Small Lung Cancer Cell Lines Research Articles

B19 New Potential Targets of Antibody-Drug Conjugates for Small-Cell Lung Carcinoma

Small-cell lung cancer (SCLC) remains one of the high-grade malignancies, whereas non-small cell lung cancer benefits from molecular target drugs or immune checkpoint inhibitors as a result of investigating driver mutations and immune microenvironment. However, novel driver mutation was not identified through genome-wide sequence analyses, resulting in invariable therapeutic strategy for SCLC. Therefore, we have to shift from cytotoxic agent and molecular target drug in the care of SCLC. In recent years, different approaches to hematologic malignancies and solid tumors were established in clinical situation. Antibody-drug conjugates (ADCs) is the key technique. In this study, we aimed to search new therapeutic targets for ADCs toward a paradigm shift in treatment and research of SCLC. We sought to transmembrane proteins of SCLC as new targets for ADCs with computational-biologic approach. We demonstrated 565 genes were overexpressed on 51 small-cell lung cancer cell lines compared to 30 normal lung tissue samples by investigating gene expression profile available in open source of Cancer Cell Line Encyclopedia and National Center for Biotechnology Information (NCBI) with Human Genome U133 Plus 2.0 Array (ThermoFisher Scientific). Among the 565 genes, 31 genes manifested increased value of compensated fluorescence signal on average by 3 or more. Of the 31 genes, by investigating RNA sequence data for normal tissue in NCBI, we identified 7 genes expressed in limited organs. We adopted these 7 selected genes as candidates for new targets of ADCs. We examined these new target genes by evaluating in vitro cytotoxicity of corresponding monoclonal antibodies followed by secondary ADCs comprising PNU-159682, a derivative of nemorubicin, using SCLC cell lines with and without overexpression of these genes. Cytotoxicity assay targeting a certain transmembrane protein, one of the candidate molecules, showed distinct effect of secondary ADC, inducing a large amount of cell death in concentration-dependent manner while secondary ADC following murine IgG isotype control exhibited lack of cytotoxicity. Secondary ADC targeting the protein showed about fourfold greater potency than that using murine IgG isotype control as a primary antibody (EC50 3.3 nM versus 13.0 nM). Conversely, CRISPR-Cas9 mediated knockout of the gene showed explicit loss of the cytotoxic effect. The expression of the gene in normal organs were examined using human total RNA, which demonstrated lower expression of the gene in many organs than in brain. The distribution of the gene expression is preferable in the viewpoint of reducing side effects of the ADC, which cannot cross the blood-brain barrier. We successfully estimated new targets for ADCs by investigating membrane proteins and narrowing these proteins with computational-biologic approach. Through in vitro cytotoxicity assays, the protein -mediated ADC exhibited specific killing of SCLC cell lines overexpressing the gene, suggesting the gene can be a potential target of ADCs.

B20 Oncogene-Mediated ERK Signaling Suppresses Neuroendocrine Transcription Factors and Facilitates Cellular Transformation in Small-Cell Lung Cancer Through Chromatin Remodeling

In contrast to non-small cell lung cancer (NSCLC), small-cell lung cancer (SCLC) rarely harbors gene alterations that activate signaling through the receptor tyrosine kinase/RAS/RAF/MEK/ERK pathway. In addition, EGFR protein expression is universally lost during histologic transformation from mutant EGFR-driven lung adenocarcinoma (LUAD) to SCLC that occurs in a subset of patients that develop acquired resistance to EGFR tyrosine kinase inhibitors (TKIs), despite the original EGFR mutations being maintained in the transformed tumor. Based on these observations, we hypothesized that signaling through mitogen-activated protein kinases (MAPKs) is detrimental to SCLC tumors and suppresses the neuroendocrine (NE) differentiation program that is a hallmark of this lung cancer subtype. To test this, we induced MAPK signaling through expression of two LUAD driver oncogenes, KRASG12V and EGFRL858R, and assessed the impact on the phenotype and signaling profiles of SCLC. KRASG12V or EGFRL858R was exogenously expressed in an inducible manner in three SCLC cell lines (H2107, H82, and H524). Effects were characterized through microscopy, growth assays, gene expression and chromatin profiling, and Western blots of master NE transcription factors including insulinoma-associated protein 1 (INSM1), POU class 3 homeobox 2 (BRN2), achaete-scute hologue 1 (ASCL1), and neurogenic differentiation factor 1 (NEUROD1). Induction of mutant KRAS or EGFR caused transition from suspension to adherent phenotype that was reversed by pharmacologic inhibition of both ERK and AKT. Moreover, whereas both oncogenes downregulated NE transcription factors, effects were more prominent after KRASG12V induction, reflecting the difference in degrees of phospho-ERK levels. Inhibition of ERK completely rescued the repression of NE factors by KRASG12V induction, and partial effects were observed through inhibition of the downstream effectors MSK/RSK. Notably, KRASG12V-mediated suppression of NE factors was restored by inhibition of the histone modifiers p300/CBP or KDM5A in a cell line-specific manner. ATAC-seq analyses are currently underway to examine the changes of chromatin accessibility after KRASG12V induction +/- inhibition of ERK, MSK/RSK, or p300/CBP. In SCLC, activation of ERK and AKT by mutant KRAS or EGFR causes phenotypic transition to a NSCLC-like state, and ERK is the central hub for the regulation of NE factors. Histone modifications by hyperactivated ERK play an important role in this process and are mediated via tumor-specific mechanisms. These findings provide a biologic basis for why SCLC lacks alterations in the MAPK pathway and shed light on the underlying mechanisms of histologic transversion of SCLC to and from NSCLC, which may play a role in TKI resistance.

