Human Small Cell Lung Cancer Tumors Research Articles

Abstract 6602: Targeting the SWI/SNF complex in POU2F-POU2AF transcription factor-driven malignancies

Abstract Background: Small cell lung cancer (SCLC) is a rapidly progressing subtype of lung cancer with a high growth rate and early metastasis propensity, often resulting in a more advanced disease stage at diagnosis. A recent transcriptome analysis of human SCLC tumors revealed that SCLC could be characterized by the expression pattern of certain transcription factors (TFs), including ASCL1 (achaete-scute family bHLH transcription factor 1) and POU2F3 (POU domain class 2 transcription factor 3), exemplifying SCLC as a TF-driven malignancy. ASCL1-driven SCLC (SCLC-A) manifests a neuroendocrine phenotype, while POU2F3-driven SCLC (SCLC-P) is characterized as a tuft-cell-like variant. Recent studies revealed that POU domain class 2 transcription factors uniquely rely on coactivators to achieve their lineage-defining functions in the tuft cell and B cell lineages. In tuft-cell-like SCLC cells, the coactivators of POU2F3 (POU2AF2 and POU2AF3) endow POU2F3 with a critical transactivation domain by forming a master regulator complex, which supports enhancer-mediated cancer-promoting gene activation in SCLC-P cells. This indicates a potential therapeutic vulnerability of tuft-cell-like SCLC whereby strategies aimed at blocking POU2F3-POU2AF2/3 function may lead to clinical benefit. Methods: We conducted a domain-targeted CRISPR screen to identify druggable targets for SCLC-P, followed by pharmacological validation in preclinical SCLC models. Multi-omics techniques, including ATAC-seq, ChIP-seq, RNA-seq, and RIME, were employed to elucidate the SWI/SNF complex's regulatory influence on the POU2F-POU2AF axis. Results: Here we identified that the POU2F3 molecular subtype of SCLC (SCLC-P) exhibits an exquisite dependence on the activity of the SWI/SNF epigenetic complex. SCLC-P cell lines were sensitive to low nanomolar levels of a SWI/SNF ATPase degrader when compared to other molecular subtypes of SCLC. Co-factors of POU2F were found to interact with components of the SWI/SNF complex. The POU2F3 transcription factor complex is evicted from chromatin upon SWI/SNF ATPase degradation. A novel, orally bioavailable SWI/SNF ATPase PROTAC degrader demonstrated preferential efficacy in SCLC-P relative to the SCLC-A subtype. The SWI/SNF ATPase PROTAC degrader did not alter normal tuft cell numbers in lung or colon, nor did it exhibit toxicity in mice. B cell malignancies which displayed a dependency on the POU2F co-factor, POU2AF1, were also remarkably sensitive to SWI/SNF ATPase degradation. In a POU2AF1-dependent, disseminated murine model of multiple myeloma, AU-24118 had an enhanced survival benefit as compared to pomalidomide, an approved treatment for multiple myeloma. Conclusions: Taken together, our studies suggest that POU2F-POU2AF driven malignancies have an intrinsic dependence on the SWI/SNF complex representing a striking therapeutic vulnerability. Citation Format: Lanbo Xiao, Tongchen He, Olaf Klingbeil, Young Eleanor, Xiaoli Wu, Yuanyuan Qiao, Abhijit Parolia, Sanjana Eyunni, Rahul Mannan, Somnath Mahapatra, NamHoon Kim, Heng Zheng, Fengyun Su, Xuhong Cao, Susanta Samajdar, Murali Ramachandra, Christopher R. Vakoc, Arul M. Chinnaiyan. Targeting the SWI/SNF complex in POU2F-POU2AF transcription factor-driven malignancies [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 6602.

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.

The synthetic non pungent capsaicin analog Arvanil displays robust anti‐tumor activity in human small cell lung cancer

The nutritional compound capsaicin is the hot spicy pungent ingredient of chili peppers. Capsaicin is a pain‐relieving agent and is found in several over‐the counter pain medications. However, recent studies have shown that capsaicin has profound anti‐neoplastic effects in several types of human cancers. The applications of capsaicin as a clinically useful drug are limited by its unpleasant side effects, such as gastric irritation, stomach cramps and burning sensation. This has led to extensive research focused on the identification and rational design of second‐generation capsaicin analogs, which possess greater bioactivity than capsaicin. Our laboratory performed an SAR study using a panel of non‐pungent capsaicin analogs and identified Arvanil as the hit compound. Arvanil triggered robust apoptosis in a panel of human small cell lung cancer (SCLC). The growth‐suppressive activity of Arvanil was confirmed in chicken chorioallantoic membrane (CAM) models. Finally, the dietary administration of Arvanil potently decreased the growth rate of human SCLC tumors xenotransplanted in athymic mice. The pro‐apoptotic activity of Arvanil was mediated by the TRPV1 receptor and the calpain pathway. Non‐pungent capsaicin analogs like Arvanil may be promising drug candidates for the management and treatment of human SCLC.

