NSCLC Tumor Cells Research Articles

Abstract 3774: BGB324, a selective small-molecule inhibitor of receptor tyrosine kinase AXL, targets tumor immune suppression and enhances immune checkpoint inhibitor efficacy

Abstract The AXL receptor tyrosine kinase is associated with poor overall survival in a wide spectrum of cancers. AXL signaling is important for tumor cell plasticity related to epithelial-to-mesenchymal transition (EMT), immune escape and intrinsic resistance to cytotoxic lymphocytes. AXL is expressed on several cells associated with the tumor immune microenvironment, including natural killer (NK) cells, dendritic cells and a subset of tumor-associated myeloid cells. AXL signaling enhances secretion of immune-suppressive cytokines from innate immune cells that limit antitumor immunity. Hence AXL resides uniquely at the nexus between tumor and microenvironmental antitumor immune suppression mechanisms. BGB324, a selective clinical-stage small-molecule Axl kinase inhibitor, is currently being evaluated in combination with pembrolizumab in three phase II clinical trials in patients with TNBC (NCT03184558), NSCLC (NCT03184571) and melanoma (NCT02872259). We show that BGB324 targets immune suppression mechanisms in the tumor microenvironment that improve immunotherapy in different murine tumor models. BGB324 treatment reduces myeloid-derived suppressor cells and tumor-associated macrophages, and lowers CCL11, IL-7, IL-1β, and IL-6 in murine pancreatic cancer models. This altered immune landscape is associated with increased tumor infiltration of NK and CD8+ T cells and enhanced therapy responses. Further, BGB324 targets tumor intrinsic immune resistance and enhances human CD8+ T cell and NK-cell mediated NSCLC tumor cell lysis. We are currently using high-dimensional mass cytometry analysis (CyTOF) to map adaptive Axl-dependent immune suppression during immune checkpoint blockade. Collectively these results highlight a prominent function for AXL in resistance to immune therapy and support continued clinical translation of combining BGB324 with immune checkpoint inhibitors to improve cancer treatment. Citation Format: Kjersti Davidsen, Katarzyna Wnuk-Lipinska, Wenting Du, Magnus Blø, Agnete Engelsen, Stephane Terry, Stacey D´mello, Maria Lie, Jing Kang, Linn Hodneland, Sebastien Bougnaud, Kristina Aguilera, Oddbjørn Straume, Salem Chouaib, Rolf A. Brekken, Gro Gausdal, James B. Lorens. BGB324, a selective small-molecule inhibitor of receptor tyrosine kinase AXL, targets tumor immune suppression and enhances immune checkpoint inhibitor efficacy [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 3774.

Telmisartan generates ROS-dependent upregulation of death receptor 5 to sensitize TRAIL in lung cancer via inhibition of autophagy flux.

Telmisartan broadly used for the treatment of hypertension that is also known for its anticancer properties. TRAIL has the potential to kill tumor cells with minimal toxicity in normal cells by binding to death receptors, DR4 and DR5. Unfortunately, these TRAIL-death receptors have failed as most human cancers are resistant to TRAIL-mediated apoptosis. In this study, we evaluated telmisartan as a novel TRAIL-DR5-targeting agent with the aim of rendering TRAIL-based cancer therapies more active. Herein, we demonstrated that telmisartan could sensitize TRAIL and enhance NSCLC tumor cell death. The molecular mechanism includes the blocking of AMPK phosphorylation causes inhibition of autophagy flux by telmisartan resulting in ROS generation leading to death receptor (DR5) upregulation and subsequent activation of the caspase cascade by TRAIL treatment. Furthermore, using chloroquine and siATG5 significantly enhances ROS production and application of the ROS scavenger N-acetyl-cysteine (NAC) rescues the cells undergoing apoptosis by abrogating the expression of DR5 and finally the caspase cascade. Additionally, NAC treatment also maintains autophagy flux and makes the cells unresponsive to TRAIL. In summary, telmisartan in combination with TRAIL exhibits enhanced cytotoxic capacity toward lung cancer cells, thereby providing the potential for effective and novel therapeutic approaches to treat lung cancer.

IFNγ mRNA signature (IFNγ sig), circulating tumor DNA (ctDNA), and survival in NSCLC or urothelial cancer (UC) treated with durvalumab (D).

