SCLC Cell Lines Research Articles

Abstract 2557: Slit2 inhibits small cell lung cancer by targeting tumor microenvironment

Abstract Small cell lung cancer (SCLC) is the deadliest subtype of lung cancer with a 5-year survival rate of <5% in extensive-state disease and is designated by US Congress as a “recalcitrant cancer”. Recently approved by FDA, immune checkpoint blockade (ICB) immunotherapy showed marginally improved survival and only in a few patients, suggesting an inert immune tumor microenvironment (TME) and the existence of SCLC subtypes. Therefore, there is a significant need to develop more effective therapies for SCLC. In addition to the nearly universal biallelic inactivation of Rb and Tp53 genes in human SCLC tumors, comprehensive genome-wide screening studies have identified major co-occurring mutations in PTEN, CREBBP, EP300, SLIT2, MLL, COBL, and EPHA7 genes. Among them, SLIT2 showed the highest level of genomic abnormalities defined by a pronounced clustering of mutations and frequent genomic loss. However, the role of Slit2 in SCLC has not been defined yet. By using genetically engineered human SCLC cells overexpressing Slit2 or recombinant Slit2 (rSlit2) protein, we show that Slit2 inhibits SCLC cells proliferation, migration, and invasion properties and tumor sphere formation ability of drug-resistant SCLC cells in vitro. The mechanistic studies have identified inhibition of AKT and β-catenin mediated signaling pathways by Slit2 overexpression. Next, we implanted human SCLC cell lines into the immunocompromised NSG mice subcutaneously followed by treatment with rSlit2 or sterile saline. Analysis of tumor volume over the 4 weeks showed that rSlit2 treatment significantly reduced the growth of SCLC tumors. Further analysis of tumors showed that rSlit2 treated tumors harbor less M2-like CD206+ macrophages. Overall, these results suggest that Slit2 possesses tumor suppressive properties and could be developed as a novel therapeutic agent against SCLC. Citation Format: Dinesh K. Ahirwar, Manish Charan, Swati Misri, Konstantin Shilo, Ramesh Ganju. Slit2 inhibits small cell lung cancer by targeting tumor microenvironment [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 2557.

Abstract 2153: ATR inhibition augments the efficacy of the lurbinectedin in small cell lung cancer

Abstract Small cell lung cancer (SCLC) is a recalcitrant disease with only a 9% five-year survival rate. SCLCs display intertumoral heterogeneity; high neuroendocrine (NE) tumors evolve with treatment and time in a largely MYC driven manner to a chemotherapy resistant non-NE state. Lurbinectedin, an RNA Pol-II inhibitor which causes DNA damage through binding ERCC5 was approved as second line therapy in 2020, with an overall response rate of 35% in patients with relapsed SCLC. However, these responses are short-lived and the majority of patient still succumb to their disease. A trial testing the combination of lurbinectedin and doxorubicin failed to improve survival compared with standard-of-care. We assessed lurbinectedin in combination with 43 other agents in SCLC cells. The top synergistic hit was the clinically relevant ATR inhibitor berzosertib which was 4-fold more synergistic than doxorubicin. Compared with lurbinectedin alone, the combination with berzosertib caused a 3.5-fold increase in DNA damage while significantly reducing the ability of cells to sense this damage by inhibiting γH2AX induction. This led to continued DNA replication even in the presence of lurbinectedin induced DNA damage, causing mitotic catastrophe and cell death. The combination was highly synergistic in 6 of 9 SCLC cell lines tested. Synergy was inversely correlated with p21 RNA and protein expression. The canonical role of p21 is to inhibit G1/S transition, and we found that thymidine induced G1 arrest inhibited synergy, while p21 knock-down increased the synergy. Interestingly, we observed synergy was inversely correlated with NE differentiation. In a high NE high p21 mouse model of SCLC, lurbinectedin alone was highly effective, with the combination showing little improvement. Conversely in a non-NE low p21 mouse model, lurbinectedin treatment alone was less effective, however the addition of berzosertib was synergistic and significantly improved efficacy. Overall, this work demonstrated berzosertib augments lurbinectedin efficacy in the recalcitrant non-NE SCLC subsets, and supported the development of a clinical trial testing lurbinectedin and berzosertib in SCLC patients NCT04802174. Citation Format: Christopher William Schultz, Yang Zhang, Haiqing Fu, Yasuhisa Murai, Darawalee Wangsa, Devon Atkinson, Liton Saha, Brian Elenbaas, Chien-Fei Lee, Astrid Zimmerman, Thomas Ried, Frank Zenke, Zoe Weaver, Rajaa Elmeskini, Yves Pommier, Anish Thomas. ATR inhibition augments the efficacy of the lurbinectedin in small cell lung cancer [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 2153.

Abstract 2565: CDK9 inhibitor KB-0742 is active in preclinical models of small-cell lung cancer

