Small Lung Carcinoma Cell Line Research Articles

NDRG2 acts as a negative regulator of the progression of small-cell lung cancer through the modulation of the PTEN-AKT-mTOR signalling cascade

N-myc downstream-regulated gene 2 (NDRG2) has been recognised as a negative regulator of the progression of numerous tumours, yet its specific role in small-cell lung carcinoma (SCLC) is not fully understood. The purpose of the current study was to investigate the biological role and mechanism of NDRG2 in SCLC. Initial investigation using the Gene Expression Omnibus (GEO) dataset revealed marked downregulation of NDRG2 transcripts in SCLC. The decreased abundance of NDRG2 in SCLC was verified by examining clinical specimens. Increasing NDRG2 expression in SCLC cell lines caused significant changes in cell proliferation, cell cycle progression, colony formation, and chemosensitivity. NDRG2 overexpression decreased the levels of phosphorylated PTEN, AKT and mTOR. In PTEN-depleted SCLC cells, the upregulation of NDRG2 did not result in any noticeable impact on AKT or mTOR activation. Additionally, the reactivation of AKT reversed the antitumour effects of NDRG2 in SCLC cells. Notably, increasing NDRG2 expression retarded the growth of SCLC cell-derived xenografts in vivo. In conclusion, NDRG2 serves as an inhibitor of SCLC, and its cancer-inhibiting effects are achieved through the suppression of AKT/mTOR via the activation of PTEN. This work suggests that NDRG2 is a potential druggable target for SCLC treatment.

All That Is Small Is Not a Small-Cell Carcinoma: Thoracic SMARCA4-Deficient Undifferentiated Tumors Masquerading as SCLC.

Small-cell lung carcinoma (SCLC) cell lines have been widely utilized as a preclinical model of this highly aggressive disease. However, since their creation decades ago, novel tumor entities have been defined that might clinicopathologically mimic SCLC, which notably includes thoracic SMARCA4-deficient undifferentiated tumor (SMARCA4-UT). Multiomic reassessment of the presumed SCLC cell lines with high YAP1 expression reveals that nearly all of these tumors represent unsuspected SMARCA4-UT. See related article by Ng et al., p. 1846.

Abstract 3734: Neuroendocrine differentiation (ND) in sensitivity of neuroendocrine tumor (NET) cells to ONC201/TIC10 cancer therapeutic

Abstract Neuroendocrine tumors (NETs) harbor neuroendocrine differentiation (ND) with specific markers including protein gene product 9.5 (PGP9.5) and Chromogranin A (CgA). In prostate cancers (PC), ND is induced by BRN2/SOX2 transcription factors. NET-like cells with low or absent androgen receptor (AR) signaling cause hormone therapy resistance and poor prognosis in PC. Small cell lung carcinoma (SCLC), a high-grade NET, presents with metastasis early and has poor survival. ONC201/TIC10 is a small molecule inducer of TRAIL signaling in clinical trials. ONC201 antagonizes dopamine D2 or D3 receptors (DRD2/DRD3) and is an agonist of mitochondrial caseinolytic protease P (ClpP) resulting in activation of DR5/TRAIL-dependent apoptosis involving the integrated stress response (ISR). ONC201 is active in various malignancies including H3K27M-mutated glioma and NETs expressing high levels of DRD2. We hypothesized that altered BRN2/SOX2 may impact NET apoptosis by ONC201 through the ISR and TRAIL/DR5. We analyzed the expression of neuroendocrine markers PGP9.5, CgA, SOX2, and BRN2, as well as markers of TRAIL signaling pathway markers ATF4, DR5, ClpP, ClpX, and DRD2/DRD3 in PC and SCLC cell lines (N=6) ± treatment with ONC201. Specifically, we compared pre-treatment protein expression levels with the IC50. Our results reveal that DU145 (IC50=3.11μM), PC3 (IC50=3.02μM), and LNCaP (IC50=1.33μM) are ONC201 sensitive. H1417 SCLC expresses CgA, unlike PC3 and DU145. PGP9.5 is expressed in these lines. PGP9.5 is expressed in PC3, DU145, H1417, and H1048 but not in LNCaP and 22RV1. BRN2 is expressed in PC3, H1417, and H1048 but not DU145, LNCaP, or 22RV1. ClpX is expressed in all 6 lines but at lower levels in SCLC. ClpP is expressed in the 6 lines. DR5 is expressed at higher levels in PC3, DU145, LNCaP, and 22RV1 PC versus H1417 and H1048 SCLC. SOX2 is expressed at high levels in H1417 cells. These results are establishing the landscape of ND in PC and SCLC lines for further experimentation and testing of our hypothesis. To characterize the association of BRN2 dysregulation with ONC201 sensitivity, we are performing BRN2/SOX2 knockdown experiments using siRNA and evaluating effects towards ONC201 sensitivity. Our results provide insights into molecular mechanisms of ND in PC and SCLC sensitivity to ONC201. We are also currently working to overexpress BRN2 and SOX2 in cell lines to analyze the impact on neuroendocrine differentiation and ONC201 sensitivity. Citation Format: Elizabeth C. Ding, Wafik S. El-Deiry. Neuroendocrine differentiation (ND) in sensitivity of neuroendocrine tumor (NET) cells to ONC201/TIC10 cancer therapeutic. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3734.