51 Background: Associations between early reduction in plasma ctDNA, pretreatment tumoral IFNγ sig, liver metastases and outcomes, and between tumor mutational burden (TMB) and CD274 (PD-L1) mRNA or IFNγ sig in TCGA were evaluated in NSCLC and UC pts treated with D. Methods: Pts received 10 mg/kg Q2W of D in a Phase 1/2 study in advanced solid tumors. RNAseq measured a 4-gene IFNγ sig; top tertile was IFNγ sig+. Pts with PD-L1 expression (Ventana SP263) ≥ 25% tumor cells in NSCLC or ≥ 25% tumor or immune cells in UC were PD-L1+. 70 genes were assayed for DNA variants (Guardant360) in plasma ctDNA pre/posttreatment. TCGA was used to calculate TMB; ≥ median TMB was high. Results: IFNγ sig+ NSCLC or UC pts had higher response and longer median PFS and OS compared with PD-L1+, PD-L1- and IFNγ sig- pts (Table). Responders showed significant decreases in ctDNA mean variant allele frequency (VAF) posttreatment with D; pts with progressive disease showed increased VAF. Pts with decreased VAF at week 6 had longer median PFS and OS compared with those with VAF increases. VAF changes were not associated with IFNγ sig. NSCLC without liver metastases had higher IFNγ sig (P < 0.001) than pts with liver metastases. In TCGA NSCLC and UC, IFNγ sig+ correlated with high TMB; CD274 mRNA did not. Conclusions: IFNγ sig correlated with outcomes and with TMB in NSCLC and UC. CtDNA VAFs were reduced in NSCLC or UC responders after treatment and correlated with longer survival, suggesting utility as an early indicator of clinical benefit. Clinical trial information: NCT01693562. [Table: see text]

83P - The impact of co-culture of NSCLC tumor cells and fibroblasts on drug response

83P - The impact of co-culture of NSCLC tumor cells and fibroblasts on drug response

Abstract 3794: Selective isolation of epithelial to mesenchymal transitioned circulating tumor cells in NSCLC using novel magnetic nanocubes

Abstract The 5-year survival rate for early to advanced NSCLC patients is 10-5%. Once the tumor metastasizes, the survival rate often drops below 1%. One of the major challenges is to select the patient for appropriate therapy, and the selection process involves invasive biopsy that is difficult to recurrently perform during treatment. Recent studies have shown that liquid biopsy is an attractive alternative. Liquid biopsy involves analysis of either CTCs or ctDNA to understand the tumor therapy. Isolating entire tumor cells provide an opportunity to perform whole genome sequencing for in depth understanding of the tumor. However, frequent changes in cancer signaling and acquired mutations during treatment lead to drug resistance that cannot be diagnosed using current CTC techniques. These oncogenic biochemical modifications that are often associated with metastasis involve upregulation of epithelial to mesenchymal transition (EMT) pathway that change the elasticity of tumor cells for easy shedding into the blood stream. Consequently, EMT transition leads to depletion in epithelial markers such as EpCAM and cytokeratin that are to be targeted. This effect largely limits the use of present technologies utilizing EpCAM as a marker to isolate the CTCs. We hypothesized that these cells can be captured by targeting surface markers that are overexpressed in EM transitioned CTCs. For example, markers such as EGFR and HER2 have been shown to be overexpressed pre and post EMT tumors and therefore can present as a new strategy for non-invasive diagnosis before and during treatment. In this study we have shown that EGFR and HER2 receptors are overexpressed in EGFR and KRAS mutated NSCLC cells. We artificially activated EMT in NSCLC cells and compared the surface biomarker concentrations. Furthermore, we used protein analysis data using western blotting to identify our targets after EMT. Based on the biomarker concentrations, we designed and developed magnetic nanocubes (MNC) surface attached with antibodies to target the selected biomarkers to capture EMT CTCs. The capture efficiency of these magnetic nanocubes was compared in multiple cell lines (HCC827 and A549). Overall we could achieve capture as low as 10 spiked cells in our study. In conclusion, cancer markers such as EGFR and HER-2 that are highly expressed in tumors, once shed as CTCs can be targeted for diagnosis. Therefore, we have developed a novel method to capture EMT CTCs with high selectivity and this method presents a minimally-invasive method for real-time monitoring of patients during drug treatment. Citation Format: Dhananjay Suresh, Shreya Ghoshdastidar, Soumavo Mukherjee, Abilash Gangula, Anandhi Upendran, Raghuraman Kannan. Selective isolation of epithelial to mesenchymal transitioned circulating tumor cells in NSCLC using novel magnetic nanocubes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3794. doi:10.1158/1538-7445.AM2017-3794

Abstract 3720: Biological and clinical relevance of PD-L1 expression in tumor and inflammatory cells in NSCLC

