Chemoresistance In Small Cell Lung Cancer Research Articles

Super-enhancer MYCNOS-SE promotes chemoresistance in small cell lung cancer by recruiting transcription factors CTCF and KLF15.

Small cell lung cancer (SCLC) is an aggressive form of lung cancer that often becomes resistant to chemotherapy. Understanding the molecular mechanisms of chemoresistance is crucial for identifying effective therapeutic targets. In this study, we used RNA-Seq to identify highly expressed molecules associated with chemoresistance. We also performed H3K27Ac and ATAC-Seq binding analyses to identify super-enhancers (SE) and their corresponding transcription factors. Both in vitro and in vivo experiments were conducted to examine the impact of these molecules and clinical samples were collected to establish their prognostic value. Our findings revealed elevated expression of MYCNOS, which exhibited chemoresistant properties in both in vitro and in vivo models of SCLC. We identified MYCNOS-SE as a significant SE in SCLC that regulates the distal target gene MYCNOS. This SE recruits transcription factors CTCF and KLF15 to regulate MYCNOS expression. Additionally, MYCNOS, an antisense of MYCN, was found to modulate chemotherapy sensitivity through the NOTCH pathway. This study highlights the significance of SE -regulated target genes as markers for chemoresistance in SCLC. Furthermore, it suggests that MYCNOS could serve as a predictor to identify patients who may benefit from NOTCH inhibitors. These findings provide valuable insights for future studies aimed at developing therapeutic strategies targeting these identified pathways.

Targeting TREX1 Induces Innate Immune Response in Drug-Resistant Small-Cell Lung Cancer.

Small-cell lung cancer (SCLC) is the most lethal type of lung cancer. Paradoxically, this tumor displays an initial exquisite response to chemotherapy; however, at relapse, the tumor is highly resistant to subsequent available therapies. Here, we report that the expression of three prime repair exonuclease 1 (TREX1) is strongly induced in chemoresistant SCLCs. Assay for transposase-accessible chromatin using sequencing and chromatin immunoprecipitation sequencing revealed a significant increase in chromatin accessibility and transcriptional activity of TREX1 gene locus in chemoresistant SCLCs. Analyses of human SCLC tumors and patient-derived xenografts (PDX) also showed an increase in TREX1 expression in postchemotherapy samples. TREX1 depletion caused the activation of cyclic GMP-AMP synthase stimulator of interferon gene pathway due to cytoplasmic accumulation of damage-associated double-stranded DNA, inducing immunogenicity and enhancing the sensitivity of drug-resistant cells to chemotherapy. These findings suggest TREX1 upregulation may partially contribute to the survival of resistant cells, and its inhibition may represent a promising therapeutic strategy to enhance antitumor immunity and potentiate the efficacy of chemotherapy and/or immunotherapy in chemoresistant SCLCs. Significance: In this study, we show that targeting TREX1 induces an innate immune response and resensitizes SCLC cells to chemotherapy, representing a promising novel target for "immunologically" cold tumors, such as SCLC.

PROTAC EZH2 degrader-1 overcomes the resistance of podophyllotoxin derivatives in refractory small cell lung cancer with leptomeningeal metastasis

BackgroundLeptomeningeal metastasis (LM) of small cell lung cancer (SCLC) is a highly detrimental occurrence associated with severe neurological disorders, lacking effective treatment currently. Proteolysis-targeting chimeric molecules (PROTACs) may provide new therapeutic avenues for treatment of podophyllotoxin derivatives-resistant SCLC with LM, warranting further exploration.MethodsThe SCLC cell line H128 expressing luciferase were mutated by MNNG to generate H128-Mut cell line. After subcutaneous inoculation of H128-Mut into nude mice, H128-LM and H128-BPM (brain parenchymal metastasis) cell lines were primarily cultured from LM and BPM tissues individually, and employed to in vitro drug testing. The SCLC-LM mouse model was established by inoculating H128-LM into nude mice via carotid artery and subjected to in vivo drug testing. RNA-seq and immunoblotting were conducted to uncover the molecular targets for LM.ResultsThe SCLC-LM mouse model was successfully established, confirmed by in vivo live imaging and histological examination. The upregulated genes included EZH2, SLC44A4, VEGFA, etc. in both BPM and LM cells, while SLC44A4 was particularly upregulated in LM cells. When combined with PROTAC EZH2 degrader-1, the drug sensitivity of cisplatin, etoposide (VP16), and teniposide (VM26) for H128-LM was significantly increased in vitro. The in vivo drug trials with SCLC-LM mouse model demonstrated that PROTAC EZH2 degrader-1 plus VM26 or cisplatin/ VP16 inhibited H128-LM tumour significantly compared to VM26 or cisplatin/ VP16 alone (P < 0.01).ConclusionThe SCLC-LM model effectively simulates the pathophysiological process of SCLC metastasis to the leptomeninges. PROTAC EZH2 degrader-1 overcomes chemoresistance in SCLC, suggesting its potential therapeutic value for SCLC LM.