Abstract 1560: Orally bioavailable macrocycles that target cyclins A and B RxL motifs cause tumor regression in xenograft models and in vitro show activity across multiple cancer types

Abstract RB and E2F genes are frequently altered in cancer. Cyclin/Cdk (cyclin-dependent kinase) complexes regulate the activity of RB and E2F to drive cell cycle progression and ensure fidelity of DNA replication. A critical subset of their substrates (Rb, E2F, Cdc6, etc.) requires a short linear motif (RxL) that interacts with the hydrophobic patch on cyclins. Peptides that disrupt the cyclin/RxL interaction were found to be synthetically lethal in cancer cell lines displaying Rb-E2F dysregulation (Chen, 1999). Using structure-guided design, cell-permeable macrocycles were synthesized that selectively inhibit RxL-mediated binding to cyclins A and B. Preclinical studies with an orally bioavailable cyclin A/B RxL inhibitor show activity in cell lines with Rb-E2F pathway dysregulation, and inhibition of tumor growth and regression in xenograft models of small cell lung carcinoma (SCLC). RB-null mutations occur with high frequency in SCLC tumors (Febres-Aldana 2022). Previously we reported (AACR April 2022 Abs.#5379) sensitivity of a large proportion of SCLC cell lines to cyclin RxL inhibitors. Further studies have shown that such compounds have growth inhibitory activity in cell lines representing multiple types of solid tumors and hematological malignancies. Sensitivity of the cell lines to these macrocyclic inhibitors is associated with increased expression of genes involved in the E2F pathway, consistent with the proposed Rb/cyclin/E2F pathway synthetic lethality mechanism. Disruption of RxL binding to both cyclin A and B is required for sensitivity. Cyclin A/B RxL inhibitors disrupt the interactions of E2F and Cdc6 with cyclin A/Cdk complexes in co-immunoprecipitation studies and have EC50 values consistent with biochemical and cellular activity. Cyclin A/B RxL inhibition results in accumulation of cells in the G2/M phase of the cell cycle which leads to apoptosis both in vitro and in vivo. A separate study (Singh et al. AACR 2023) utilizing a CRISPR screen identified that genes of the spindle assembly checkpoint (SAC) are required for the synthetic lethality induced by the cyclin A/B RxL inhibitors. The inhibitors assessed in vitro for off-target activity showed negligible activity in a panel of 468 kinases and in safety panels that include GPCRs, ion channels, and transporters. In a 14-day tolerability study, neither neutropenia nor significant effects in other blood cell counts were noted. Cyclin A/B RxL inhibitors cause significant tumor regression via oral administration in SCLC xenograft models. Given their compelling characteristics, we are progressing the development of these orally bioavailable macrocyclic cyclin A/B RxL inhibitors to the clinic. Citation Format: Catherine E. Gleason, Pablo D. Garcia, Ranya Odeh, Frances Hamkins-Indik, Daphne He, Meisam Nosrati, Gavin Situ, Roberta Sala, Bernard Levin, Li-Fen Liu, Evelyn W. Wang, Siegfried Leung, Breena Fraga, Andrew T. Bockus, Justin Shapiro, Nathan Dupper, Chinmay Bhatt, Kai Yang, Megan DeMart, Sammy Metobo, James Aggen, Peadar Cremin, Ramesh Bambal, Constantine Kreatsoulas, David J. Earp, Rajinder Singh. Orally bioavailable macrocycles that target cyclins A and B RxL motifs cause tumor regression in xenograft models and in vitro show activity across multiple cancer types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1560.

