Classic Small Cell Lung Cancer Research Articles

EP14.02-004 Barasertib Inhibits the Growth of SCLC Cell Lines and Transformed SCLC Patient Derived Model In Vitro and In Viv

Approximately 5% to 10% of EGFR (epidermal growth factor receptor) -mutant non-small cell lung cancers (NSCLCs) develops an acquired resistance to EGFR tyrosine kinase inhibitors with transformation into small-cell lung cancer (SCLC). After the transformation, clinical behavior mimics classic SCLC on many aspects, with frequent but transient responses to platinum-etoposide (EP) or taxanes, modest progression free survival and rare response to immune checkpoint inhibitors. Recently, four SCLC subtypes, defined largely by differential expression of transcription factors ASCL1, NEUROD1, and POU2F3 or low expression of all three transcription factor signatures, were suggested (Gay CM et al, Cancer Cell 2021) (SCLC-A, N, P and I, respectively).

Identification and Characterization of the Molecular Mechanisms of SCLC Chemo-Radiation Resistance

Small cell lung cancer (SCLC) is among the most aggressive form of lung malignancies and accounts for 15-20% of all lung cancers. It has the tendency to metastasize early, thus limited-stage SCLC patients still receive systemic treatment with chemo-radiotherapy (chemoXRT) for their localized disease. SCLC is exceptionally sensitive to chemoXRT and exhibits high response rates; however, the recurrence rate is > 80% and patients relapse with tumors that resist further treatments. Elucidating mechanisms of chemoXRT resistance in SCLC is needed to develop improved therapies and positively impact patient outcomes.To better interrogate mechanisms of chemoXRT resistance, we developed a SCLC patient-derived xenograft (PDX) in vivo system for the major molecular subtypes of SCLC (classic and variant). We also established a novel PDX organoid (PDO) model to study the molecular underpinnings of chemoXRT resistance in SCLC.PDX tumor bearing mice were treated with: 1) vehicle control; 2) cisplatin plus etoposide (EP); 3) radiotherapy (XRT); and 4) both EP/XRT. A major response was observed within the EP/XRT arm compared to vehicle or single therapy arms. Whole transcriptome profiling among all treatment arms revealed molecular pathways and biological processes associated with chemoXRT resistance. By comparing our data with two previous SCLC patient cohort studies, we identified gene candidates for functional validation of chemoXRT resistance (i.e., ST6GAL1, TNIK and SOHLH2). To enable real-time cellular and molecular analysis of PDX behavior ex vivo and to validate SCLC chemoXRT resistance candidate genes, classic and variant SCLC organoids will be used. PDOs can be cultured up to 30 days in medium and parental PDX molecular subtype phenotypes are maintained as shown by comparative genomic hybridization, RNA-seq and protein marker analyses. Additionally, PDOs are capable of forming tumors in vivo.Genetically manipulated SCLC PDOs will be utilized as a tool to identify and validate candidates for chemoXRT resistance to be used as biomarkers and targets to combat chemoXRT resistance in SCLC.

Cell-autonomous immune gene expression is repressed in pulmonary neuroendocrine cells and small cell lung cancer

Small cell lung cancer (SCLC) is classified as a high-grade neuroendocrine (NE) tumor, but a subset of SCLC has been termed “variant” due to the loss of NE characteristics. In this study, we computed NE scores for patient-derived SCLC cell lines and xenografts, as well as human tumors. We aligned NE properties with transcription factor-defined molecular subtypes. Then we investigated the different immune phenotypes associated with high and low NE scores. We found repression of immune response genes as a shared feature between classic SCLC and pulmonary neuroendocrine cells of the healthy lung. With loss of NE fate, variant SCLC tumors regain cell-autonomous immune gene expression and exhibit higher tumor-immune interactions. Pan-cancer analysis revealed this NE lineage-specific immune phenotype in other cancers. Additionally, we observed MHC I re-expression in SCLC upon development of chemoresistance. These findings may help guide the design of treatment regimens in SCLC.