HMOX1 regulates ferroptosis via mic14 and its impact on chemotherapy resistance in small-cell lung cancer.

This study aimed to investigate the role and molecular mechanism of heme oxygenase-1 (HMOX1) in chemotherapy resistance in small-cell lung cancer (SCLC). Employed bioinformatics, qPCR, and Western Blot to assess HMOX1 levels in SCLC versus normal tissues and its prognostic relevance. CCK-8, flow cytometry, and thiobarbituric acid assays determined HMOX1's impact on SCLC chemosensitivity, ferroptosis markers, lipid peroxidation, and mic14's role in chemoresistance. In the GSE40275 and GSE60052 cohorts, HMOX1 expression was downregulated in SCLC tissues compared to normal tissues. Higher HMOX1 expression was associated with improved prognosis in the Sun Yat-sen University Cancer Hospital cohort and GSE60052 cohort. The RNA and protein levels of HMOX1 were reduced in drug-resistant SCLC cell lines compared to chemosensitive cell lines. Upregulation of HMOX1 increased chemosensitivity and reduced drug resistance in SCLC, while downregulation of HMOX1 decreased chemosensitivity and increased drug resistance. Upregulation of HMOX1 elevated the expression of ferroptosis-related proteins ACSL4, CD71, Transferrin, Ferritin Heavy Chain, and Ferritin Light Chain, while decreasing the expression of GPX4 and xCT. Conversely, downregulation of HMOX1 decreased the expression of ACSL4, CD71, Transferrin, Ferritin Heavy Chain, and Ferritin Light Chain, while increasing the expression of GPX4 and xCT. Upregulation of HMOX1 promoted cellular lipid peroxidation, whereas downregulation of HMOX1 inhibited cellular lipid peroxidation. Upregulation of HMOX1 reduced the RNA level of mic14, while downregulation of HMOX1 increased the RNA level of mic14. mic14 exhibited inhibitory effects on cellular lipid peroxidation in SCLC cells and contributed to reduced chemosensitivity and increased drug resistance in chemoresistant SCLC cell lines. HMOX1 plays a role in ferroptosis by regulating mic14 expression, thereby reversing chemoresistance in SCLC.

Identification of Hsp90 inhibitors from Ananas comosus potential phytochemicals for lung cancer treatment

Lung cancer is a significant global health issue, with thousands of lives lost each year. One potential approach to preventing lung cancer is the use of heat shock protein 90 (Hsp90) inhibitors, which have been shown to induce substantial cell death in both chemo-sensitive and chemo-resistant small cell lung cancer cells. In this study, we conducted in silico computational molecular docking of pineapple phytochemicals with Hsp90 to investigate their potential inhibitory effects on Hsp90 and, consequently, their ability to prevent lung cancer. Our findings demonstrate that the phytochemicals found in Ananas comosus, specifically caffeic acid, ferulic acid, 4-Hydroxycinnamic acid, Sinapic acid, and D-Galacturonic Acid, exhibit notable inhibitory activity against Hsp90. Beyond their Hsp90 inhibition, these phytochemicals also demonstrate promising biological activities, serving as effective agents against neoplastic conditions, particularly lung cancer, and displaying pro-apoptotic properties. Moreover, the ADME (Absorption, Distribution, Metabolism, and Excretion) and toxicological assessments conducted on these compounds yielded satisfactory results. These findings suggest that pineapple phytochemicals may have potential in the development of novel therapeutic strategies for lung cancer prevention.

Abstract A066: TUSC2 immunogene therapy enhances checkpoint blockade through increased cytotoxic immune activation in chemo-resistant small cell lung cancer (SCLC) in humanized mice

