 had the strongest correlation with distant relapse-free survival, locoregional relapse-free survival, and disease-free survival in patients with NSCLC. We confirmed that knockdown of RAD50 sensitized NSCLC cells to radiation and that upregulation of RAD50 increased radioresistance in in vitro experiments.Conclusions: Upregulated RAD50 may be a predictor of radioresistance in patients with lung cancer who received radiotherapy. Clin Cancer Res; 24(2); 341–50. ©2017 AACR.

PDF file - 66K, CDH1 probes vary in their accuracy and dynamic range

Supplemental Figure 2. VEGFR TKIs inhibit the migration of NSCLC cells with KDR CNGs.

Purpose: VEGF pathway inhibitors have been investigated as therapeutic agents in the treatment of non–small cell lung cancer (NSCLC) because of its central role in angiogenesis. These agents have improved survival in patients with advanced NSCLC, but the effects have been modest. Although VEGFR2/KDR is typically localized to the vasculature, amplification of KDR has reported to occur in 9% to 30% of the DNA from different lung cancers. We investigated the signaling pathways activated downstream of KDR and whether KDR amplification is associated with benefit in patients with NSCLC treated with the VEGFR inhibitor vandetanib.Methods: NSCLC cell lines with or without KDR amplification were studied for the effects of VEGFR tyrosine kinase inhibitors (TKI) on cell viability and migration. Archival tumor samples collected from patients with platinum-refractory NSCLC in the phase III ZODIAC study of vandetanib plus docetaxel or placebo plus docetaxel (N = 294) were screened for KDR amplification by FISH.Results: KDR amplification was associated with VEGF-induced activation of mTOR, p38, and invasiveness in NSCLC cell lines. However, VEGFR TKIs did not inhibit proliferation of NSCLC cell lines with KDR amplification. VEGFR inhibition decreased cell motility as well as expression of HIF1α in KDR-amplified NSCLC cells. In the ZODIAC study, KDR amplification was observed in 15% of patients and was not associated with improved progression-free survival, overall survival, or objective response rate for the vandetanib arm.Conclusions: Preclinical studies suggest KDR activates invasion but not survival pathways in KDR-amplified NSCLC models. Patients with NSCLC whose tumor had KDR amplification were not associated with clinical benefit for vandetanib in combination with docetaxel. Clin Cancer Res; 22(8); 1940–50. ©2015 AACR.

Supplementary Table S1. List of all primers and oligos used in this study. Supplementary Table S2. Subcellular staining locations of 12 candidate marker proteins in tumor microarray. Supplementary Table S3. Staining results for 17 candidate marker protein variables in the tumor microarray. Supplementary Table S4. Univariate and multivariate analyses of associations between clinical variables and RAD50 expression for OS, LRRFS, DMFS and DFS among 127 NSCLC patients. Supplementary Table S5. Univariate and multivariate analyses of associations between all the 17 markers and OS, LRRFS, DMFS and DFS among 127 NSCLC patients.

PDF file - 175K

PDF file - 50K, Mesenchymal lines are sensitive to Axl inhibition by SGI-7079

Supplemental Figure 1. (A) Calu-1 cells were treated with VEGF (50 ng/ml) for 15 minutes and phospho-p38 levels were evaluated by Western blotting. VEGF treatment resulted in activation of p38, and this was blocked with the addition of VEGFR TKIs axitinib or sorafenib. (B) A549, H23, and Calu-1 cells express minimal levels of VEGFR1 as determined by ELISA assay. SKNAS neuroblastoma cells serve as a positive control.

PDF file - 214K, The EMT signature genes

PDF file - 71K, Percentage tumor volume compared to control, survival and therapeutic efficacy at days 17 and 38 post-treatment

PDF file - 51K, EMT status of commonly mutated genes in NSCLC

Supplementary Figure S1. Q-Q plot of NSCLC cell line radiosensitivity distribution. Supplementary Figure S2. Kaplan-Meier curve of in-field recurrence free survival in high-RAD50 and low-RAD50 tumors in all patients (Suppl. Fig. S2A) and in stage III patients (Suppl. Fig. S2B). Supplementary Figure S3. Column graph of RAD50 H-score in patients with or without neoadjuvant chemotherapy. Supplementary Figure S4. Cell cycle distribution of H23-SAM-RAD50 and H23-SAM-Control cells, with or without radiation.

In the TCGA NSCLC dataset there was a significantly greater prevalence of p53 mutations in patients with KDR CNG compared to KDR CNG negative patients (p = 0.001).