Anticancer Activity of ZnO Nanoparticles against Human Small-Cell Lung Cancer in an Orthotopic Mouse Model.

Small-cell lung cancer, a highly malignant form of lung cancer, often responds to first-line treatments but relapses in most cases with resistance to further treatments. We tested zinc oxide (ZnO) nanoparticles against small-cell lung cancer and other cancer cell lines, in light of reported anticancer effects in vitro Because of a strong safety record, ZnO nanoparticles are frequently used in biomedical research, including in cellular imaging and drug delivery, and have been used for many years in several commercial products such as skin care agents. Strikingly, ZnO nanoparticles were genotoxic against small-cell lung cancer cells, resulting in low viability, even in cells orthotopically grafted onto mouse models. However, the nanoparticles were less cytotoxic against normal lung-derived cells and did not elicit observable adverse effects after intravenous administration. ZnO nanoparticles were also found to induce highly reactive oxygen species and DNA leakage from nuclei. This study is the first comprehensive evaluation of the anticancer effects of ZnO nanoparticles in vitro and in vivo and highlights new therapeutic opportunities against small-cell lung cancer.

Expression of neuro-oncological ventral antigen-1 in small-cell lung cancer and its value in pathological diagnosis.

BackgroundTo investigate the expression of neuro-oncological ventral antigen-1 (Nova1) in small-cell lung cancer (SCLC) and its value in clinical pathological diagnosis of SCLCs.MethodsNova1 expression in the SCLC cell line H446, lung adenocarcinoma cell line A549, squamous cell carcinoma (SCC) cell line H226 and bronchial epithelial cell line BEAS-2B were examined by RT-PCR. It’s expression in paraffin specimens of 100 SCLC cases, 15 specimens of lung adenocarcinoma cases and 15 specimens of lung SCC cases, as well as adjacent normal tissue was determined by immunohistochemistry. The positive rate of Nova1 in SCLC tissue was compared with those of traditional neuroendocrine markers CD56, Syn and CgA.ResultsNova1 expression in SCLC cell line H446 was significantly higher than that of bronchial epithelial cell line BEAS-2B and lung adenocarcinoma cell line A549 and SCC cell line H226. The positive rate of Nova1 in SCLC tissue was similar to that of CD56 but higher than those of Syn and CgA. Nova1 was neither expressed in the cancer tissue nor in the adjacent normal tissue of the 15 cases of SCC and the 15 cases of adenocarcinoma. In all six cases of combined SCLC, Nova1 was expressed in the SCLC component, whereas markers of SCC or adenocarcinoma were absent.ConclusionsNova1 can be used as a novel neuroendocrine marker for pathological diagnosis of SCLC in clinical practice with high sensitivity and specificity, in addition to its role as an independent prognostic marker of overall survival and tumour recurrence, as suggested by previous studies.

High expression of GLI1 is associated withbetter survival in advanced SCLC.

Aberrant Sonic hedgehog (Shh) pathway signaling has been described in small cell lung cancer (SCLC), as well discrepancies, when analyzing expression of pathway components in SCLC cell lines vs tumor biopsies. Shh key component GLI1was evaluated in advanced SCLC and data correlated with patient survival. GLI1expression was analyzed by quantitative real-time polymerase chain reaction in pre-treatment fresh frozen tumor biopsies of 12advanced SCLC patients and mRNA level of GLI1was compared in short-term vs long-term survivor's samples (stratified by median survival, independent samples t-test). Expression of GLI1 mRNA was significantly higher in long-term (> 9.6months, n = 6) survivor's biopsies than in short-term (≤ 9.6months, n = 6) survivors (p = 0.0196, 95%CI: 0.000016to0.000147, two-tailed independent samples t-test). High GLI1mRNA expression in SCLC was found to be positive prognostic marker associated with longer survival. Further research is needed for validation of these results due to the small number of patients in the study.

Non-small cell lung cancer-small cell lung cancer transformation as mechanism of resistance to tyrosine kinase inhibitors in lung cancer

Mutated or rearranged driver kinases in non-small cell lung cancer (NSCLC) cells are clinically amenable to treatment with tyrosine kinase inhibitors (TKIs) resulting in prolonged survival and significant benefit compared to cytotoxic chemotherapy. The most frequent genomic alterations are observed for epidermal growth factor receptor and anaplastic lymphoma kinase, which can be blocked by a range of specific TKIs in sequence. In clinics, resistance to TKIs emerges after approximately one year and comprises secondary mutations of the kinases (on-target) or alternative pathways circumventing the original kinase (off-target) alterations. A special feature of NSCLC is the occurrence of histological transformation to small cell lung cancer (SCLC) in up to 14% of cases, which, in general, is accompanied by resistance to the original TKIs. SCLC transformed tumors may be treated with the classical platinum/etoposide regimen but thus far there are no definitive guidelines. Four transformed pleural SCLC lines in our lab indicate the presence of a gradual NSCLC-SCLC shift with overlapping drug sensitivities. In conclusion, the treatment of NSCLC-SCLC transformed cancer cells would need a better chemosensitivity assessment using functional genomics to guide further therapy.