Abstract Immunotherapy has been effective but has had limited effect in clinical trials for SCLC. We identified TUSC2, a potent tumor suppressor gene, as a novel immunogene, which mediates both apoptosis and induces a strong antitumor immune response. TUSC2 expression is reduced in almost all SCLC tumors and absent in 41%. We investigated the antitumor immune response to TUSC2 gene therapy in combination with immune checkpoint blockade (ICB) against a chemo-resistant inflamed SCLC tumors in humanized mice. Colony formation was markedly reduced in platinum resistant inflamed H841 cells transfected with TUSC2. Humanized mice were generated by transplanting fresh human cord blood derived CD34 stem cells into sub-lethally irradiated NSG mice(hu-NSG). The level of engraftment of human CD45, CD3 T, CD19 B, NK cells was verified before tumor implantation at &amp;gt; 25%. Subcutaneous tumors were developed in hu-NSG mice and treated with intravenous injections of TUSC2 loaded cationic lipid nanoparticles (quaratusugene ozeplasmid abbreviated quar oze) with or without pembrolizumab (pembro; anti-PD1). A dramatic antitumor effect was mediated by quar oze, whereas pembro remained ineffective. A synergistic antitumor effect was found when quar oze was combined with pembro. The antitumor effect of the quar oze + pembro was associated with increased infiltration of human TILs, CD3 T, cytotoxic T, and NK cells, and a decreased number of human regulatory T (Treg) cells, and PD1 expressing exhausted CD8 T cells in the tumor microenvironment. Quar oze was also combined with atezolizumab (atezo; anti-PD-L1) and the antitumor effect on H841 lung metastases in hu-NSG mice was tested. Metastases were treated with quar oze and atezo alone or in combination. Although quar oze alone and atezo alone showed a strong antitumor effect in controlling metastases, a greater antitumor effect was found when quar oze was combined with atezo. Infiltration of human CD45, CD3 T, CD8 T and NK cells increased in the quar oze and quar oze +atezo treated groups compared with others. A significantly higher number of CD8 T effector memory and lower number of naïve T cells were found in the quar oze +atezo which were associated with increased activated CD8 T cells (CD69+, PD1+ CD8 T cells). HLA-DR+ dendritic cells were increased, whereas myeloid derived suppressor cells were strongly inhibited by the combination. Quar oze was also combined with carboplatin+atezo which showed enhanced antitumor efficacy compared with carbo+atezo or quar oze alone. When quar oze was combined with carboplatin, a greater effect was found in inhibiting H841 cell growth, colony formation, and induction of apoptosis. Quar oze was combined with NPRL2, another tumor suppressor gene, in H841 tumors in humanized mice. Synergistic antitumor efficacy of the dual gene therapy occurred with the quar oze +NPRL2 treatment. Taken together, these data suggest that TUSC2 gene therapy in combination with ICB induces strong antitumor activity on a chemo-resistant inflamed SCLC through cytotoxic CD8 T and NK cell activation. Citation Format: Ismail M Meraz, Mourad Majidi, Renduo Song, Feng Meng, Lihui Gao, Qi Wang, Jing Wang, Elizabeth Shpall, Mark Berger, Hemant Kumar, Jack A Roth. TUSC2 immunogene therapy enhances checkpoint blockade through increased cytotoxic immune activation in chemo-resistant small cell lung cancer (SCLC) in humanized mice [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A066.

Extracellular Vesicles Derived circSH3PXD2A Inhibits Chemoresistance of Small Cell Lung Cancer by miR-375-3p/YAP1.

Small cell lung cancer (SCLC) is a subtype of lung cancer with high malignancy and poor prognosis. Rapid acquisition of chemoresistance is one of the main reasons leading to clinical treatment failure of SCLC. Studies have indicated that circRNAs participate in multiple processes of tumor progression, including chemoresistance. However, the molecular mechanisms of circRNAs driving the chemoresistance of SCLC are not well specified. The differentially expressed circRNAs were screened by transcriptome sequencing of chemoresistant and chemosensitive SCLC cells. The EVs of SCLC cells were isolated and identified by ultracentrifugation, Western blotting, transmission electron microscopy, nanoparticle tracking analysis and EVs uptake assays. The expression levels of circSH3PXD2A in serum and EVs of SCLC patients and healthy individuals were detected by qRT‒PCR. The characteristics of circSH3PXD2A were detected by Sanger sequencing, RNase R assay, nuclear-cytoplasmic fraction assay, and fluorescence in situ hybridization assay. The mechanisms of circSH3PXD2A inhibiting SCLC progression were studied by bioinformatics analysis, chemoresistance assay, proliferation assay, apoptosis assay, transwell assay, pull-down assay, luciferase reporting assay, and mouse xenograft assay. It was identified that the circSH3PXD2A was a prominently downregulated circRNA in chemoresistant SCLC cells. The expression level of circSH3PXD2A in EVs of SCLC patients was negatively associated with chemoresistance, and the combination of EVs-derived circSH3PXD2A and serum ProGRP (Progastrin-releasing peptide) levels had better indications for DDP-resistant SCLC patients. CircSH3PXD2A inhibited the chemoresistance, proliferation, migration, and invasion of SCLC cells through miR-375-3p/YAP1 axis in vivo and in vitro. SCLC cells cocultured with EVs secreted by circSH3PXD2A-overexpressing cells exhibited decreased chemoresistance and cell proliferation. Our results manifest that EVs-derived circSH3PXD2A inhibits the chemoresistance of SCLC through miR-375-3p/YAP1 axis. Moreover, EVs-derived circSH3PXD2A may serve as a predictive biomarker for DDP-resistant SCLC patients.