Supplementary Methods

PDF file - 27K, SGI-7079 and erlotinib do not adversely affect animal body weight

PDF file - 96K, Structure of pyrrolopyrimidine AXL inhibitor SGI-7079

Loss-of-function somatic mutations of STK11, a tumor suppressor gene encoding LKB1 that contributes to the altered metabolic phenotype of cancer cells, is the second most common event in lung adenocarcinomas and often co-occurs with activating KRAS mutations. Tumor cells lacking LKB1 display an aggressive phenotype, with uncontrolled cell growth and higher energetic and redox stress due to its failure to balance ATP and NADPH levels in response to cellular stimulus. The identification of effective therapeutic regimens for patients with LKB1-deficient non–small cell lung cancer (NSCLC) remains a major clinical need. Here, we report that LKB1-deficient NSCLC tumor cells displayed reduced basal levels of ATP and to a lesser extent other nucleotides, and markedly enhanced sensitivity to 8-Cl-adenosine (8-Cl-Ado), an energy-depleting nucleoside analog. Treatment with 8-Cl-Ado depleted intracellular ATP levels, raised redox stress, and induced cell death leading to a compensatory suppression of mTOR signaling in LKB1-intact, but not LKB1-deficient, cells. Proteomic analysis revealed that the MAPK/MEK/ERK and PI3K/AKT pathways were activated in response to 8-Cl-Ado treatment and targeting these pathways enhanced the antitumor efficacy of 8-Cl-Ado. Implications: Together, our findings demonstrate that LKB1-deficient tumor cells are selectively sensitive to 8-Cl-Ado and suggest that therapeutic approaches targeting vulnerable energy stores combined with signaling pathway inhibitors merit further investigation for this patient population.

Inactivation of p53 is present in almost every tumor, and hence, p53-reactivation strategies are an important aspect of cancer therapy. Common mechanisms for p53 loss in cancer include expression of p53-negative regulators such as MDM2, which mediate the degradation of wildtype p53 (p53α), and inactivating mutations in the TP53 gene. Currently, approaches to overcome p53 deficiency in these cancers are limited. Here, using non–small cell lung cancer and glioblastoma multiforme cell line models, we show that two alternatively spliced, functional truncated isoforms of p53 (p53β and p53γ, comprising exons 1 to 9β or 9γ, respectively) and that lack the C-terminal MDM2-binding domain have markedly reduced susceptibility to MDM2-mediated degradation but are highly susceptible to nonsense-mediated decay (NMD), a regulator of aberrant mRNA stability. In cancer cells harboring MDM2 overexpression or TP53 mutations downstream of exon 9, NMD inhibition markedly upregulates p53β and p53γ and restores activation of the p53 pathway. Consistent with p53 pathway activation, NMD inhibition induces tumor suppressive activities such as apoptosis, reduced cell viability, and enhanced tumor radiosensitivity, in a relatively p53-dependent manner. In addition, NMD inhibition also inhibits tumor growth in a MDM2-overexpressing xenograft tumor model. These results identify NMD inhibition as a novel therapeutic strategy for restoration of p53 function in p53-deficient tumors bearing MDM2 overexpression or p53 mutations downstream of exon 9, subgroups that comprise approximately 6% of all cancers.

Common mechanisms for p53 loss in cancer include expression of MDM2 or the human papilloma virus (HPV)-encoded E6 protein which both mediate degradation of wild-type (WT) p53 (p53α). Here, we show that two alternatively-spliced, functional, truncated isoforms of p53 (p53β and p53γ, containing exons 1-9 of the p53 gene) can be markedly upregulated by pharmacologic or genetic inhibition of nonsense mediated decay (NMD), a regulator of aberrant mRNA stability. These isoforms lack the MDM2 binding domain and hence have reduced susceptibility to MDM2-mediated degradation. In MDM2-overexpressing cells bearing wildtypeTP53gene, NMD blockade increased p53β/γ expression and p53 pathway activation, enhanced radiosensitivity, and inhibited tumor growth. A similar pattern was observed in HPV+cancer cells and in cancer cells with p53 mutations downstream of exon 9. These results identify a novel therapeutic strategy for restoration of p53 function in tumors rendered p53 deficient through MDM2 overexpression, HPV infection, or certain p53 mutations.

HPV-positive (HPV+) head and neck squamous cell carcinoma (HNSCC) tumors typically have p53 loss due to the activity of the human papillomavirus (HPV)-encoded E6 protein and the E6-associated protein (HPVE6-AP) which mediate the degradation of wild-type (WT) p53 (p53α). The loss of p53 is thought to be a major contributor to the pathogenesis of HPV+ HNSCC, which comprise approximately 35% of all HNSCC. Currently, standard care for HPV+HNSCC includes radiation and chemotherapy. However long-term toxicity related to these treatments is a concern, and there is a need for newer therapeutic strategies. Previously, we reported that two alternatively spliced, functional truncated isoforms of p53 (p53β and p53γ, comprising of exons 1 to 9β or 9γ, respectively) are degraded by nonsense-mediated decay (NMD), a regulator of aberrant mRNA stability. Here, using HPV+HNSCC cell line models, we show that NMD inhibition rescues p53β/γ isoforms and activates p53 pathway. Furthermore, we show that p53β/γ isoforms are more stable compared to p53α in these cells, with reduced vulnerabililty to HPVE6-AP- mediated degradation, and that p53β/γ isoforms contribute to increased expression of p53 transcriptional targets p21 and PUMA following NMD inhibition. Consistent with p53 pathway activation, NMD inhibition enhanced radiosensitivity of HNSCC cells. NMD inhibition attenuated colony forming ability and disrupted cell cycle progression. To evaluate the therapeutic implications of NMD inhibition, we assessed the in vivo growth of HPV+ UMSCC47 tumors. Nude mice were injected with UMSCC47 cells either subcutaneously or orthotopically in the tongue and randomized to receive vehicle or with an NMD inhibitor. In both tumor models, we observed a significant reduction in tumor volume with NMD inhibition as compared to the vehicle-treated animals. To investigate whether NMD inhibition induced the expression of p53β/γ isoforms and activated the p53 pathway in vivo, we collected tumor tissues from animals and evaluated expression of p53 isoforms and transcriptional targets by RT-PCR. We observed increased expression of p53γ, p21, GADD45A and PUMA mRNAs in NMD inhibitor treated UMSCC47 tumors, compared to their respective vehicle treated controls. These results identify NMD inhibition as a novel therapeutic strategy for restoration of p53 function in major subgroups of p53-deficient HPV+ HNSCC tumors. Citation Format: Jayanthi Gudikote, Tina Cascone, Alissa Poteete, Piyada Sitthideatphaiboon, Sonia Patel, Yan Yang, Fahao Zhang, Lerong Li, Li Shen, Monique Nilsson, Phillip Jones, Jing Wang, Jean-Christophe Bourdon, Faye M. Johnson, John V. Heymach. Targeting nonsense-mediated decay restores p53 function in HPV-associated head and neck cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5733.