Abstract 2699: Targeting an ubiquitin-activating enzyme in small-cell lung cancer (SCLC)

Abstract Background: Despite valiant efforts to improve patient outcomes, small cell lung cancer (SCLC) (~15-17% of lung cancer) remains an aggressive lung cancer, with an overall five-year survival of 7%. Current first-line therapy (chemotherapy+/-radiation) has not changed in 30+ years. TAK-243, an inhibitor of the ubiquitin activating enzyme (UAE), E1 (encoded by UBA1), poses a novel approach for cancer therapy. TAK-243 limits the formation of ubiquitin conjugates that ultimately mediate many cellular processes, including DNA repair signalling and protein degradation. Consequently, TAK-243 may disrupt cancer-specific dependencies of UAE to induce malignant cell death, and potentiate radiation-induced DNA damage. The discovery publication revealed that TAK-243 is especially potent in 2 SCLC cell-lines compared to healthy breast and fibroblastic tissues. With these limited data, the potential benefit of this novel agent as monotherapy and in combination with chemotherapy for SCLC has yet to be elucidated. Methods: We assessed the anti-proliferative effect of TAK-243 monotherapy in a large panel (n = 10) of variant and classical SCLC cell-lines, including assorted MYC family member amplifications and different UBA1 mRNA expression levels. TAK-243 EC50 single agent doses were determined by treating cell-lines with incremental concentrations of TAK-243 (0-1μM). For combinatorial analysis, TAK-243 (0-1μM) was administered to cell-lines alongside cisplatin/etoposide (CE) at cell-line specific optimized doses yielding 30-35% kill by CE. Short-term efficacy was determined by a cell viability resazurin assay, 48 hours after treatment initiation. ΔEC50 was measured as the single agent EC50 subtracted by the EC50 of TAK-243+CE. Positive ΔEC50 values show potential synergy between TAK-243 and CE. Results: Single-agent therapy. SCLC cell-lines demonstrated a range of sensitives to TAK-243 monotherapy after 48 hours. The cell-lines tested are ranked from highest to lowest sensitivity: NCI-H82, NCI-H69, NCI-H1618, LX22 (ex-vivo cultured PDX), NCI-H1092, NCI-H446, SHP77 and NCI-H196; with EC50 values ranging from 18nM to 80nM. However, NCI-H526, NCI-H889, and SBC-5 were non-responsive to therapy at 1μM. Combination therapy. TAK-243 upon combination treatment with CE revealed ΔEC50 values ranging from 5.23-14.54nM. NCI-H82, NCI-H69, SHP77 and NCI-H1092 demonstrated potential chemosensitivity to combination treatment. Conclusions: These data begin to hint at synergy between TAK-243 and CE, where lower doses of TAK-243 may be required for certain SCLC cell-lines when used in combination. These preliminary results suggest that TAK-243 may have the potential to improve the current SCLC standard therapies in molecular defined subgroups. These results are being confirmed in additional cell-lines and through in vivo experiments using patient-derived xenograft models of SCLC. *S.M and M.K.A contributed equally to this work Citation Format: Safa Majeed, Mansi K. Aparnathi, Lifang Song, Aaron D. Schimmer, Ming S. Tsao, Geoffrey Liu, Benjamin H. Lok. Targeting an ubiquitin-activating enzyme in small-cell lung cancer (SCLC) [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 2699.

Oncolytic virotherapy for small-cell lung cancer induces immune infiltration and prolongs survival.

Oncolytic virotherapy has been proposed as an ablative and immunostimulatory treatment strategy for solid tumors that are resistant to immunotherapy alone; however, there is a need to optimize host immune activation using preclinical immunocompetent models in previously untested common adult tumors. We studied a modified oncolytic myxoma virus (MYXV) that shows high efficiency for tumor-specific cytotoxicity in small-cell lung cancer (SCLC), a neuroendocrine carcinoma with high mortality and modest response rates to immune checkpoint inhibitors. Using an immunocompetent SCLC mouse model, we demonstrated the safety of intrapulmonary MYXV delivery with efficient tumor-specific viral replication and cytotoxicity associated with induction of immune cell infiltration. We observed increased SCLC survival following intrapulmonary MYXV that was enhanced by combined low-dose cisplatin. We also tested intratumoral MYXV delivery and observed immune cell infiltration associated with tumor necrosis and growth inhibition in syngeneic murine allograft tumors. Freshly collected primary human SCLC tumor cells were permissive to MYXV and intratumoral delivery into patient-derived xenografts resulted in extensive tumor necrosis. We confirmed MYXV cytotoxicity in classic and variant SCLC subtypes as well as cisplatin-resistant cells. Data from 26 SCLC human patients showed negligible immune cell infiltration, supporting testing MYXV as an ablative and immune-enhancing therapy.