Abstract 3854: TNIK inhibition as a novel therapeutic in cMyc high/TTF1 low SCLC

Abstract Background: Small cell lung cancer (SCLC) is a highly lethal malignancy, with rapidly acquired chemotherapy resistance. Some studies have reported that Wnt signaling pathway activation promoted cell proliferation and was correlated with chemo-resistance in SCLC. None of the therapies targeting Wnt pathway components of the transmembrane and the cytoplasm have been successful in a clinical application due to toxicity and insufficient efficacy. However, targeting Wnt signaling inside the nucleus has been drawing increasing attention as cancer therapeutics. TRAF2 and NCK-interacting protein kinase (TNIK), which interacts with downstream effectors, TCF4/β-catenin transcriptional complex, is an essential activator of Wnt target genes. TNIK is highly expressed in several cancers for cell proliferation, thus TNIK is expected as a novel druggable target. On the other hand, the question remains whether TNIK is a critical target in SCLC. We hypothesize that a TNIK inhibitor has potent anti-tumor effects in SCLC and its promising biomarkers exist. Methods: We used 29 SCLC cell lines including all four subtypes defined by differential expression of transcription factors ASCL1, NEUROD1, POU2F3, and inflamed gene signature (SCLC-A, N, P, and I, respectively) to evaluate the effect of a TNIK inhibitor, NCB-0846 in vitro. We correlated NCB-0846 IC50 values with proteomic profiling (Reverse Phase Protein Array, RPPA) data. Protein expression was examined by western blotting. Results: NCB-0846 markedly reduced cell proliferation in SCLC-N and P cell lines. There was a strong positive correlation between cMyc and the efficacy of NCB-0846 (r=-0.484, P&amp;lt;0.01), while a negative correlation between TTF1 and the efficacy (r=0.569, P&amp;lt;0.01). Additionally, NCB0846 decreased the expression of cMyc in cMyc-high/TTF-1 low SCLC cells. Conclusions: These findings indicate that TNIK inhibitors may be a new personalized molecular-targeted therapy in cMyc-high/TTF-1-low SCLC. Citation Format: Azusa Tanimoto, Robert J. Cardnell, Benjamin B. Morris, Kavya Ramkumar, Shen Li, Qi Wang, Allison C. Stewart, Carl Michael Gay, Jing Wang, Lauren Averett Byers. TNIK inhibition as a novel therapeutic in cMyc high/TTF1 low SCLC. [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 3854.

Abstract 6188: Exportin 1 inhibition synergizes with lurbinectedin by altering the response to DNA damage in neuroendocrine lung tumors

Abstract Background: Neuroendocrine lung cancer carcinomas (Lung NECs), which include large cell neuroendocrine carcinomas (LCNECs) and small cell lung cancer (SCLC) tumors, are particularly aggressive lung neoplasms with limited clinical therapeutic options. Patients with lung NECs are treated with platinum-based chemotherapy as the first line of treatment, but they promptly develop resistance. Lurbinectedin has recently been approved by the FDA as a second-line treatment for SCLC due to its efficacy in metastatic chemorelapsed SCLC patients. However, there is still a large percentage of SCLC patients who do not respond to lurbinectedin and the overall survival of those who benefit from it remains very low (&amp;lt;6 months). Previous work from our group had shown that exportin-1 (XPO1) was a driver of chemoresistance in SCLC and its inhibition with selinexor resentitized to first- and second-line chemotherapies in naïve and chemorelapsed SCLC patients derived xenografts, respectively. Importantly, selinexor is an FDA-approved drug for the treatment of relapsed multiple myeloma and is currently undergoing clinical testing in multiple solid tumors. Methods: We performed synergy proliferation assays in vitro. DNA damage/repair pathways were analyzed by western blotting. Cell cycle and apoptosis were analyzed by flow cytometry. Results: We explored the cytotoxic capacity of the combination of lurbinectedin with selinexor in cell linesfrom both SCLC (N=2) and LCNECs (N=3) in synergy in vitro assays. Selinexor strongly synergized with lurbinectedin in both SCLC and LCNEC settings and induced a potent increase in the DNA damage marker γ-H2A, reflective of increased DNA damage induced by the combination treatment. Mechanistically, this combination altered DNA damage and DNA repair mechanisms by decreasing CHK1 and CHK2 protein levels in short-term treatments, suggestive of the impairment of DNA response. Additionally, selinexor reduced the expression of MLH, a key regulator of DNA mismatch repair in monotherapy and in combination with lurbinectedin. Consistent with these results, selinexor in combination with lurbinectedin also induced a significant increase in apoptotic cells and promoted cell cycle arrest. Conclusions: These data indicates that inhibition of exportin 1 with selinexor in combination with lurbinectedin is a promising therapeutic strategy in lung NECs. Subsequent In vivo testing in patient-derived xenograft to study efficacy and toxicity of this combination will provide preclinical rationale for clinically exploring this combination in patients with lung NECs who have failed chemotherapy regimen. Citation Format: Esther Redin Resano, Charles Rudin, Alvaro Quintanal-Villalonga. Exportin 1 inhibition synergizes with lurbinectedin by altering the response to DNA damage in neuroendocrine lung tumors. [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 6188.