Purpose: Although postoperative radiotherapy is often used to maintain local control after surgical resection and chemotherapy for locally advanced non–small cell lung cancer (NSCLC), both locoregional failure and distant metastasis remain problematic. The mechanisms of therapeutic resistance remain poorly understood. Experimental Design: We used reverse-phase protein arrays (RPPA) to profile the baseline expression of 170 total and phosphorylated proteins in 70 NSCLC cell lines to categorize pathways that may contribute to radiation resistance. Significant markers identified by RPPA were further analyzed in tissue microarrays (TMA) of specimens from 127 patients with NSCLC who had received surgery before receiving postoperative radiotherapy. Cox regression analysis and log-rank tests were used to identify potential predictive factors. We then validated the biological function of the markers in NSCLC cell lines in vitro. Results: Of the 170 proteins or phospho-proteins profiled, a subset of 12 proteins was found to correlate with radiation response parameters. TMA analysis of the 12 proteins showing the greatest differences in expression in the RPPA analysis demonstrated that RAD50 had the strongest correlation with distant relapse-free survival, locoregional relapse-free survival, and disease-free survival in patients with NSCLC. We confirmed that knockdown of RAD50 sensitized NSCLC cells to radiation and that upregulation of RAD50 increased radioresistance in in vitro experiments. Conclusions: Upregulated RAD50 may be a predictor of radioresistance in patients with lung cancer who received radiotherapy. Clin Cancer Res; 24(2); 341–50. ©2017 AACR.

Purpose: VEGF pathway inhibitors have been investigated as therapeutic agents in the treatment of non–small cell lung cancer (NSCLC) because of its central role in angiogenesis. These agents have improved survival in patients with advanced NSCLC, but the effects have been modest. Although VEGFR2/KDR is typically localized to the vasculature, amplification of KDR has reported to occur in 9% to 30% of the DNA from different lung cancers. We investigated the signaling pathways activated downstream of KDR and whether KDR amplification is associated with benefit in patients with NSCLC treated with the VEGFR inhibitor vandetanib. Methods: NSCLC cell lines with or without KDR amplification were studied for the effects of VEGFR tyrosine kinase inhibitors (TKI) on cell viability and migration. Archival tumor samples collected from patients with platinum-refractory NSCLC in the phase III ZODIAC study of vandetanib plus docetaxel or placebo plus docetaxel (N = 294) were screened for KDR amplification by FISH. Results: KDR amplification was associated with VEGF-induced activation of mTOR, p38, and invasiveness in NSCLC cell lines. However, VEGFR TKIs did not inhibit proliferation of NSCLC cell lines with KDR amplification. VEGFR inhibition decreased cell motility as well as expression of HIF1α in KDR-amplified NSCLC cells. In the ZODIAC study, KDR amplification was observed in 15% of patients and was not associated with improved progression-free survival, overall survival, or objective response rate for the vandetanib arm. Conclusions: Preclinical studies suggest KDR activates invasion but not survival pathways in KDR-amplified NSCLC models. Patients with NSCLC whose tumor had KDR amplification were not associated with clinical benefit for vandetanib in combination with docetaxel. Clin Cancer Res; 22(8); 1940–50. ©2015 AACR.

