Human Small Cell Lung Cancer Cell Lines Research Articles

CDK9 inhibition as an effective therapy for small cell lung cancer

Treatment-naïve small cell lung cancer (SCLC) is typically susceptible to standard-of-care chemotherapy consisting of cisplatin and etoposide recently combined with PD-L1 inhibitors. Yet, in most cases, SCLC patients develop resistance to first-line therapy and alternative therapies are urgently required to overcome this resistance. In this study, we tested the efficacy of dinaciclib, an FDA-orphan drug and inhibitor of the cyclin-dependent kinase (CDK) 9, among other CDKs, in SCLC. Furthermore, we report on a newly developed, highly specific CDK9 inhibitor, VC-1, with tumour-killing activity in SCLC. CDK9 inhibition displayed high killing potential in a panel of mouse and human SCLC cell lines. Mechanistically, CDK9 inhibition led to a reduction in MCL-1 and cFLIP anti-apoptotic proteins and killed cells, almost exclusively, by intrinsic apoptosis. While CDK9 inhibition did not synergise with chemotherapy, it displayed high efficacy in chemotherapy-resistant cells. In vivo, CDK9 inhibition effectively reduced tumour growth and improved survival in both autochthonous and syngeneic SCLC models. Together, this study shows that CDK9 inhibition is a promising therapeutic agent against SCLC and could be applied to chemo-refractory or resistant SCLC.

Abstract 606: The PLK1 inhibitor, onvansertib, synergizes with paclitaxel in small cell lung cancer

Abstract Background: Small cell lung cancer (SCLC) is an aggressive neuroendocrine tumor with rapid disease progression and poor patient survival. Therapeutic options for SCLC remain a challenge due to limited understanding of both the biology of the disease and the major determinants of treatment response. Key transcription factors associated with unique biological phenotypes of SCLC include ASCL1, NEUROD1, YAP1, and POU2F3. Previously, we demonstrated that onvansertib, a highly specific inhibitor of the PLK1 Ser/Thr kinase, was effective in SCLC preclinical models, leading to its ongoing clinical development for relapsed/refractory SCLC patients (NCT05450965). Here we further explored potential strategies to enhance onvansertib activity by assessing its in vitro and in vivo activity in combination treatments. Methods: Human SCLC cell lines were treated for 72 hr with onvansertib in combination with paclitaxel, lurbinectedin or the ATR inhibitors BAY1895344 and AZD6738. Effect on cell proliferation was analyzed by WST1 and CellTiterGlo assays. In vivo activity of onvansertib in combination with paclitaxel was tested in 3 SCLC patient-derived xenograft (PDX) models either cisplatin-sensitive (TKO5- ASCL1 subtype) or cisplatin-resistant (TKO2-ASCL1 subtype, TKO8-NEUROD1 subtype). Tumor-bearing nude mice were treated with vehicle, onvansertib (50 mg/kg, oral, 4 times/week), paclitaxel (15 mg/kg, IP, once a week) or the combination for 5 weeks. Mice were followed for tumor growth and survival (defined as time to reach tumor volume greater than 2000 mm3). Results: The combination of onvansertib and paclitaxel inhibited cell proliferation synergistically in cell lines from 3 SCLC subtypes (ASCL1, NEUROD1, and YAP1), while the combinations with lurbinectedin or ATR inhibitors were not synergistic. We further examined the combination of onvansertib and paclitaxel in SCLC PDXs. The combination exhibited potent anti-tumor activity, resulting in significantly greater tumor growth inhibition and increase in survival compared to the monotherapies in all 3 PDXs. The combination was well tolerated with no observed body weight loss. While onvansertib and paclitaxel had negligible single agent activity in cisplatin-resistant TK02 PDX and TKO8 PDX models, the combination induced durable complete responses in 25% and 100% of treated mice respectively. Conclusion: The PLK1 inhibitor onvansertib in combination with paclitaxel showed synergy in multiple SCLC cell lines in vitro and potent anti-tumor activity in cisplatin-sensitive and -resistant SCLC PDX models in vivo. Tumor tissue analysis is underway to detail the mechanisms of this interaction. Citation Format: John C. Schmitz, Guojing Zhang, Abbe Pannucci, Tod Smeal, Chu-Chiao Wu, Maya Ridinger, Taofeek K. Owonikoko. The PLK1 inhibitor, onvansertib, synergizes with paclitaxel in small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 606.