Abstract 5145: Targeting TREX1 induces innate immune response in chemoresistant small cell lung cancer

Abstract Small cell lung cancer (SCLC) is the most lethal type of lung cancer. The uniqueness of this tumor consists of an initial exquisite response to chemotherapy. However, at relapse, which occurs nearly in all patients, the tumor is resistant to all available therapies, causing a premature death of the patient. Despite the addition of immune checkpoint blockade (ICB) therapy to standard chemotherapy, response rates are modest and only a very small fraction of SCLC patients responds to these therapies. The mechanistic basis for acquired chemoresistance and impaired immunogenicity in SCLC remains to be elucidated. Here, we report that the expression of three prime repair exonuclease 1 (TREX1) is strongly induced in chemoresistant SCLCs. ATAC-seq and ChIP-seq analyses revealed a significant increase in chromatin accessibility of TREX1 gene locus in drug resistant SCLC cells. Depletion of TREX1 in SCLC cells caused the activation of cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway due to the cytoplasmic accumulation of DNA damage-associated double-stranded DNA (dsDNA) and micronuclei. Furthermore, targeting TREX1 induced an innate immune response and re-sensitized chemoresistant SCLC cells to chemotherapy. These findings suggest that TREX1 upregulation may be an important mediator of cGAS/STING pathway inactivation in cancer, contributing to the survival of resistant cells, and its inhibition may represent a promising therapeutic strategy to potentiate the efficacy of chemotherapy and immunotherapy in chemoresistant and immunologically ‘cold’ tumors, such as SCLC. Citation Format: Takahiko Murayama, Navin R. Mahadevan, Tetsuo Tani, Xueying Ma, Hideo Watanabe, David A. Barbie, Israel Cañadas. Targeting TREX1 induces innate immune response in chemoresistant small cell lung cancer. [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 5145.

Identifying CDC7 as a synergistic target of chemotherapy in resistant small-cell lung cancer via CRISPR/Cas9 screening

There is currently a lack of efficacious treatments for patients with chemo-resistant small-cell lung cancer (SCLC), leading to poor prognoses. We examined a chemo-resistant SCLC cell line using genome-wide CRISPR/Cas9 screening and identified serine/threonine kinase cell division cycle 7 (CDC7) as a potential synergistic target. Silencing CDC7 in chemo-resistant SCLC cells decreased the IC50 and improved the efficacy of chemotherapy. Based on the highest single agent model, the CDC7 inhibitor XL413 had a synergistic effect with both cisplatin and etoposide in chemo-resistant SCLC cells, but had no such effect in chemo-sensitive SCLC cells; the combination of XL413 and chemotherapy significantly inhibited cell growth. Western blot and flow cytometry showed that the combined treatments increased apoptosis, whereas XL413 alone had little effect on apoptosis. An analysis of cell cycle and cyclin protein levels indicated that the combination of XL413 and chemotherapy-induced G1/S phase arrest and DNA damage in chemo-resistant SCLC cells. Xenografted tumor and histoculture drug response assays using patient-derived xenografts showed that XL413 improved the efficacy of chemotherapy in vivo and with SCLC tissues. These results suggest that XL413 exerts a synergistic effect with chemotherapy on chemo-resistant SCLC.

Ligand-independent EphA2 contributes to chemoresistance in small-cell lung cancer by enhancing PRMT1-mediated SOX2 methylation.

Chemoresistance is the crux of clinical treatment failure of small-cell lung cancer (SCLC). Cancer stem cells play a critical role in therapeutic resistance of malignant tumors. Studies have shown that the role of erythropoietin-producing hepatocellular A2 (EphA2) in tumors is complex. This study aimed to test the hypothesis that ligand-independent activation of EphA2 modulates chemoresistance by enhancing stemness in SCLC. We verified that EphA2 was activated in chemoresistance sublines in a ligand-independent manner rather than a ligand-dependent manner. Ligand-independent EphA2 enhanced the expression of stemness-associated biomarkers (CD44, Myc, and SOX2), accelerated epithelial-mesenchymal transition (EMT) and reinforced self-renewal to drive the chemoresistance of SCLC, while the P817H mutant EphA2 neutralized intrinsic function. Co-immunoprecipitation (co-IP) and GST-pull down experiments were conducted to verify that EphA2 directly interacted with PRMT1. Moreover, EphA2 increased the expression and activity of PRMT1. Whereafter, PRMT1 interacted with and methylated SOX2 to induce stemness and chemoresistance in SCLC. Pharmacological inhibition of EphA2 showed a synergistic anti-tumor effect with chemotherapy in preclinical models, including patient-derived xenograft (PDX) models. These findings highlight, for the first time, that the EphA2/PRMT1/SOX2 pathway induces chemoresistance in SCLC by promoting stemness. EphA2 is a potential therapeutic target in SCLC treatment.

Identification of key genes and pathway related to chemoresistance of small cell lung cancer through an integrative bioinformatics analysis.