Introduction: Blockade of immune checkpoints has improved clinical outcomes for patients with metastatic non-small cell lung cancer (NSCLC), but its role in the perioperative setting for early-stage disease is unclear. We generated preclinical models of resectable NSCLC expressing an antigen that permits quantitative assessment of the immune response and compared survival, tumor recurrence, and immune response after neoadjuvant or adjuvant immunotherapy. Experimental Procedures: We transfected murine 344SQ NSCLC cells with an ovalbumin-expression plasmid to generate OVA+ cells that can be identified with an antibody against the peptide SIINFEKL bound to H-2Kb of MHC-I. We implanted 344SQ-OVA+ cells in the flank of syngeneic mice and then randomized mice with established tumors to either 3 doses of neoadjuvant IgG, anti-PD-1, anti-CTLA-4, anti-PD-1+anti-CTLA-4, or to observation. Primary tumors were resected in all mice 2 days after mice in the neoadjuvant group had received their last dose of therapy. Two days post-surgery, mice in the observation group were treated with 3 doses of adjuvant IgG, anti-PD-1, anti-CTLA-4, anti-PD-1+anti-CTLA-4. Mice were euthanized 4 weeks after injection or when moribund and their survival recorded and lung metastases counted. We determined the composition of CD3+CD8+ tumor-infiltrating lymphocytes (TILs) with flow cytometry using tetramers against SIINFEKL-specific T cell receptors and immunohistochemical staining of tumors. Results: Single agent and combined immunotherapy significantly prolonged survival compared to controls in both neoadjuvant and adjuvant groups (p Conclusions: Neoadjuvant combined immune checkpoint blockade is superior to adjuvant immunotherapy at prolonging survival, reducing distal recurrence and inducing anti-tumor immunity in preclinical models of resectable NSCLC. Citation Format: Tina Cascone, Haifa Hamdi, Fahao Zhang, Alissa Poteete, Lerong Li, Courtney W. Hudgens, Leila J. Williams, Qiuyu Wu, Jayanthi Gudikote, Weiyi Peng, Patrick Hwu, Jing Wang, Michael Tetzlaff, William N. William, John V. Heymach. Superior efficacy of neoadjuvant compared to adjuvant immune checkpoint blockade in non-small cell lung cancer [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 1719.

The T-cell receptor (TCR) and immunoglobulin (Ig) genes are unique among vertebrate genes in that they undergo programmed rearrangement, a process that allows them to generate an enormous array of receptors with different antigen specificities. While crucial for immune function, this rearrangement mechanism is highly error prone, often generating frameshift or nonsense mutations that render the rearranged TCR and Ig genes defective. Such frame-disrupting mutations have been reported to increase the level of TCRbeta and Igmicro pre-mRNA, suggesting the hypothesis that RNA processing is blocked when frame disruption is sensed. Using a chimeric gene that contains TCRbeta sequences conferring this upregulatory response, we provide evidence that pre-mRNA upregulation is neither frame- nor translation-dependent; instead, several lines of evidence suggested that it is the result of disrupted cis elements necessary for efficient RNA splicing. In particular, we identify the rearranging VDJ(beta) exon as being uniquely densely packed with exonic-splicing enhancers (ESEs), rendering this exon hypersensitive to mutational disruption. As the chimeric gene that we developed for these studies generates unusually stable nuclear pre-mRNAs that accumulate when challenged with ESE mutations, we suggest it can be used as a sensitive in vivo system to identify and characterize ESEs.

The T-cell receptor (TCR) locus undergoes programmed rearrangements that frequently generate premature termination codons (PTCs). The PTC-bearing transcripts derived from such nonproductively rearranged genes are dramatically downregulated by the nonsense-mediated decay (NMD) pathway. Here, we show that depletion of the NMD factor UPF3b does not impair TCRbeta NMD, thereby distinguishing it from classical NMD. Depletion of the related factor UPF3a, by itself or in combination with UPF3b, also has no effect on TCRbeta NMD. Mapping experiments revealed the identity of TCRbeta sequences that elicit a switch to UPF3b dependence. This regulation is not a peculiarity of TCRbeta, as we identified many wild-type genes, including one essential for NMD, that transcribe NMD-targeted mRNAs whose downregulation is little or not affected by UPF3a and UPF3b depletion. We propose that we have uncovered an alternative branch of the NMD pathway that not only degrades aberrant mRNAs but also regulates normal mRNAs, including one that participates in a negative feedback loop controlling the magnitude of NMD.

The molecular underpinnings that drive the heterogeneity of KRAS-mutant lung adenocarcinoma are poorly characterized. We performed an integrative analysis of genomic, transcriptomic, and proteomic data from early-stage and chemorefractory lung adenocarcinoma and identified three robust subsets of KRAS-mutant lung adenocarcinoma dominated, respectively, by co-occurring genetic events in STK11/LKB1 (the KL subgroup), TP53 (KP), and CDKN2A/B inactivation coupled with low expression of the NKX2-1 (TTF1) transcription factor (KC). We further revealed biologically and therapeutically relevant differences between the subgroups. KC tumors frequently exhibited mucinous histology and suppressed mTORC1 signaling. KL tumors had high rates of KEAP1 mutational inactivation and expressed lower levels of immune markers, including PD-L1. KP tumors demonstrated higher levels of somatic mutations, inflammatory markers, immune checkpoint effector molecules, and improved relapse-free survival. Differences in drug sensitivity patterns were also observed; notably, KL cells showed increased vulnerability to HSP90-inhibitor therapy. This work provides evidence that co-occurring genomic alterations identify subgroups of KRAS-mutant lung adenocarcinoma with distinct biology and therapeutic vulnerabilities. Significance: Co-occurring genetic alterations in STK11/LKB1, TP53, and CDKN2A/B—the latter coupled with low TTF1 expression—define three major subgroups of KRAS-mutant lung adenocarcinoma with distinct biology, patterns of immune-system engagement, and therapeutic vulnerabilities. Cancer Discov; 5(8); 860–77. ©2015 AACR. This article is highlighted in the In This Issue feature, p. 783