Abstract Background: High PD-L1 expression has been shown to be associated with improved clinical outcomes to anti-PD-1/L1 therapies in NSCLC and other indications. However, only a fraction of the PD-L1 high patients (pts) respond. PD-L1 can be induced by IFNG and expressed in tumor cells (TC) and inflammatory cells (IC). Improving our ability to predict patient benefit from anti-PD-1/L1 therapies requires a better understanding of associations between PD-L1 expression in TC and/or IC and outcome. Here we explore the relationship between patterns of IHC PD-L1 expression in TC and IC, gene expression, and clinical outcome. Methods: CP1108/NCT01693562 was a nonrandomized phase 1/2 trial evaluating durvalumab in pts with advanced NSCLC or other solid tumors. As of 29 APR 2016, 368 previously treated NSCLC pts received durvalumab ≤12 months with a median 18.8 months follow up. Pts with ≥25% TC or IC were scored TC+ and/or IC+, respectively. Results: TC+ PD-L1 pts (includes IC+ and IC- PD-L1 pts) had improved survival compared to TC- PD-L1 pts. TC+ and IC+ PD-L1 pts had: improved survival compared to TC+/IC-, TC-/IC+, or TC-/IC- PD-L1 pts. However, prevalence of TC+/IC+ was lower than TC+. Twenty-one genes significantly differed between TC+/IC+, TC+/IC-, TC-/IC+, and TC-/IC- PD-L1 patient subsets, the vast majority being well-known IFNG-inducible genes and mostly over-expressed in the TC+/IC+ subset. Conclusions: TC+ and IC+ PD-L1 pts had the highest levels of IFNG-inducible gene expression, a key biological feature that distinguishes PD-L1 IHC positive from negative pts. Thus, in addition to PD-L1 IHC, the predictive value of IFNG should be investigated in additional relevant studies Selection CriteriaPrevalence of Biomarker Positive BiomarkerPositive/negative median OSlog-rank pHRcox pTC+158/276 (57%)15.67/7.730.00910.690.046TC+/IC+70/276 (25%)25.63/8.377.46E-060.345.24E-05TC+/IC-88/276 (32%)10.5/13.970.0941.50.032TC-/IC+50/276 (18%)9.07/13.230.830.960.84TC-/IC-68/276 (25%)5.77/14.030.000841.660.012 All comers: median OS: 11.2 months (N=304) Citation Format: Carlos Bais, Chris Morehouse, Brandon W. Higgs, Rebelatto Marlon, Keith Steele, Xiaoping Jin, Li Shi, Susana Korolevich, Ashok Gupta, Koustubh Ranade. Biological and clinical relevance of PD-L1 expression in tumor and inflammatory cells in NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3720. doi:10.1158/1538-7445.AM2017-3720

Abstract 626: BGB324, a selective small molecule inhibitor of receptor tyrosine kinase AXL, abrogates tumor intrinsic and microenvironmental immune suppression and enhances immune checkpoint inhibitor efficacy in lung and mammary adenocarcinoma models

Abstract The AXL receptor tyrosine kinase is associated with poor overall survival in a wide spectrum of cancers including lung and breast adenocarcinomas. AXL signaling is an important regulator of tumor plasticity related to epithelial-to-mesenchymal transition (EMT) and stem cell traits that drive metastasis and drug resistance. Signaling via AXL is also a key suppressor of the anti-tumor innate immune response, and AXL is expressed on several cells associated with the tumor immune microenvironment including natural killer (NK) cells and tumor-associated macrophages. Hence AXL resides uniquely at the nexus between tumor and microenvironmental anti-tumor immune suppression mechanisms. We report that BGB324, a selective clinical-stage small molecule Axl kinase inhibitor, enhances the effect of immune checkpoint blockade in aggressive adenocarcinoma models with limited immunogenicity by targeting both tumor intrinsic and microenvironmental immune suppression. Immune therapy with anti-CTLA4/PD1 in the 4T1 model increased AXL and EMT-marker expression correlating with a lack of response. Combination with BGB324 resulted in durable primary tumor clearance versus anti-CTLA4/PD1 alone. In a separate study, BGB324 + anti-CTLA4 treatment resulted in significant long-term primary tumor clearance while no response was observed with anti-CTLA4 treatment alone. The extensive metastasis to the lung, liver and spleen characteristic of the 4T1 model was not detected in animals responding to the combination treatment. Importantly, responding animals rejected orthotopic 4T1 tumor cell re-challenge, demonstrating sustained tumor immunity. In the LL2 Lewis Lung model, BGB324 in combination with anti-PD1/PDL1 significantly prevented tumor growth compared to treatment with anti-PD1/PDL1. Tumors from mice treated with BGB324 in combination with immune checkpoint inhibitors displayed reduced EMT traits, altered cytokine expression, enhanced tumor infiltration of effector cells and decreased number of mMDSC. Also, BGB324 significantly reduced IL10 secretion by isolated human macrophages and enhanced human NK-cell mediated NSCLC tumor cell lysis. Collectively these results support a prominent role for AXL in resistance to immune therapy and support clinical translation of combining BGB324 with immune checkpoint inhibitors to improve cancer treatment. Citation Format: Katarzyna Wnuk-Lipinska, Kjersti Davidsen, Magnus Blø, Agnete Engelsen, Jing Kang, Linn Hodneland, Maria Lie, Sebastien Bougnaud, Kristina Aguilera, Lavina Ahmed, Agata Rybicka, Eline Milde Nævdal, Paulina Deyna, Anna Boniecka, Straume Oddbjørn, Salem Chouaib, Rolf Brekken, Gro Gausdal, James B. Lorens. BGB324, a selective small molecule inhibitor of receptor tyrosine kinase AXL, abrogates tumor intrinsic and microenvironmental immune suppression and enhances immune checkpoint inhibitor efficacy in lung and mammary adenocarcinoma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 626. doi:10.1158/1538-7445.AM2017-626