Abstract Small-cell lung cancer (SCLC) is a highly aggressive malignancy, with a 5-year survival rate below 5%. In contrast to non-small cell lung cancer, in which treatment advancements have shown much success, there has not been significant progress in the development of new therapies for SCLC over the last 40 years. Thus, an urgent need exists to identify new and effective pharmacological interventions for these patients. Recent advancements in the understanding of the pathology of the disease has shown that SCLC tumorigenesis and evolution is governed by increased expression of neuroendocrine-associated and other proto-oncogenic transcription factors such as ASCL1, NEUROD1, and MYC. Thus, targeting transcription may be an effective therapeutic strategy. Cyclin-dependent kinase 9 (CDK9) is a serine-threonine kinase that is involved in transcriptional elongation through the phosphorylation of the RNA polymerase II. Additionally, CDK9 interacts with transcription factors such as MYC to promote activation of target genes. Inhibition of CDK9 results in disrupted transcriptional networks and inhibition of cancer cell proliferation and survival, making CDK9 an attractive target for the treatment of SCLC. We have developed a highly selective and orally bioavailable inhibitor of CDK9, KB-0742. Here we present the evaluation of KB-0742 activity in preclinical models of SCLC. Using the Broad PRISM platform, we evaluated the sensitivity of 24 SCLC cell lines. We observed a correlation between MYC copy number amplification and sensitivity with amplified lines having smaller AUC values than non-amplified lines (Mann-Whitney-Wilcoxon; P>0.05). We observed a similar trend of lower IC50 values with increasing levels of MYC expression in a separate cell-line screen that included 5 SCLC lines (Pearson’s correlation; P=0.71). We then evaluated KB-0742 activity in a panel of 6 patient-derived organoid (PDO) SCLC models with different treatment histories and compared to 4 standard-of-care (SOC) compounds. KB-0742 was more active than the SOC compounds, with maximum inhibition rates of over 90% for 5 of the 6 models. In a separate study of 4 treatment-naïve PDO models, KB-0742 was active in 3 different transcription factor-driven subtypes of SCLC, and the response correlated significantly with c-MYC and MYCL expression. Lastly, we used 4 patient-derived xenograft (PDX) models to evaluate KB-0742 activity in vivo. The tumor growth inhibition (TGI) rate ranged from 54% to 92%, with tumor regressions observed in 2 of the 4 models, and 1 model showed greater TGI with KB-0742 than the SOC. These data support the development of KB-0742 as a potential treatment for SCLC. Patients with relapsed or refractory solid tumors or non-Hodgkin lymphoma are currently being enrolled in a phase 1/2 clinical trial of KB-0742 (NCT04718675) with an expansion arm for SCLC being planned after the recommended phase 2 dose is identified. Citation Format: Melinda A. Day, Douglas C. Saffran, Tressa Hood, Nikolaus Obholzer, Akanksha Pandey, Charles Y. Lin, Pavan Kumar, Jorge DiMartino. CDK9 inhibitor KB-0742 is active in preclinical models of small-cell lung cancer [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 2565.

Abstract 3473: Use of DNA methylation from tumor and plasma to identify four major small cell lung cancer subtypes with distinct biology and therapeutic vulnerabilities

Abstract Small cell lung cancer (SCLC) is a highly aggressive cancer with limited treatment options and generally poor prognosis. Treatment of SCLC has not considerably changed over the last decades with therapeutic options focusing on unselected populations. Although in the past SCLC was thought to be a relatively homogenous malignancy, recent reports from our group and others identified four major distinct subgroups of SCLC, each with different therapeutic vulnerabilities. Three of the subtypes are defined by the expression of a specific transcription factor, ASCL1 (SCLC-A), NEUROD1 (SCLC-N) and POU2F3 (SCLC-P) while the fourth subtype is defined by an inflamed phenotype (SCLC-I). While our initial subtyping of SCLC is based on a gene expression signature comprised of ~1300 genes, which makes routine implementation challenging, we hypothesized that DNA methylation as a proxy to gene expression might be a more suitable approach for biomarker development in SCLC. We assembled a cohort of 105 SCLC formalin-fixed paraffin embedded (FFPE) samples (82/105 Stage > IIIb) and performed matched RNA-Sequencing (RNAseq) and methylation profiling using reduced-representation bisulfite sequencing (RRBS). To validate our findings and expand our analysis across different sample types, we profiled a panel of 59 fully characterized SCLC cell lines as well as 68 patient-derived xenograft models. We found that methylation levels differ markedly between the four subtypes, with the SCLC-N presenting with a hypermethylated phenotype and the SCLC-P with a hypomethylated phenotype across the genome, highlighting the profound differences in the underlying epigenetic regulation among the SCLC subtypes and supporting DNA methylation analysis as a potential readout for identifying SCLC subtypes. Furthermore, in order to subtype the clinical SCLC samples, we developed a predictive model using an extreme gradient boost model using RNA expression and DNA methylation, respectively, to allow the classification with 94.5% accuracy in the tissue testing cohort. Using a cohort of matched plasma samples, we further demonstrated that the DNA methylation differences were indeed preserved in cell-free DNA (cfDNA) allowing subtype classification with an accuracy of 87.5%. These data indicate that DNA methylation can be used for reliable subtyping of SCLC in tissue and in liquid biopsy samples. In summary, using a large cohort of predominantly extensive stage SCLC clinical samples, we were able to identify profound differences in DNA methylation that can be exploited as a novel biomarker for the classification of SCLC into four distinct subtypes with both tissue biopsy and non-invasive using plasma. Considering the previously shown therapeutic vulnerabilities of the four subtypes, these findings will enable the rapid initiation of personalized clinical trials in SCLC. Citation Format: Simon Heeke, Carl M. Gay, Marcos R. Estecio, Allison Stewart, Hai Tran, Bingnan Zhang, Ximing Tang, Gabriela Raso, Kyle Concannon, Luana Guimaraes De Sousa, Whitney E. Lewis, Monique Nilsson, Yuanxin Xi, Lixia Diao, Qi Wang, Jianjun Zhang, Jing Wang, Ignacio I. Wistuba, Lauren A. Byers, John V. Heymach. Use of DNA methylation from tumor and plasma to identify four major small cell lung cancer subtypes with distinct biology and therapeutic vulnerabilities [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 3473.