Junctional adhesion molecule 3 is a potential therapeutic target for small cell lung carcinoma

There are few effective therapies for small cell lung carcinoma (SCLC); thus, we need to develop novel and efficacious treatments. We hypothesized that an antibody–drug conjugate (ADC) could be a promising option for SCLC. Several publicly available databases were used to demonstrate the extent to which junctional adhesion molecule 3 (JAM3) mRNA was expressed in SCLC and lung adenocarcinoma cell lines and tissues. Three SCLC cell lines, Lu-135, SBC-5, and Lu-134 A, were selected and examined for JAM3 protein expression by flow cytometry. Finally, we examined the response of the three SCLC cell lines to a conjugate between an anti-JAM3 monoclonal antibody HSL156 (developed in-house) and the recombinant protein DT3C, which consists of diphtheria toxin lacking the receptor-binding domain but containing the C1, C2, and C3 domains of streptococcal protein G. In silico analyses revealed that JAM3 mRNA was expressed higher in SCLC cell lines and tissues than in those of lung adenocarcinoma. As expected, all the three SCLC cell lines examined were positive for JAM3 at the mRNA and protein levels. Consequently, control SCLC cells, but not JAM3-silenced ones, were highly sensitive to HSL156-DT3C conjugates, resulting in dose- and time-dependent decreased viability. Finally, silencing JAM3 alone suppressed the growth of all SCLC cell lines examined. Taken together, these findings suggest that an ADC targeting JAM3 could represent a new approach to treating SCLC patients.

Abstract P078: Cancer cell derived extracellular vesicles convey protein signatures of small cell lung carcinoma

Abstract Extracellular vesicles (EVs) are known effectors of cell signaling and exchange implicated in cancer development and progression, as well as remodeling and immunomodulation of the tumor microenvironment. EVs are detected in various biofluids and are considered to convey signature features of the cells from which they originate. Clinical assessment of plasma-derived EV molecular contents therefore offers promise of “liquid biopsies” for detection or subtyping of cancers as well as dynamic reporting of tumor status during and after treatment. To date, there have been no comprehensive analyses aimed at interrogating the diverse repertoire of EV protein cargoes in small cell lung carcinoma (SCLC). Using plasmas from a cohort of 18 SCLC cases, 16 lung adenocarcinoma (LUAD) cases and 34 controls matched based on smoking status, age, and sex, EVs were enriched via affinity capture using magnetic beads functionalized with phosphatidyl serine binding protein TIM4 in addition to single-step density flotation ultracentrifugation to deplete abundant protein background. In-depth proteomic profiling was performed on enriched EVs via nano liquid chromatography / high-resolution ion-mobility mass spectrometry. Profiles were intersected with proteomic profiles generated from intratumor EVs dissociated from 12 SCLC patient-derived xenograft (PDX) mouse models as well as EVs isolated from cell-conditioned medias of a panel of 17 SCLC cell lines. A total of 496 proteins were quantified in TIM4pos EVs. Unsupervised hierarchical clustering and principal component analyses showed that TIM4pos EV protein cargoes differentiate SCLC from LUAD and matched controls. Differential analyses identified 117 proteins that were elevated (AUC> 0.60) in TIM4pos SCLC EVs compared to controls. Of these 117 features, 101 (86%) were also elevated (AUC> 0.60) in TIM4pos SCLC EVs compared to TIM4pos LUAD EVs. Intersection of proteins elevated in TIM4pos SCLC EVs with proteomic profiles of intratumor EVs from SCLC PDX models and conditioned media-derived EVs from SCLC cell lines yielded 67 and 92 overlapping proteins, respectively. Overlapping proteins were characterized by YAP1-associated signatures of cytoskeletal re-arrangement and epithelial-to-mesenchymal transition and included a prevalence of neuronal tubulin and actin family members, YWHA family members, and annexins. We developed and applied an optimized EV isolation and proteomics workflow to profile EV protein cargoes and establish circulating EV-associated protein signatures of SCLC with biomarker potential. Our integrated analyses identified novel EV-associated proteins for detection of SCLC that are associated with oncogenic drivers. These studies demonstrate that EVs harbor cancer cell disseminated signatures and that continued efforts to query the EV proteome towards discovery of novel SCLC biomarkers are warranted. Citation Format: Jody Vykoukal, Taketo Kato, Hiroyuki Katayama, Allison Stewart, Ehsan Irajizad, Yining Cai, Fu-Chung Hsiao, Jennifer B. Dennison, Edwin J. Ostrin, Hai T. Tran, Carl M. Gay, Lauren A. Byers, Johannes F. Fahrmann, Samir M. Hanash. Cancer cell derived extracellular vesicles convey protein signatures of small cell lung carcinoma. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr P078.

Proteomics analysis of meclofenamic acid-treated small cell lung carcinoma cells revealed changes in cellular energy metabolism for cancer cell survival.