1171P - The SP142 PD-L1 IHC assay for atezolizumab (atezo) reflects pre-existing immune status in NSCLC and correlates with PD-L1 mRNA

PD-L1 expression as measured by the SP142 PD-L1 IHC assay is associated with improved efficacy with atezo in patients with NSCLC, including overall survival. In capturing PD-L1 on both tumor cells (TC) and tumor-infiltrating immune cells (IC), this assay is able to determine preexisting immune status, underscoring the predictive value of PD-L1 IHC. In this study, we further investigated intratumoral immune infiltration status by measuring T effector (Teff) gene signature and correlated PD-L1 mRNA expression with SP142 PD-L1 IHC. Using the SP142 IHC assay, procured samples from NSCLC tumors were scored according to the percent of TC expressing PD-L1 (TC0 < 1%; TC1 ≥ 1% and < 5%; TC2 ≥ 5% and < 50%; TC3 ≥ 50%). IC were scored as percentage of tumor area (IC0 < 1%; IC1 ≥ 1% and < 5%; IC2 ≥ 5% and < 10%; IC3 ≥ 10%). Four mutually exclusive groups increasing in PD-L1 expression (TC0 and IC0, TC1 or IC1, TC2 or IC2, TC3 or IC3) were designated. mRNA levels for PD-L1 and Teff signature were measured in the same NSCLC specimens using the Fluidigm platform and correlated with each of the PD-L1 IHC sub-groups. In 86 NSCLC specimens, mRNA levels for PD-L1 increased incrementally with higher PD-L1 IHC status, from 0 median expression for TC0 and IC0 tumors to 3.14 median expression for TC3 or IC3 tumors. A similar trend was seen for the Teff signature, with values increasing from 0 to 1.65 median expression with increasing IHC cutoffs (Table). Results from the SP142 PD-L1 IHC assay associates with PD-L1 and Teff mRNA transcripts, reflecting pre-existing immunity associated with adaptive PD-L1 expression on IC as well as intrinsic PD-L1 expression on TC in NSCLC, thus providing an independent orthogonal correlation of IHC to gene expression. The value of mRNA gene expression as a predictive marker of efficacy for atezo in NSCLC is being studied in ongoing randomized trials of atezo.Tabled 1PD-L1 IHC vs PD-L1 mRNA and T effector gene signaturePD-L1 IHCPD-L1 mRNAT EffectoranMedianb expression95% CInMedianb expression95% CITC0 and IC0460-0.22, 0.36460-0.39, 0.16TC1 or IC1220.890.53, 1.66220.730.25, 0.95TC2 or IC2112.662.49, 2.85111.530.55, 1.57TC3 or IC373.141.4, 4.2971.650.46, 1.95a Includes CD8A, GZMA, GZMB, EOMES, CXCL9, CXCL10, TBX21. b Median expressions are log scale expression (-ΔCT) relative to the median of TC0 and IC0 group. Larger values indicate higher expression. Open table in a new tab

MiR-652-3p is upregulated in non-small cell lung cancer and promotes proliferation and metastasis by directly targeting Lgl1.

Our previous study found that miR-652-3p is markedly upregulated in the serum of patients with NSCLC and suggesting that miR-652-3p is a potential biomarker for the early diagnosis of NSCLC. In this study, we detected the expression of miR-652-3p in NSCLC tumor tissues and cell lines and investigated the effect of miR-652-3p on the proliferation and metastasis of NSCLC cells. Our results showed that the expression of miR-652-3p was significantly upregulated in tumor tissues of 50 patients with NSCLC, and it was significantly higher in patients with positive lymph node metastasis, advanced TNM stage and poor prognosis. Using functional analyses by overexpressing or suppressing miR-652-3p in NSCLC cells, we demonstrated that miR-652-3p promoted cell proliferation, migration, invasion and inhibited cell apoptosis. Moreover, the lethal(2) giant larvae 1 (Lgl1) was identified as a direct and functional target of miR-652-3p. Overexpression or knockdown of miR-652-3p led to decreased or increased expression of Lgl1 protein, and the binding site mutation of LLGL1 3′UTR abrogated the responsiveness of the luciferase reporters to miR-652-3p. Overexpression of Lgl1 partially attenuated the function of miR-652-3p. Collectively, these results revealed that miR-652-3p execute a tumor-promoter function in NSCLC through direct binding and regulating the expression of Lgl1.