Myc manipulates the miRNA content and biologic functions of small cell lung cancer cell-derived small extracellular vesicles.

MYC genes are amplified/overexpressed in 20% of SCLCs, showing that Myc and Myc-dependent cellular mechanisms are strong candidates as therapeutic targets in SCLC. Small extracellular vesicles support the carcinogenesis process by acting as messengers delivering nucleic acids and proteins-moreover, no reports associate Myc and the functional effect of small extracellular vesicles in small cell lung cancer. After the effects of small extracellular vesicles (sEVs) obtained from H82 and H209 cells on HUVEC and MRC-5 cells were observed, the Myc-dependent effect of the sEVs on oncogenic potentials was further evaluated by manipulating Myc expression via lentiviral vectors in H82 and H209 cells. Then, small extracellular vesicles of Myc-manipulated SCLC cells were isolated using sEVs isolation reagents. Finally, HUVEC and MRC5 cells were treated with SCLC-derived small extracellular vesicles. Cellular activity of recipient normal lung cells was investigated by cell growth assay, wound healing assay, and transwell assay. miRNA composition changes in small extracellular vesicles and SCLC cells were investigated using miRNA microarray and QRT-PCR assay. Our results indicated that normal lung cells treated with SCLC-derived small extracellular vesicles had higher proliferation, migration capability than non-treated counterparts. Additionally, after investigating the potential effects of small extracellular vesicles derived from Myc-dysregulated SCLC cell lines, we further evaluated the Myc-dependent miRNA composition in the small extracellular vesicles. The present study revealed that Myc regulates hsa-miR-7, hsa-miR-9, hsa-miR-125b, hsa-miR-181a_2, hsa-miR-455, hsa-miR-642, and hsa-miR-4417 expressions in SCLC cell lines, not only in cellular but also in exosomal content. Small extracellular vesicles and MYC are essential targets for therapeutic strategy in SCLC. Our study revealed that the expression level of MYC can affect the function of sEVs and encapsulate the miRNA composition in SCLC. Besides, small extracellular vesicles derived from SCLC cells can modulate normal lung cells.

Targeting Lysine-Specific Demethylase 1 Rescues Major Histocompatibility Complex Class I Antigen Presentation and Overcomes Programmed Death-Ligand 1 Blockade Resistance in SCLC

IntroductionSCLC is a highly aggressive neuroendocrine tumor that is characterized by early acquired therapeutic resistance and modest benefit from immune checkpoint blockade (ICB). Repression of the major histocompatibility complex class I (MHC-I) represents a key mechanism driving resistance to T cell-based immunotherapies. MethodsWe evaluated the role of the lysine-specific demethylase 1 (LSD1) as a determinant of MHC-I expression, functional antigen presentation, and immune activation in SCLC in vitro and in vivo through evaluation of both human SCLC cell lines and immunocompetent mouse models. ResultsWe found that targeted inhibition of LSD1 in SCLC restores MHC-I cell surface expression and transcriptionally activates genes encoding the antigen presentation pathway. LSD1 inhibition further activates interferon signaling, induces tumor-intrinsic immunogenicity, and sensitizes SCLC cells to MHC-I–restricted T cell cytolysis. Combination of LSD1 inhibitor with ICB augments the antitumor immune response in refractory SCLC models. Together, these data define a role for LSD1 as a potent regulator of MHC-I antigen presentation and provide rationale for combinatory use of LSD1 inhibitors with ICB to improve therapeutic response in SCLC. ConclusionsEpigenetic silencing of MHC-I in SCLC contributes to its poor response to ICB. Our study identifies a previously uncharacterized role for LSD1 as a regulator of MHC-I antigen presentation in SCLC. LSD1 inhibition enables MHC-I–restricted T cell cytolysis, induces immune activation, and augments the antitumor immune response to ICB in SCLC.

Abstract A005: Transcriptional profiling of tumor heterogeneity and chemoresistance in small cell lung cancer

Abstract Small Cell Lung Cancer (SCLC) is often a heterogeneous tumor, where multiple populations of phenotypically different cells exist and contribute differentially to tumor dynamics. This tumor is characterized by a very low 2- year survival rate, high metastatic rates, and rapid acquisition of chemoresistance. Beyond first-line chemotherapy, targeted treatment options are scarce, and none take in to consideration the complex genetics of the tumor. The heterogeneous nature of this tumor makes it difficult to study and to treat, as current attempts to understand tumor dynamics use a bulk approach that averages the contribution of all cells within a tumor. This work uses a cellular barcoding lineage tracing approach combined with single-cell RNA sequencing (scRNA-seq) to understand tumor heterogeneity and evolution of SCLC in both a xenograft of human SCLC cell lines and an in vivo system using a genetically modified mouse model with alterations in the same driver genes as the majority of humans with SCLC. Tumors are sampled pre-growth, post-growth and metastasis, and pre- and post- chemotherapy. By paring the barcode populations that exist in samples over time, in metastasis, or in chemoresistance, a clonal evolution map of growth, metastasis, and chemoresistance can be generated, based on shared barcodes and convergent or divergent transcriptomic profiles. In validation of scRNA-seq data, we have identified the cancer testis antigens (CTA) PAGE5 and GAGE2A as markers of chemoresistance in human SCLC. CTAs have been successfully targeted in other tumor types and may be a promising avenue for targeted therapy in SCLC. In mouse tumors, we have identified an AP-1-high population of early tumor cells that are maintained throughout the course of the disease. The knowledge gained in this work will pave the way for the identification of new therapeutic targets for SCLC. Citation Format: Hannah Wollenzien, Yohannes Afeworki, Michael S. Kareta. Transcriptional profiling of tumor heterogeneity and chemoresistance in small cell lung cancer [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr A005.