Aberrant mRNAs harboring premature termination codons (PTCs or nonsense codons) are degraded by the nonsense-mediated mRNA decay (NMD) pathway. mRNAs transcribed from genes that naturally acquire PTCs during lymphocyte development are strongly downregulated by PTCs. Here we show that a signal essential for this robust mRNA downregulatory response is efficient RNA splicing. Strong mRNA downregulation can be conferred on a poor NMD substrate by either strengthening its splicing signals or removing its weak introns. Efficient splicing also strongly promotes translation, providing a molecular explanation for enhanced NMD and suggesting that efficient splicing may have evolved to enhance both protein production and RNA surveillance. Our results suggest simple approaches for increasing protein expression from expression vectors and treating human genetic diseases caused by nonsense and frameshift mutations.

The nonsense-mediated decay (NMD) RNA surveillance pathway detects and degrades mRNAs containing premature termination codons (PTCs). T-cell receptor (TCR) and immunoglobulin transcripts, which commonly harbor PTCs as a result of programmed DNA rearrangement during normal development, are down-regulated much more than other known mammalian gene transcripts in response to nonsense codons. Here, we demonstrate that this is not because of promoter or cell type but instead is directed by regulatory sequences within the rearranging VDJ exon and immediately flanking intron sequences of a Vbeta8.1 TCR-beta gene. Insertion of these sequences into a heterologous gene elicited strong down-regulation (30-fold) in response to PTCs, indicating that this region is sufficient to trigger robust down-regulation. The rearranging Vbeta5.1 exon and the flanking intron sequences from another member of the TCR-beta family also triggered strong down-regulation, suggesting that down-regulatory-promoting elements are a conserved feature of TCR genes. Importantly, we found that the Vbeta8.1 down-regulatory-promoting element was position dependent, such that it failed to function when positioned downstream of a PTC. To our knowledge, this is the first class of down-regulatory elements identified that act upstream of nonsense codons.

e23222Background: MEK inhibitors have limited success in cancer treatment because tumors circumvent their action. Here, we report broad proteomic analysis of NSCLC cell lines after treatment with t...

Purpose: Epithelial–mesenchymal transition (EMT) has been associated with metastatic spread and EGF receptor (EGFR) inhibitor resistance. We developed and validated a robust 76-gene EMT signature using gene expression profiles from four platforms using non–small cell lung carcinoma (NSCLC) cell lines and patients treated in the Biomarker-Integrated Approaches of Targeted Therapy for Lung Cancer Elimination (BATTLE) study. Experimental Design: We conducted an integrated gene expression, proteomic, and drug response analysis using cell lines and tumors from patients with NSCLC. A 76-gene EMT signature was developed and validated using gene expression profiles from four microarray platforms of NSCLC cell lines and patients treated in the BATTLE study, and potential therapeutic targets associated with EMT were identified. Results: Compared with epithelial cells, mesenchymal cells showed significantly greater resistance to EGFR and PI3K/Akt pathway inhibitors, independent of EGFR mutation status, but more sensitivity to certain chemotherapies. Mesenchymal cells also expressed increased levels of the receptor tyrosine kinase Axl and showed a trend toward greater sensitivity to the Axl inhibitor SGI-7079, whereas the combination of SGI-7079 with erlotinib reversed erlotinib resistance in mesenchymal lines expressing Axl and in a xenograft model of mesenchymal NSCLC. In patients with NSCLC, the EMT signature predicted 8-week disease control in patients receiving erlotinib but not other therapies. Conclusion: We have developed a robust EMT signature that predicts resistance to EGFR and PI3K/Akt inhibitors, highlights different patterns of drug responsiveness for epithelial and mesenchymal cells, and identifies Axl as a potential therapeutic target for overcoming EGFR inhibitor resistance associated with the mesenchymal phenotype. Clin Cancer Res; 19(1); 279–90. ©2012 AACR.