Magnetic nanocubes for selective capture of circulating tumor cells in NSCLC

Motivation: Lung cancer is the leading cause of cancer death, and lung adenocarcinoma (LUAD) is its predominant histological subtype. Fifteen single nucleotide polymorphisms (SNPs) have been significantly associated with LUAD risk in genome-wide association studies (GWAS). However, because most GWAS SNPs reside in non-coding regions and are co-inherited with hundreds of SNPs in linkage disequilibrium (LD), which SNPs play a causal role in disease development remains largely unknown. We hypothesized that some of these SNPs affect oncogenic transcriptional programs by modulating the activity of gene enhancers in alveolar epithelial cells (AECs), the purported cells of origin for LUAD. Methods: To test this hypothesis, we overlaid epigenomic features of primary human AECs over the locations of index LUAD risk SNPs and associated high LD SNPs. Luciferase assays for enhancer activity were performed for candidate SNPs that were predicted to disrupt transcription factor (TF) binding sites in loci marked by features of active enhancers. Expression quantitative trait loci (eQTL) analysis was also performed using The Cancer Genome Atlas (TCGA) dataset and the online Genotype-Tissue Expression (GTEx) Portal to identify potential target genes of each SNP-enhancer pair. Results: Thirty-three LUAD risk-associated SNPs mapped to putative AEC enhancer regions. TF binding site prediction suggests that numerous SNPs might alter the binding affinity of TFs implicated in lung cancer, including RXRA and NKX2-1. Luciferase assays indicate that two of the SNPs significantly affect enhancer activity in lung cancer cell lines. eQTL analyses link each of the putative enhancers to candidate target genes, including both known oncogenes and genes not previously associated with lung cancer. Conclusions: Taken together, our analyses provide new mechanistic insight into long-known associations between non-coding SNPs and LUAD outcomes, and may ultimately yield more effective and personalized strategies for lung cancer risk assessment, prevention, and treatment. Magnetic nanocubes for selective capture of circulating tumor cells in NSCLC

Functional lung adenocarcinoma risk SNPs identified through positional integration with human alveolar epithelial cell epigenomes

Motivation: Lung cancer is the leading cause of cancer death, and lung adenocarcinoma (LUAD) is its predominant histological subtype. Fifteen single nucleotide polymorphisms (SNPs) have been significantly associated with LUAD risk in genome-wide association studies (GWAS). However, because most GWAS SNPs reside in non-coding regions and are co-inherited with hundreds of SNPs in linkage disequilibrium (LD), which SNPs play a causal role in disease development remains largely unknown. We hypothesized that some of these SNPs affect oncogenic transcriptional programs by modulating the activity of gene enhancers in alveolar epithelial cells (AECs), the purported cells of origin for LUAD. Methods: To test this hypothesis, we overlaid epigenomic features of primary human AECs over the locations of index LUAD risk SNPs and associated high LD SNPs. Luciferase assays for enhancer activity were performed for candidate SNPs that were predicted to disrupt transcription factor (TF) binding sites in loci marked by features of active enhancers. Expression quantitative trait loci (eQTL) analysis was also performed using The Cancer Genome Atlas (TCGA) dataset and the online Genotype-Tissue Expression (GTEx) Portal to identify potential target genes of each SNP-enhancer pair. Results: Thirty-three LUAD risk-associated SNPs mapped to putative AEC enhancer regions. TF binding site prediction suggests that numerous SNPs might alter the binding affinity of TFs implicated in lung cancer, including RXRA and NKX2-1. Luciferase assays indicate that two of the SNPs significantly affect enhancer activity in lung cancer cell lines. eQTL analyses link each of the putative enhancers to candidate target genes, including both known oncogenes and genes not previously associated with lung cancer. Conclusions: Taken together, our analyses provide new mechanistic insight into long-known associations between non-coding SNPs and LUAD outcomes, and may ultimately yield more effective and personalized strategies for lung cancer risk assessment, prevention, and treatment. Magnetic nanocubes for selective capture of circulating tumor cells in NSCLC

Abnormal Expression of miR-21 and miR-95 in Cancer Stem-Like Cells is Associated with Radioresistance of Lung Cancer

This study demonstrated that miR-21 and miR-95 expression were significantly higher in the ALDH1+CD133+subpopulation than in the ALDH1―CD133― subpopulation of lung cancer cells. Combined delivery of anti-miR-21 and anti-miR-95 by calcium phosphate nanoparticles significantly inhibited tumor growth in a xenograft tumor model and sensitized radiotherapy. The anti-miRNAs significantly reduced miR-21 and miR-95 levels, increased PTEN, SNX1, and SGPP1 protein expression, but reduced Akt Ser473 and Thr308 phosphorylation. ALDH1+CD133+ subpopulation of NSCLC tumor cells confers radioresistance due to high expression of miR-21 and miR-95. Targeting inhibition of miR-21 and miR-95 can inhibit tumor growth through elevating PTEN, SNX1, and SGPP1 expression and inhibiting Akt phosphorylation.