Abstract 955: Combined inhibition of MEK and mTOR pathways is effective in NRAS Q61K mutant small cell lung cancer

Abstract Background: Small cell lung cancer (SCLC) is a heterogeneous disease, consisting of a phenotypically diverse population of cells, often described as neuroendocrine (NE) and non-NE. Though previous studies suggest that aberrant RAS signaling is involved in the transition from NE to non-NE profile, the characteristics of SCLC with RAS mutations have not been fully examined due to the rarity of the occurrence and the lack of resources available to study its biology. Methods: We created a cell line (DFCI168) from the pleural effusion of the SCLC patient with a rare NRASQ61K mutation. A patient-derived xenograft (PDX) model from this patient was successfully established by subcutaneous implantation of cancer cells in NSG mice. The efficacy of MEK and mTOR inhibitors was evaluated in DFCI168 and the commercially available lung cancer cell lines with NRAS Q61 mutations (SW1271, H1299, H2087, H2347). The Chou-Talalay combination index was used to evaluate drug synergy. Retroviral NRASQ61K transduction was performed on a classical SCLC cell line Glc16 to examine whether NRAS activation leads to acquisition of non-NE features. Results: DFCI168 showed non-NE/mesenchymal features both morphologically and biologically, which is exemplified by an elongated spindle shape, mesenchymal marker (VIM) expression and silenced NE markers (SYP, ASCL1) expression. NRASQ61K transduction on Glc16 achieved the transition from NE to non-NE/mesenchymal phenotype. Although DFCI168 showed marked sensitivity to MEK inhibitors both in vitro (trametinib IC50=2nmol/L, selumetinib IC50=50nmol/L) and in vivo, the tumors regrew after withdrawal of treatment with trametinib in xenograft mice, and caused sustained activation of the mTOR pathway during MEK inhibitor treatment in vitro. The combination of MEK and mTOR inhibitors (trametinib and TORIN2) prevented mTOR activation at 100nmol/L and yielded synergistic effects (CI=0.61). The commercially available lung cancer cell lines (SW1271, H1299, H2087, H2347) also responded synergistically to the trametinib/TORIN2 combination except H1299, a cell line with a known PTEN promoter methylation. Conclusion: We demonstrate that the oncogenic NRAS mutation defines a distinct molecular subset of SCLC, and is sufficient to cause the phenotypic switch from NE to non-NE/mesenchymal profile. Such tumors are likely to benefit from the dual targeted therapy with MEK and mTOR inhibitors. Our findings highlight the future potential of targeted therapies for SCLC patients with NRAS activating mutations. Citation Format: Atsuko Ogino, Mika Lin, Prafulla C. Gokhale, Margaret K. Wilkens, Jihyun Choi, Antonio Calles, Man Xu, Marzia Capelletti, Geoffrey Oxnard, Nathanael S. Gray, Paul Kirschmeier, Pasi A. Jänne. Combined inhibition of MEK and mTOR pathways is effective in NRAS Q61K mutant small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 955.

Capsaicin synergizes with camptothecin to induce increased apoptosis in human small cell lung cancers via the calpain pathway

Small cell lung cancer (SCLC) is characterized by excellent initial response to chemotherapy and radiation therapy with a majority of the patients showing tumor shrinkage and even remission. However, the challenge with SCLC therapy is that patients inevitably relapse and subsequently do not respond to the first line treatment. Recent clinical studies have investigated the possibility of camptothecin-based combination therapy as first line treatment for SCLC patients. Conventionally, camptothecin is used for recurrent SCLC and has poor survival outcomes. Therefore, drugs which can improve the therapeutic index of camptothecin should be valuable for SCLC therapy. Extensive evidence shows that nutritional compounds like capsaicin (the spicy compound of chili peppers) can improve the anti-cancer activity of chemotherapeutic drugs in both cell lines and animal models. Statistical analysis shows that capsaicin synergizes with camptothecin to enhance apoptosis of human SCLC cells. The synergistic activity of camptothecin and capsaicin is observed in both classical and variant SCLC cell lines and, in vivo, in human SCLC tumors xenotransplanted on chicken chorioallantoic membrane (CAM) models. The synergistic activity of capsaicin and camptothecin are mediated by elevation of intracellular calcium and the calpain pathway. Our data foster hope for novel nutrition based combination therapies in SCLC.