PAX9 Determines Epigenetic State Transition and Cell Fate in Cancer.

Abnormalities in genetic and epigenetic modifications can lead to drastic changes in gene expression profiles that are associated with various cancer types. Small cell lung cancer (SCLC) is an aggressive and deadly form of lung cancer with limited effective therapies currently available. By utilizing a genome-wide CRISPR-Cas9 dropout screen in SCLC cells, we identified paired box protein 9 (PAX9) as an essential factor that is overexpressed in human malignant SCLC tumor samples and is transcriptionally driven by the BAP1/ASXL3/BRD4 epigenetic axis. Genome-wide studies revealed that PAX9 occupies distal enhancer elements and represses gene expression by restricting enhancer activity. In multiple SCLC cell lines, genetic depletion of PAX9 led to significant induction of a primed-active enhancer transition, resulting in increased expression of a large number of neural differentiation and tumor-suppressive genes. Mechanistically, PAX9 interacted and cofunctioned with the nucleosome remodeling and deacetylase (NuRD) complex at enhancers to repress nearby gene expression, which was reversed by pharmacologic HDAC inhibition. Overall, this study provides mechanistic insight into the oncogenic function of the PAX9/NuRD complex epigenetic axis in human SCLC and suggests that reactivation of primed enhancers may have potential therapeutic efficacy in treating SCLC expressing high levels of PAX9. SIGNIFICANCE: A genome-wide screen in small cell lung cancer reveals PAX9/NuRD-mediated epigenetic enhancer silencing and tumor progression, supporting the development of novel personalized therapeutic approaches targeting the PAX9-regulated network.

Real-world multiomic characterization of small cell lung cancer subtypes to reveal differential expression of clinically relevant biomarkers.

8508 Background: The dominant expression of four lineage-defining transcription factors ( ASCL1, NEUROD1, YAP1, or POU2F3) has enabled the classification of small cell lung cancer (SCLC) into four subtypes (SCLC-A/N/Y/P, respectively). Emerging evidence suggests that YAP1 expression is associated with a T-cell inflamed phenotype, and SCLC has significant intra-tumor heterogeneity mediated by MYC-driven activation of NOTCH signaling. We performed a large-scale analysis of real-world SCLC patient samples to examine the expression of clinically relevant biomarkers across SCLC subtypes. Methods: Comprehensive molecular profiling of 437 small cell lung neuroendocrine tumors (including 7.3% high-grade neuroendocrine lung carcinomas) was performed using next-generation DNA sequencing (592-gene panel), RNA sequencing (whole transcriptome), and immunohistochemistry at Caris Life Sciences (Phoenix, AZ). Tumors were stratified into 5 subgroups (SCLC-A/N/Y/P and -mixed) based on the relative expression of the four transcription factors. RNA expression of key genes and previously validated immune signatures (T-cell inflamed, NK cell, and STING pathway signatures) were evaluated across subgroups. Significance was tested by Chi-square, Fisher’s exact test, or Mann-Whitney U test. Results: Median age of the study cohort was 66 years (IQR: 59-72) and 50.6% of patients were female. The majority (67.3%) of samples were derived from metastatic sites. Stratification of tumors by expression resulted in 35.7% SCLC-A, 17.6% SCLC-N, 21.1% SCLC-Y, 6.4% SCLC-P, and 19.2% SCLC-mixed samples. Compared to tumors from metastatic sites, YAP1 expression was significantly increased (p < 0.001) in primary tumors. Amongst the 14 tumors obtained from the CNS, SCLC-N (36%, n = 5) was the most common subtype identified. dMMR/MSI-high (negative MMR protein expression/ ≥46 altered loci per tumor) was rare overall (0.5%, n = 2); TMB (median of 9-10 mut/Mb) was similar between the SCLC subtypes. SCLC-Y was associated with the highest expression of T-cell inflamed, NK cell and STING pathway signatures (p < 0.0001 each). MYC and NOTCH gene expression ( NOTCH1/2/3/4) strongly correlated with YAP1 expression. Analysis of co-mutations revealed that EGFR-sensitizing mutations (L858R and Exon 19 deletions) were recurrent (5.2%, n = 4) in SCLC-N tumors. The expression of SNF11, SSTR2, and MYC varied significantly among SCLC subtypes (p < 0.001 each), with the highest median expression of SNF11 and SSTR2 observed in SCLC-N, while MYC expression was highest in SCLC-P. Conclusions: Our analysis represents the largest real-world dataset of human SCLC tumors profiled by whole transcriptomic sequencing. The differential expression of immune genes and predictive biomarkers across SCLC subtypes may inform therapeutic vulnerabilities for rational and personalized treatment approaches in SCLC.