Abstract 5480: NSCLC cells with LKB1 mutation are vulnerable to energetic and oxidative stresses induced by 8-Chloroadenosine

Abstract 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&amp;lt;0.05). Knockdown LKB1 resulted in increased sensitivity to 8-Cl-Ado in Calu6 cells (p&amp;lt;0.05). Moreover, 8-Cl-Ado treatment caused a significant decrease in cellular ATP levels in LKB1 mutant cells. However, in A549 and H460 cells overexpressing wild-type LKB1, ATP levels were not decreased. Similarly, in H661 cells (wild-type LKB1) 8-Cl-Ado treatment did not affect ATP levels, whereas when LKB1 was knocked down by shRNA 8-Cl-Ado treatment reduced intracellular ATP levels. Furthermore, 8-Cl-Ado increased ROS levels in A549 cells but not in A549 cells overexpressing wild-type LKB1. Conclusion: Our result indicates that LKB1 mutant NSCLC cells are vulnerable to energetic and oxidative stresses induced by 8-Cl-Ado. LKB1 played a protecting role in cells under 8-Cl-Ado treatment. 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

Pseudo-cyclization through intramolecular hydrogen bond enables discovery of pyridine substituted pyrimidines as new Mer kinase inhibitors.

Abnormal activation or overexpression of Mer receptor tyrosine kinase has been implicated in survival signaling and chemoresistance in many human cancers. Consequently, Mer is a promising novel cancer therapeutic target. A structure-based drug design approach using a pseudo-ring replacement strategy was developed and validated to discover a new family of pyridinepyrimidine analogues as potent Mer inhibitors. Through SAR studies, 10 (UNC2250) was identified as the lead compound for further investigation based on high selectivity against other kinases and good pharmacokinetic properties. When applied to live cells, 10 inhibited steady-state phosphorylation of endogenous Mer with an IC50 of 9.8 nM and blocked ligand-stimulated activation of a chimeric EGFR-Mer protein. Treatment with 10 also resulted in decreased colony-forming potential in rhabdoid and NSCLC tumor cells, thereby demonstrating functional antitumor activity. The results provide a rationale for further investigation of this compound for therapeutic application in patients with cancer.

Increased VEGFR-2 Gene Copy Is Associated with Chemoresistance and Shorter Survival in Patients with Non–Small-Cell Lung Carcinoma Who Receive Adjuvant Chemotherapy

VEGF receptor-2 (VEGFR-2 or kinase insert domain receptor; KDR) is a known endothelial target also expressed in NSCLC tumor cells. We investigated the association between alterations in the KDR gene and clinical outcome in patients with resected non-small-cell lung carcinoma (NSCLC; n = 248). KDR copy number gains (CNG), measured by quantitative PCR and fluorescence in situ hybridization, were detected in 32% of tumors and associated with significantly higher KDR protein and higher microvessel density than tumors without CNGs. KDR CNGs were also associated with significantly increased risk of death (HR = 5.16; P = 0.003) in patients receiving adjuvant platinum-based chemotherapy, but no differences were observed in patients not receiving adjuvant therapy. To investigate potential mechanisms for these associations, we assessed NSCLC cell lines and found that KDR CNGs were significantly associated with in vitro resistance to platinum chemotherapy as well as increased levels of nuclear hypoxia inducible factor-1α (HIF-1α) in both NSCLC tumor specimens and cell lines. Furthermore, KDR knockdown experiments using small interfering RNA reduced platinum resistance, cell migration, and HIF-1α levels in cells bearing KDR CNGs, providing evidence for direct involvement of KDR. No KDR mutations were detected in exons 7, 11, and 21 by PCR-based sequencing; however, two variant single nucleotide polymorphism genotypes were associated with favorable overall survival in adenocarcinoma patients. Our findings suggest that tumor cell KDR CNGs may promote a more malignant phenotype including increased chemoresistance, angiogenesis, and HIF-1α levels, and that KDR CNGs may be a useful biomarker for identifying patients at high risk for recurrence after adjuvant therapy, a group that may benefit from VEGFR-2 blockade.