Small cell lung carcinoma (SCLC) is a highly aggressive cancer with low survival rate. Although initial response to chemotherapy in SCLC patients is well-rated, the treatments applied after the disease relapses are not successful. Drug resistance is accepted to be one of the main reasons for this failure. Therefore, there is an urgent need for new treatment strategies for SCLC. Meclofenamic acid, a nonsteroidal anti-inflammatory drug, has been shown to have anticancer effects on various types of cancers via different mechanisms. The aim of this study was to investigate the alterations that meclofenamic acid caused on a SCLC cell line, DMS114 using the tools of proteomics namely two-dimensional gel electrophoresis coupled to MALDI-TOF/TOF and nHPLC coupled to LC-MS/MS. Among the proteins identified by both methods, those showing significantly altered expression levels were evaluated using bioinformatics databases, PANTHER and STRING. The key altered metabolism upon meclofenamic acid treatment appeared to the cellular energy metabolism. Glycolysis was suppressed, whereas mitochondrial activity and oxidative phosphorylation were boosted. The cells underwent metabolic reprogramming to adapt into their new environment for survival. Metabolic reprogramming is known to cause drug resistance in several cancer types including SCLC. The identified differentially regulated proteins in here associated with energy metabolism hold value as the potential targets to overcome drug resistance in SCLC treatment.

P2.10-04 Immunologic Subtype of Small Cell Lung Carcinoma Dictates Susceptibility to NK Cell-Mediated Cytotoxicity

Small cell lung carcinoma (SCLC) comprises approximately 15% of all lung cancer cases and SCLC patients initially respond to cytotoxic chemotherapy, but resistance inevitably emerges. Despite recent incorporation of immune checkpoint blockade (ICB) into first line treatment, predictive biomarkers of response remain lacking and patients with SCLC continue to have a poor prognosis with limited treatment options. Notably, recent transcriptomic profiling of SCLC cell lines and patient samples has indicated significant inter-tumoral and intra-tumoral heterogeneity.

Abstract 5379: Macrocycles inhibiting RxL-mediated binding of substrates to Cyclin A are synthetic lethal in Rb mutated small cell lung carcinoma (SCLC)

Abstract Inhibition of substrate binding to Cyclin A has been postulated to be synthetically lethal in retinoblastoma (Rb) mutated cancers (Chen et al 1999). While compounds that target the cyclin-associated kinases are approved for clinical use, attempts to target the protein-protein interactions between cyclins and their substrates, such as E2F, have so far been unsuccessful. Using structure-guided design we have developed cell-permeable macrocycle compounds that selectively inhibit the RxL-mediated binding of substrates to Cyclin A. We used NCI-H1048, an Rb-/- SCLC cell line, to study the effects of these Cyclin A targeting macrocyclic compounds on cell proliferation, apoptosis, and cell cycle regulation. Proliferation of H1048 cells was inhibited ~90% at 100nM of compound A whereas no inhibition was observed at up to 10 uM in WI-38 cells, a human non tumor lung fibroblast cell line, or with an inactive isomer of compound A in NCI-H1048 cells. Cyclin A inhibition in H1048 cells resulted in accumulation of cells at G2/M, an increased proportion of cells with 4C DNA content by FACS, hyperphosphorylation of RPA and upregulation of both phosphorylated histone H3 (pHH3) and phosphorylated FOXM1 (pFOXM1). Substantial apoptosis (10 to 20-fold) measured using caspase 3/7 activity was induced after just 4 hours incubation of H1048 cells with Cyclin A inhibitors. In vivo efficacy studies in a murine H1048 xenograft model showed reduction in tumor growth. We analyzed sensitivity to these compounds in tumor cell lines representing multiple indications and observed inhibition of cell proliferation at 1 uM or below in 6 out of 10 SCLC cell lines, where all of the responding cell lines were Rb dysfunctional. Sensitivity to these compounds in a larger panel of SCLC and other cancer cell lines is currently under investigation to further evaluate the broader utility of cyclin inhibition. Macrocycle inhibitors that inhibit Cyclin A RxL-mediated substrate binding have demonstrated G2/M accumulation and apoptosis in Rb -/- SCLC in vitro consistent with synthetic lethality, and anti-proliferative effects in vitro and in vivo. Inhibiting cyclins is mechanistically distinct from strategies that inhibit cell cycle kinases, and is expected to be clinically useful, hence the compound series is being optimized for clinical development. Citation Format: Pablo D. Garcia, Catherine E. Gleason, Miles Membreno, Frances Hamkins-Indik, Gavin Situ, Bernard Levin, Evelyn W. Wang, Siegfried Leung, Breena Fraga, Andrew Bockus, James Aggen, David Spellmeyer, David J. Earp, Rajinder Singh. Macrocycles inhibiting RxL-mediated binding of substrates to Cyclin A are synthetic lethal in Rb mutated small cell lung carcinoma (SCLC) [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 5379.