The Impact of High-mobility Group Box Mutation of T-cell Factor 4 on Its Genomic Binding Pattern in Non–small Cell Lung Cancer

T-cell factor 4 (TCF-4) is determined to play a crucial role in Wnt/β-catenin signaling pathway activation. The mutations and alternative splice isoforms of TCF-4 can cause cancers and other diseases. The high-mobility group (HMG) box domain of TCF-4 contributes to interacting with DNA motif for transcriptional regulation. However, the impact of the mutations within HMG box of TCF-4 on the genomic binding pattern is poorly investigated. Herein, we generated non–small cell lung cancer (NSCLC) cell line A549 with stably overexpressed TCF-4 with HMG box hot spot mutation (10th exon partial deletion), and conducted TCF-4 and β-catenin chromatin immunoprecipitation sequence to explore the differential genomic binding patterns. Our results revealed that TCF-4 lost 19365 but gained 1724 peaks, and β-catenin lost 4035 but gained 5287 peaks upon mutant TCF-4 compared with the wild type (log2FC > 1 or < -1, FDR<0.01). The transcriptional levels of the genes associated with these differential peaks such as H3F3C, KRT1, KRT14, MMp1, and MMP15 were all found to strongly change responding to TCF-4 binding (P < 0.05). Furthermore, A549 cells with TCF-4 mutation displayed a more compromising tumor characterization on cell proliferation and invasion. Our data determined the important role of TCF-4 in gene transcription controlling and provided the gain function evidence of TCF-4 caused by the TCF-4 mutation in NSCLC.

Abstract B064: The Aurora kinase A-inhibitor, alisertib, is a potential candidate for combination with immunotherapy in small cell lung cancer

Abstract Background: Small cell lung cancer (SCLC) is characterized by a loss of TP53 and RB1 and by amplification of MYC in about 25% of patients. Our group and others have shown that MYC-driven SCLC cell lines are vulnerable to Aurora Kinase A (AURKA) inhibition. Alisertib, a selective AURKA inhibitor, has also shown clinical efficacy in MYC-positive relapsed SCLC patients in combination with paclitaxel in a phase II trial (NCT02038647). AURKA regulates DNA chromosome alignment during cell division and thus its inhibition results in mitotic stress and subsequently DNA damage. We have previously demonstrated that DNA damage response (DDR) inhibitors synergize with immune checkpoint inhibition in SCLC in vivo models by activating a STING-mediated innate immune response. Based on these findings, we hypothesized that AURKA inhibition may activate the STING pathway and induce PD-L1 expression in SCLC. Methods: We compared AURKA gene expression among a panel of SCLC and NSCLC cell lines and, similarly, in tumor versus normal tissue in SCLC clinical dataset (Sato et al.). In parallel, we explored the landscape of STING and immune genes expression in two SCLC clinical datasets (George et al. and Sato et al.).We tested human and GEMM-derived SCLC cell lines for changes in immune and DDR-related protein expression by Western blot analysis after treatment with the AURKA-i alisertib. Results: AURKA gene expression was higher in SCLC cell lines as compared to NSCLC cell lines (p=0.023) and in SCLC tumors compared to normal tissue (FC=1.6; p&amp;lt;0.001), thus making AURKA an attractive target in SCLC. Furthermore, in SCLC clinical datasets, we found that CXCL10 and CCL5, the two downstream chemokines of STING pathway, are highly expressed in about 30% SCLC tumors and their expression is significantly associated with CMYC (Spearman Rho&amp;gt;0.45, p&amp;lt;0.001), possibly due to high baseline levels of replication stress and also to CD274, CD8 and other markers and chemokines suggestive of an inflamed immune microenvironment. We observed that in vitro treatment with alisertib (1µM for 72 hours) further increases the level of phospho-H2AX protein, a marker of DNA damage, along with PD-L1. Furthermore, alisertib treatment resulted in STING pathway activation, as indicated by increased phospho-STING_S366 and phospho-TBK1_S172 protein levels, in both human and murine SCLC cells. Conclusions: Our results demonstrate the potential of alisertib, the selective AURKA inhibitor, in modulation of PD-L1 and STING pathway activation in SCLC cells, thus supporting the rationale for in vivo testing with immunotherapy drugs. In particular, evaluation of this strategy in MYC-positive SCLC models could open new therapeutic investigations in this highly resistant SCLC subgroup. Citation Format: Carminia M. Della Corte, Liz Lauren Ajpacaja, Robert J Cardnell, Carl M Gay, Lixia Diao, Qi Wang, Kavya Ramkumar, Allison C. Stewart, Rebecca B. Kow, Hannah E Stumpf, You-Hong Fan, Jing Wang, John V Heymach, Lauren A. Byers. The Aurora kinase A-inhibitor, alisertib, is a potential candidate for combination with immunotherapy in small cell lung cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B064. doi:10.1158/1535-7163.TARG-19-B064

Abstract LB-B13: Lurbinectedin down-regulates ASCL1 transcription factor in Small Cell Lung Cancer (SCLC)