Targeting LSD1 rescues MHC class I antigen presentation and promotes immune checkpoint blockade response in small cell lung cancer

Abstract Purpose Small cell lung cancer (SCLC) is an aggressive tumor with modest clinical response to immune checkpoint blockade (ICB). Repression of major histocompatibility class I (MHC-I) molecules represents a key mechanism driving ICB resistance in SCLC. Recent studies have kindled interests in the lysine-specific demethylase 1 (LSD1) as an immunomodulatory target. Our study investigated the potential functions of LSD1 in regulating MHC-I antigen presentation in SCLC. Method We employed the inhibitor ORY-1001 and RNA interference to assess changes in MHC-I expression in SCLC cell lines by flow cytometry. We then performed RNA-seq to characterize whole transcriptomic changes in SCLC cells following LSD1 inhibition. To explore effects of targeting LSD1 on T cell cytolysis, we co-cultured SCLCs engineered to present endogenous peptides with pre-activated cognate CD8+ T cells. Finally, we treated immunocompetent mice bearing syngeneic SCLC tumors with ORY-1001 and/or anti-PD-L1 to evaluate tumor growth and characterize intratumor immune activities. Result We first demonstrated that targeting LSD1 restores MHC-I cell surface expression, transcriptionally activates APP-regulatory genes, and activates tumor intrinsic immunity. We further show that LSD1 inhibition of SCLC cells enables for efficient MHC-I-restricted T cell-mediated cytolysis. Finally, the addition of LSD1 inhibitor to anti-PD-L1 therapy significantly augments intratumor CD8+ T cell infiltrates and sensitizes refractory SCLC model to ICB response. Conclusion Our data define a potent regulatory role of LSD1 in MHC-I antigen presentation and provide support for targeting LSD1 to overcome immune evasion in SCLC. Supported by NIH NCI R01 CA197936 (CMR); U24 CA213274 (CMR); the Druckenmiller Center for Lung Cancer Research (CMR, TS); NIH/NCI R01 CA258784 (TS); Congressionally Directed Medical Research Programs (DOD-IITRA) LC190161 (TS); Parker Institute for Cancer Immunotherapy grant (TS); LCFA-BMS/ILC Foundation Young Investigator Research Awards in Translational Immuno-oncology (TS); the Geoffrey Beene Foundation (EMN); The Yasuda Medical Foundation (HT); NIH K08 CA-248723 (AC); and the Van Andel Institute – Stand Up to Cancer Epigenetics Dream Team grant (CMR). Stand Up to Cancer is a division of the Entertainment Industry Foundation. Research grants are administered by the American Association for Cancer Research, the Scientific Partner of SU2C. We also acknowledge the use of the MSKCC Flow Cytometry Core Facility, the MSKCC Antitumor Assessment Core, and the Integrated Genomics Core, which are funded by MSKCC Support Grant/Core Grant (P30 CA008748).

High mRNA expression of POU2F3 in small cell lung cancer cell lines predicts the effect of lurbinectedin

BackgroundSmall cell lung cancer (SCLC) is a progressive disease with a poor prognosis. Recently, a method to classify SCLC by the expression status of four transcription factors, ASCL1, NEUROD1, POU2F3, and YAP1, was proposed. Here, we investigated the potential relationships between expression of these four transcription factors and the effect of lurbinectedin.MethodsmRNA and protein expression of ASCL1, NEUROD1, POU2F3, and YAP1 were quantified in eight SCLC cell lines and analyzed for potential correlations with drug sensitivity. In addition, ASCL1, NEUROD1, POU2F3, and YAP1 expression were evaluated in 105 resected cases of high‐grade neuroendocrine carcinoma of the lung, including 59 resected cases of SCLC.ResultsBased on the results of qRT‐PCR and western blot analyses, the eight SCLC cell lines examined were classified into NEUROD1, POU2F3, and YAP1 subtypes, as well as five ASCL1 subtypes. There were no correlations between cell line subtype classification and drug sensitivity to cisplatin, etoposide, or lurbinectedin. Next, we compared relative mRNA expression levels of each transcription factor with drug sensitivity and found that the higher the mRNA expression level of POU2F3, the lower the IC50 of lurbinectedin. Evaluation of resected SCLC tissue revealed that the composition of subtypes defined by the relative dominance of ASCL1, NEUROD1, POU2F3, and YAP1 was as follows: 61% ASCL1, 15% NEUROD1, 14% POU2F3, 5% YAP1, and 5% all‐negative.ConclusionIn our experiments, high mRNA expression of POU2F3 in SCLC cell lines correlated with the effect of lurbinectedin.

Inhibition of XPO1 Sensitizes Small Cell Lung Cancer to First- and Second-Line Chemotherapy.