BackgroundSmall cell lung cancer (SCLC), the most malignant of all the lung cancer subtypes, is characterized by drug resistance. This study sought to explore the key genes and pathways associated with the chemoresistance of SCLC.MethodsThe drug sensitivity of chemosensitive and chemoresistance SCLC cell lines was measured by Cell Counting Kit-8 assays. The total RNA from chemosensitive cell line H69 and chemoresistance cell line H69AR cells was extracted and subjected to messenger RNA (mRNA) and long non-coding RNA (lncRNA) microarray analyses. The differentially expressed genes (DEGs) and the differentially expressed lncRNAs (DELs) were screened out with a threshold of a |log fold change | ≥1 and an adjusted P value <0.05. A protein-protein interaction network was constructed, and hub genes were screened out. A lncRNA-mRNA co-expression network was also constructed. Gene Ontology and Kyoto Encyclopedia of Genes, Genomes enrichment analyses and Cis-regulatory element analyses were conducted on the DEGs and the top 10 upregulated DEL-co-expressed DEGs. The expression of the key genes was further analyzed in the GSE149507 data set and validated in H69/H69AR and H446/H446DDP cells by quantitative polymerase chain reaction assays.ResultsThe microarray results showed that a total of 609 mRNAs and 394 lncRNAs were differentially expressed in the chemoresistant SCLC cells. The mammalian target of rapamycin (mTOR) signaling pathway was enriched among the DEGs, the top 10 upregulated DEL-co-expressed DEGs, and the NCRNA00173-co-expressed DEGs, which included IGF1, INS, WNT6, WNT11, WNT2B, and SESN2. IGF1, WNT2B, and SESN2 were downregulated, and WNT11 was upregulated in the SCLC tumor tissues in the GSE149507 data set. Further, IGF1, WNT6, WNT11, and WNT2B were lowlier expressed and SESN2 and NCRNA00173 were more highly expressed in the chemoresistant cells than sensitive cells.ConclusionsThe top 10 upregulated DELs containing NCRNA00173 may be involved in the regulation of drug resistance in SCLC. These DELs may regulate the genes related to the mTOR signaling pathway. These genes may also be biomarkers and potential targets for the treatment of SCLC.

HuR affects chemoresistance of small cell lung cancer by regulating FGFRL1 expression.

Human antigen R (HuR), an RNA-binding protein, has been demonstrated to serve an oncogenic role in various types of cancer. Fibroblast growth factor receptor-like 1 (FGFRL1) has been shown to regulate small cell lung cancer (SCLC) chemoresistance. In the present study, the role of HuR in chemoresistance of SCLC, as well as its possible molecular mechanism involving FGFRL1, was explored by reverse transcription-quantitative PCR, western blotting, Cell Counting Kit-8 assay, flow cytometry and RNA immunoprecipitation. The results revealed that HuR expression levels were markedly upregulated in drug-resistant SCLC cell lines (H69AR and H446DDP) compared with in the parental cell lines (H69 and H446). Knockdown of HuR in drug-resistant SCLC cells enhanced drug sensitivity, cell apoptosis and cell cycle arrest. Furthermore, molecular mechanism studies indicated that HuR could bind and regulate FGFRL1 expression levels to increase FGFRL1 mRNA stability. Taken together, the present study suggested that HuR may mediate chemoresistance of SCLC by regulating FGFRL1 expression. HuR may represent a prognostic predictor and a potential target for overcoming chemoresistance in SCLC.

Combinatory therapy of MRP1-targeted photoimmunotherapy and liposomal doxorubicin promotes the antitumor effect for chemoresistant small cell lung cancer

Small cell lung cancer (SCLC), considered a mortal recalcitrant cancer, is a severe healthcare issue because of its poor prognosis, early metastasis, drug resistance and limited clinical treatment options. In our previous study, we established a MRP1-targeted antibody-IR700 system (Mab-IR700) for near infrared photoimmunotherapy (NIR-PIT) which exhibited a promising therapeutic effect on drug resistant H69AR cells both in vitro and in vivo, though the tumor growth suppression effect did not last long with a single round of PIT treatment. To achieve a better anticancer effect, we have combined Mab-IR700-mediated NIR-PIT with liposomal doxorubicin (Doxil®) and investigated the in vitro and in vivo cytotoxicity by using a H69AR/3T3 cell co-culture model in which 3T3 cells were used to mimic stromal cells. Cytotoxicity experiments demonstrated the specificity of Mab-IR700 to H69AR cells, while cytotoxicity and flow cytometry experiments confirmed that H69AR cells were doxorubicin-resistant. Compared with Mab-IR700-mediated PIT or Doxil-mediated chemotherapy, the combination therapy exhibited the best cell killing effect in vitro and superior tumor growth inhibition and survival prolongation effect in vivo. Super enhanced permeability and retention (SUPR) effect was observed in both co-culture spheroids and tumor-bearing mice. Owing to an approximately 9-fold greater accumulation of Doxil within the tumors, NIR-PIT combined with Doxil resulted in enhanced antitumor effects compared to NIR-PIT alone. This photoimmunochemotherapy is a practical strategy for the treatment of chemoresistant SCLC and should be further investigated for clinical translation.