Abstract 3842: Neuroendocrine differentiation and treatment of human SCLC: NRF2 has a role

Abstract SCLC is a lethal form of lung cancer and highly metastatic disease with limited therapeutic options. Four main neuro and non-neuroendocrine SCLC molecular subtypes have been identified which further complicate the efforts of finding targeted therapies. NRF2 is a transcription factor that is known to protect cells from oxidative damage by regulating more than 500 cytoprotective genes. NRF2 is frequently mutated in non-small cell lung cancer (NSCLC), however, whether alterations occur in small cell lung cancer (SCLC) is not clear due to limited sequencing data. Recently, our group has discovered a previously unrecognized role of NRF2 signaling pathway in the development of SCLC using a novel genetically engineered mouse model (GEMM). In this study, we have investigated the role of NRF2 in the progression and regression of human SCLC. Human SCLC cell lines were generated with NRF2E79Q/+ mutation (one of the most common mutations found in human tumors) to allow the constitutive activation of NRF2 in these cells. Results from hSCLC cell lines showed that NRF2 activation in NRF2-negative SCLCs can significantly inhibit their growth. Further, human SCLC gene expression data showed reduced levels of a NRF2 signature as hSCLC neuroendocrine SCLC transforms to non-neuroendocrine subtypes. As an anti-inflammatory pathway, NRF2 is known to inhibit immune cell gene signature. Our in vivo and invitro studies showed that NRF2 inhibits immune cell infiltration in SCLC; and increases PDL1 protein expression. Currently, we are investigating the effect of NRF2 activation in hSCLC growth, neuroendocrine differentiation and immune gene activation in vitro and in vivo. We are also using immune checkpoint inhibitors to determine the best therapeutic options for each SCLC subtype. Citation Format: Samera Hamad, Hansa Joshi, Yahui Li, Helen Toma, Nasrine Bendjilali, Stuart Jefferys, David Corcoran, Weam Elbezanti, Ben Major, Weissman Bernard, Francis Spitz. Neuroendocrine differentiation and treatment of human SCLC: NRF2 has a role [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3842.

Abstract A019: ATR inhibitor efficacy depends on CD8+ T-cell recruitment and MHC class-I upregulation via intratumoral STING pathway activation in small cell lung cancer

Abstract Introduction: Small cell lung cancer (SCLC) is the most aggressive subtype of lung cancer. Recent clinical trials of immune checkpoint blockade (ICB) combined with chemotherapy delivered only very modest benefits. Using genetic screens, we identified inhibition of G2/M cell cycle regulator, ataxia telangiectasia, and rad3 related (ATR), as a promising therapeutic target in small cell lung cancer (SCLC). In this study, we investigated the effect of targeting ataxia telangiectasia and rad3-related (ATR), the primary activator of the replication stress response, on the immune microenvironment in SCLC. We further confirm the efficacy of combining ATR inhibition with immune checkpoint blockade in SCLC. Methods: In this study, we performed genetic and pharmacological inhibition of ATR in a panel of human and murine SCLC cell lines. Furthermore, we investigated the effect of ATR inhibition either alone or in combination with PD-L1 blockade in multiple immunocompetent mouse models of SCLC. The downstream effects of ATR inhibition was assessed by bulk RNA sequencing, multicolor flow cytometry, western blot analysis and real-time qRT-PCR. The SCLC clinical samples from treatment naïve and patients treated with an ATR inhibitor we analysis by single cell and bulk RNA sequencing to ascertain the effect of ATR on immune subsets. Results: In multiple immunocompetent SCLC mouse models, ATR inhibition (ATRi) remarkably enhanced the anti-tumor effect of PD-L1 blockade. We next tested the ATR inhibition either alone or in combination with PD-L1 in the second-line regimen for SCLC. We observed that ATR inhibition in combination with PD-L1 blockade significantly reduced tumor volume and prolonged survival of aggressive mice models when compared to PD-L1 alone. Targeting ATR enhanced the expression of PD-L1, activated the cGAS/STING pathway, induced the expression of Type I and II interferon pathways, and caused significant infiltration of cytotoxic and memory/effector T-cells into tumors. Interestingly, ATRi also led to significant induction of MHC class I in SCLC in vitro and in vivo models. Single-cell analysis of patient samples confirms immunosuppressed phenotype in SCLC. Analysis of pre-and post-treatment clinical samples from a proof-of-concept study of a first-in-class ATR inhibitor, M6620 (VX970, berzosertib), and TOP1 inhibitor topotecan, in patients with relapsed SCLCs, validated the induction of MHC class I and interferon pathway genes, for the first time in this disease. Conclusion: Our findings highlight ATRi as a potentially transformative vulnerability of SCLC, paving the way for combination clinical trials with anti-PD-L1. We are the first to show that ATR inhibition can activate MHC class I in SCLC clinical samples. Given the increasing importance of immunotherapy for the management of SCLC and that ATR inhibitors are already in clinical trials, combining an ATR inhibitor with PD-L1 blockade may offer a particularly attractive strategy for the treatment of SCLC and contribute to the rapid translation of this combination into the clinic. Citation Format: Triparna Sen. ATR inhibitor efficacy depends on CD8+ T-cell recruitment and MHC class-I upregulation via intratumoral STING pathway activation in small cell lung cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr A019.

Cisplatin-induced Pyroptosis Enhances the Efficacy of PD-L1 Inhibitor in Small-Cell Lung Cancer via GSDME/IL12/CD4Tem Axis.