Abstract The reported clinical activity of lurbinectedin in Small Cell Lung Cancer (SCLC) prompted us to investigate the selective mechanism of this drug in SCLC cell lines. Since lurbinectedin binds the DNA minor groove modifying DNA structure, we hypothesized that it can interfere with transcription regulation. Using SHP-77 SCLC cell line, we investigated the effect of lurbinectedin on transcriptomic profile by microarray approach, at drug concentration that reduced the cell growth rate by 50%. We found only 89 differently expressed genes (DEG), 31 up- regulated and 58 down-regulated, after 6 hours of treatment whereas there was a dramatic change in gene expression profiles after 24 hours (4529 DEGs of which 1982 up-regulated and 2547 down-regulated) and 48 hours (5457 DEGs of which 2368 up-regulated and 3089 down-regulated) of exposure. Among the genes that were clearly down-regulated at both 24 and 48 hours, there was ASCL1, a gene that encodes a transcription factor required for proper development of neuronal and pulmonary neuroendocrine cells. ASCL1 regulates stemness and cell cycle progression and it is increased in high grade neuroendocrine tumours including SCLC. Western blot analysis confirmed strong reduction of the ASCL1 protein level in SHP-77 cell line. From literature data we retrieved the ChIP-Seq target genes of ASCL1 derived from different SCLC cell lines and compared them to the list of genes at basal condition in SHP-77 cell line. Then we computed Pearson correlation between the expression of ASCL1 and these targets and filtered only strong positive correlations. Through this approach, we finally selected 213 target genes of ASCL1 many of which were coherently inhibited after 24 and 48 hours of treatment. These genes were mainly involved in MAPK and Wnt signalling pathways, both crucial for SCLC. The mechanism of transcription modulation driven by ASCL1 after lurbinectedin treatment is under investigation by ChIP-Seq technology. Moreover, work is in progress to extend our findings on others SCLC cell lines and in sub-lines in which ASCL1 was silenced. We tested whether lurbinectedin shared a similar effect on transcriptomic profile as other known drugs: by dividing up and down regulated genes and testing their correlation to the drug-related gene profiles provided by the tool Enrichr, we found that lurbinectedin has a high correlation to the topoisomerase I inhibitor class. This finding deserves further experimental verifications also in view of potential clinical interest in the combination of lurbinectedin and topoisomerase I inhibitors. Citation Format: Laura Mannarino, Ilaria Craparotta, Federica Mirimao, Nicolò Panini, Monica Lupi, Giulia Protti, Roberta Frapolli, Sergio Marchini, Maurizio D'Incalci. Lurbinectedin down-regulates ASCL1 transcription factor in Small Cell Lung Cancer (SCLC) [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-B13. doi:10.1158/1535-7163.TARG-19-LB-B13

Abstract B013: Associations of epigenome-wide DNA methylation patterns with chemosensitivity and chemoresistance of small cell lung cancer cell lines

Abstract Small cell lung cancer (SCLC), an aggressive neuroendocrine type of lung cancer, rapidly acquires resistance to treatment. SCLC progression, lineage differentiation, and resistance to therapy have been suggested to involve epigenetic processes. To date, epigenetic links connecting SCLC DNA methylation patterns to drug response and the ways in which these links are mediated by gene expression remain unclear. In order to understand how DNA methylation may affect SCLC response to chemotherapy, we performed an epigenome-wide association study of 66 SCLC cell lines. We used Illumina Infinium MethylationEPIC BeadChip to measure methylation of 866,091 probes. We examined how methylation of probes and gene regions was associated with SCLC in vitro response to 526 antitumor agents. We also identified associations of epigenetic variation with drug response which may be mediated by regulation of gene expression. A potentially important strong association was observed for TREX1, which encodes the 3’ exonuclease I (DNase III) that is involved in resolution of chromatin bridges and has a potential role in chromothripsis. Increased methylation and low expression of TREX1 were associated with SCLC cell line sensitivity to multiple Aurora kinase inhibitors AZD-1152, SCH-1473759, SNS-314, and TAK-901, as well as to the CDK inhibitor R-547, Vertex ATR inhibitor Cpd 45, and the mitotic spindle disruptor vinorelbine. TREX1 upregulation has been previously associated with resistance of other cancers to DNA damaging agents and with DNA repair or DNA degradation after drug exposure. In our analysis, when compared to other cancer categories, TREX1 in SCLC cell lines had low mRNA expression and increased DNA methylation upstream of its transcription start site, which may provide a possible molecular mechanism for SCLC sensitivity to Aurora kinase inhibitors. CEP350 and MLPH, which are involved in centrosome machinery and microtubule tracking, were associated with several Aurora kinase inhibitors and other agents. Among other examples, EPAS1 (HIF2A) was associated with several Aurora kinase inhibitors, the PLK1 inhibitor GSK-461364, and the Bcl-2 inhibitor ABT-737. Methylation of KDM1A, encoding the histone modifier lysine demethylase 1A (LSD1), was associated with PLK1 inhibitors and the KSP inhibitor SB-743921. IGFBP5, which is expressed in the tuft cell-like SCLC subtype, was associated with the mTOR inhibitor INK-128. Upstream regions of MDM2 and DLL3, a Notch pathway regulator overexpressed in ASCL1-high SCLC tumors, were associated with Bcl-2 inhibitors. Methylation and expression of YAP1, a SCLC lineage driver regulating the Hippo pathway, were correlated with the MTOR inhibitor rapamycin. Among non-neuroendocrine lineage markers, EPHA2 was associated with Aurora kinase inhibitors and a PLK1 inhibitor, and CD151 with Bcl-2 inhibitors. Increased methylation upstream of SLFN11 was correlated with resistance to DNA damaging agents, which is likely mediated by SLFN11 expression. The 5’ UTR region of the epigenetic modifier EZH2 was associated with Aurora kinase inhibitors and the FGFR inhibitor BGJ-398. These and multiple other associations identified in this study provide a novel understanding of epigenetic mechanisms which may modulate SCLC response to chemotherapy, and suggest potential molecular targets for combination therapies. This research was supported in part with federal funds from the National Cancer Institute, NIH, under contract HHSN261200800001E. Citation Format: Julia Krushkal, Thomas Silvers, Dmitriy Sonkin, Suleyman Vural, John Connelly, Sudhir Varma, Paul S. Meltzer, William C. Reinhold, Annamaria Rapisarda, David Evans, Yves Pommier, Beverly A. Teicher. Associations of epigenome-wide DNA methylation patterns with chemosensitivity and chemoresistance of small cell lung cancer cell lines [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B013. doi:10.1158/1535-7163.TARG-19-B013