P1.06-005 An International Cohort of Patients with Small Cell Lung Cancer after a Non-Small Cell Lung Carcinoma Oncogene or Non-Oncogene Addicted

Phenotypic transformation from Non-small cell lung cancer (NSCLC) to small cell lung cancer (SCLC) is a resistance mechanism in tyrosine kinase inhibitors (TKIs) treated EGFR mutant tumors. SCLC is also however less frequently diagnosed in patients without EGFR mutations treated with chemotherapy. These transformations are rare and little is known about the clinical and therapeutic characteristics of these patients. In this study we describe and compare the characteristics of SCLC arising from mutant or non-mutant NSCLC. We performed a multicentric retrospective collection (27 centers in France and Italy) of cases. Between 2005 and 2015, patients with stage III or IV NSCLC with a secondary transformation to SCLC with histological proof were included. Forty seven cases of SCLC transformation were collected, 34 in EGFR mutant and 13 in non. Most of the patients (n=37, 82%) were stage IV, (n=27, 57%) female and (n=26, 76%) had an exon 19 mutation. The last treatment before transformation was a TKI in 23 (68%) cases in the mutant group and in 3 (23%) patients (erlotinib) in the non-mutant. Median time to SCLC transformation was 17 [IQR, 11-29] months in the mutant group and 26 [IQR 23-36] months in the other (p=0.03). Molecular analyses were not performed in the non-mutant group, 25 (74%) had molecular analyses in the EGFR mutant. A driver mutation was identified in 22/25 (88%) patients: in most of the cases the same as the initial, 1 case of ALK fusion and 1 of PI3K mutation. Thirty patients (88%) received at least one line of treatment after transformation in the mutant group, in all cases a platinum-etoposide (P-E) chemotherapy. Median survival from initial diagnosis in the EGFR mutant group was significantly worse 28 [17-41] months vs 49 [36-118] months in the non EGFR mutant group (p=0.01). After transformation, the same tendency was observed with a median survival of 8 [3-12] months for the EGFR mutated patients vs 13 [6-15] months for the non EGFR mutated patients (p=0.06). SCLC that occurs in EGFR mutant treated by TKIs is more aggressive than classic SCLC, and differs on epidemiological characteristics. These transformed SCLC are not fully explained and we need to define the molecular characteristics of this cohort, before and after transformation and if funded the whole genome sequencing of the tumors to understand this TKIs mechanism of resistant.

Loss of somatostatin receptor 2 expression reduces small cell lung cancer growth and alters cellular metabolism

Small cell lung cancer (SCLC) is a high grade poorly differentiated neuroendocrine carcinoma of the lung responsible for w15% of diagnosed lung cancers and up to 25% of lung cancer deaths. Treatment paradigms in small cell lung cancer have not changed significantly in the last 20 years. Advances in targeted therapies for SCLC are sorely needed. Somatostatin receptors (SSTR) are neuroendocrine associated G protein-coupled receptors associated with multiple tumor types and which have effects on cell cycling, angiogenesis, apoptosis, and growth factors. They canonically signal by inhibition of adenylate cyclase, calcium influx, and act through downstream MAPK and Akt as well as other downstream kinases. We evaluated SSTR2 expression by IHC staining and western blotting of multiple cell lines and tumor tissues and found high expression in multiple neuroendocrine lung carcinomas including classical and variant SCLC lines. Given the high level of SSTR2 expression found in most SCLC lines, we hypothesized that this signaling pathway stimulates growth and survival of these tumor cells. We followed these preliminary studies with an assessment of 98 SCLC patients whose tumor IHC was subdivided into high or low SSTR2 expressing tumors by continuous IHC scoring. Low SSTR2 tumors had a better prognosis with a median survival in limited stage disease of 36 months compared to 12 months in the SSTR2 high expressing SCLCs suggesting that SSTR2 expression has clinical relevance in SCLC progression. The hazard ratio was 0.45 with a p<0.05. This led to further experiment to interrogate the mechanism and function of SSTR2 in SCLC. We established multiple stable SSTR2 shRNA knockdown lines including bronchial carcinoid, and multiple adherent SCLC lines. SSTR2 knockdown led to up to 3 fold changes in cell viability in vitro with reduced proliferation in multiple cell culture lines and constructs as well as apoptosis in H1048 cells. Metabolic testing with Seahorse mito stress kits concurred with significant differences in mitochondrial respiration and metabolic activity in the SSTR2 knockdown cell lines with a slightly increased basal metabolic rate and a significant greater than 75% increase in maximal respiration compared to the control scrambled construct cell line. We are actively pursuing metabolic studies in SCLC and will expand this work to evaluate ADP/ATP and AMP/ATP ratios to determine if aberrant energy homeostasis may contribute to the observed cell death in this metabolically active cell population. Collectively, this data brings new interest to SSTR2 signaling as a pathway target for therapy in a subset of SCLC and suggests potential metabolic targets in SCLC.