The combination therapy of platinum-based chemotherapy and PD-L1 inhibitors but not the single anti-PD-L1 therapy has significantly improved the prognosis of patients with small-cell lung cancer (SCLC). However, the synergistic mechanism of combination therapy has not been fully elucidated. In this work, we identified a positive correlation between the expression of pyroptosis-related proteins Gasdermin E (GSDME) and the survival rates of patients with SCLC. Importantly, it was shown that human SCLC cell lines with high expression of GSDME showed more sensitivity to cisplatin, as well as cisplatin plus anti-PD-L1 treatment both in vitro and in vivo. Mechanically, cisplatin induced the activation of GSDME and the release of cytokines including IL-12, which enhance the expression of IFN-γ in T cells in the tumor immune microenvironment (TME) and subsequently improve anti-PD-L1 response. Altogether, our work demonstrates that cisplatin could induce GSDME-dependent cell pyroptosis to improve the response of anti-PD-L1 therapy though switching the TME from "cold" to "hot" in SCLC, indicating GSDME as a response biomarker for combination therapy of anti-PD-L1 and chemotherapy, as well as a potential target to sensitize the response to PD-L1 inhibitor therapy in future.

Molecular and Pathologic Characterization of YAP1-Expressing Small Cell Lung Cancer Cell Lines Leads to Reclassification as SMARCA4-Deficient Malignancies.

The classification of small cell lung cancer (SCLC) into distinct molecular subtypes defined by ASCL1, NEUROD1, POU2F3, or YAP1 (SCLC-A, -N, -P, or -Y) expression, paves the way for a personalized treatment approach. However, the existence of a distinct YAP1-expressing SCLC subtype remains controversial. To better understand YAP1-expressing SCLC, the mutational landscape of human SCLC cell lines was interrogated to identify pathogenic alterations unique to SCLC-Y. Xenograft tumors, generated from cell lines representing the four SCLC molecular subtypes, were evaluated by a panel of pathologists who routinely diagnose thoracic malignancies. Diagnoses were complemented by transcriptomic analysis of primary tumors and human cell line datasets. Protein expression profiles were validated in patient tumor tissue. Unexpectedly, pathogenic mutations in SMARCA4 were identified in six of eight SCLC-Y cell lines and correlated with reduced SMARCA4 mRNA and protein expression. Pathologist evaluations revealed that SMARCA4-deficient SCLC-Y tumors exhibited features consistent with thoracic SMARCA4-deficient undifferentiated tumors (SMARCA4-UT). Similarly, the transcriptional profile SMARCA4-mutant SCLC-Y lines more closely resembled primary SMARCA4-UT, or SMARCA4-deficient non-small cell carcinoma, than SCLC. Furthermore, SMARCA4-UT patient samples were associated with a YAP1 transcriptional signature and exhibited strong YAP1 protein expression. Together, we found little evidence to support a diagnosis of SCLC for any of the YAP1-expressing cell lines originally used to define the SCLC-Y subtype. SMARCA4-mutant SCLC-Y cell lines exhibit characteristics consistent with SMARCA4-deficient malignancies rather than SCLC. Our findings suggest that, unlike ASCL1, NEUROD1, and POU2F3, YAP1 is not a subtype defining transcription factor in SCLC. See related commentary by Rekhtman, p. 1708.

Targeting CDH17 with Chimeric Antigen Receptor-Redirected T Cells in Small Cell Lung Cancer.

Chimeric antigen receptor T cell (CAR-T) therapy stands as a precise and targeted approach in the treatment of malignancies. In this study, we investigated the feasibility of targeting Cadherin 17 (CDH17) with CDH17 CAR-T cells as a therapeutic modality for small cell lung cancer (SCLC). CDH17 expression levels were assessed in human SCLC tumor tissues and cell lines using qPCR and Western blot. Subsequently, we established CDH17 CAR-T cells and assessed their cytotoxicity by co-culturing them with various SCLC cell lines at different effector-to-target (E:T) ratios, complemented by ELISA assays. To ascertain the specificity of CDH17 CAR-T cells, we conducted experiments on SCLC cells with and without CDH17 expression (shRNAs). Furthermore, we employed an SCLC xenograft model to evaluate the in vivo efficacy of CDH17 CAR-T cells. Our results revealed a significant upregulation of CDH17 in both SCLC tissues and cell lines. CDH17 CAR-T cells exhibited robust cytotoxic activity against SCLC cells in vitro, while demonstrating no cytotoxicity towards CDH17-deficient SCLC cells and HEK293 cells that lack CDH17 expression. Importantly, the production of IFN-γ and TNF-α by CDH17 CAR-T cells correlated with their cytotoxic potency. Additionally, treatment with CDH17 CAR-T cells significantly decelerated the growth rate of SCLC-derived xenograft tumors in vivo. Remarkably, no significant difference in body weight was observed between the control group and the group treated with CDH17 CAR-T cells. The preclinical data open further venues for the clinical use of CDH17 CAR-T cells as an immunotherapeutic strategy for SCLC treatment.

