Human Small-cell Lung Cancer Cell 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.

Six unprecedented steroidal glycosides from Allium atropurpureum bulbs and their cytotoxicities against SBC-3 human small-cell lung cancer cells

Six unprecedented steroidal glycosides from Allium atropurpureum bulbs and their cytotoxicities against SBC-3 human small-cell lung cancer cells

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.

Entinostat Enhances the Efficacy of Chemotherapy in Small Cell Lung Cancer Through S-phase Arrest and Decreased Base Excision Repair.

Acquired chemoresistance is a frequent event in small cell lung cancer (SCLC), one of the deadliest human malignancies. Histone deacetylase inhibitors (HDACi) have been shown to synergize with different chemotherapeutic agents including cisplatin. Accordingly, we aimed to investigate the dual targeting of HDAC inhibition and chemotherapy in SCLC. The efficacy of HDACi and chemotherapy in SCLC was investigated both in vitro and in vivo. Synergistic drug interactions were calculated based on the HSA model (Combenefit software). Results from the proteomic analysis were confirmed via ICP-MS, cell-cycle analysis, and comet assays. Single entinostat- or chemotherapy significantly reduced cell viability in human neuroendocrine SCLC cells. The combination of entinostat with either cisplatin, carboplatin, irinotecan, epirubicin, or etoposide led to strong synergy in a subset of resistant SCLC cells. Combination treatment with entinostat and cisplatin significantly decreased tumor growth in vivo. Proteomic analysis comparing the groups of SCLC cell lines with synergistic and additive response patterns indicated alterations in cell-cycle regulation and DNA damage repair. Cell-cycle analysis revealed that cells exhibiting synergistic drug responses displayed a shift from G1 to S-phase compared with cells showing additive features upon dual treatment. Comet assays demonstrated more DNA damage and decreased base excision repair in SCLC cells more responsive to combination therapy. In this study, we decipher the molecular processes behind synergistic interactions between chemotherapy and HDAC inhibition. Moreover, we report novel mechanisms to overcome drug resistance in SCLC, which may be relevant to increasing therapeutic success.

Determination of Structure and Cytotoxicity of Ten Undescribed Steroidal Glycosides from Allium cristophii × A. macleanii 'Globemaster'.

'Globemaster' is an ornamental hybrid cultivar whose parent plants are Allium cristophii and A. macleanii. The chemical constituents of 'Globemaster' bulbs have not yet been examined; thus, a systematic phytochemical investigation was undertaken herein. A series of chromatographic separations of the MeOH extract of 'Globemaster' bulbs afforded 27 steroidal glycosides (1-27), which are classified into 23 spirostanol glycosides (1-8 and 11-25), two furostanol glycosides (9 and 26), a pregnane glycoside (10), and a cholestane glycoside (27). The structures of the hitherto undescribed compounds (1-10) were determined from the two-dimensional NMR spectroscopic data and hydrolysis. The cytotoxicity of the isolated compounds (1-27) toward HL-60 human promyelocytic leukemia cells, A549 human adenocarcinoma lung cancer cells, and SBC-3 human small-cell lung cancer cells was evaluated. Compounds 8, 22, 23, 24, and 26 exhibited cytotoxicity toward all cell lines in a dose-dependent manner, with IC50 values in the 1.3-49 µM range.

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.

Does oridonin inhibit the growth of small-cell lung cancer?

Research Question: Can oridonin inhibit small cell lung cancer growth by blocking the Notch signalling? Purpose: Small cell lung cancer (SCLC) is an aggressive illness with a low 5-year survival rate. Oridonin is a Chinese medicine extracted from the leaves of the Rabdosia rubescens, a traditional Chinese medicinal herb, which has been proven to have many medical effects in opposition to the tumour. Notch signalling is an essential pathway in multicellular organisms and transfers information into living organisms. Methods: This study will use a human small-cell lung cancer cell line (H1688). Migration assay will test the influence of oridonin on cell migration. A Xenograft mouse tumour model is created to determine the effect of oridonin on tumour growth. Annexin V will test cell apoptosis, and a western blot is used to test whether Notch signalling is activated. All the assays are repeated three times, and the statistics are analyzed by calculating the means and doing the student's t-test. Possible results: There are three main possible results:(1) Oridonin can inhibit SCLC growth by blocking the notch signalling. (2) Oridonin can inhibit the tumour growth of SCLC cells but not by blocking notch signalling. (3) Oridonin cannot inhibit tumour growth but can block the notch signalling. Conclusion: The study will show whether oridonin can inhibit the growth of SCLC by blocking the notch signalling. It will provide a new method of treatment for those with SCLCs.