Small cell lung cancer (SCLC) is an aggressive malignancy characterized by early metastasis and extreme lethality. The backbone of SCLC treatment over the past several decades has been platinum-based doublet chemotherapy, with the recent addition of immunotherapy providing modest benefits in a subset of patients. However, nearly all patients treated with systemic therapy quickly develop resistant disease, and there is an absence of effective therapies for recurrent and progressive disease. Here we conducted CRISPR-Cas9 screens using a druggable genome library in multiple SCLC cell lines representing distinct molecular subtypes. This screen nominated exportin-1, encoded by XPO1, as a therapeutic target. XPO1 was highly and ubiquitously expressed in SCLC relative to other lung cancer histologies and other tumor types. XPO1 knockout enhanced chemosensitivity, and exportin-1 inhibition demonstrated synergy with both first- and second-line chemotherapy. The small molecule exportin-1 inhibitor selinexor in combination with cisplatin or irinotecan dramatically inhibited tumor growth in chemonaïve and chemorelapsed SCLC patient-derived xenografts, respectively. Together these data identify exportin-1 as a promising therapeutic target in SCLC, with the potential to markedly augment the efficacy of cytotoxic agents commonly used in treating this disease. SIGNIFICANCE: CRISPR-Cas9 screening nominates exportin-1 as a therapeutic target in SCLC, and exportin-1 inhibition enhances chemotherapy efficacy in patient-derived xenografts, providing a novel therapeutic opportunity in this disease.

CAR NK-92 cells targeting DLL3 kill effectively small cell lung cancer cells in vitro and in vivo.

Small cell lung cancer (SCLC) is characterized by a high relapse rate, drug tolerance, and limited treatment choices. Chimeric antigen receptor (CAR)-modified NK cells represent a promising immunotherapeutic modality for cancer treatment. However, their potential applications have not been explored in SCLC. Delta-like ligand 3 (DLL3) has been reported to be overexpressed in SCLC and may be a rational target for CAR NK immunotherapy. In this study, we developed DLL3-specific NK-92 cells and explored their potential in the treatment of SCLC. A coculture of DLL3+ SCLC cell lines with DLL3-CAR NK-92 cells exhibited significant in vitro cytotoxicity and cytokine production. DLL3-CAR NK-92 cells induced tumor regression in an H446-derived pulmonary metastasis tumor model under a good safety threshold. The potent antitumor activities of DLL3-CAR NK-92 cells were observed in subcutaneous tumor models of SCLC. Moreover, obvious tumor-infiltrated DLL3-CAR NK-92 cells were detected in DLL3+ SCLC xenografts. These findings indicate that DLL3-CAR NK-92 cells might be a potential strategy for the treatment of SCLC.

FK228 potentiates topotecan activity against small cell lung cancer cells via induction of SLFN11.

The response rate of topotecan, as a second-line chemotherapeutic drug for small cell lung cancer, is ~20%. DNA/RNA helicase SLFN11 (schlafen family member 11), a member of the Schlafen (SLFN) family, is a crucial determinant of response to many DNA damaging agents, expression of SLFN11 tends to augment the antitumor effects of the commonly used DNA-targeting agents. In the present study we investigated how SLFN11 expression regulated the sensitivity of small cell lung cancer to topotecan. We showed that SLFN11 expression levels were positively associated with the sensitivity to topotecan in a panel of seven SCLC cell lines. Topotecan treatment induced different patterns of the DNA response network in SCLC cells: DNA damage response (DDR) was more prominently activated in SLFN11-deficient SCLC cell line H82 than in SLFN11-plentiful SCLC cell line DMS273, whereas topotecan induced significant accumulation of p-Chk1, p-RPA2 and Rad51 in H82 cells, but not in DMS273 cells. We unraveled that SLFN11 expression was highly negatively correlated to the methylation of the SLFN11 promoter. HDAC inhibitors FK228 and SAHA dose-dependently increased SLFN11 expression through suppressing DNA methylation at the SLFN11 promoter, thereby sensitizing SCLC cells to topotecan. Finally, we assessed the methylation status of the SLFN11 promoter in 27 SCLC clinical specimens, and found that most of the clinical samples (24/27) showed DNA methylation at the SLFN11 promoter. In conclusion, it is feasible to combine topotecan with FK228 to improve the response rate of topotecan in SCLC patients.

CCL19 associates with lymph node metastasis and inferior prognosis in patients with small cell lung cancer

ObjectiveSmall cell lung cancer (SCLC) is a systemic disease and most patients have metastases at diagnosis. Better understanding of the underlying mechanisms of SCLC metastasis may provide potential approach to improve clinical outcome. MethodsHTG Edge-seq was used to identify the differential gene expression between primary SCLC lesions and paired metastatic lymph nodes (LN). Overall survival (OS) analysis was performed in patients with different levels of plasma CCL19 concentration. Invasion, migration, proliferation, apoptosis and angiogenesis ability of SCLC cells and function of CD8 + T cells were evaluated in vitro to investigate the mechanism of CCL19 in promoting metastasis. ResultsFour chemokines (CCL19, CCL21, CCL8, CCR1) were the most differentially expressed between primary lesions and metastatic LN. CCL19 was further investigated because its mRNA and protein level expression were also validated in four SCLC cell lines (H446, H69, H82, H196). Higher plasma CCL19 was associated with late lymph node (N3) metastasis (training cohort P = 0.044, validation cohort P = 0.020) and shorter OS (training cohort P = 0.040, validation cohort P = 0.047) in SCLC patients. Silencing CCL19 inhibited SCLC cell migration, invasion, proliferation and HUVECs tube formation. Furthermore, we found that CCL19 could decrease percentage of CD8 + Ki67 + and CD8 + GZMB + T cells and increase proportion of CD8 + PD1 + T cells. ConclusionCCL19 was associated with LN metastasis and poor prognosis in patients with SCLC. Its expression promoted tumor progression and metastasis and impaired the function of CD8 + T cells, suggesting CCL19 might be a potential target for SCLC.