Abstract B021: PARP16 is a novel target of talazoparib which contributes to synergy with adavosertib in SCLC

Abstract Background: Small cell lung cancer (SCLC) is a highly aggressive type of neuroendocrine tumor with a low survival rate, signifying an unmet medical need. Although PARP inhibitors (PARPis) display single-agent anticancer activity in SCLC, the underlying biology is poorly understood. In the present study, we determined the differential efficacy of multiple clinically relevant PARPis and identified a novel non-canonical and actionable PARPi target in SCLC. Methods: A panel of SCLC cell lines was analyzed for sensitivity to PARPis using the Cell Titer Glow (CTG) viability assay. Chemical proteomics and western blotting were performed to investigate the targets of PARPis. The effect of PARP16 on cell viability was analyzed using RNA interference. Drug combination screening for inhibition of viability was performed, network-wide signaling changes were determined using phosphoproteomics, and efficacy of drug combinations was determined in ex vivo PDX clonogenic assays. Results: Compared to other clinically relevant PARPis (rucaparib, olaparib, niraparib) talazoparib displayed the highest potency across a panel of SCLC cell lines, including cells negative for SLFN11, which has been suggested to indicate PARPi sensitivity. Subsequent chemical proteomics with SLFN11-negative SCLC cells identified the non-canonical PARP family member PARP16 as a unique talazoparib target, along with the eminent PARP1. Transient knockdown of PARP16 significantly reduced cell survival in certain SCLC cell lines, particularly in combination with olaparib. Furthermore, drug combination screens revealed synergistic activity of talazoparib when combined with the WEE1 inhibitor adavosertib in SCLC cells, which was more pronounced than the combination of adavosertib with olaparib, which has no activity against PARP16. Silencing of PARP16, in combination with olaparib and adavosertib, with enhanced cell killing, and global phosphoproteomics identified network-wide signaling crosstalk inhibition by the talazoparib/adavosertib drug combination. Conclusion: Our data suggest that in addition to the well-studied PARP1 targeting mechanism, PARP16, a non-canonical member of the PARP family, is likely involved in talazoparib’s overall mechanism of action and may constitute a novel actionable target in SCLC. Citation Format: Vinayak Palve, Clare Knezevic, Yunting Luo, Xueli Li, Slivia Novakova, Eric Welsh, Bin Fang, Fumi Kinose, Eric B Haura, Alvaro N Monteiro, John M Koomen, Harshani R Lawrence, Uwe Rix. PARP16 is a novel target of talazoparib which contributes to synergy with adavosertib in SCLC [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B021. doi:10.1158/1535-7163.TARG-19-B021

Abstract C033: HPN328: An anti-DLL3 T cell engager for treatment of small cell lung cancer

Abstract HPN328: An Anti-DLL3 T Cell Engager for Treatment of Small Cell Lung Cancer Delta-like protein 3 (DLL3) is a Notch inhibitory ligand is expressed in more than 70% of small cell lung cancers (SCLCs), while there is little to no surface expression in normal adult tissues outside of the CNS *1. SCLC is an aggressive neuroendocrine tumor that represents about 15 percent of all lung cancers. Although often responsive to standard of care treatment, relapse is common, with a median progression-free survival of 2–3 months and median overall survival (OS) of 8–13 months and 5-year OS rate of &amp;lt;5%*2. DLL3 thus represents an attractive potential target for T cell–redirecting immunotherapy in SCLC. HPN328 is a tri-specific T cell activating construct (TriTAC) consisting of three binding domains: a N-terminal single chain Fv (scFv) that binds to CD3ϵ of the T cell receptor (TCR), a middle single domain antibody (sdAb) that binds to human serum albumin (HSA) to extend the half-life, and a C-terminal sdAb that binds to human DLL3. HPN328 is designed to simultaneously engage DLL3 on a target SCLC cell and CD3 on a T cell resulting in T cell activation, proliferation, and the eventual lysis of the target lung cancer cell. HPN328 is a highly stable single polypeptide of ~ 53 kDa expressed in CHO cells. It binds to human and cynomolgus monkey DLL3, albumin, and CD3ϵ with similar affinities. Flow cytometry analysis of T cells from various normal donors and a panel of DLL3 positive and DLL3 negative tumor cell lines confirmed binding of HPN328 to its native targets expressed on the cell surface. HPN328 induces potent killing of DLL3 expressing SCLC cell lines in vitro. In co-cultures of T cells from normal human donors, target tumor cells, and HSA, HPN328 mediated dose-dependent and DLL3-dependent cytotoxicity. Concomitant with target tumor cell killing, HPN328 also mediated dose-dependent upregulation of CD25 and CD69 on T cells in the TDCC co-cultures when DLL3 positive tumor cells were present. Consistent with the mechanism of action of CD3-based T cell engaging molecules, T cell derived cytokines, including TNFα, IL-2 and IFNγ, were detected. Nonclinical in vivo properties of HPN328 were evaluated in an NCI-H82 SCLC established tumor model. When administered to mice bearing human SCLC xenografts and human T cells, HPN328 eradicated subcutaneous tumors. In a single dose pilot toxicity study in cynomolgus monkeys, HPN328 was well tolerated at 1 and 10 mg/kg. Pharmacodynamic changes such as transient cytokine elevation mainly at the high dose were observed, consistent with the expected mechanism of action of T cell engagers. There were no clinically significant or adverse test article-related changes in hematology or clinical chemistry, and no apparent adverse findings at terminal and recovery necropsy. HPN328 exhibited linear PK properties in the given dose range with a serum half-life of 64 to 85 hours. Pharmacokinetic analysis supports weekly administration of HPN328 in humans. Preclinical and nonclinical characterization suggests that HPN328 is a highly efficacious, safe, and novel therapeutic candidate. A first-in-human phase 1 clinical trial is planned to evaluate HPN328 in SCLC. *1. Saunders, L et al. (2015) Sci Transl Med. 7(302): 302ra136. *2. Navarro, A et al. (2017) Transl Lung Cancer Res. 6(1): S78–S83. Citation Format: Wade H Aaron, Richard Austin, Manasi Barath, Evan Callihan, Michael Cremin, Thomas Evans, Maria Gamez, Vaishnavi Ganti, Golzar Hemmati, Kathryn Kwant, Che-Leung Law, Bryan Lemon, Llewelyn Lao, Mary Ellen Molloy, Jessica O’Rear, Laura Sun, Holger Wesche, Stephen Yu, Timothy Yu. HPN328: An anti-DLL3 T cell engager for treatment of small cell lung cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C033. doi:10.1158/1535-7163.TARG-19-C033