Diagnostic value of abnormal chromosome 3p genes in small‑cell lung cancer

The diagnosis of small cell lung carcinoma (SCLC) remains a great challenge. Changes in chromosome 3p (chr3) genes are usually observed in the pathogenesis of lung cancer, which suggests that these chr3 genes may be a diagnostic marker in the early stage of SCLC. The present study explored the diagnostic value of the chr3 gene in SCLC using Bioinformatics. Furthermore, reverse transcription-quantitative PCR (RT-qPCR) was used to reveal the expression patterns of diagnostic biomarkers in human pulmonary alveolar epithelial cells and in the SCLC cell line NCI-H146. A total of 33 differentially expressed (DE) chr3 genes and 1,156 module genes associated with clinical features of patients with SCLC were identified and functional enrichment analysis indicated that all these genes were significantly enriched in cell cycle terms. The area under the receiver operating characteristic curve demonstrated that the overlapping genes of the DE-chr3 and module genes, namely cell division cycle 25 A (CDC25A), FYVE and coiled-coil domain autophagy adaptor 1 (FYCO1) and lipid raft linker 1 (RFTN1), were relatively accurate in distinguishing normal from SCLC samples, and may thus be considered diagnostic biomarkers. CDC25A was overexpressed in SCLC samples, while FYCO1 and RFTN1 were highly expressed in normal samples, as evidenced by the RT-qPCR results. Single-gene gene set enrichment analysis suggested that the diagnostic biomarkers were significantly associated with cell cycle, ATP-binding cassette transporter, immune cell differentiation, immune response and multiple respiratory disease pathways. Furthermore, a total of 141 drugs were predicted by The Comparative Toxicogenomics Database to be able to modulate the expression of the diagnostic biomarkers, of which 8 drugs were shared among the three aforementioned diagnostic biomarkers. The present study identified three novel and powerful diagnostic biomarkers for SCLC based on chr3 genes. Suggestions for the development and selection of drugs for clinical treatment based on diagnostic biomarkers were also provided.

Antagonist G-targeted liposomes for improved delivery of anticancer drugs in small cell lung carcinoma

Ligand-mediated targeted liposomes have the potential to increase therapeutic efficacy of anticancer drugs. This work aimed to evaluate the ability of antagonist G, a peptide targeting agent capable of blocking the action of multiple neuropeptides, to selectivity improve targeting and internalization of liposomal formulations (long circulating liposomes, LCL, and stabilized antisense lipid particles containing ionizable amino lipid, SALP) to H69 and H82 small cell lung carcinoma (SCLC) cell lines. Antagonist G-targeted LCL and SALP were prepared by two different methods (either by direct covalent linkage at activated PEG grafted onto the liposomal surface or by post-insertion of DSPE-PEG-antagonist-G-conjugates into pre-formed liposomes). Association of the liposomal formulations with target SCLC cells was studied by fluorescence microscopy using fluorescence-labelled liposomes and confirmed quantitatively with [3H]-CHE-labelled liposomes. An antisense oligodeoxynucleotide against the overexpressed oncogene c-myc(as(c-myc)) was efficiently loaded into SALP formulations, the encapsulation efficiency decreased due to the inclusion of the targeting ligand. Also, liposome size was affected by as(c-myc) physical chemical properties. The amount of antagonist G linked to the surface of the liposomal formulations was dependent on the coupling method and lipid composition used. Covalent attachment of antagonist G increased liposomes cellular association and internalization via receptor-mediated and clathrin-dependent endocytosis, as assessed in SCLC cell lines. Biodistribution studies in healthy mice revealed a preferential lung accumulation of antagonist G–targeted SALP as compared to the non-targeted counterpart. Lung levels of the former were up to 3-fold higher 24 h after administration, highlighting their potential to be used as delivery vectors for SCLC treatment.

De novo deoxyribonucleotide biosynthesis regulates cell growth and tumor progression in small-cell lung carcinoma

Deoxyribonucleotide biosynthesis from ribonucleotides supports the growth of active cancer cells by producing building blocks for DNA. Although ribonucleotide reductase (RNR) is known to catalyze the rate-limiting step of de novo deoxyribonucleotide triphosphate (dNTP) synthesis, the biological function of the RNR large subunit (RRM1) in small-cell lung carcinoma (SCLC) remains unclear. In this study, we established siRNA-transfected SCLC cell lines to investigate the anticancer effect of silencing RRM1 gene expression. We found that RRM1 is required for the full growth of SCLC cells both in vitro and in vivo. In particular, the deletion of RRM1 induced a DNA damage response in SCLC cells and decreased the number of cells with S phase cell cycle arrest. We also elucidated the overall changes in the metabolic profile of SCLC cells caused by RRM1 deletion. Together, our findings reveal a relationship between the deoxyribonucleotide biosynthesis axis and key metabolic changes in SCLC, which may indicate a possible link between tumor growth and the regulation of deoxyribonucleotide metabolism in SCLC.