Abstract 2436: Heterogeneity of neurogenic transcription factor expression in small cell lung cancer

Abstract SCLC is an aggressive neuroendocrine (NE) cancer with a 7% 5-year survival rate. Molecular characterization of this disease has lagged behind other cancer types and standard of care treatment has remained largely unchanged for over three decades. SCLC is treated as a clinically homogenous disease, but evidence of its intra- and inter-tumor heterogeneity is increasing. Part of this heterogeneity is attributed to expression of neurogenic transcription factors (TFs) ASCL1 and NEUROD1, which correlate with SCLC pathogenesis and NE status, and are now included in a consensus for SCLC classification [1]. Our Circulating tumor cell patient Derived eXplant (CDX) models recapitulate disease heterogeneity in terms of neurogenic TF expression; furthermore, we recently identified a previously unrecognized subset within our CDX model panel expressing another neurogenic TF, ATOH1, mostly within NEUROD1 expressing models [2]. I am investigating the roles of the three NE TFs in SCLC CDX, to refine disease classification and explore rationale for new therapeutic approaches stratified to SCLC phenotypes. To do so, we have characterized ASCL1 and NEUROD1 expression in CDX models with multiplex immunofluorescence and flow cytometry. We have begun to interrogate the biology of ASCL1 and NEUROD1 in established SCLC cell lines via CRISPR- and shRNA-mediated genetic depletion of these TFs. Furthermore, we have recently developed antibodies to ATOH1 to support interrogation of its under-researched role in SCLC biology. We have found that CDX models express ASCL1 (30/38), NEUROD1 (3/38) and ATOH1 together with NEUROD1 (4/38) [2]. ASCL1 and NEUROD1 are co-expressed in several CDX but (with the exception of one CDX model) in different cell subpopulations. In contrast with published literature, genetic depletion of ASCL1 and NEUROD1 in established SCLC cell lines did not influence cell proliferation and response to chemotherapy. I have recently generated specific antibodies to ATOH1 and ongoing studies are focused on characterization of ATOH1 expression in CDX models and its impact on the pathogenesis and chemotherapy responses in SCLC. In conclusion, ASCL1 and NEUROD1 are expressed in a mutually exclusive way in CDX models, with the exception of one CDX; manipulation of their expression does not influence cell proliferation or response to chemotherapy in SCLC cell lines. Studies looking at the consequences of genetic depletion of these TFs, including ATOH1, in CDX models are underway. [1] Rudin et al., Nature Reviews Cancer, 2019 [2] Simpson et al., Nature Reviews Cancer, 2020 Citation Format: Alessia Catozzi, Mitchell Revill, Fiona Blackhall, Kristopher Frese, Kathryn Simpson, Caroline Dive. Heterogeneity of neurogenic transcription factor expression in small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2436.

Abstract 1022: Characterization of lurbinectedin as a single agent and in combinations with DNA damage response inhibitor for the treatment and bio-marker discovery of SCLC

Abstract Small-cell lung cancer (SCLC) is an aggressive neuroendocrine lung malignancy which is primarily driven by loss of function of tumor suppressor gene TP53 and RB1 and accounts for around 13-15% of all lung cancers. For metastatic SCLC the standard-of-care, first-line therapy is combination of platinum-based therapy with etoposide or with irinotecan. Though response rate in first-line chemotherapy in SCLC is very high, relapse is almost universal. For decades topotecan, a topoisomerase-I inhibitor was the only FDA approved second-line treatment in SCLC. However, on June 15th, 2020 the FDA approved lurbinectedin for treatment of patients with metastatic SCLC with disease progression from platinum-based chemotherapy and designated it as an orphan drug. Lurbinectedin (PM01183) is a synthetic analog of the natural marine-based tetrahydroisoquinoline, trabectedin which comes from the sea-squirt species Ecteinascidia turbinate. Lurbinectedin blocks transcription by inhibiting the activity of RNA-polymerase-II and inducing its specific degradation by the ubiquitin/proteasome machinery, also inducing DNA damage. Emerging data from our group, and others supports that SCLC is transcriptionally addicted via one of three main transcription factors ASCL1 (A), NeuroD1 (N) & POU2F3 (P), which may contribute to the promising results observed in SCLC trials to date with lurbinectedin. However, there are currently no established biomarkers to predict SCLC sensitivity or resistance to lurbinectedin. Furthermore, little is known regarding molecular changes in SCLC cells or other tumors upon exposure to lurbinectedin. In this preclinical study we investigated the therapeutic efficacy of lurbinectedin in large number of profiled SCLC cell lines representing the four SCLC subtypes (A, N, P and I) to identify candidate markers of drug sensitivity and resistance. In 12 human-derived and 3 mouse model-derived SCLC cell lines, the majority of cell lines were highly sensitive to lurbinectedin at a very low doses (median IC50 0.52 nM, range 0.08-1.84nM). We also observed increased markers of DNA damage following treatment in sensitive cell lines (e.g., γH2AX, ChK1, RPA32) by western blot. Notably, no subtype specific difference in lurbinectedin sensitivity was observed among the four subtypes (A, N, P and I) of SCLC cells. However, cell lines with higher SLFN11 expression were more sensitive to lurbinectedin. Western blot experiments showed significant increase in phosphorylation of γH2AX, ChK1, RPA32 in lurbinectedin treated SCLC cells compared to DMSO treatment. Together our preliminary data confirm lurbinectedin as a potent treatment option for SCLC, and support a rationale for potential combinations with other DNA damaging agents. Citation Format: Kiran Kundu, Robert J. Cardnell, Li Shen, C. Allison Stewart, Kasey Cargill, Carl M. Gay, Jing Wang, Lauren A. Byers. Characterization of lurbinectedin as a single agent and in combinations with DNA damage response inhibitor for the treatment and bio-marker discovery of SCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1022.