AMPAR receptor inhibitors suppress proliferation of human small cell lung cancer cell lines.

Small cell lung cancer (SCLC) is a neuroendocrine tumor with poor prognosis. Neuroendocrine tumors possess characteristics of both nerve cells and hormone-secreting cells; therefore, targeting the neuronal properties of these tumors may lead to the development of new therapeutic options. Among the endogenous signaling pathways in the nervous system, targeting the glutamate pathway may be a useful strategy for glioblastoma treatment. Perampanel, an antagonist of the synaptic glutamate α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR), has been reported to be effective in patients with glioblastoma. In this study, we aimed to investigate the antitumor effects of AMPAR antagonists in human SCLC cell lines. We performed to examine the expression of AMPAR using Western blot and immunohistochemical analysis. The antitumor effects of AMPAR antagonists on human SCLC cell lines were investigated in vitro and in vivo. We also analyzed the signaling pathway of AMPAR antagonists in SCLC cell lines. Statistical analysis was performed by the GraphPad Prism 6 software. We first examined the expression of endogenous AMPAR in six human SCLC cell lines, detecting AMPAR proteins in all of them. Next, we tested the anti-proliferative effect of two AMPAR antagonists, talampanel and cyanquixaline, using SCLC cells in vitro and in vivo. Both AMPAR antagonists inhibited cell proliferation and mitogen-activated protein kinase (MAPK) phosphorylation in SCLC cells in vitro. Further, we observed reduced proliferation of implanted cell lines in an in vivo setting, assessed by Ki-67 immunohistochemistry. Additionally, using immunohistochemical analysis we confirmed AMPAR protein expression in human SCLC samples. AMPAR may be a potential therapeutic target for SCLC.

Abstract LB233: Preclinical analysis identifies predictive biomarker and potential pathways of resistance to lurbinectedin treatment in small cell lung cancer

Abstract Background: Small cell lung cancer (SCLC) is an exceptionally aggressive disease with limited treatment options that typically result in transient responses. SCLC is responsible for approximately 250,000 deaths globally per year. Major hurdles to improving SCLC treatment include the development of rapid chemo-resistance and limited second-line therapies. Lurbinectedin is FDA approved as a second-line treatment for SCLC but shows a response in a subset of patients. Therefore, improved mechanistic understanding and identifying predictive biomarkers of lurbinectedin treatment is a major unmet clinical need. Methods: We treated SCLC cell lines and patient-derived xenograft (PDX) models from all major SCLC subtypes with lurbinectedin. Lurbinectedin-mediated changes in signaling pathways were studied by bulk RNA sequencing, western blot, and flow cytometry. Anti-tumor efficacy and toxicity studies were performed in vivo. Result: All human and PDX-derived SCLC cell lines showed sensitivity to lurbinectedin at a nano-molar concentration ranging from 1.905 to 30 nM. Bulk RNA-seq analysis showed lurbinectedin induced changes in neuroendocrine phenotype, DNA damage response and tumor progression markers in vitro. Single agent treatment of lurbinectedin showed remarkable anti-tumor efficacy in an ASCL1-driven PDX model. RNA sequencing analysis identified modulation of genes in multiple signaling pathways including PI3K-AKT, apoptosis and EMT to be significantly associated with the lurbinectedin response in PDX models. Conclusion: There is an immediate need to understand the subsets of SCLC that would be most sensitive to lurbinectedin and identify predictive biomarkers. We demonstrate MYC as a predictive biomarker for lurbinectedin response. We are the first to show that single agent lurbinectedin shows remarkable anti-tumor efficacy in a ASCL1-driven PDX models of SCLC. Furthermore, our pre and post-lurbinectedin treatment transcriptomic analysis identify the pathways that may contribute to primary or acquired resistance to lurbinectedin in SCLC. Finally, we identity candidate targets that would guide the design of future combination clinical trials with lurbinectedin in SCLC. Citation Format: Subhamoy Chakraborty, Charles Coleman, Deniz Demircioglu, Dan Hasson, Triparna Sen. Preclinical analysis identifies predictive biomarker and potential pathways of resistance to lurbinectedin treatment in small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB233.

Abstract 2145: High SLFN11 expression correlates with sensitivity to lurbinectedin in small cell lung cancer (SCLC) models