Prognostic Impacts of Angiopoietins in NSCLC Tumor Cells and Stroma: VEGF-A Impact Is Strongly Associated with Ang-2

IntroductionAngiopoietins and their receptor Tie-2 are, in concert with VEGF-A, key mediators in angiogenesis. This study evaluates the prognostic impact of all known human angiopoietins (Ang-1, Ang-2 and Ang-4) and their receptor Tie-2, as well as their relation to the prognostic expression of VEGF-A.Methods335 unselected stage I-IIIA NSCLC-patients were included and tissue samples of respective tumor cells and stroma were collected in tissue microarrays (TMAs). Immunohistochemistry (IHC) was used to semiquantitatively evaluate the expression of markers in duplicate tumor and stroma cores.Principal FindingsIn univariate analyses, low tumor cell expression of Ang-4 (P = 0.046) and low stromal expressions of Ang-4 (P = 0.009) and Ang-2 (P = 0.017) were individually associated with a poor survival. In the multivariate analysis, low stromal Ang-2 (HR 1.88; CI 95% 1.15-3.08) and Ang-4 (HR 1.47, CI 95% 1.02–2.11, P = 0.04) expressions were independently associated with a poor prognosis. In patients with high tumor cell expression of Ang-2, a concomitantly high tumor VEGF-A expression mediated a dramatic survival reduction (P<0.001). In the multivariate analysis of patients with high Ang-2 expression, high tumor VEGF-A expression appeared an independent poor prognosticator (HR 6.43; CI 95% 2.46–16.8; P<0.001).ConclusionsIn tumor cells, only Ang-4 expression has prognostic impact in NSCLC. In tumor stroma, Ang-4 and Ang-2 are independently associated with survival. The prognostic impact of tumor cell VEGF-A in NSCLC appears strongly associated with a concomitantly high tumor cell expression of Ang-2.

EGFR and COX-2 protein expression in non-small cell lung cancer and the correlation with clinical features

BackgroundTo evaluate the expression of EGFR and COX-2 and their correlation with prognosis in NSCLCMethodsThe paraffin embedded tumor samples of 50 NSCLC patients receiving radical resection were analyzed immunohistochemically for EGFR and COX-2 expression and their prognostic values were explored.ResultsThe positive rate of EGFR protein in NSCLC tumor cells was 46%, which was significantly higher than its expression in normal lung (p = 0.0234) and paracancerous tissues (p = 0.020). EGFR expression was significantly higher in nodal positive than in nodal negative patients (p = 0.04). The mean survival time for EGFR positive patients (31 months) was significantly lower than that for patients with EGFR negative expression (48 months) (p = 0.008,). In patients receiving post-operation thoracic irradiation, the mean survival time for EGFR positive patients was significantly lower than that for patients without EGFR positive expression (25 vs. 48 months, P = 0.004). The positive rate of COX-2 protein expression in NSCLC tumor cells was 90%, which was significantly higher than that in normal tissue(p = 0.00) and paracancerous tissue (p = 0.00). There was no correlation between COX-2 expression and patient survival, and no correlation between COX-2 and EGFR protein expression (P = 0.555).ConclusionsCOX-2 and EGFR are over-expressed in NSCLC. EGFR is an independent prognostic factor and a predictive factor for radiotherapy response in NSCLC.

Abstract 5045: The Hh inhibitor IPI-926 inhibits relapse of a non-small cell lung cancer xenograft model following treatment with an EGF-R targeted tyrosine kinase inhibitor

Abstract The Hedgehog (Hh) pathway plays a key role in multiple types of cancer, either by mutational activation of the pathway in tumor cells or by ligand-dependent signaling to the tumor cells or paracrine signaling between tumor cells and the tumor microenvironment. We have previously shown in a primary model of SCLC that the Hh pathway inhibitor IPI-926 prevents tumor relapse after regression of the tumor with chemotherapy (Travaglione et al., AACR 2009). To investigate whether tumor regression with a targeted agent could elicit a similar response as that seen with chemotherapy, two NSCLC lines (H1650 and HCC827), with activating mutations in the EGFR were evaluated. NSCLC cell lines bearing mutant EGFR are exquisitely sensitive to EGFR kinase inhibitors. Upon treatment with gefitinib in vitro, HCC827 increased the expression of Indian and Sonic hedgehog ligands 10-fold, as measured by qRT-PCR. When grown in mice as subcutaneous xenografts, H1650 tumors regressed following treatment with the EGFR inhibitor gefitinib (40 mg/kg every day) for 7 days. The gefitinib-treated mice were then administered either vehicle or IPI-926 at 40 mg/kg every other day for three weeks, and in this setting IPI-926 treatment led to a 65% decrease (p=0.0104) in tumor volume compared to vehicle-treated mice. Conversely, tumors that had not been regressed with the EGFR TKI showed no response to the Hh pathway inhibitor, similar to our previous results in SCLC with chemotherapy. Studies are ongoing to define the mechanism of action of IPI-926 in this setting. When grown in mice as subcutaneous xenografts, these tumor cells produce Shh ligand in conjunction with stromal Gli-1 expression in the murine stroma. Stromal Gli-1 expression is inhibited by treatment with IPI-926, indicating that Hh ligand produced by NSCLC tumor cells signals to stroma in a paracrine manner. Together with our previous results in SCLC, these data suggest that therapeutic inhibition of the Hh signaling pathway might be an important strategy to extend progression free survival in patients who initially respond to therapy but later relapse, and that this paradigm is applicable to multiple cancer types and treatment modalities. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5045.