Signatures of plasticity and immunosuppression in a single-cell atlas of human small cell lung cancer.

8509 Background: Small cell lung cancer (SCLC) is an aggressive malignancy that includes subtypes defined by differential expression of ASCL1, NEUROD1, and POU2F3 (SCLC-A, -N, and -P, respectively), which are associated with distinct therapeutic vulnerabilities. The emerging consensus on SCLC subtypes has led to new questions, such as whether subtypes are associated with different disease stages, metastatic potential, or immune microenvironments; whether there is plasticity between subtypes; and whether novel SCLC phenotypes exist. Single cell RNA sequencing (scRNA-seq) offers a unique opportunity to address these questions by dissecting intratumoral transcriptional heterogeneity and the surrounding tumor microenvironment (TME). However, efforts to apply this technology to human SCLC tumors have been limited, as these tumors are infrequently resected. Methods: We have optimized protocols to process both surgical resections and biopsies to construct the first single-cell atlas of SCLC patient tumors (N = 21), with comparative lung adenocarcinoma (LUAD) and normal lung data. We leverage computational methods including diffusion maps and non-negative matrix factorization to perform a deep annotation of SCLC phenotypes and the surrounding immune TME. We perform validation experiments using flow cytometry, Vectra, and immunohistochemistry in independent SCLC cohorts, as well as genetic manipulation in preclinical SCLC models. Results: Our data reveals substantial transcriptional heterogeneity in SCLC both within and across tumors and confirms a pro-metastatic gene program in SCLC-N subtype characterized by epithelial-mesenchymal transformation and axonogenesis. Beyond known subtypes, we discover a PLCG2-high tumor cell population with stem-like, pro-metastatic features that recurs across subtypes and predicts significantly worse overall survival. Manipulation of PLCG2 expression in cells confirms correlation with key metastatic markers. Treatment and subtype are associated with substantial phenotypic changes in the SCLC immune microenvironment, with greater T-cell dysfunction in SCLC-N than SCLC-A. Moreover, the recurrent, PLCG2-high subclone is associated with exhausted CD8+ T-cells and a pro-fibrotic, immunosuppressive monocyte/macrophage population, suggesting possible tumor-immune coordination to promote metastasis. Conclusions: This atlas of SCLC illustrates how canonical subtypes and a novel PLCG2-high recurrent tumor subclone enlist diverse gene programs to create tumor heterogeneity and facilitate metastasis in a profoundly immunosuppressed TME. Our dataset provides further insight into tumor and immune biology in SCLC at single-cell resolution, with potential implications for design of novel targeted therapies and immunotherapeutic approaches.

Delta-like 1 homolog (DLK1) as a possible therapeutic target and its application to radioimmunotherapy using 125I-labelled anti-DLK1 antibody in lung cancer models (HOT1801 and FIGHT004)

ObjectivesDelta-like 1 homolog (DLK1) is a non-canonical Notch ligand known to be expressed in several cancers but whose role in lung cancer is not yet fully understood. We sought to confirm DLK1 expression in small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC), and to examine DLK1’s clinical significance. Furthermore, we examined the possible utility of DLK1 as a novel target in radioimmunotherapy (RIT). MethodsWe retrospectively assessed the correlation between clinical features and DLK1 expression by immunohistochemistry in resected specimens from 112 patients with SCLC and 101 patients with NSCLC. Moreover, we performed cell and animal experiments, and examined the possibility of RIT targeting DLK1 in SCLC using iodine-125 (125I) -labeled anti-DLK1 antibody, knowing that 125I can be replaced with the alpha-particle-emitter astatine-211 (211At). ResultsIn SCLC and NSCLC, 20.5 % (23/112) and 16.8 % (17/101) of patients (respectively) had DLK1-positive tumors. In NSCLC, DLK1 expression was associated with recurrence-free survival (P < 0.01) but not with overall survival. In SCLC, there was no association between DLK1 expression and survival. In addition, 125I-labeled anti-DLK1 antibody specifically targeted DLK1 on human SCLC tumor cell lines. Furthermore, 125I-labeled anti-DLK1 antibody was incorporated into tumor tissue in a mouse model. ConclusionA proportion of SCLC and NSCLC exhibits DLK1 expression. As a clinical feature, DLK1 expression could be a promising prognostic factor for recurrence in patients with resected NSCLC. In addition, DLK1 could serve as a new therapeutic target, including RIT, as suggested by our pilot study using a radiolabeled anti-DLK1 antibody in SCLC.