Abstract Background: Lurbinectedin monotherapy received accelerated FDA approval for adults with metastatic SCLC with disease progression on or after platinum-based chemotherapy. Lurbinectedin binds to GC-rich areas of gene promoters, blocks RNA-polymerase-II and induces degradation via ubiquitin/proteasome machinery. DNA binding of lurbinectedin results in double stranded DNA breaks and DNA damage that leads to cellular apoptosis. Schlafen 11 (SLFN11), a putative DNA/RNA helicase irreversibly binds to DNA replication forks resulting in replication block and is a predictive biomarker of response to therapeutics that elicit DNA damage including cisplatin, topoisomerase I/II inhibitors and PARP inhibitors. Objective: Evaluate the role of SLFN11 expression in predicting response to lurbinectedin, a DNA damaging agent, in human SCLC cell lines and in vivo models. Methods: Cytotoxicity assays: SCLC cell lines DMS 53, DMS 114, NCI-H69, NCI-H82, NCI-H196, NCI-H209, NCI-H211, NCI-H446, NCI-H526, NCI-H841, NCI-H889, NCI-H1048, and SHP-77 were tested with lurbinectedin dose range from 100 nM to 0.01 nM. Cell viability was determined by measuring intracellular ATP to determine percent cellular viability and IC50 of lurbinectedin. SLFN11 expression of 13 SCLC cell lines were retrieved as RNAseq values from the DepMap database. RNAseq value >2 was assigned as SLFN11 high, values <2 assigned as low. Western blot analysis was used to confirm protein expression. The difference in 10log IC50 between SLFN11 high and low lines is determined using Mann-Whitney test. In vivo efficacy studies: High SLFN11 (NCI-H1048) and low SLFN11 (NCI-H889 and NCI-H69) SCLC cell lines were used for xenograft studies in BALB/c nude mice. Mice were randomly allocated to 4 groups (n=8) as tumors reached a volume of 100 - 150 mm3. Groups received either vehicle or lurbinectedin at doses of 0.06, 0.12 or 0.18 mg/kg IV, QWx 3 weeks. Immunohistochemistry was used to assess SLFN11 protein expression on xenograft tissue. Results and Conclusions: Cell viability assays in 13 SCLC models confirm high SLFN11 expressing cell lines are 4-fold more sensitive to lurbinectedin compared to low SLFN11 expressing cell lines (ΔpIC50 = 0.64; p = 0.0451). The in vivo efficacy data confirms that the SLFN11 high NCI-H1048 model is more responsive with 90% TGI compared to 35% observed in SLFN11 low models (NCI-H889 and NCI-H69) at the highest lurbinectedin dose (p-value = 0.006). Efficacy data confirms correlation to SLFN11 protein expression, consistent with the RNA level association. Both in vitro cytotoxicity and in vivo efficacy studies in SCLC models confirm that high SLFN11 expression predicts response to lurbinectedin. Cell viability studies demonstrate that a limited set of SLFN11 low expressing cell lines are sensitive to lurbinectedin, suggesting a role for other genes. Proteomics and genomics work is underway to define additional response biomarkers Citation Format: Aparna Gupta, Kedar S. Vaidya, Janneke J.T.M. Melis, Robert Hauptschein, Graham Brock, Robin Humphreys. High SLFN11 expression correlates with sensitivity to lurbinectedin in small cell lung cancer (SCLC) models [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 2145.

Onionin A inhibits small-cell lung cancer proliferation through suppressing STAT3 activation induced by macrophages-derived IL-6 and cell–cell interaction with tumor-associated macrophage

Tumor-associated macrophage (TAM)-derived IL-6 is involved in small-cell lung cancer (SCLC) progression and chemoresistance via the activation of signal transducer and activator of transcription 3 (STAT3) in the tumor microenvironment. This study aimed to identify natural compounds that suppress cell–cell interactions between TAMs and SCLC cells by inhibiting STAT3 activation. We used a library of natural compounds to identify candidate agents possessing anti-SCLC effects by inhibiting macrophage-induced tumor proliferation. SBC-3 and SBC-5, human SCLC cell lines, were used for in vitro experiments. Furthermore, we assessed the efficacy of these candidate agents in a murine xenograft model of human SCLC. Among the natural compounds examined, onionin A (ONA) inhibited IL-6-induced STAT3 activation and SCLC cell proliferation. ONA also reduced the secretion of IL-6 from macrophages and interfered with the direct effect of cell–cell interactions between macrophages and SCLC cells. Furthermore, ONA administration suppressed tumor progression in a tumor-bearing mouse model. ONA was identified as the most useful candidate for targeting cell–cell interactions between cancer cells and TAMs for anti-SCLC therapy.

In-depth proteomic analysis reveals unique subtype-specific signatures in human small-cell lung cancer.