RB loss in resistant EGFR mutant lung adenocarcinomas that transform to small-cell lung cancer.

Tyrosine kinase inhibitors are effective treatments for non-small-cell lung cancers (NSCLCs) with epidermal growth factor receptor (EGFR) mutations. However, relapse typically occurs after an average of 1 year of continuous treatment. A fundamental histological transformation from NSCLC to small-cell lung cancer (SCLC) is observed in a subset of the resistant cancers, but the molecular changes associated with this transformation remain unknown. Analysis of tumour samples and cell lines derived from resistant EGFR mutant patients revealed that Retinoblastoma (RB) is lost in 100% of these SCLC transformed cases, but rarely in those that remain NSCLC. Further, increased neuroendocrine marker and decreased EGFR expression as well as greater sensitivity to BCL2 family inhibition are observed in resistant SCLC transformed cancers compared with resistant NSCLCs. Together, these findings suggest that this subset of resistant cancers ultimately adopt many of the molecular and phenotypic characteristics of classical SCLC.

Selective Tropism of Seneca Valley Virus for Variant Subtype Small Cell Lung Cancer

We assessed the efficacy of Seneca Valley virus (SVV-001), a neuroendocrine cancer-selective oncolytic picornavirus, in primary heterotransplant mouse models of small cell lung cancer (SCLC), including three lines each of classic and variant SCLC. Half-maximal effective concentrations for cell lines derived from three variant heterotransplants ranged from 1.6×10(-3) (95% confidence interval [CI] = 1×10(-3) to 2.5×10(-3)) to 3.9×10(-3) (95% CI = 2.8×10(-3) to 5.5×10(-3)). Sustained tumor growth inhibition in vivo was only observed in variant lines (two-sided Student t test, P < .005 for each). Doses of 10(14) vp/kg were able to completely and durably eradicate tumors in a variant SCLC heterotransplant model in two of six mice. Gene expression profiling revealed that permissive lines are typified by lower expression of the early neurogenic transcription factor ASCL1 and, conversely, by higher expression of the late neurogenic transcription factor NEUROD1. This classifier demonstrates a sensitivity of .89, specificity of .92, and accuracy of .91. The NEUROD1 to ASCL1 ratio may serve as a predictive biomarker of SVV-001 efficacy.

Outcomes in recurrent small-cell lung cancer after one to four chemotherapy lines: A retrospective study of 300 patients

Standard treatment of small-cell lung cancer (SCLC) is a combination of etoposide and platinum for patients with extensive disease, associated with radiotherapy for patients with limited disease (LD). Therapeutic strategies for relapse, although well characterized, are disappointing. Between 1997 and 2009, 300 patients were treated for SCLC at Grenoble University Hospital. We analyzed patients’ characteristics and outcomes at different treatment steps, to determine prognostic factors and propose subsequent treatment strategies according to “sensitive”, “resistant” or “refractory” status established after first-line treatment (L1). The median patient age was 63.2 years, 46.3% had LD, and 23% were female. The objective response rate (ORR) to first-line chemotherapy was 73% [CI95%: 67.6–77.9] and median survival was 13 months. After L1, comparison between “refractory” and “sensitive” groups showed more extensive disease (76.6% vs. 34.3%, p=0.003), poorer Performance Status (PS 0–1: 48.4% vs. 67.8%, p=0.008), more endocrine paraneoplastic syndrome (18.7% vs. 8.4%, p=0.03) and more composite histology (17.2% vs. 4.9%, p=0.004) in “refractory” patients. After second line (L2), ORR was 55.8% [CI95%: 45.2–66.0] in “sensitive”, 18.2% [CI95%: 8.2–32.7] in “resistant”, and 14.7% [CI95%: 4.9–31.0] in “refractory” groups; with partial response only for the last two groups. After L3 and L4, ORR was 24.0% [CI95%: 14.9–35.2] in “sensitive”, 9.1% [CI95%: 11.2–29.2] in “resistant” with partial response only. No response was observed for “refractory”. After L1, the median survival was respectively 23, 10 and 6.4 months for “sensitive”, “resistant” and “refractory” groups (p<0.001). Multivariate analysis showed that LD and classical SCLC histology were positive predictors of belonging to the “sensitive” group. Positive factors for survival were sensitivity to L1, PS 0–1, LD, Charlson score <4, no endocrine paraneoplastic syndrome and no occupational exposure. Limited disease is the major predictive factor for sensitivity to treatments and survival. Factors linked to the patients’ clinical presentation also impact on survival. With currently recommended drugs, the “sensitivity” of the patient determined by the response to L1 indicates that it is pointless to treat “sensitive” with L4, “resistant” with L3 and “refractory” with L2, except for a few selected patients after multidisciplinary group discussion.