Abstract 1108: Evaluation of heme inhibitory therapy in combination with chemotherapy drugs on SCLC progression

Abstract Lung cancer is the leading cause of cancer-related death in the US. It is mainly divided in two types: small cell lung cancer and non-small cell lung cancer. About 85-90% of cases are classified as non-small cell lung cancer (NSCLC). Small cell lung cancer (SCLC) is an aggressive neuroendocrine lung tumor representing 15% of lung cancers. Despite the advent of targeted therapies and chemotherapies, effective therapies for lung cancer are an unlikely outcome for most patients. SCLC is initially highly responsive to cisplatin and etoposide chemotherapy but, in almost every case, becomes rapidly chemo resistant. However, the mechanisms underlying initial sensitivity and subsequent resistance are not well investigated. Thus, alternative approaches and combination therapy is required for treating SCLCs. Recent studies in our lab show that NSCLC cells demonstrate elevated levels of heme via increased heme uptake and synthesis, and elevated oxidative phosphorylation (OXPHOS) and ATP generation which fuels NSCLC proliferation. Also, limiting heme via inhibition of heme uptake using heme sequestering peptides (HeSP2) can delay growth and progression of NSCLC tumors. This HeSP2 can also effectively inhibit angiogenesis, normalize tumor vasculature, and alleviate tumor hypoxia in NSCLC tumors and thereby inhibit lung tumor growth and progression. Tumor heterogeneity is critical for tumor growth, metastasis, and acquired therapeutic resistance. To understand if heme can effectively reduce tumor cell proliferation and growth and improve tumor microenvironment, we treated chemo resistant SCLCs with HeSP2 and in combination with commonly used chemo drugs cisplatin and etoposide. Our data shows that HesP2 can strongly inhibit cell proliferation and decreases oxygen consumption rate along with ATP generation in SCLCs. To gain insights into mechanism by which the heme sequestering peptides limit tumor growth and progression, we implanted subcutaneous or SCLC cell lines in NOD/SCID mice. Mice were treated with saline (control), HeSP2, HeSP2 and cisplatin, HeSP2 and etoposide. Tumor growth was monitored using caliper measurements. Tumors tissues were harvested, processed and paraffin embedded for immunohistochemistry (IHC) and histology experiments. These results indicate that heme sequestration can be a powerful strategy for suppressing lung tumors likely drug-resistant tumors that rely on oxidative phosphorylation for survival. Citation Format: Adnin Ashrafi, Narges Salamat, Li Zhang. Evaluation of heme inhibitory therapy in combination with chemotherapy drugs on SCLC progression [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 1108.

Exportin 1 as a therapeutic target against chemoresistance in small cell lung cancer.

e20613 Background: Small cell lung cancer (SCLC) is an extremely aggressive disease comprising around 13% of lung cancer cases and responsible for approximately 250,000 deaths globally per year. A major cause of SCLC poor prognosis is the sparse treatment options available, typically resulting in only transient responses. The backbone of SCLC treatment over the past several decades has been platinum-based doublet chemotherapy, with the recent addition of immunotherapy to first-line chemotherapy showing only limited benefit in a small subset of patients. Major hurdles to improving SCLC treatment include development of rapid chemoresistance and ineffective second line therapies. The identification of more durably effective therapeutic strategies is a major unmet clinical need. Methods: We performed an in vitro CRISPR screen in SCLC cell lines to identify potential therapeutic targets to sensitize to chemotherapy, including short-term cultured cell lines derived from patient-derived xenografts (PDXs). Candidate hits were validated genetically and pharmacologically with in vitro and in vivo. Signaling pathways involved in phenotypes observed were studied by RNA sequencing and western blot, and toxicity studies were performed in vivo to assess the safety of the agents at pharmacologically effective doses. We performed immunohistochemistry (IHC) to assess expression of candidate targets in tissue microarrays (TMAs). Results: Our CRISPR screen revealed the nuclear exporter XPO1 (Exportin 1) as a promising target sensitizing to chemotherapy, independently of the SCLC subtype. Importantly, exportin 1 is a therapeutic target in hematological malignancies, counting with a potent and selective inhibitor, selinexor, approved for clinical use. Combination of selinexor with cisplatin or irinotecan demonstrated high synergy in vitro and exquisite efficacy i n vivo in an array of chemonäive and chemoresistant SCLC PDXs, respectively, including all major SCLC subtypes. These chemotherapy-sensitizing effects were associated with the ability of Exportin 1 to impair chemotherapy-induced AKT overactivation, potentially occurring as a cytoprotective/anti-apoptotic mechanism in response to chemotherapy. The combinations were well tolerated in mice. We found that XPO1 mRNA expression was higher in SCLC than in any other solid tumor type or hematological malignancies, which we were able to confirm at the protein level in clinical TMAs. Conclusions: Exportin 1 inhibition strongly enhances sensitivity of SCLC tumors to cisplatin and irinotecan, used in first line and second line treatment of SCLC tumors, respectively. These results provide preclinical rationale for the combination of selinexor with first line and second line chemotherapy in SCLC. The clinical availability of selinexor will allow immediate clinical translation of these results in a disease setting with extremely limited therapeutic options.