BackgroundSmall‐cell lung cancer (SCLC) molecular subtypes have been primarily characterized based on the expression pattern of the following key transcription regulators: ASCL1 (SCLC‐A), NEUROD1 (SCLC‐N), POU2F3 (SCLC‐P) and YAP1 (SCLC‐Y). Here, we investigated the proteomic landscape of these molecular subsets with the aim to identify novel subtype‐specific proteins of diagnostic and therapeutic relevance.MethodsPellets and cell media of 26 human SCLC cell lines were subjected to label‐free shotgun proteomics for large‐scale protein identification and quantitation, followed by in‐depth bioinformatic analyses. Proteomic data were correlated with the cell lines’ phenotypic characteristics and with public transcriptomic data of SCLC cell lines and tissues.ResultsOur quantitative proteomic data highlighted that four molecular subtypes are clearly distinguishable at the protein level. The cell lines exhibited diverse neuroendocrine and epithelial–mesenchymal characteristics that varied by subtype. A total of 367 proteins were identified in the cell pellet and 34 in the culture media that showed significant up‐ or downregulation in one subtype, including known druggable proteins and potential blood‐based markers. Pathway enrichment analysis and parallel investigation of transcriptomics from SCLC cell lines outlined unique signatures for each subtype, such as upregulated oxidative phosphorylation in SCLC‐A, DNA replication in SCLC‐N, neurotrophin signalling in SCLC‐P and epithelial–mesenchymal transition in SCLC‐Y. Importantly, we identified the YAP1‐driven subtype as the most distinct SCLC subgroup. Using sparse partial least squares discriminant analysis, we identified proteins that clearly distinguish four SCLC subtypes based on their expression pattern, including potential diagnostic markers for SCLC‐Y (e.g. GPX8, PKD2 and UFO).ConclusionsWe report for the first time, the protein expression differences among SCLC subtypes. By shedding light on potential subtype‐specific therapeutic vulnerabilities and diagnostic biomarkers, our results may contribute to a better understanding of SCLC biology and the development of novel therapies.

A Fully-Human Antibody Specifically Targeting a Membrane-Bound Fragment of CADM1 Potentiates the T Cell-Mediated Death of Human Small-Cell Lung Cancer Cells.

Small-cell lung cancer (SCLC) is the most aggressive form of lung cancer and the leading cause of global cancer-related mortality. Despite the earlier identification of membrane-proximal cleavage of cell adhesion molecule 1 (CADM1) in cancers, the role of the membrane-bound fragment of CAMD1 (MF-CADM1) is yet to be clearly identified. In this study, we first isolated MF-CADM1-specific fully human single-chain variable fragments (scFvs) from the human synthetic scFv antibody library using the phage display technology. Following the selected scFv conversion to human immunoglobulin G1 (IgG1) scFv-Fc antibodies (K103.1–4), multiple characterization studies, including antibody cross-species reactivity, purity, production yield, and binding affinity, were verified. Finally, via intensive in vitro efficacy and toxicity evaluation studies, we identified K103.3 as a lead antibody that potently promotes the death of human SCLC cell lines, including NCI-H69, NCI-H146, and NCI-H187, by activated Jurkat T cells without severe endothelial toxicity. Taken together, these findings suggest that antibody-based targeting of MF-CADM1 may be an effective strategy to potentiate T cell-mediated SCLC death, and MF-CADM1 may be a novel potential therapeutic target in SCLC for antibody therapy.

Expression patterns and prognostic relevance of subtype-specific transcription factors in surgically resected small-cell lung cancer: an international multicenter study.

The tissue distribution and prognostic relevance of subtype‐specific proteins (ASCL1, NEUROD1, POU2F3, YAP1) present an evolving area of research in small‐cell lung cancer (SCLC). The expression of subtype‐specific transcription factors and P53 and RB1 proteins were measured by immunohistochemistry (IHC) in 386 surgically resected SCLC samples. Correlations between subtype‐specific proteins and in vitro efficacy of various therapeutic agents were investigated by proteomics and cell viability assays in 26 human SCLC cell lines. Besides SCLC‐A (ASCL1‐dominant), SCLC‐AN (combined ASCL1/NEUROD1), SCLC‐N (NEUROD1‐dominant), and SCLC‐P (POU2F3‐dominant), IHC and cluster analyses identified a quadruple‐negative SCLC subtype (SCLC‐QN). No unique YAP1‐subtype was found. The highest overall survival rates were associated with non‐neuroendocrine subtypes (SCLC‐P and SCLC‐QN) and the lowest with neuroendocrine subtypes (SCLC‐A, SCLC‐N, SCLC‐AN). In univariate analyses, high ASCL1 expression was associated with poor prognosis and high POU2F3 expression with good prognosis. Notably, high ASCL1 expression influenced survival outcomes independently of other variables in a multivariate model. High POU2F3 and YAP1 protein abundances correlated with sensitivity and resistance to standard‐of‐care chemotherapeutics, respectively. Specific correlation patterns were also found between the efficacy of targeted agents and subtype‐specific protein abundances. In conclusion, we investigated the clinicopathological relevance of SCLC molecular subtypes in a large cohort of surgically resected specimens. Differential IHC expression of ASCL1, NEUROD1, and POU2F3 defines SCLC subtypes. No YAP1‐subtype can be distinguished by IHC. High POU2F3 expression is associated with improved survival in a univariate analysis, whereas elevated ASCL1 expression is an independent negative prognosticator. Proteomic and cell viability assays of human SCLC cell lines revealed distinct vulnerability profiles defined by transcription regulators. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Notch pathway inhibition mediated by arsenic trioxide depletes tumor initiating cells in small cell lung cancer.