Abstract 3355: Comparison of CD133, uPAR, and CXCR4 expression in lung cancer cell lines under normal and spheroid culture conditions

Abstract Background: Cancer stem cells (CSC) have been identified in several solid tumors including breast, brain, and recently lung cancers, etc. In the CSC model, CSCs are thought to be able to self-renew extensively, grow as spheroids in vitro, and be resistant to conventional cancer therapies. CD133, uPAR and CXCR4 have been reported as putative markers that can be used to mark enriched cancer stem cell populations. Although CD133, uPAR and CXCR4 have been implicated in CSC biology and identification, their application as lung CSC (LCSC) markers have not been fully explored. In this study we examined and compared CD133, uPAR and CXCR4 expression in non-spheroid cells and CSC-like spheroid cells derived from several lung and non-lung cancer cell lines. Methods: We cultured four classic small cell lung cancer (SCLC) lines, two variant SCLC lines, three non-small cell lung cancer (NSCLC) lines, one primary lung cancer line derived in our lab, three lymphoma cell lines, one thymoma cell line, and one breast cancer cell line. Cell lines were maintained in RPMI or DMEM with 10% fetal calf serum. Spheroids were cultured and maintained in low-attachment flasks in serum free media supplemented with EGF and FGF. CD133, CXCR4, and uPAR expression were evaluated by immunostaining followed by flow cytometry analysis. Results: Under spheroid culture conditions, CD133 expression tends to remain the same or increases in most of the cell lines tested. SCLC and lymphoma lines tend to express more CD133 than NSCLC and non-lung cancer lines in both normal and spheroid culture conditions. uPAR expression is generally low, however when detectable, it only shows up in cell lines grown in spheroid culture conditions. CXCR4 expression generally increases in spheroid culture conditions with greater expression seen in SCLC lines and lymphoma lines. Conclusion: Spheroid cells tend to exhibit higher CD133 expression than cells under regular culture conditions. This finding supports the idea that CD133 is enriched in LCSC populations. SCLC and lymphoma lines in normal and spheroid culture conditions express more CD133, uPAR, and CXCR4. This does not appear to be tied to adherent growth properties as two adherent SCLC lines displayed no striking difference in expression compared with other non-adherent SCLC lines. However the tendency for SCLC cells and lymphoma cells to grow in regular culture conditions as clumps may offer some insight into the increased expression of these markers. 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 3355.

Investigation of peptomimetics of GD2 antibodies as targeted therapy against human small cell lung cancer in-vitro

13128 Background: GD2 is a well-established target that has been validated for neuroblastoma and small cell lung cancer. The therapeutic and diagnostic use of monoclonal antibodies directed to GD2 in small cell lung cancer is well documented. It has been shown that the binding of GD2 monoclonal antibodies alone can induce growth suppression and cell death of small cell lung cancer cells in-vitro. Our laboratory has developed synthetic small molecule peptomimetics as ligands of GD2. Peptomimetics have favorable in-vivo pharmacological properties compared to antibodies with no immunogenicity, longer half-lives, low toxicity, good tissue penetration, biodistribution and high target selectivity. This study proposed to determine the efficacy of peptomimetics of GD2 antibodies against small cell lung cancer cells in-vitro. Methods: 2 human cell lines were studied. H69 is a classic small cell lung cancer and H82 is a morphological variant small cell lung cancer both of which have been reported in the literature to express GD2. Cell surface expression of ganglioside GD2 was analyzed by flow cytometry (FACScan, BD Biosciences) using GD2 mAB 3F8 and GD2 mAB ME361. Cell proliferation was assessed using standard MTT assays with serum containing medium and cultured for approximately 3 doubling times for each cell line. The cell lines were exposed to increasing doses of GD2 specific peptomimetic to a maximum of 25 uM with controls including serum containing media with and without a GD2 negative peptomimetic and assessed for cell proliferation. Results: GD2 expression was confirmed for both cell lines- H69 and H82 using FACs. Exposure of the GD2 specific peptomimetic clearly caused growth suppression on the range of 35–40% when compared to controls. A dose response relationship was demonstrated with a plateau beyond 10 uM concentrations. Each experiment repeated ≥ 3 occasions. Conclusions: We have shown that attachment of GD2 specific peptomimetics can cause decreased cell proliferation in 2 small cell lung cancer cell lines H69 and H82. We have shown that there is a dose response relationship by which these compounds reduce cell viability. Peptomimetics of GD2 antibodies show promise as a targeted therapy for small cell lung cancer in-vitro and warrant further study. [Table: see text]