Ascl1-induced Wnt11 regulates neuroendocrine differentiation, cell proliferation, and E-cadherin expression in small-cell lung cancer and Wnt11 regulates small-cell lung cancer biology

The involvement of Wnt signaling in human lung cancer remains unclear. This study investigated the role of Wnt11 in neuroendocrine (NE) differentiation, cell proliferation, and epithelial-to-mesenchymal transition (EMT) in human small-cell lung cancer (SCLC). Immunohistochemical staining of resected specimens showed that Wnt11 was expressed at higher levels in SCLCs than in non-SCLCs; 58.8% of SCLC, 5.2% of adenocarcinoma (ADC), and 23.5% of squamous cell carcinoma tissues stained positive for Wnt11. A positive relationship was observed between Achaete-scute complex homolog 1 (Ascl1) and Wnt11 expression in SCLC cell lines, and this was supported by transcriptome data from SCLC tissue. The expression of Wnt11 and some NE markers increased after the transfection of ASCL1 into the A549 ADC cell line. Knockdown of Ascl1 downregulated Wnt11 expression in SCLC cell lines. Ascl1 regulated Wnt11 expression via lysine H3K27 acetylation at the enhancer region of the WNT11 gene. Wnt11 controlled NE differentiation, cell proliferation, and E-cadherin expression under the regulation of Ascl1 in SCLC cell lines. The phosphorylation of AKT and p38 mitogen-activated protein kinase markedly increased after transfection of WNT11 into the SBC3 SCLC cell line, which suggests that Wnt11 promotes cell proliferation in SCLC cell lines. Ascl1 plays an important role in regulating the Wnt signaling pathway and is one of the driver molecules of Wnt11 in human SCLC. Ascl1 and Wnt11 may employ a cooperative mechanism to control the biology of SCLC. The present results indicate the therapeutic potential of targeting the Ascl1–Wnt11 signaling axis and support the clinical utility of Wnt11 as a biological marker in SCLC.

Role of Focal Adhesion Kinase in Small-Cell Lung Cancer and Its Potential as a Therapeutic Target.

Small-cell lung cancer (SCLC) represents 15% of all lung cancers and it is clinically the most aggressive type, being characterized by a tendency for early metastasis, with two-thirds of the patients diagnosed with an extensive stage (ES) disease and a five-year overall survival (OS) as low as 5%. There are still no effective targeted therapies in SCLC despite improved understanding of the molecular steps leading to SCLC development and progression these last years. After four decades, the only modest improvement in OS of patients suffering from ES-SCLC has recently been shown in a trial combining atezolizumab, an anti-PD-L1 immune checkpoint inhibitor, with carboplatin and etoposide, chemotherapy agents. This highlights the need to pursue research efforts in this field. Focal adhesion kinase (FAK) is a non-receptor protein tyrosine kinase that is overexpressed and activated in several cancers, including SCLC, and contributing to cancer progression and metastasis through its important role in cell proliferation, survival, adhesion, spreading, migration, and invasion. FAK also plays a role in tumor immune evasion, epithelial-mesenchymal transition, DNA damage repair, radioresistance, and regulation of cancer stem cells. FAK is of particular interest in SCLC, being known for its aggressiveness. The inhibition of FAK in SCLC cell lines demonstrated significative decrease in cell proliferation, invasion, and migration, and induced cell cycle arrest and apoptosis. In this review, we will focus on the role of FAK in cancer cells and their microenvironment, and its potential as a therapeutic target in SCLC.

P1.12-01 CD81-HOTTIP Regulating Loop Enhances Myc-Mediated Chemoresistance in Small Cell Lung Cancer via ERK Signaling Pathway