Small cell lung cancer (SCLC) is the most malignant type of lung cancer. We previously reported that arsenic trioxide (As2O3) inhibited tumor initiating cells (TICs) of SCLC in vitro. In the present study, we aimed to identify the above effect in vivo and shed light on its underlying mechanism. TICs were enriched by culturing human SCLC cell line as sphere cells in specified serum-free medium. The expression of stem cell markers, CD133 and CD44, and the in vivo tumorigenicity of both TICs and their parental cells were examined. To demonstrate the inhibitory effect of As2O3 on TICs, cell proliferation, clone formation and sphere formation assays were performed. CD133 and Notch pathway-related factors were also measured after As2O3 treatment. Xenograft models were established by injecting TICs into nude mice. Mice were treated with As2O3 for 14days. Afterwards, the tumor volume and the expression of CD133 and Notch1 were evaluated. TICs obtained by the above-mentioned method showed elevated levels of stem cell markers and increased tumorigenicity compared with their parental cells. As2O3 treatment largely inhibited TICs proliferation, sphere formation and clonogenic capacity. As2O3 also reduced the expression of CD133 and down-regulated Notch pathway in TICs. Furthermore, As2O3 potently inhibited tumor growth, decreased the expression of CD133 and down-regulated Notch1 in tumors originating from TICs. Our data demonstrate that As2O3 has a remarkable inhibitory effect on TICs of SCLC both in vitro and in vivo, and the mechanism might involve the down-regulation of Notch pathway.

SLFN11 biomarker status predicts response to lurbinectedin as a single agent and in combination with ATR inhibition in small cell lung cancer.

BackgroundLurbinectedin recently received FDA accelerated approval as a second line treatment option for metastatic small cell lung cancer (SCLC). However, there are currently no established biomarkers to predict SCLC sensitivity or resistance to lurbinectedin or preclinical studies to guide rational combinations.MethodsDrug sensitivity was assayed in proliferation assays and xenograft models. Baseline proteomic profiling was performed by reverse-phase protein array. Lurbinectedin-induced changes in intracellular signaling pathways were assayed by Western blot.ResultsAmong 21 human SCLC cell lines, cytotoxicity was observed following lurbinectedin treatment at a low dose (median IC50 0.46 nM, range, 0.06–1.83 nM). Notably, cell lines with high expression of Schlafen-11 (SLFN11) protein, a promising biomarker of response to other DNA damaging agents (e.g., chemotherapy, PARP inhibitors), were more sensitive to single-agent lurbinectedin (FC =3.2, P=0.005). SLFN11 was validated as a biomarker of sensitivity to lurbinectedin using siRNA knockdown and in xenografts representing SLFN11 high and low SCLC. Replication stress and DNA damage markers (e.g., γH2AX, phosphorylated CHK1, phosphorylated RPA32) increased in SCLC cell lines following treatment with lurbinectedin. Lurbinectedin also induced PD-L1 expression via cGAS-STING pathway activation. Finally, the combination of lurbinectedin with the ataxia telangiectasia and Rad3-related protein (ATR) inhibitors ceralasertib and berzosertib showed a greater than additive effect in SLFN11-low models.ConclusionsTogether our data confirm the activity of lurbinectedin across a large cohort of SCLC models and identify SLFN11 as a top candidate biomarker for lurbinectedin sensitivity. In SLFN11-low SCLC cell lines which are relatively resistance to lurbinectedin, the addition of an ATR inhibitor to lurbinectedin re-sensitized otherwise resistant cells, confirming previous observations that SLFN11 is a master regulator of DNA damage response independent of ATR, and the absence of SLFN11 leads to synthetic lethality with ATR inhibition. This study provides a rationale for lurbinectedin in combination with ATR inhibitors to overcome resistance in SCLC with low SLFN11 expression.

Notch signaling and efficacy of PD-1/PD-L1 blockade in relapsed small cell lung cancer

Immune checkpoint blockade (ICB) benefits only a small subset of patients with small cell lung cancer (SCLC), yet the mechanisms driving benefit are poorly understood. To identify predictors of clinical benefit to ICB, we performed immunogenomic profiling of tumor samples from patients with relapsed SCLC. Tumors of patients who derive clinical benefit from ICB exhibit cytotoxic T-cell infiltration, high expression of antigen processing and presentation machinery (APM) genes, and low neuroendocrine (NE) differentiation. However, elevated Notch signaling, which positively correlates with low NE differentiation, most significantly predicts clinical benefit to ICB. Activation of Notch signaling in a NE human SCLC cell line induces a low NE phenotype, marked by increased expression of APM genes, demonstrating a mechanistic link between Notch activation, low NE differentiation and increased intrinsic tumor immunity. Our findings suggest Notch signaling as a determinant of response to ICB in SCLC.

Curcumin-Induced Global Profiling of Transcriptomes in Small Cell Lung Cancer Cells.