Abstract 1128: Selective, novel, small molecule PRMT5 inhibitors for treatment of cancer

Abstract Introduction: Arginine methylation deregulation in cancer has been well studied and PRMT5 which modulates dimethylation of arginine has emerged as an attractive therapeutic strategy in various cancer types, including lung cancer, lymphoma, glioblastoma, pancreatic cancers, etc. PRMT5 is over-expressed in multiple cancers leading to repression of tumor suppressor genes and genetic studies have identified it is a validated target in lymphoma. Recently, dysregulation of the splicing machinery in cancers has been identified to be one of the therapeutic vulnerabilities for PRMT5 inhibition, especially in glioblastoma. Therefore, inhibitors selectively targeting PRMT5 could be of high clinical value, especially in cancers with defects in spliceosome machinery. Methods: Rational design and structure based drug design were used to identify novel PRMT5 inhibitors. To assess in vitro potency, flash plate based activity assay was used. Cell based activity of these inhibitors was assessed by measuring the symmetrical dimethylation of known cellular protein SmD3 by ELISA and Western blotting. Long term cell proliferation assays were used to assess the functional effect of PRMT5 inhibition. Tumor growth inhibition was measured in orthotopic glioblastoma model in mice. Results: One of the lead PRMT5 inhibitors had an in vitro potency of 3 nM in the biochemical assay which translated well in the cell based SDMA ELISA where the EC50 was <10 nM. Anti-proliferative activity of this molecule in lymphoma, leukaemia, SCLC, pancreatic, lung and glioblastoma cell lines ranged from ~16 nM to 1035 nM. This molecule showed acceptable in vitro ADME properties in terms of aqueous solubility and metabolic stability and excellent oral bioavailability in rodent pharmacokinetics. In Z-138 mantle cell lymphoma xenograft model, oral administration of the lead compound at 50 mg/kg resulted in strong and complete (~95 %) tumour growth inhibition and with a concomitant complete inhibition of SDMA. The lead compound was well tolerated with no reduction in body weight at the tested doses. Interestingly, this molecule showed excellent brain exposure sufficient to achieve target engagement for 10h and significant tumour growth inhibition of orthotopic brain tumors by oral dosing. Repeat dose non-GLP study in rodents clearly demonstrated the safety of this molecule. Conclusion: Given the therapeutic importance of PRMT5 in glioblastoma and other lymphomas, this molecule will be extremely valuable in treating these cancers both as a standalone therapy and in combination with other standard of care agents. Citation Format: Dhanalakshmi Sivanandhan, Sridharan Rajagopal, Naveen Sadhu M, Chandru Gajendran, Saravanan Vadivelu, Natarajan Tamizharasan, Indu N. Swamy, Santhosh Viswakarma, Amir Siddiqui, Saif wahid, Mohammed Zainuddin, Rudresh G, Prashanthi Daram, Ramchandraiah Gosu, Dinesh Tiagaraj, Shivani Garapaty, Sreekala Nair, Namratha Kapoor. Selective, novel, small molecule PRMT5 inhibitors for treatment of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1128.

Abstract 3107: HSB-1216, A Novel Agent for the Treatment of Chemotherapy-Resistant ES-SCLC

Abstract Small cell lung cancer (SCLC) is a highly malignant form of lung cancer that accounts for 15% of lung cancer cases. The tumor has a tendency to disseminate early resulting in ~ 85% patients being diagnosed with extensive stage disease (ES-SCLC). Chemotherapy has been the mainstay of treatment for many decades, until recently when immunotherapeutic agents have been approved as first-line agents. However, resistance to anti-cancer drugs represents the main cause of cancer-related deaths. Thus, new therapeutic approach to suppress chemotherapy-resistant ES-SCLC constitutes a significant unmet medical need. We have generated novel biodegradable QUATRAMERTM polymeric nanoparticles comprised of a polyethylene glycol (PEG)-polypropylene glycol (PPG)-PEG- polylactic acid (PLA)-tetra-block copolymer for the intracellular delivery of salinomycin (HSB-1216). Here we studied the HSB-1216 activity in a resistant SCLC cell line (NCI-H69AR). Compared to cisplatin and irinotecan, a second-line agent often used in ES-SCLC, HSB-1216 exhibited a ~7-fold and ~2-fold decrease in IC50in the aggressive and resistant variant of NCI-H69AR cell line and ~30-fold and ~18-fold decrease in a non-resistant NCI-H69 SCLC line. The tumorsphere formation assay has become an essential tool for quantifying cancer stem cells (CSC) and testing of anti-cancer agents. We used this assay to estimate an effect of HSB-1216 on NCI-H69AR tumorspheres .Our results demonstrated significant dose-dependent inhibition of tumorosphere formation in response to HSB-1216. In addition, exposure to HSB-1216 resulted in substantial decrease in number of CD44+/CD24- cells in comparison to untreated control. Furthermore, significant reduction in the expression of CSC markers such as ALDH1 and Oct3/Oct4 was seen when chemotherapy-resistant NCI-H69AR cells were treated with HSB-1216.Taken together, based on these findings, HSB-1216 is being developed as a novel agent in the treatment of second-line ES-SCLC in chemo-resistant tumors, where a gap exists in current standards of care. Further work of this novel agent is planned to outline its effect when combined with targeted and non-targeted therapies in both first- and third-line SCLC. Citation Format: Anees Mohammad, Sachchidanand Tiwari, Neha Mehrotra, Bozena Korczak, Steve Laumas, Sireesh Appajosyula, Harpal Singh, Surender M. Kharbanda. HSB-1216, A Novel Agent for the Treatment of Chemotherapy-Resistant ES-SCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3107.