The majority of small cell lung cancer (SCLC) patients who are treated with platinum-based chemotherapy will eventually develops into drug resistance. Currently, there are no effective durable therapies for patients with resistant recurrent SCLC. High expression of CD81 is associated with SCLC chemoresistance and poor clinical outcome. Here, our goal is to study the underlying mechanism and define a novel signaling pathway controlled by CD81 -modulating SCLC chemotherapy resistance. The expression of CD81, HOTTIP, and MYCBP were examined in human SCLC tissues, patient-derived xenograft (PDX) models, cell lines derived xenograft tumor model, and SCLC cell lines. The correlations between CD81 and HOTTIP were also confirmed by RNA immunoprecipitation and luciferase reporter assay. Functional analysis of HOTTIP, MYCBP, and ERK signaling was investigated in SCLC cell lines and xenografts. Knockdown CD81 enhanced cell responsiveness to chemotherapeutic drugs in SCLC and decreased the expression of HOTTIP, as well as inhibited the intra-nuclear translocation of MYCBP/MYC complex. Conversely, overexpression of CD81 decreased the chemotherapeutic reactivity and increased the expression of HOTTIP and promoting nuclear translocation of MYCBP/MYC complex. CD81 upregulation of HOTTIP requires the transcription factor TCF4. Further analysis demonstrated that CD81-mediated activation of ERK signaling pathway and inhibition of β-catenin degradation leads to promotion of β-catenin transfer to the nucleus. Nuclear β-catenin enhances transcriptional activity of TCF4 and then promotes the transcription of HOTTIP. Synergistically, HOTTIP could bind and increase the nucleus transduction of the MYCBP, form a HOTTIP/MYCBP/MYC complex in nucleus, resulting in increased MYC levels in nucleus. Nuclear MYC further mediates chemoresistance and promotes CD81 expression, forming a positive feedback regulatory pathway in SCLC. In addition, in vivo experiments showed that inhibitors that target the CD81-HOTTIP feedback regulatory loop combined with chemotherapeutic drugs can reverse the chemotherapeutic resistance of SCLC. Our findings suggest a novel feedback regulatory pathway dominated by CD81 and HOTTIP controlling SCLC chemotherapeutic resistance, which could serve as a new therapeutic targets for this devastating disease.

S02.06 New Developments in SCLC and Neuroendocrine Tumors

In an IASLC memorial earlier this year, Dr. Gazdar was very appropriately described as “a true giant in the field of lung cancer.” Dr. Gazdar’s profound impact on our field continues to be felt both in the clinic and at the bench. As a world-renowned molecular and clinical pathologist, he played a key role in setting the standards for classifying human lung cancers. As a scientist, he established ∼400 human cancer cell lines. These included a large number of molecularly-annotated non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) cell lines that have led to countless, practice-changing biological discoveries. As a founding-father of lung cancer research, he had a remarkable depth of knowledge in lung cancer biology and pathology. He shared his expertise through hundreds of publications. However, he also gave generously of himself (and his knowledge) to collaborators and mentees – serving as an advisor and teacher to almost everyone in the field who had the opportunity to be around him. Having contributed to key discoveries over five decades, Dr. Gazdar intuitively understood which new developments were likely to make the biggest impact and what questions we should be asking next. This was especially true for small cell lung cancer (SCLC) and other neuroendocrine tumors, where despite years of research there had been relatively few advances in the clinic. In a review published a year before his death, Dr. Gazdar and his co-authors shared their excitement for the recent worldwide resurgence of SCLC research – which they describe as “The Second Golden Age” of SCLC research.1 Several new developments in SCLC and neuroendocrine tumors have contributed to a better understanding of the disease and have shown promise for translational application. These include the discovery of (1) significant heterogeneity between patients with SCLC, (2) the plasticity of SCLC over time, (3) the role of intra-tumoral heterogeneity in metastasis and resistance, and (4) the identification of new therapeutic targets, immunotherapy approaches, and candidate biomarkers for SCLC. Going forward, opportunities and unmet needs in SCLC include enrolling patients onto clinical trials that can identify therapeutic vulnerabilities among specific SCLC subtypes; deeply profiling relapsed SCLC (through new models and patient specimen profiling); and extending our understanding of the immune microenvironment in SCLC. Given the pace and impact of recent discoveries in SCLC, it is no surprise that Dr. Gazdar and his co-authors concluded that “…while the past has been bleak, the future offers great promise.” 1. Gazdar AF, Bunn PA, Minna JD. Small-cell lung cancer: what we know, what we need to know and the path forward. Nat Rev Cancer 2017;17:725-37. Heterogeneity, biomarkers, SCLC

Chemical composition, in vitro antibacterial and antioxidant potential of Omani Thyme essential oil along with in silico studies of its major constituent

Thyme is an evergreen shrub that has been utilized in traditional medicine and culinary packages for centuries because of its aroma and medicinal properties. The medicinal properties of thyme leaves are attributed to its volatile constituents. The study aimed to identify the volatile constituents in the oil extracted from the Thyme sp grown in Oman and to evaluate its in vitro antibacterial and antioxidant activity. Fresh Thyme leaves were collected from Muscat, Sultanate of Oman in the month of September 2018. Thyme oil was isolated from leaves by hydro-distillation. The volatile chemical constituents present in the thyme oil were analyzed by Gas Chromatograph coupled with Mass Spectrometer. The ability of isolated oil to scavenge free radicals was evaluated by an in vitro DPPH assay method while antimicrobial activity was tested against S. aureus and E. coli bacterial strains by disc diffusion method. The bioactivity of the major constituent of the oil was predicted with the help of PASS and CLC-pred software. Molecular docking studies were performed by docking server. GC–MS analysis of thyme oil revealed the presence of 11 components. Carvacrol and γ-terpinene were identified as the major volatile constituents in thyme oil. Thyme oil inhibited 71.57% of DPPH radicals at 40 µg/mL concentration. Thyme oil displayed the better antimicrobial activity than the standard drug ampicillin against both the bacteria. Antiseptic, antiinfective, reductant and antimutagenic bioactivities of carvacrol as predicted by PASS support the in vitro experimental results. CLC-pred showed carvacrol to be active against Metastatic melanoma, Oligodendroglioma and Non small cell lung cancer cell lines. The antioxidant and antimicrobial activity of Omani thyme oil could be due to its high content of carvacrol. Omani thyme oil seems to be a viable alternative source of natural antimicrobial agent(s) and warrants further studies to ascertain its therapeutic spectrum of biological studies.