Protein neddylation as a therapeutic target in pulmonary and extrapulmonary small cell carcinomas.

Small cell lung carcinoma (SCLC) is among the most lethal of all solid tumor malignancies. In an effort to identify novel therapeutic approaches for this recalcitrant cancer type, we applied genome-scale CRISPR/Cas9 inactivation screens to cell lines that we derived from a murine model of SCLC. SCLC cells were particularly sensitive to the deletion of NEDD8 and other neddylation pathway genes. Genetic suppression or pharmacological inhibition of this pathway using MLN4924 caused cell death not only in mouse SCLC cell lines but also in patient-derived xenograft (PDX) models of pulmonary and extrapulmonary small cell carcinoma treated ex vivo or in vivo. A subset of PDX models were exceptionally sensitive to neddylation inhibition. Neddylation inhibition suppressed expression of major regulators of neuroendocrine cell state such as INSM1 and ASCL1, which a subset of SCLC rely upon for cell proliferation and survival. To identify potential mechanisms of resistance to neddylation inhibition, we performed a genome-scale CRISPR/Cas9 suppressor screen. Deletion of components of the COP9 signalosome strongly mitigated the effects of neddylation inhibition in small cell carcinoma, including the ability of MLN4924 to suppress neuroendocrine transcriptional program expression. This work identifies neddylation as a regulator of neuroendocrine cell state and potential therapeutic target for small cell carcinomas.

A unique small cell lung carcinoma disease progression model shows progressive accumulation of cancer stem cell properties and CD44 as a potential diagnostic marker

ObjectivesCancer stem cells (CSCs) have been implicated in disease progression of aggressive cancers including small cell lung carcinoma (SCLC). Here, we have examined the possible contribution of CSCs to SCLC progression and aggressiveness. Materials and methodsGLC-14, GLC-16 and GLC-19 SCLC cell lines derived from one patient, representing increasing progressive stages of disease were used. CSC marker expressions was determined by RT-qPCR and western blotting analyses, and heterogeneity was studied by CSC marker expression by immunofluorescence microscopy and flow cytometry. Colony formation assays were used to assess stem cell properties and therapy sensitivity. ResultsIncreasing expression of stem cell markers MYC, SOX2 and particularly CD44 were found in association with advancing disease. Single and overlapping expression of these markers indicated the presence of different CSC populations. The accumulation of more homogeneous double- and triple-positive CSC populations evolved with disease progression. Functional characterization of CSC properties affirmed higher proficiency of colony forming ability and increased resistance to γ-irradiation in GLC-16 and GLC-19 compared to GLC-14. GLC-19 colony formation was significantly inhibited by a human anti-CD44 antibody. ConclusionThe progressive increase of MYC, SOX2 and particularly CD44 expression that was accompanied with enhanced colony forming capacity and resistance in the in vitro GLC disease progression model, supports the potential clinical relevance of CSC populations in malignancy and disease relapse of SCLC.

Increased expression of REV7 in small cell lung carcinomas and its association with tumor cell survival and proliferation.

REV7 is involved in multiple biological processes including DNA damage tolerance, cell cycle regulation and gene expression, and is an accessory subunit of the mutation-prone DNA polymerase ζ. It has been reported that REV7 expression is associated with poor prognosis in several human cancers. The aim of this study is to investigate the significance of REV7 in lung carcinogenesis. Immunohistochemical analyses of surgically resected lung cancer specimens revealed that REV7 shows an increased expression in small cell lung carcinomas (SCLCs) when compared with other histological types of lung carcinoma. Association between REV7 expression levels and clinicopathological factors was investigated using SCLC cases with or without surgical resection. Our analyses revealed that high REV7 expression significantly correlated with tumor cell proliferation, assessed by Ki-67 labeling indices, and was negatively associated with distant metastasis and extensive-stage disease. No significant association was detected between REV7 expression and other factors, including prognosis or response to chemoradiotherapy in SCLC. Increase in REV7 expression in SCLC was confirmed using SCLC cell lines. In addition, siRNA-mediated depletion of REV7 activated the apoptotic pathway and suppressed cell growth in SCLC cells. These results suggest that REV7 plays an important role in tumor cell survival and proliferation in SCLC.