Antagonist G-mediated targeting and cytotoxicity of liposomal doxorubicin in NCI-H82 variant small cell lung cancer.

The aim of the present study was to characterize the interactions of antagonist G (H-Arg-D-Trp-N(me)Phe-D-Trp-Leu-Met-NH 2)-targeted sterically stabilized liposomes with the human variant small cell lung cancer (SCLC) H82 cell line and to evaluate the antiproliferative activity of encapsulated doxorubicin against this cell line. Variant SCLC tumors are known to be more resistant to chemotherapy than classic SCLC tumors. The cellular association of antagonist G-targeted (radiolabeled) liposomes was 20-30-fold higher than that of non-targeted liposomes. Our data suggest that a maximum of 12,000 antagonist G-targeted liposomes were internalized/cell during 1-h incubation at 37 masculine C. Confocal microscopy experiments using pyranine-containing liposomes further confirmed that receptor-mediated endocytosis occurred, specifically in the case of targeted liposomes. In any of the previously mentioned experiments, the binding and endocytosis of non-targeted liposomes have revealed to be negligible. The improved cellular association of antagonist G-targeted liposomes, relative to non-targeted liposomes, resulted in an enhanced nuclear delivery (evaluated by fluorimetry) and cytotoxicity of encapsulated doxorubicin for incubation periods as short as 2 h. For an incubation of 2 h, we report IC50 values for targeted and non-targeted liposomes containing doxorubicin of 5.7 +/- 3.7 and higher than 200 micro M doxorubicin, respectively. Based on the present data, we may infer that receptors for antagonist G were present in H82 tumor cells and could mediate the internalization of antagonist G-targeted liposomes and the intracellular delivery of their content. Antagonist G covalently coupled to liposomal drugs may be promising for the treatment of this aggressive and highly heterogeneous disease.

In vitro chemo- and radio-resistance in small cell lung cancer correlates with cell adhesion and constitutive activation of AKT and MAP kinase pathways.

Most small cell lung cancer (SCLC) patients relapse within 12 months of starting combination chemotherapy plus radio-therapy, due to the development of acquired chemo- and radio-resistance. This phenomenon relates to the induction of tumour differentiation, resulting in apoptosis-resistant, morphologically variant (v-SCLC) cells, which lack the neuroendocrine expression of classic (c-) SCLC cells. In this study spontaneously adherent SCLC sublines were shown by differential gene expression analysis to provide an in vitro model of variant differentiation in SCLC, with down-regulation of neuroendocrine markers and up-regulation of epithelial differentiation markers cyclin D1, endothelin, the cell adhesion molecules CD 44 and integrin subunits alpha2, beta3 and beta4. The sensitivity of adherent SCLC sublines to etoposide, cyclophosphamide and gamma radiation was significantly diminished relative to parent suspension cell lines. Western blot analysis using phosphorylation-specific antibodies to Akt and MAP kinase revealed markedly elevated activation in adherent SCLC sublines, paralleled by increased levels of phosphorylated Bad protein and activated NF-kappaB. Subcultivation of the adherent sublines on uncoated surfaces reversed their adherent phenotype immediately and under these conditions Akt activity reverted to low levels. These results suggest that c-SCLC cells can differentiate spontaneously to v-SCLC and that the associated cellular adhesion may trigger Akt-dependent inhibition of apoptosis in SCLC cells, thus leading to acquired chemo- and radio-resistance.