Introduction The development of more effective treatment strategies for LUAC bearing activating mutations in KRAS is hampered by the biological heterogeneity of KRAS-mutant tumors. The molecular underpinnings that drive this process are poorly characterized. Here, we implemented an integrated approach to the discovery of biologically distinct subsets of KRAS-mutant LUAC and explored their molecular vulnerabilities. Methods Our datasets consisted of 68 KRAS-mutant tumors from TCGA, 88 additional chemo-naive KRAS-mutant LUACs (PROSPECT and Chitale datasets) and 36 platinum-refractory LUACs from the BATTLE-2 clinical trial. Non-negative matrix factorization (NMF) consensus clustering was applied to RNASeq data as previously described. Signature enrichment was assessed using Gene Set Enrichment Analysis (GSEA). Results NMF consensus clustering identified three robust subsets of KRAS-mutant LUAC that were reproducible across diverse clinical datasets of early-stage, chemotherapy-naive and metastatic, chemo-refractory tumors. Distinct KRAS-mutant alleles were not differentially represented in the three subgroups (P = 0.3). In contrast, the subgroups were dominated, respectively, by co-occurring genetic events in STK11/LKB1 (termed the KL subgroup) (P = 1.03e−05), TP53 (KP) (P = 3.8e-06) and low expression of TTF1 coupled with frequent CDKN2A/B inactivation (KC) (P = 0.004 and P = 0.002). Distinct patterns of intracellular signaling were detected in the three subsets. KL tumors showed evidence of LKB1-AMPK pathway inactivation and adaptation to energetic, proteotoxic and oxidative stress, the latter exemplified by near ubiquitous inactivation of KEAP1 and up-regulation of a NRF2-driven antioxidant signature. KP tumors carried a higher somatic mutation load and were characterized by prominent inflammation and up-regulation of several immune checkpoint effector molecules, including PD-L1. KC tumors frequently displayed a GI-like differentiation program, suppression of MTORC1 signaling and elevated wild-type p53 transcriptional output. Using a large panel of KRAS-mutant NSCLC cell lines we detected co-mutation-dependent patterns of drug sensitivity. Specifically, KL cell lines showed enhanced sensitivity to several structurally distinct HSP90 inhibitors. These results were confirmed in panels of isogenic cell lines. Mechanistically, treatment with ganetespib resulted in concurrent degradation of several molecules with established role in supporting the fitness of LKB1-deficient cells. Conclusions Our work identifies three major subsets of KRAS-mutant LUAC - dominated by co-occurring genetic events - with distinct biology and therapeutic vulnerabilities. Citation Format: Ferdinandos Skoulidis, Lauren Byers, Lixia Diao, Vassiliki Papadimitrakopoulou, Pan Tong, Julie Izzo, Carmen Behrens, Humam Kadara, Edwin R. Parra, Jaime Rodriguez-Canales, Jianjun Zhang, Uma Giri, Jayanthi Gudikote, Maria Angelica Cortez, Chao Yang, You Hong Fan, Michael Peyton, Luc Girard, Kevin R. Coombes, Carlo Toniatti, Timothy P. Heffernan, Murim Choi, Garrett M. Frampton, Vincent Miller, John N. Weinstein, Roy S. Herbst, Kwok-Kin Wong, Jianhua Zhang, Padmanee Sharma, Gordon M. Mills, Waun Ki Hong, John D. Minna, James P. Allison, Andrew Futreal, Jing Wang, Ignacio Wistuba, John V. Heymach. Co-occurring genomic alterations define major subsets of KRAS-mutant lung adenocarcinoma (LUAC) with distinct biology and therapeutic vulnerabilities. [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 968. doi:10.1158/1538-7445.AM2015-968

Mammalian messenger ribonucleic acid (mRNA)-binding proteins regulate gene expression at several steps including pre-mRNA processing, nucleocytoplasmic export and degradation. Keywords: pre-mRNA processing; mRNA transport; mRNA stability; RNA splicing; RNA-binding proteins; nonsense-mediated mRNA decay; exon-junction complex

Nonsense-mediated decay (NMD) is an RNA surveillance mechanism that degrades mRNAs containing premature termination (nonsense) codons. The second signal for this pathway in mammalian cells is an intron that must be at least approximately 55 nucleotides downstream of the nonsense codon. Although the functional significance of this '-55 boundary rule' is not known, it is widely thought to reflect the important role of an exon junction protein complex deposited just upstream of exon-exon junctions after RNA splicing. Here we report that a T-cell receptor (TCR)-beta gene did not conform to this rule. Rather than a definitive boundary position, nonsense codons had a polar effect, such that nonsense codons distant from the terminal downstream intron triggered robust NMD and proximal nonsense codons caused modest NMD. We identified a region of the TCR-beta gene that conferred this boundary-independent polar expression pattern on a heterologous gene. Collectively, our results suggest that TCR-beta transcripts contain one or more sequence elements that elicit an unusual NMD response triggered by a novel second signal that ultimately causes boundary-independent polar regulation. TCR genes may have evolved this unique NMD response because they frequently acquire nonsense codons during normal development.

Background: It has been demonstrated that loss of LKB1 is associated with a higher likelihood of tumor recurrence after surgery and shorter overall survival in early stage NSCLC. We sought to determine whether NSCLC tumor cells with loss of LKB1 are preferentially sensitive or resistant to various therapeutic agents. We tested 23 NSCLC cell lines, and observed that LKB1 mutant NSCLC cells are highly resistant to standard chemotherapy as well as EGFR inhibitors. In contrast, LKB1 mutant NSCLC cells are highly sensitive to 8-Chloroadenosine (8-Cl-Ado). 8-Cl-Ado is an RNA-directed nucleoside analogue, which depletes cellular ATP and triggers apoptosis in sensitive cells. LKB1 is known to play a key role in balancing cellular energy consumption and production. Therefore, we hypothesize that LKB1 mutant NSCLC cells are vulnerable to 8-Cl-Ado induced energetic stress. Method: Cellular ATP level was determined by LC-MS and luminescent based methods. Cell proliferation was measured by Celltiter-Glo assay. IC50 values were calculated by Calcusyn. Oxidative stress was measured by CellRox assay. Result: NSCLC cell lines with LKB1 mutation were significantly more sensitive to 8-Cl-Ado than wild type cell lines (p=0.024). We transfected H460 and A549 cells (both harboring mutant LKB1) with wild-type LKB1. Re-expression of LKB1 rendered cells resistant to 8-Cl-Ado (P Citation Format: Chao Yang, Maria Angelica Cortez, Uma Giri, Jayanthi Gudikote, William G. Wierda, Varsha Gandhi, Lauren Averett Byers, John V. Heymach. NSCLC cells with LKB1 mutation are vulnerable to energetic and oxidative stresses induced by 8-Chloroadenosine. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5480. doi:10.1158/1538-7445.AM2014-5480