DrugSniper, a Tool to Exploit Loss-Of-Function Screens, Identifies CREBBP as a Predictive Biomarker of VOLASERTIB in Small Cell Lung Carcinoma (SCLC).

The development of predictive biomarkers of response to targeted therapies is an unmet clinical need for many antitumoral agents. Recent genome-wide loss-of-function screens, such as RNA interference (RNAi) and CRISPR-Cas9 libraries, are an unprecedented resource to identify novel drug targets, reposition drugs and associate predictive biomarkers in the context of precision oncology. In this work, we have developed and validated a large-scale bioinformatics tool named DrugSniper, which exploits loss-of-function experiments to model the sensitivity of 6237 inhibitors and predict their corresponding biomarkers of sensitivity in 30 tumor types. Applying DrugSniper to small cell lung cancer (SCLC), we identified genes extensively explored in SCLC, such as Aurora kinases or epigenetic agents. Interestingly, the analysis suggested a remarkable vulnerability to polo-like kinase 1 (PLK1) inhibition in CREBBP-mutant SCLC cells. We validated this association in vitro using four mutated and four wild-type SCLC cell lines and two PLK1 inhibitors (Volasertib and BI2536), confirming that the effect of PLK1 inhibitors depended on the mutational status of CREBBP. Besides, DrugSniper was validated in-silico with several known clinically-used treatments, including the sensitivity of Tyrosine Kinase Inhibitors (TKIs) and Vemurafenib to FLT3 and BRAF mutant cells, respectively. These findings show the potential of genome-wide loss-of-function screens to identify new personalized therapeutic hypotheses in SCLC and potentially in other tumors, which is a valuable starting point for further drug development and drug repositioning projects.

Comparative proteogenomics profiling of non-small and small lung carcinoma cell lines using mass spectrometry.

BackgroundEvidences indicated that non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC) might originate from the same cell type, which however ended up to be two different subtypes of lung carcinoma, requiring different therapeutic regimens. We aimed to identify the differences between these two subtypes of lung cancer by using integrated proteome and genome approaches.Methods and MaterialsTwo representative cell lines for each lung cancer subtype were comparatively analysed by quantitative proteomics, and their corresponding transcriptomics data were obtained from the Gene Expression Omnibus database. The integrated analyses of proteogenomic data were performed to determine key differentially expressed proteins that were positively correlated between proteomic and transcriptomic data.ResultThe proteomics analysis revealed 147 differentially expressed proteins between SCLC and NSCLC from a total of 3,970 identified proteins. Combined with available transcriptomics data, we further confirmed 14 differentially expressed proteins including six known and eight new lung cancer related proteins that were positively correlated with their transcriptomics data. These proteins are mainly involved in cell migration, proliferation, and invasion.ConclusionThe proteogenomic data on both NSCLC and SCLC cell lines presented in this manuscript is complementary to existing genomic and proteomic data related to lung cancers and will be crucial for a systems biology-level understanding of the molecular mechanism of lung cancers. The raw mass spectrometry data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD015270.

Significance of achaete-scute complex homologue 1 (ASCL1) in pulmonary neuroendocrine carcinomas; RNA sequence analyses using small cell lung cancer cells and Ascl1-induced pulmonary neuroendocrine carcinoma cells.

ASCL1 is one of the master transcription factors of small cell lung carcinoma (SCLC). To investigate the significance of ASCL1 in pulmonary neuroendocrine carcinoma, we performed 2 comparative RNA-seq studies between H69 (ASCL1-positive, classical type SCLC) and H69AR (ASCL1-negative, variant type SCLC) and between ASCL1-transfected A549 adenocarcinoma cell lines (A549(ASCL1+) cell lines) and A549(control) cell lines. RNA-seq analyses revealed that 940 genes were significantly different between the H69 and H69AR cell lines, and 728 between the A549(ASCL1+) and A549(control) cell lines. In total, 120 common genes between these analyses were selected as candidate ASCL1-related genes, and included genes with various cellular functions, such as neural development, secretion, growth, and morphology. Their expression degrees in three classical and two variant SCLC cell lines, two A549(ASCL1+) and two A549(control) cell lines were subjected to quantitative PCR analyses. Since the candidate ASCL1-related genes were strongly expressed in the classical SCLC and A549(ASCL1+) cell lines and more weakly expressed in the variant SCLC and A549(control) cell lines, the ASCL1-related 7 molecules INSM1, ISL1, SYT4, KCTD16, SEZ6, MS4A8, and COBL were further selected. These molecules suggested diverse functions for A549(ASCL1+): INSM1 and ISL1 are transcription factors associated with neuroendocrine differentiation, while SYT4, KTCD16, and SEZ6 may be related to neurosecretory functions and MS4A8 and COBL to cell growth and morphology. An immunohistochemistry of these seven molecules was performed on lung carcinoma tissues and the xenotransplanted tumors of A549(ASCL1+), and they were preferentially and positively stained in ASCL1-postive tumor tissues.