A growth factor antagonist as a targeting agent for sterically stabilized liposomes in human small cell lung cancer

The ability of a growth factor antagonist, [ d-Arg 6, d-Trp 7,9- N mePhe 8]-substance P(6–11), named antagonist G, to selectively target polyethylene glycol-grafted liposomes (known as sterically stabilized liposomes) to a human classical small cell lung cancer (SCLC) cell line, H69, was examined. Our results showed that radiolabeled antagonist G-targeted sterically stabilized liposomes (SLG) bound to H69 cells with higher avidity than free antagonist G and were internalized (reaching a maximum of 13 000 SLG/cell), mainly through a receptor-mediated process, likely involving clathrin-coated pits. This interaction was confirmed by confocal microscopy to be peptide- and cell-specific. Moreover, it was shown that SLG significantly improved the nuclear delivery of encapsulated doxorubicin to the target cells, increasing the cytotoxic activity of the drug over non-targeted liposomes. In mice, [ 125I]tyraminylinulin-containing SLG were long circulating, with a half-life of 13 h. Use of peptides like antagonist G to promote binding and internalization of sterically stabilized liposomes, with their accompanying drug loads, i.e., anticancer drugs, genes or antisense oligonucleotides, into target cells has the potential to improve therapy of SCLC.

Transition from suspension to adherent growth is accompanied by tissue factor expression and matrix metalloproteinase secretion in a small cell lung cancer cell line.

Small cell lung cancer (SCLC) is a very malignant tumor known to grow aggressively and to metastasize early. It is well established that metastasis generally involves both tumor cell adhesion and proteolytic degradation of the extracellular matrix. However, SCLC cells cultured in vitro, such as the classic SCLC cell line NCI-H69, grow in floating aggregates and express only negligible proteolytic activity. In this report, we show that NCI-H69 cells can be selected for adherent growth. In contrast to parental suspension cells, the adherent cells were found to express tissue factor as well as gelatinolytic activity, attributable to matrix metalloproteinases 2 and 9. Such a switch of tumor cell characteristics, if it could occur in SCLC patients, might add to the understanding of the steps involved in the spreading of this highly metastatic type of lung cancer.

Suppression subtractive hybridization to identify gene expressions in variant and classic small cell lung cancer cell lines.

Small Cell Lung Cancer (SCLC), a clinically aggressive cancer, accounts for approximately 25% of primary lung cancers. We carried out suppression subtractive hybridization (SSH), a PCR-based method for cDNA subtraction, between the human classic, NCI-H69 and variant, more aggressive NCI-N417 SCLC cell lines to isolate and characterize variable expression of genes, which may be responsible for differential degree of tumorigenicity of SCLC. Using NCI-N417 as a tester, we obtained 28 differentially expressed cDNA clones from a total of 60 arbitrarily picked clones. Among the 28 cDNA clones, 4 were unknown genes, 2 were fatty acid binding protein (FABP) with specific identification of mRNA for mammary-derived growth inhibitor (MDGI), 1 was human alpha-enolase, 4 were ribosomal proteins, 2 were structural genes, vimentin and moesin (membrane-organizing extension spike protein), and 9 were homologous with murine leukemia viruses, whereas 2 others had enhanced expression in NCI-H69 and A549 cell lines, and 4 were cell surface proteins and murine type C retrovirus. Expression of FABP/MDGI was significantly high in NCI-H417, which may influence mitosis and cell growth as implicated in other tissues, contrary to the conclusion drawn for the role of MDGI in human breast cancer. Higher expression of ribosomal proteins in NCI-N417 compared to NCI-H69 may have a role in differential tumorigenicity and metastatic ability. Further, we obtained 14 differentially expressed cDNA clones by reversing the tester and driver, using NCI-H69 as a tester. Of these 14 differential cDNAs, 5 were unknown genes, 2 were specific for keratins, others had similarities with protease inhibitor, human BAC clone, Alu RNA binding protein, and tumor expression-enhanced gene. Characterization of these differentially expressed cDNA clones will provide useful information in understanding of the genes responsible for differential tumorigenicity of SCLC.

Spectrum of neuroendocrine differentiation in lung cancer cell lines featured by cytomorphology, markers, and their corresponding tumors.

Lung cancer cell lines which show features of neuroendocrine (NE) differentiation can be divided into 4 types which have distinct clinicopathologic correlates: classic small cell lung cancer (SCLC), variant SCLC, pulmonary carcinoid, and non-small cell lung cancer with NE features (NSCLC-NE). These cell lines form a spectrum regarding their degree of NE differentiation which ranges from high levels seen in carcinoid cell lines to very low which is typical of the variant SCLC. A careful comparison of the properties of tumors and their cell lines and correlating these data with the clinical history of the patient has markedly enhanced the relevance of cell lines as models for NE biology and lung carcinogenesis.