Abstract 2335: Targeting MYC-enhanced glycolysis in small cell lung cancer

Abstract Small cell lung cancer (SCLC) accounts for approximately 15% of lung cancer cases with a poor five year survival rate of 6%. The National Cancer Institute has designated SCLC as one of two “recalcitrant” cancers and urges translational research to advance treatment options. In our effort to maximize treatment benefits, we have aimed our investigation at uncovering metabolic differences contributing to disease progression that may be targeted through specific pathway inhibition. Although metabolism is relatively unstudied in SCLC, metabolic reprogramming is recognized as a hallmark of cancer and can be regulated by several different mechanisms. Notably, the transcription factor MYC is overexpressed in 30% SCLC tumors and is known to modulate the balance between two major pathways of metabolism: glycolysis and mitochondrial respiration. While mitochondrial respiration is the preferred source of energy production in terminally differentiated cells, many cancer cells switch to become predominantly glycolytic in an effort to generate energy quickly and produce biomolecules and electron carriers required for proliferation—a phenomena known as the Warburg Effect. Since MYC is expressed in only a subset of highly aggressive SCLC, we applied bimodal separation to both patient and cell line SCLC datasets to establish defined MYC subsets (MYCLow; MYCHigh) and performed gene ontology pathway analysis to genes that were significantly upregulated among the MYC-expressing samples. This revealed that 37% of significantly upregulated genes were linked to metabolic processes and further investigation showed many of those genes were linked to the glycolysis pathway. We also confirmed upregulation of protein expression through reverse phase protein array (RPPA). With glycolysis proving to be increased in MYCHigh samples, we simultaneously characterized pathway utilization in cell lines at baseline and in the presence of a potent glycolysis inhibitor. MYCHigh cell lines exhibited greater glucose consumption and lactate secretion, which was significantly decreased by glycolytic inhibition. Likewise, mitochondrial analysis revealed lower oxygen consumption and ATP production that were further reduced in the presence of the glycolysis inhibitor. Although mitochondrial density was unchanged regardless of MYC expression or glycolytic inhibition, reactive oxygen species (ROS) generation was greatly enhanced in the MYCHigh subset upon suppression of glycolysis. Lastly, SCLC xenografts derived from a MYCHigh SCLC cell line showed significantly slower tumor growth whereas MYCLow derived xenografts exhibited no significant difference in tumor growth. Together, these data provide evidence of metabolic differences among SCLC subsets such that MYC expression induces reliance on glycolysis, which can be targeted for therapeutic intervention. Citation Format: Kasey R. Cargill, C. Allison Stewart, Elizabeth M. Park, Robert J. Cardnell, You Hong Fan, Qi Wang, Lixia Diao, Wai Kin Chan, Philip L. Lorenzi, Jing Wang, Lauren A. Byers. Targeting MYC-enhanced glycolysis in small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2335.

Abstract 1906: Relationship between neuroendocrine and immune gene expression in small cell lung cancer

Abstract Small cell lung cancer (SCLC) is classified as a high-grade neuroendocrine (NE) tumor, but a subset of SCLC has been termed “variant” due to the loss of NE characteristics. In this study, we computed NE scores for patient-derived SCLC cell lines and xenografts, as well as human tumors. We aligned NE properties with transcription factor-defined molecular subtypes. Then we investigated the different immune phenotypes associated with high and low NE scores. We found repression of immune response genes as a shared feature between classic SCLC and pulmonary neuroendocrine cells of the healthy lung. With loss of NE fate, variant SCLC tumors regain cell-autonomous immune gene expression and exhibit higher tumor-immune interactions. Pan-cancer analysis revealed this NE lineage-specific immune phenotype in other cancers. Additionally, we observed MHC I re-expression in SCLC upon development of chemoresistance. These findings provide a new framework to guide design of treatment regimens in SCLC. Citation Format: Ling Cai, Hongyu Liu, Fang Huang, Junya Fujimoto, Luc Girard, Yang Xie, John Minna, Adi Gazdar. Relationship between neuroendocrine and immune gene expression in small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1906.

Possible involvement of the Hedgehog and PDPK1-Akt pathways in the growth and migration of small-cell lung cancer.

BackgroundSmall-cell lung cancer (SCLC) accounts for approximately 15% to 20% of all lung cancers, and it is the leading cause of tumor-related deaths globally. This study explored the molecular mechanisms underlying the development of SCLC.MethodsThe correlations of phosphoinositide-dependent kinase-1 (PDPK1), p-Akt, and Hedgehog expression with patient characteristics were analyzed using SCLC specimens, and their expression was measured in BEAS-2B cells (control) and the SCLC cell lines H82, H69, H446, H146, and H526. Transfection experiments were performed to inhibit or activate gene expression in cells. We then measured the proliferation and migration of H146 cells.ResultsPDPK1, p-Akt, and Hedgehog expression was significantly higher in SCLC tissues, and their expression was correlated with patient characteristics. p-Akt expression was significantly correlated with Hedgehog expression. In H146 cells, PDPK1 and p-Akt were significantly upregulated. Silencing of PDPK1 or Akt and inhibition of Hedgehog significantly inhibited the proliferation and migration of H146 cells. PDPK1 and Akt affected Hedgehog expression, but Hedgehog did not affect PDPK1 or p-Akt expression.ConclusionsThe interaction between the PDPK1–Akt pathway and the Hedgehog pathway influences the prognosis, growth, and migration of SCLC.