Comparative analysis of TTF-1 binding DNA regions in small-cell lung cancer and non-small-cell lung cancer.

Thyroid transcription factor‐1 (TTF‐1, encoded by the NKX2‐1 gene) is highly expressed in small‐cell lung carcinoma (SCLC) and lung adenocarcinoma (LADC), but how its functional roles differ between SCLC and LADC remains to be elucidated. Here, we compared the genome‐wide distributions of TTF‐1 binding regions and the transcriptional programs regulated by TTF‐1 between NCI‐H209 (H209), a human SCLC cell line, and NCI‐H441 (H441), a human LADC cell line, using chromatin immunoprecipitation‐sequencing (ChIP‐seq) and RNA‐sequencing (RNA‐seq). TTF‐1 binding regions in H209 and H441 cells differed by 75.0% and E‐box motifs were highly enriched exclusively in the TTF‐1 binding regions of H209 cells. Transcriptome profiling revealed that TTF‐1 is involved in neuroendocrine differentiation in H209 cells. We report that TTF‐1 and achaete‐scute homolog 1 (ASCL1, also known as ASH1, an E‐box binding basic helix–loop–helix transcription factor, and a lineage‐survival oncogene of SCLC) are coexpressed and bound to adjacent sites on target genes expressed in SCLC, and cooperatively regulate transcription. Furthermore, TTF‐1 regulated expression of the Bcl‐2 gene family and showed antiapoptotic function in SCLC. Our findings suggest that TTF‐1 promotes SCLC growth and contributes to neuroendocrine and antiapoptotic gene expression by partly coordinating with ASCL1.

Abstract 2897: Dynamic transdifferentiation programs in small cell lung carcinoma

Abstract Tumors must also have the ability to rapidly adjust to a mutable environment during the lifespan of the cell. This form of phenotypic plasticity is typified by biological programs regulated by a host of transcription factors in a well-coordinated “switch” that is initiated by both cell autonomous and microenvironmental cues. We sought to study these dynamics in small cell lung carcinoma (SCLC). First, we used k-means clustering to identify discrete transcriptional programs that are represented across SCLC using gene expression values from 53 and 71 SCLC cell lines and primary tumors, respectively. To investigate these programs further, we used our inventory of 67 patient-derived xenografts (PDX) to establish an ex vivo cell culture system that reconstitutes with high fidelity the gene expression values observed in the PDXs and the primary tumors from which these cells were derived. Our data demonstrates that gene expression groupings or clusters across samples mark distinct SCLC transcriptional states, representing intertumoral variation. However, in an intriguing demonstration of intratumoral heterogeneity, the very same groupings define morphologically and phenotypically distinct subpopulations within individual samples. Subpopulations were represented by suspending aggregates of very small cells (neuroendocrine or NEn), pre-suspension aggregates that are nested above mesenchymal-like cells and appear to give rise to the suspending aggregates (proneural or PN), pleomorphic cells growing as a monolayer composed of larger cells with visible cytoplasm and spindle-like membrane extensions (mesenchymal or MS), and semi-adherent cells that are physically positioned over the latter (neuroepithelial or NEp). WES demonstrated high genetic fidelity across all subpopulations, indicating that the significant morphologic and phenotypic differences were non-genetic. RNAseq and gene ontology was applied to differentially expressed genes to inform classifications. Using network analyses, we identified transcription factors (TFs) that function as central nodes in the gene expression networks of each population. Namely, we show that ASCL1 marks a rapidly proliferating neuroendocrine population (NEn), NEUROD1 marks a primitive neural population (PN) and YAP1 marks a slow-growing mesenchymal population (MS). We also developed a promoter-reporter fluorescent protein system that positions distinctly colored fluorescent proteins under the regulatory control of the promoters of the three TFs. We show that a Markov process models the transition probability of states. Taken together, our results show that human SCLC is comprised of distinct tumor subpopulations characterized by significant, non-genetic plasticity. We posit that understanding of the SCLC intratumoral ecosystem can create new opportunities to evolutionarily steer this tumor toward improved therapeutic results. Note: This abstract was not presented at the meeting. Citation Format: Priyanka Gopal, Kevin Rogacki, Craig D. Peacock, Mohamed E. Abazeed. Dynamic transdifferentiation programs in small cell lung carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2897.