Small Cell Lung Carcinoma Cells 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.

Efficacy of endobronchial ultrasound-guided transbronchial needle aspiration in the diagnosis of mediastinal and hilar lesions.

Mediastinal and hilar lesions may be benign or malignant. Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is increasingly used for the diagnosis of these lesions as it is both minimally invasive and safe. To investigate the clinical efficacy of EBUS-TBNA in the diagnosis and differential diagnosis of mediastinal and hilar lesions. A retrospective observational study was undertaken to investigate patients diagnosed with mediastinal and hilar lymphadenopathy based on imaging at our hospital from 2020 to 2021. After evaluation, EBUS TBNA was used and data including the puncture site, postoperative pathology, and complications were recorded. Data from 137 patients were included in the study, of which 135 underwent successful EBUS TBNA. A total of 149 lymph node punctures were performed, of which 90 punctures identified malignant lesions. The most common malignancies were small-cell lung carcinoma, adenocarcinoma, and squamous cell carcinoma. Forty-one benign lesions were identified, resulting from sarcoidosis, tuberculosis, and reactive lymphadenitis, amongst others. Follow-up findings showed that 4 cases were malignant tumors, with 1 case of pulmonary tuberculosis and 1 case of sarcoidosis). Four specimens where lymph node puncture was insufficient were subsequently confirmed by other means. The sensitivity of EBUS TBNA for malignant lesions, tuberculosis and sarcoidosis in mediastinal and hilar lesions was 94.7%, 71.4%, and 93.3%, respectively. Similarly, the negative predictive values (NPV) were 88.9%, 98.5%, and 99.2%, and the accuracy was 96.3%, 98.5%, and 99.3%. EBUS TBNA is an effective and feasible approach for the diagnosis of mediastinal and hilar lesions that is minimally invasive and safe.

Phase 1 first-in-human study of PF-07257876, a novel CD47/PD-L1 bispecific checkpoint inhibitor, in patients with PD-1/PD-L1-refractory and -naïve advanced solid tumors.

2529 Background: Tumor expression of the antiphagocytic protein CD47 (integrin associated protein) may lead to immunotherapy resistance (IO-R). PF-07257876 is a novel, IgG1, Fc-active, bispecific antibody targeting CD47 and PD-L1 designed to reverse IO-R, decrease anemia risk, and eliminate a priming dose. We report a Phase 1 dose escalation study (NCT04881045) in patients (pts) with advanced solid tumors, both IO-refractory and IO-naïve. Methods: The study enrolled pts ≥18 yrs with advanced non–small-cell lung carcinoma (NSCLC) and squamous cell carcinoma of the head and neck (SCCHN) with PD-L1 expression >1% post anti-PD-1/PD-L1 therapy. Pts with other PD-L1+ tumors known to express CD47 were included (prior IO not required). Pts received PF-07257876 at escalating dose levels in 28-day cycles. Primary objectives were safety (dose-limiting toxicity [DLT] rate), tolerability, and determination of Recommended Phase 2 Dose (RP2D). Secondary and exploratory objectives included pharmacokinetics (PK), blood receptor occupancy (RO), CD47 levels by immunohistochemistry (IHC), tissue biopsy findings, and objective response rate (ORR) per RECIST v.1.1. Results: As of 20OCT2022, 18 pts were enrolled (NSCLC: n = 6; SCCHN: n = 4; other: n = 8). Median (range) age 68 (46–79) yrs. PF-07257876 was given at ascending doses, 150-2400 mg by subcutaneous (SC) injection or SC infusion every 2 weeks. 16 (89%) pts had treatment-related adverse events (TRAEs): Grades 1/2: n = 12 (67%); Grade 3: n = 1 (6%); Grade 4: n = 3 (17%); no Grade 5. Most common TRAEs: injection site reaction/erythema (n = 13, 72%), thrombocytopenia (n = 6, 33%), anemia (n = 5, 28%), and nausea (n = 4, 22%). DLTs occurred in 2 of 3 pts at the highest tested dose (Grade 4 neutropenia and thrombocytopenia, n = 1; Grade 4 thrombocytopenia, n = 1; both pts recovered). One pt with IO-R SCCHN had a confirmed partial response (duration of response 16 wks). ORR was 5.6% (1/18). Drug exposure increase after 1st dose was not dose proportional. Blood RO was > 95% for PD-L1 and > 70% for CD47 with drug exposure. All pts’ baseline CD47 IHC levels were moderate to high. Pharmacodynamics cytokine profiling showed notable dose-response increases of IFNg, TNFa, CXCL9/10, CCL3/4, without IL-6 increase. Conclusions: PF-07257876 was well tolerated with manageable toxicity. Incidence and grade of anemia or thrombocytopenia were manageable. Target engagement of CD47 and PD-L1 in peripheral blood was high. Innate and adaptive cytokine modulation was observed. Antitumor activity was modest. Expansion is ongoing at 1800 mg. Clinical trial information: NCT04881045 .

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.