P1.12-05 Microenvironment Characterization of Small Cell Lung Cancer Xenografts Implanted in Hematopoietic Humanized Mice

With the high mutational burden seen in small cell lung cancer (SCLC) there is the theoretical potential to improve SCLC outcome through immunotherapy. Recent immunotherapeutic clinical trials in SCLC have demonstrated promising results. However, to better understand the immune response and the potential role of immunotherapy in SCLC, immunocompetent models are needed. To this end, we have applied a hematopoietic humanized mouse model, Hu-CB-BRGS to investigate the mechanisms underlying SCLC immunotherapy and develop novel strategies to improve therapeutic efficacy. BALB/c-Rag2nulllll2rynullSIRPαNOD (BRGS) pups were humanized through transplantation of cord blood (CB)-derived CD34+ cells. SCLC flank tumors were initiated in BRGS mice using two characterized SCLC cell lines (CDX) and 1 patient derived xenograft (PDX). Upon verification of human T-cell chimerism in the Hu-CB-BRGS mice, SCLC tumors grown in BRGS mice were engrafted into the flanks of Hu-CB-BRGS mice by trocar transfer. Tumor growth was quantified by twice weekly measurement and harvested on reaching 1200 mm3. At harvest, tumor tissue as well as host immune organs (lymph node, spleen) were collected for immunological assessment. Humanized immune system and tumor were evaluated by flow cytometry and immunohistochemistry (IHC). Flank tumors from two CDX tumors (H82 and H187) and one PDX tumor (LX-95) were successfully developed in the Hu-CB-BRGS mice with take rates averaging 82%. SCLC tumor growth rate in Hu-CB-BRGS mice was comparable to that seen in BRGS mice. Although human T cells were well represented in the lymph nodes and spleens of the Hu-CB-BRGS mice, we detected very few tumor infiltrating immune cells in the engrafted SCLC tumors by IHC and flow cytometry as defined by CD45 and CD3, which is consistent with the observations in SCLC patient tumor tissue. Tumor cells, identified by EpCAM expression, expressed low levels of MHC class I, II and PD-L1. PD-1 was expressed by human T cells found in the lymph nodes and spleen of Hu-CB-BRGS mice, while the SCLC xenografts expressed varying levels. We demonstrate that both SCLC PDX and CDX tumors can be grown in the context of a humanized immune system within mouse recipients. SCLC Hu-CB-BRGS mice demonstrate persistence of human immune cells, including T cells and B cells in the immune organs. The xenograft tumor microenvironment included variable human immune infiltrates. The SCLC Hu-CB-BRGS mouse model may be a valuable preclinical platform for testing human specific immune-oncology therapeutics for SCLC patients.

Cisplatin bioconjugated enzymatic GNPs amplify the effect of cisplatin with acquiescence

Enzymatic gold nanoparticles (B-GNPs) have been synthesized using a natural anticancer agent bromelain (a cysteine protease) and these nanoparticles were used to bioconjugate Cisplatin (highly effective against osteosarcoma and lung cancer). Cisplatin bioconjugated bromelain encapsulated gold nanoparticles (B-C-GNPs) were found profoundly potent against same cancers at much lower concentration with minimum side effects due to the synergistic effect of bromelain. The B-C-GNPs have been observed to inhibit the proliferation of osteosarcoma cell lines Saos-2 and MG-63 with IC50 estimation of 4.51 µg/ml and 3.21 µg/ml, respectively, and against small lung cancer cell line A-549 with IC50 2.5 µg/ml which is lower than IC50 of cisplatin against same cell lines. The B-GNPs/B-C-GNPs were characterized by TEM, UV-Visible spectroscopy, Zeta potential and DLS to confirm the production, purity, crystalline nature, stability of nanoemulsion, size and shape distribution. The change in 2D and 3D conformation of bromelain after encapsulation was studied by Circular Dichroism and Fluorometry, respectively. It was found that after encapsulation, a 19.4% loss in secondary structure was observed, but tertiary structure was not altered significantly and this loss improved the anticancer activity. The confirmation of bioconjugation of cisplatin with B-GNPs was done by UV-Visible spectroscopy, TEM, FTIR, 2D 1H NMR DOSY and ICP-MS. Further, it was found that almost ~4 cisplatin molecules bound with each B-GNPs nanoparticle.

EphA2 inhibition suppresses proliferation of small-cell lung cancer cells through inducing cell cycle arrest

Small-cell lung cancer (SCLC) is characterized by one of neuroendocrine tumors, and is a clinically aggressive cancer due to its rapid growth, early dissemination, and rapid acquisition of multidrug resistance to chemotherapy. Moreover, the standard chemotherapeutic regimen in SCLC has not changed for three decades despite of the dramatic therapeutic improvement in non-SCLC. The development of a novel therapeutic strategy for SCLC has become a pressing issue. We found that expression of Eph receptor A2 (EphA2) is upregulated in three of 13 SCLC cell lines and five of 76 SCLC tumor samples. Genetic inhibition using siRNA of EphA2 significantly suppressed the cellular proliferation via induction of cell cycle arrest in SBC-5 cells. Furthermore, small molecule inhibitors of EphA2 (ALW–II–41-27 and dasatinib) also exclusively inhibited proliferation of EphA2-positive SCLC cells by the same mechanism. Collectively, EphA2 could be a promising candidate as a therapeutic target for SCLC.