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.

Structure Elucidation of 16 Undescribed Steroidal Glycosides from the Underground Parts of Agapanthus africanus and Apoptosis-Inducing Activity in Small-Cell Lung Cancer Cell.

To explore new candidates for anticancer agents from natural products, the underground parts of Agapanthus africanus, commonly used as an ornamental plant, were investigated phytochemically. As a result, 16 undescribed steroidal glycosides (1-16) were obtained, and their structures were determined mainly by NMR spectroscopic analysis and chemical transformations. The cytotoxic activities of the isolated compounds (1-16) against SBC-3 human small-cell lung cancer cells, A549 human adenocarcinoma cells, and HL-60 human promyelocytic leukemia cells were evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2-tetrazolium bromide (MTT) assay. Compound 1, a bisdesmosidic furostanol glycoside, and 10, a bisdesmosidic spirostanol glycoside, were cytotoxic to all three cell lines with IC50 values ranging from 1.2 to 13 μM. As 1 exhibited the most potent cytotoxicity against SBC-3 cells among the isolated compounds, its apoptosis-inducing activity toward SBC-3 cells was examined. Compound 1 arrested SBC-3 cells at the G2/M phase of the cell cycle and effectively induced apoptosis via an intrinsic pathway accompanied by the dissipation of membrane potential and morphological changes in mitochondria.

A multiplexed in vivo approach to identify driver genes in small cell lung cancer

Small cell lung cancer (SCLC) is a lethal form of lung cancer. Here, we develop a quantitative multiplexed approach on the basis of lentiviral barcoding with somatic CRISPR-Cas9-mediated genome editing to functionally investigate candidate regulators of tumor initiation and growth in genetically engineered mouse models of SCLC. We found that naphthalene pre-treatment enhances lentiviral vector-mediated SCLC initiation, enabling high multiplicity of tumor clones for analysis through high-throughput sequencing methods. Candidate drivers of SCLC identified from a meta-analysis across multiple human SCLC genomic datasets were tested using this approach, which defines both positive and detrimental impacts of inactivating 40 genes across candidate pathways on SCLC development. This analysis and subsequent validation in human SCLC cells establish TSC1 in the PI3K-AKT-mTOR pathway as a robust tumor suppressor in SCLC. This approach should illuminate drivers of SCLC, facilitate the development of precision therapies for defined SCLC genotypes, and identify therapeutic targets.

Cancer-Cell-Selective Targeting by Arylcyclopropylamine-Vorinostat Conjugates.

Anticancer drug delivery by small molecules offers a number of advantages over conventional macromolecular drug delivery systems. We previously developed phenylcyclopropylamine (PCPA)-drug conjugates (PDCs) as small-molecule-based drug delivery vehicles for targeting lysine-specific demethylase 1 (LSD1)-overexpressing cancers. In this study, we applied this PDC strategy to the HDAC-inhibitory anticancer agent vorinostat. Among three synthesized PCPA or arylcyclopropylamine (ACPA)-vorinostat conjugates 1, 9, and 32, conjugate 32 with a 4-oxybenzyl linker showed sufficient stability in buffer solutions, potent LSD1 inhibition, efficient LSD1-dependent vorinostat release, and potent and selective antiproliferative activity toward LSD1-expressing human breast cancer and small-cell lung cancer cell lines. These results indicate that the conjugate selectively releases vorinostat in cancer cells. A similar strategy may be applicable to other anticancer drugs.

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.