NRBF2 regulates the chemoresistance of small cell lung cancer by interacting with the P62 protein in the autophagy process

SummaryReversing chemotherapy resistance in small cell lung cancer (SCLC) is crucial to improve patient prognosis. The present study aims to investigate the underlying mechanisms in SCLC chemoresistance. We see that nuclear receptor binding factor 2 (NRBF2) is a poor prognostic factor in SCLC. The effects of NRBF2 on chemoresistance were determined in SCLC. The underlying molecular mechanisms of NRBF2 in the autophagy process in SCLC were examined. NRBF2 positively regulated autophagy, leading to drug resistance in SCLC. The MIT domain of NRBF2 directly interacted with the PB1 domain of P62. This interaction increased autophagic P62 body formation, revealing the regulatory role of NRBF2 in autophagy. Notably, NRBF2 was directly modulated by the transcription factor XRCC6. The MIT domain of NRBF2 interacts with the PB1 domain of P62 to regulate the autophagy process, resulting in SCLC chemoresistance. NRBF2 is likely a useful chemotherapy response marker and therapeutic target in SCLC.

Reducing Chemotherapy-Induced DNA Damage via nAChR-Mediated Redox Reprograming-A New Mechanism for SCLC Chemoresistance Boosted by Nicotine.

Simple SummaryTobacco smoking is the dominant risk factor for lung cancer, particularly for small cell lung cancer (SCLC). Smoking is also associated with worse clinical outcomes of SCLC. For SCLC patients, platinum-based chemotherapy (cisplatin or carboplatin), in combination with etoposide, has been the first-line therapy for decades, even with the recent introduction of immunotherapy. One key limitation of such chemotherapy is the quick acquisition of drug resistance. Here, we found that nicotine and its main metabolite, cotinine, reduced the efficacy of chemotherapies to SCLC cells and accelerated drug resistance, which could contribute to poorer survival rates in SCLC patients who continue to smoke.Up to 60% of patients with small cell lung cancer (SCLC) continue to smoke, which is associated with worse clinical outcomes. Platinum-based chemotherapies, in combination with topoisomerase inhibitors, are first-line therapies for SCLC, with rapid chemoresistance as a major barrier. We provided evidence in this study that nicotine and its major metabolite, cotinine, at physiologically relevant concentrations, reduced the efficacy of platinum-based chemotherapies and facilitated chemoresistance in SCLC cells. Mechanistically, nicotine or cotinine reduced chemotherapy-induced DNA damage by modulating cellular redox processes, with nAChRs as the upstream targets. Surprisingly, cisplatin treatment alone also increased the levels of nAChRs in SCLC cells, which served as a self-defense mechanism against platinum-based therapies. These discoveries were confirmed in long-term in vitro and in vivo studies. Collectively, our results depicted a novel and clinically important mechanism of chemoresistance in SCLC treatment: nicotine exposure significantly compromises the efficacy of platinum-based chemotherapies in SCLC treatment by reducing therapy-induced DNA damage and accelerating chemoresistance acquisition. The results also emphasized the urgent need for tobacco cessation and the control of NRT use for SCLC management.

Therapeutic targeting of the mevalonate-geranylgeranyl diphosphate pathway with statins overcomes chemotherapy resistance in small cell lung cancer.

Small cell lung cancer (SCLC) lacks effective treatments to overcome chemoresistance. Here we established multiple human chemoresistant xenograft models through long-term intermittent chemotherapy, mimicking clinically relevant therapeutic settings. We show that chemoresistant SCLC undergoes metabolic reprogramming relying on the mevalonate (MVA)-geranylgeranyl diphosphate (GGPP) pathway, which can be targeted using clinically approved statins. Mechanistically, statins induce oxidative stress accumulation and apoptosis through the GGPP synthase 1 (GGPS1)-RAB7A-autophagy axis. Statin treatment overcomes both intrinsic and acquired SCLC chemoresistance in vivo across different SCLC PDX models bearing high GGPS1 levels. Moreover, we show that GGPS1 expression is negatively associated with survival in patients with SCLC. Finally, we demonstrate that combined statin and chemotherapy treatment resulted in durable responses in three patients with SCLC who relapsed from first-line chemotherapy. Collectively, these data uncover the MVA-GGPP pathway as a metabolic vulnerability in SCLC and identify statins as a potentially effective treatment to overcome chemoresistance.