Abstract A10: Antiangiogenic therapy (VEGF-R1–3 and PDGFR inhibitor) increases tumor hemoglobin saturation and decreases interstitial pressure, and microvessel density

Abstract Methods: We evaluate the effects of pazopanib, a novel multi-targeted tyrosine kinase inhibitor, by examining its effects on tumor oxygenation, interstitial pressure (IFP), and vascularity as compared to an untreated control group. Nude mice (n=35) were injected with A-549 NSCLC tumor cells. Tumors reached an average volume of 650 mm3 before treatment with pazobanib 100mg/kg/day, Pazobanib 30mg/kg/day. or Vehicle (Control). Animals were treated daily for 14 days. Spectroscopic Measurements: A novel Skinskan spectrofluorometer was used to obtain non-invasive measurements of hemoglobin saturation (Hbsat) and total Hb for each tumor. Measurements were performed every other day, starting the day of treatment. These values were analyzed by a custom MATLAB program. IFP Measurements: IFP was measured on the last day of treatment(day 14), prior to sacrifice. Immunohistochemical staining: The number of vessels, determined by positive CD31 staining, was averaged over 6–8 image fields to obtain microvessel density (MVD). Results: Optical Spectroscopy: A significant increase in Hbsat was observed at day 8 for the low and high pazopanib dose groups, compared to control (p=0.0109, p=0.0290, respectively). After the 8th treatment day, Hbsat trended downward toward baseline. Tumor IFP: Tumor IFP for animals treated with pazopanib 100mg/kg was significantly reduced, compared to control (p=0.0026). Treatment with pazopanib 30mg/kg also demonstrated a strong trend towards IFP reduction, compared to control (p=0.0645). MVD: MVD was significantly reduced in mice treated with low and high dose pazopanib, compared to control (p=0.0271, p=0.0107, respectively). Conclusions: Treatment with pazopanib lead to a significant reduction in IFP, MVD, and increase in tumor Hb sat. This result demonstrates that pazopanib enhances efficiency of tumor perfusion which could lead to better drug distribution into the tumor, and increased of tumor Hb saturation would correlate with better oxygenation and predicts better response to radiotherapy. Work supported by a grant from GlaxoSmithKline. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A10.

Panitumumab and cetuximab epitope mapping and in vitro activity

14536 Background: Panitumumab, a fully human monoclonal antibody against epidermal growth factor receptor (EGFR), has anti- tumor activity as monotherapy in both preclinical models and clinical trials. The objective of this study was to identify the epitope on EGFR for panitumumab and compare it to that of cetuximab, a chimeric anti-EGFR Ab. Methods: The extracellular domain of EGFR (amino acids 1–618) and domains I, II, III, IV individually were expressed to determine the domain on EGFR that was necessary for panitumumab binding. Chromatography was used to compare the binding affinity of panitumumab vs cetuximab. By flow cytometry and amino acid replacement scanning, the critical residues involved in panitumumab and cetuximab binding for EGFR were determined. Using diffraction data from the panitumumab Fab’2 and published data for cetuximab, crystal structure models of EGFR and the residues determined to be critical for panitumumab or cetuximab binding were built. To evaluate in vitro activity, A549 NSCLC cells were treated with 20μg/ml of panitumumab or cetuximab for 1 hour prior to a 15-minute stimulation with known EGFR ligands (TGF-α, amphiregulin, epiregulin, HB-EGF, betacellulin, and EGF). Inhibition of ligand-induced phosphorylation of EGFR was determined using a specific anti-pEGFR (pY1068) antibody. Results: Truncation analysis by flow cytometry narrowed the binding epitopes of panitumumab and cetuximab to domain III of EGFR. Point mutations revealed that amino acids 349, 355, 412, and 438 were critical for panitumumab and cetuximab binding. A 2.9 Angstrom crystal structure of the panitumumab Fab’2 was solved. Models of the panitumumab-EGFR and cetuximab-EGFR complexes were generated and predicted that the CDRs for both light and heavy chains would be proximal to domain III of EGFR. From in vitro studies, panitumumab and cetuximab can inhibit receptor activation of all known EGFR ligands in A549 NSCLC tumor cells. Conclusion: Panitumumab and cetuximab bind to comparable surface exposed amino acids in domain III of EGFR and inhibit all known EGFR ligands, resulting in inhibition of receptor activation. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Amgen Inc. Amgen Inc.