Targeted therapies designed to inhibit the vascular endothelial growth factor (VEGF) pathway have been extensively evaluated in the treatment of malignancies including Non-Small Cell Lung Cancer (NSCLC). VEGF pathway inhibitors such as bevacizumab, or the multitargeted receptor tyrosine kinase inhibitors (TKIs) vandetanib and sorafenib, have been shown to prolong progression-free survival (PFS) and/or overall survival (OS). These benefits, however, have been modest, seen only in subsets of patients. Thus, predictive markers for identifying which patients are likely to benefit are critically needed. Although expression of VEGF receptor-2 (VEGFR-2, also known as KDR) was initially thought to primarily occur in endothelial cells, VEGFR-2 has been detected on malignant cells, including lung cancer cells, and in NSCLC, overexpression of VEGFR-2 on tumor cells is associated with a poor clinical outcome. Amplification of KDR has been detected in lung cancer specimens at a relatively high frequency (9% and 32%). The consequences of KDR copy number gains (CNGs) are not yet understood. Recently, we have shown that NSCLC cell lines with KDR copy number gains (CNGs) were associated with in vitro resistance to platinum chemotherapy, and KDR CNG predicted worse overall survival in patients who received platinum adjuvant therapy but not in untreated patients. We investigated the hypothesis that NSCLC tumor cells with KDR CNG display increased sensitivity to VEGFR TKIs compared to tumor cells without KDR CNG. In tumor cell lines with KDR CNG, treatment with exogenous VEGF ligand enhanced cell motility and this was inhibited by VEGFR blockade with TKIs. Multiple receptor tyrosine kinases have been shown to drive HIF-1α levels, and NSCLC cells with KDR CNG express elevated levels of HIF-1α in normoxic conditions compared to NSCLC cell lines without KDR CNG. Here, we show that in NSCLC cell lines with KDR CNG, VEGFR TKIs decreased protein levels of HIF-1α and HIF-1α- regulated proteins. Furthermore, we report a clinical case in which a NSCLC patient with KDR CNG as determined by SNP array had a partial response to VEGFR inhibition with sorafenib. Citation Format: Monique B. Nilsson, Tina Cascone, Jayanthi Gudikote, Emily Roarty, Lixia Diao, Andrew Koo, Sumankalai Ramachandran, Erick Riquelme, Hai Tran, Ignacio Wistuba, David P. Carbone, John Heymach. KDR amplification in NSCLC is associated with sensitivity to VEGFR tyrosine kinase inhibitors. [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 374. doi:10.1158/1538-7445.AM2013-374

Background: LKB1 is a serine/threonine kinase which is mutated in 20-30% of non-small cell lung cancers (NSCLC).In general, DNA repair pathway activation leads cellular resistant to DNA damaging agents. The loss of LKB1 leads NSCLC to be more invasive and resistant to DNA damage based therapies. We hypothesize that loss of LKB1 may contribute to DNA damage response. We investigated LKB1- mediated signaling pathways by stably overexpressing LKB1 in its mutated NSCLC cell lines, A549 and H460. Methods: Expression of 139 proteins was analyzed by reverse phase protein array (RPPA) in LKB1 overexpressing cell lines along with the appropriate vector control lines. Differences in protein expression at baseline in clones with LKB1 versus clones with vector control were assessed by unpaired T-test with Welch's correction. Ionizing radiation sensitivity (reproductive cell death) was assessed by clonogenic cell survival assay (CSA). Results: LKB1 overexpression in A549 and H460 showed a significant downregulation of genes involved in DNA damage and repair pathway, such as RAD50 (1.4-fold relative expression, p< 0.05 and 1.5-fold relative expression p Citation Format: Chao Yang, Uma Giri, Maria Angelica Cortez, Jayanthi Gudikote, YouHong Fan, Lixia Diao, Lauren A. Byers, John V. Heymach. LKB1 overexpression regulates DNA repair pathway and sensitivity to radiation in LKB1 mutant non-small cell lung cancer cell lines . [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 1589. doi:10.1158/1538-7445.AM2013-1589