BackgroundCurcumin, one of the promising candidates for supplementary therapy in cancer treatment, has been demonstrated by numerous preclinical and clinical evidence to be beneficial in treating various cancers. Apart from the critical role in a deluge of pathological processes, some mRNAs, in particular, microRNAs (miRNAs), are also involved in the anti-tumor activity. Therefore, our research focused on the possible effects of curcumin on small cell lung cancer (SCLC) cells and drew a comprehensive transcriptomes profile by high throughput sequencing to understand the molecular mechanism of curcumin as an anti-tumor agent.MethodsFirst, we calculated the apoptosis rate of H446 cells (a human SCLC cell line) cultured with curcumin. The high output sequencing uncovered the altered expression profile of genes and miRNAs. KEGG analysis selected the potential signal pathway associated with the antiproliferative property of curcumin. Finally, miRNAs significantly changed, as well as the regulatory roles of those miRNAs in cell apoptosis were determined.ResultThe apoptosis rate of H446 cells increased under the elevated concentration of curcumin treatment. And cell cycle-related genes downregulated in the curcumin-treated cells. Besides, miRNA-548ah-5p of a high level acted as a negative role in the anticarcinogenic activity of curcumin.ConclusionOur findings not only enriched the understanding of anti-tumor activity initiated by curcumin through figuring out the downregulated cell cycle-related pathways but also shed light on its novel therapeutic application.

Effective ferroptotic small-cell lung cancer cell death from SLC7A11 inhibition by sulforaphane.

Small-cell lung cancer (SCLC) is a highly aggressive cancer with poor prognosis, due to a lack of therapeutic targets. Sulforaphane (SFN) is an isothiocyanate derived from cruciferous vegetables and has shown anticancer effects against numerous types of cancer. However, its anticancer effect against SCLC remains unclear. The present study aimed to demonstrate the anticancer effects of SFN in SCLC cells by investigating cell death (ferroptosis, necroptosis and caspase inhibition). The human SCLC cell lines NCI-H69, NCI-H69AR (H69AR) and NCI-H82 and the normal bronchial epithelial cell line, 16HBE14o- were used to determine cell growth and cytotoxicity, evaluate the levels of iron and glutathione, and quantify lipid peroxidation following treatment with SFN. mRNA expression levels of cystine/glutamate antiporter xCT (SLC7A11), a key component of the cysteine/glutamate antiporter, were measured using reverse transcription-quantitative PCR, while the levels of SLC7A11 protein were measured using western blot analysis. Following the addition of SFN to the cell culture, cell growth was significantly inhibited, and cell death was shown in SCLC and multidrug-resistant H69AR cells. The ferroptotic effects of SFN were confirmed following culture with the ferroptosis inhibitor, ferrostatin-1, and deferoxamine; iron levels were elevated, which resulted in the accumulation of lipid reactive oxygen species. The mRNA and protein expression levels of SLC7A11 were significantly lower in SFN-treated cells compared with that in the control cells (P<0.0001 and P=0.0006, respectively). These results indicated that the anticancer effects of SFN may be caused by ferroptosis in the SCLC cells, which was hypothesized to be triggered from the inhibition of mRNA and protein expression levels of SLC7A11. In conclusion, the present study demonstrated that SFN-induced cell death was mediated via ferroptosis and inhibition of the mRNA and protein expression levels of SLC7A11 in SCLC cells. The anticancer effects of SFN may provide novel options for SCLC treatment.

Combinations of proteasome inhibitors with obatoclax are effective for small cell lung cancer.

Proteasome inhibitors, bortezomib (BTZ), and carfilzomib (CFZ) are approved drugs for hematological malignancies, but lack anticancer activities against most solid tumors. Small cell lung cancer (SCLC) is a very aggressive neuroendocrine carcinoma of the lungs demanding effective therapy. In this study we investigated whether BTZ or CFZ combined with obatoclax (OBX), an antagonist for MCL-1 and a pan-BCL family inhibitor, could cause synergistic growth inhibition of SCLC cells. We showed that combined application of BTZ or CFZ with OBX caused synergistic growth inhibition of human SCLC cell lines (H82, H526, DMS79, H196, H1963, and H69) than single agent alone. Both BTZ-OBX and CFZ-OBX combinations displayed marked synergism on inducing apoptosis (~50% increase vs BTZ or CFZ alone). A comprehensive proteomics analysis revealed that BTZ preferentially induced the expression of MCL-1, an antiapoptotic protein, in SCLC cells. Thus, proteasome inhibitor-OBX combinations could specifically induce massive growth inhibition and apoptosis in SCLC cells. Subsequent proteome-wide profiling analysis of activated transcription factors suggested that BTZ- or CFZ-induced MCL-1 upregulation was transcriptionally driven by FOXM1. In nude mice bearing in SCLC H82 xenografts, both BTZ-OBX, and CFZ-OBX combinations exhibited remarkable antitumor activities against SCLC tumors evidenced by significant reduction of tumor size and the proliferation marker Ki-67 signals in tumor tissues as compared with single agent alone. Thus, proteasome inhibitor-OBX combinations are worth immediate assessments for SCLC in clinical settings.