Novel Silver Complexes Based on Phosphanes and Ester Derivatives of Bis(pyrazol-1-yl)acetate Ligands Targeting TrxR: New Promising Chemotherapeutic Tools Relevant to SCLC Management

Bis(pyrazol-1-yl)acetic acid (HC(pz)2COOH) and bis(3,5-dimethyl-pyrazol-1-yl)acetic acid (HC(pzMe2)2COOH) were converted into the methyl ester derivatives 1 (LOMe) and 2 (L2OMe), respectively, and were used for the preparation of silver(I) complexes 3-5. The Ag(I) complexes were prepared by the reaction of AgNO3 and 1,3,5-triaza-7-phosphaadamantane (PTA) or triphenylphosphine (PPh3) with LOMe and L2OMe in methanol solution. All Ag(I) complexes showed a significant in vitro antitumor activity, proving to be more effective than the reference drug cisplatin in the in-house human cancer cell line panel containing examples of different solid tumors. Compounds were particularly effective against the highly aggressive and intrinsically resistant human small-cell lung carcinoma (SCLC) cells, either in 2D and 3D cancer cell models. Mechanistic studies revealed their ability to accumulate into cancer cells and to selectively target Thioredoxin (TrxR), thus leading to redox homeostasis unbalance and ultimately inducing cancer cell death through apoptosis.

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.

PFKFB3 regulates cancer stemness through the hippo pathway in small cell lung carcinoma

PFKFB3 (6-phosphofructo-2-kinase) is the rate-limiting enzyme of glycolysis and is overexpressed in several human cancers that are associated with poor prognosis. High PFKFB3 expression in cancer stem cells promotes glycolysis and survival in the tumor microenvironment. Inhibition of PFKFB3 by the glycolytic inhibitor PFK158 and by shRNA stable knockdown in small cell lung carcinoma (SCLC) cell lines inhibited glycolysis, proliferation, spheroid formation, and the expression of cancer stem cell markers CD133, Aldh1, CD44, Sox2, and ABCG2. These factors are also associated with chemotherapy resistance. We found that PFK158 treatment and PFKFB3 knockdown enhanced the ABCG2-interacting drugs doxorubicin, etoposide, and 5-fluorouracil in reducing cell viability under conditions of enriched cancer stem cells (CSC). Additionally, PFKFB3 inhibition attenuated the invasion/migration of SCLC cells by downregulating YAP/TAZ signaling while increasing pLATS1 via activation of pMST1 and NF2 and by reducing the mesenchymal protein expression. PFKFB3 knockdown and PFK158 treatment in a H1048 SCLC cancer stem cell-enriched mouse xenograft model showed significant reduction in tumor growth and weight with reduced expression of cancer stem cell markers, ABCG2, and YAP/TAZ. Our findings identify that PFKFB3 is a novel target to regulate cancer stem cells and its associated therapeutic resistance markers YAP/TAZ and ABCG2 in SCLC models.

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.

Identification of distinct N-glycosylation patterns on extracellular vesicles from small-cell and non–small-cell lung cancer cells

Asparagine-linked glycosylation (N-glycosylation) of proteins in the cancer secretome has been gaining increasing attention as a potential biomarker for cancer detection and diagnosis. Small extracellular vesicles (sEVs) constitute a large part of the cancer secretome, yet little is known about whether their N-glycosylation status reflects known cancer characteristics. Here, we investigated the N-glycosylation of sEVs released from small-cell lung carcinoma (SCLC) and non–small-cell lung carcinoma (NSCLC) cells. We found that the N-glycans of SCLC-sEVs were characterized by the presence of structural units also found in the brain N-glycome, while NSCLC-sEVs were dominated by typical lung-type N-glycans with NSCLC-associated core fucosylation. In addition, lectin-assisted N-glycoproteomics of SCLC-sEVs and NSCLC-sEVs revealed that integrin αV was commonly expressed in sEVs of both cancer cell types, while the epithelium-specific integrin α6β4 heterodimer was selectively expressed in NSCLC-sEVs. Importantly, N-glycomics of the immunopurified integrin α6 from NSCLC-sEVs identified NSCLC-type N-glycans on this integrin subunit. Thus, we conclude that protein N-glycosylation in lung cancer sEVs may potentially reflect the histology of lung cancers.

The Effect of VPA Treatment on Radiolabeled DOTATATE Uptake: Differences Observed In Vitro and In Vivo

Background: To improve peptide receptor radionuclide therapy (PRRT), we aimed to enhance the expression of somatostatin type-2 receptors (SSTR2) in vitro and in vivo, using valproic acid (VPA). Methods: Human NCI-H69 small-cell lung carcinoma cells were treated with VPA, followed by [111In]In-DOTATATE uptake studies, RT-qPCR and immunohistochemistry analysis. Furthermore, NCI-H69 xenografted mice were treated with VPA or vehicle, followed by [177Lu]Lu-DOTATATE injection. Biodistribution studies were performed, and tissues were collected for further analysis. Results: VPA significantly increased SSTR2 expression in vitro. In animals, a statistically significant increased [177Lu]Lu-DOTATATE tumoral uptake was observed when VPA was administered eight hours before [177Lu]Lu-DOTATATE administration, but increased tumor SSTR2 expression levels were lacking. The animals also presented significantly higher [177Lu]Lu-DOTATATE blood levels, as well as an elevated renal tubular damage score. This suggests that the enhanced tumor uptake was presumably a consequence of the increased radiotracer circulation and the induced kidney damage. Conclusions: VPA increases SSTR2 expression in vitro. In vivo, the observed increase in tumoral [177Lu]Lu-DOTATATE uptake is not caused by SSTR2 upregulation, but rather by other mechanisms, e.g., an increased [177Lu]Lu-DOTATATE circulation time and renal toxicity. However, since both drugs are safely used in humans, the potential of VPA to improve PRRT remains open for investigation.

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.