The phosphoinositide 3-kinase (PI3K) signaling pathway is the most commonly dysregulated pathway in many human cancers, including non-small cell lung cancer (NSCLC). Therefore, targeting PI3K signaling has become a promising approach in cancer therapy. Although studies have indicated development of resistance to PI3K inhibitors, the molecular mechanism of drug resistance is largely unknown in NSCLC. Here, we sought to identify new potential biomarkers and pathways that confer resistance to PI3K inhibitors in NSCLC. To this end, we used reverse-phase protein array (RPPA) to assess the expression levels and activation status of 137 proteins involved in signaling pathways implicated in lung cancer. Seventy-four NSCLC cell lines were treated with the PI3K inhibitor BAY 80-6946 and lysates were collected for proteomic analysis. First, we evaluted the association between drug sensitivity (IC50) and RPPA expression levels at baseline in sixty NSCLC cell lines. Correlation test was applied to each marker vs drug and Wilcox Rank test was performed to compare the smallest and largest one third of all the cell lines. We found increased phosphorylation and inactivation of pro-apoptotic protein Bad and phospho-LKB1 in the resistant cell lines. An inverse correlation was found for phosphorylation and inactivation of c-Src. Interestingly, activation of EGFR and increased levels of PTCH and PKCα were found in the resistant cell lines. Next, paired t-test was applied to each marker comparing the difference between drug and control treated cells. As expected, treatment with BAY 80-6946, significantly downregulates components of PI3K/mTOR pathway, as illustrated by decreased levels of phospho-Akt (-4.28-fold relative expression) and phospho-S6 ribosomal protein (-9.64-fold), phospho-p70S6K (-3.47-fold) and phospho-mTOR (-1.42-fold). We observed elevated expression of negative regulators of mTOR pathway, LKB1 (1.11-fold), AMPKα (1.12-fold) and phospho-AMPKα (12-fold). On the other hand, our analysis identified new potential pathways of resistance that are activated upon PI3K inhibition. We found increased levels of phospho-EGFR (P Citation Format: Maria A. Cortez, Lauren Averett Byers, You Hong Fan, Lixia Diao, Philip Groth, Julianne Paul, Jing Wang, Uma Giri, Jayanthi Gudikote, Hai Tran, Kevin Coombes, John D. Minna, Ningshu Liu, John V. Heymach. Proteomic analysis reveals Src/Stat and EGFR/MAPK pathways as potential mechanism of resistance to PI3K inhibitors in lung cancer. [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 2498. doi:10.1158/1538-7445.AM2013-2498

Background: MEK inhibitors such as BAY86-9766 are a new class of agents that show promise in the treatment of non-small cell lung cancer (NSCLC). The downstream effects of MEK inhibition in NSCLC have not been fully elucidated. We performed a broad proteomic analysis to determine which signaling pathways were modulated by BAY86-9766 treatment and how these pathways correlate with sensitivity. Methods: We treated 109 lung cancer cell lines with MEK inhibitor BAY86-9766 at a concentration of 2200 nM. Drug sensitivity was determined by CellTiter-Glo assay and cell lines were classified as sensitive or resistant based on whether their IC50 values were in the highest or lowest 1/3rd of those tested. Using paired t-tests, we compared pre- versus post-treatment protein levels in the overall group and between the sensitive vs. resistant cell lines. Results: MEK inhibitor BAY86-9766 was effective in reducing phosphorylation of direct downstream signaling molecules pMAPK (p Conclusions: We have performed broad proteomic analysis on cell lines treated with MEK inhibitor BAY86-9766 to determine which pathways are modulated by treatment and how they might relate to sensitivity. We conclude that MEK inhibition with BAY86-9766 may exert some of its effects by suppressing mTOR activity via the LKB1/AMPK pathway, and that the degree of modulation of the AMPK pathway correlates with sensitivity. This mechanism may involve decreased activation of p90RSK by MAPK, which leads to decreased degradation and increased activity of LKB1, and thereby increased AMPK activity and decreased mTOR signaling. This works suggests a rational basis for combinations of targeted agents to overcome resistance, such as combinations of MEK inhibitors with mTOR inhibitors or PI3 kinase inhibitors. Citation Format: Kathryn A. Gold, Lauren A. Byers, You Hong Fan, Lixia Diao, Philip Groth, Julianne Paul, Jing Wang, Uma Giri, Jayanthi Gudikote, Hai T. Tran, Kevin R. Coombes, John D. Minna, Ningshu Liu, John V. Heymach. Proteomic analysis of effects of MEK inhibition with BAY86-9766 on LKB1/AMPK and mTOR pathway in lung cancer cell lines. [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 1045. doi:10.1158/1538-7445.AM2013-1045

10612 Background: LKB1 is a serine/threonine kinase which is mutated in 20-30% of non-small cell lung cancers (NSCLC) and functions as a tumor suppressor by activating AMPK. Loss of LKB1 by point mutation or deletion suppresses AMPK, leading to increased mTOR signaling. We investigated the signaling pathways modulated by LKB1 mutations and by mTOR inhibition in NSCLC. Methods: Protein expression in cell lines was measured by reverse phase protein array. Differences in protein expression at baseline in LKB1 wild-type versus mutant cell lines and the effects of protein modulation by treatment were assessed by ANOVA. Results: LKB1 mutant cell lines had lower expression of phosphorylated AMPK and TSC (p