Guanosine triphosphate links MYC-dependent metabolic and ribosome programs in small-cell lung cancer.

MYC stimulates both metabolism and protein synthesis, but how cells coordinate these complementary programs is unknown. Previous work reported that, in a subset of small-cell lung cancer (SCLC) cell lines, MYC activates guanosine triphosphate (GTP) synthesis and results in sensitivity to inhibitors of the GTP synthesis enzyme inosine monophosphate dehydrogenase (IMPDH). Here, we demonstrated that primary MYChi human SCLC tumors also contained abundant guanosine nucleotides. We also found that elevated MYC in SCLCs with acquired chemoresistance rendered these otherwise recalcitrant tumors dependent on IMPDH. Unexpectedly, our data indicated that IMPDH linked the metabolic and protein synthesis outputs of oncogenic MYC. Coexpression analysis placed IMPDH within the MYC-driven ribosome program, and GTP depletion prevented RNA polymerase I (Pol I) from localizing to ribosomal DNA. Furthermore, the GTPases GPN1 and GPN3 were upregulated by MYC and directed Pol I to ribosomal DNA. Constitutively GTP-bound GPN1/3 mutants mitigated the effect of GTP depletion on Pol I, protecting chemoresistant SCLC cells from IMPDH inhibition. GTP therefore functioned as a metabolic gate tethering MYC-dependent ribosome biogenesis to nucleotide sufficiency through GPN1 and GPN3. IMPDH dependence is a targetable vulnerability in chemoresistant MYChi SCLC.

Abstract 4558: Antitumor activity of AMG757, a half-life extended (HLE) bispecific T-cell engager (BiTE®) immune therapy targeting DLL3, in human PDX and orthotopic mouse models of small cell lung cancer (SCLC)

Abstract SCLC is a highly aggressive neuroendocrine tumor with poor prognosis and limited therapeutic options. Delta-like ligand 3 (DLL3) is a tumor-associated antigen that is highly specific for SCLC; expression is elevated in tumors but minimal and mainly cytoplasmic in normal tissues. AMG 757 is an HLE BiTE® immune therapy that is designed to redirect T-cell cytotoxicity to cancer cells by binding to DLL3 on the surface of cancer cells and CD3 on T cells. AMG 757 is being evaluated in a phase 1 clinical trial in patients with relapsed/refractory SCLC (NCT03319940). We evaluated the efficacy and pharmacodynamic (PD) effects of AMG 757 in three preclinical models of SCLC that express DLL3. We used the LXFS 1129 patient-derived xenograft (PDX) model to assess AMG 757 efficacy in established, subcutaneously implanted tumors similar to human SCLC tumors with their associated stroma. We also developed two orthotopic models of SCLC (SHP-77 and H82) to assess AMG 757 efficacy and PD at biologically relevant sites. In the SHP-77 orthotopic model, cells injected intravenously trafficked to the lungs where they formed diffuse tumors similar to primary SCLC tumors. In the H82 orthotopic model, cells injected intravenously formed discrete metastatic lesions in the liver, mimicking a major site for SCLC metastasis. In each model, mice bearing established tumors received a single infusion of human T cells and were then dosed with AMG 757 or a control HLE BiTE molecule. In the LXFS 1129 PDX model, treatment with AMG 757 induced complete tumor regression in 8 out of 10 mice, whereas treatment with the control HLE BiTE molecule showed no tumor growth inhibition. In the orthotopic SHP-77 model, treatment with AMG 757 led to significant tumor growth inhibition in the lungs relative to treatment with a control HLE BiTE molecule; the bioluminescence (BLI) signal decreased to near the limit of detection 22 days after start of treatment. AMG 757 antitumor activity was associated with increased T-cell trafficking and expansion in tumors; a single dose of AMG 757 significantly increased the number of human CD4+ and CD8+ cells in lungs as assessed by flow cytometry. In the orthotopic H82 model, AMG 757 treatment completely cleared visible lesions from the liver, as assessed by a decrease in BLI to near-background levels and macroscopic analysis of the liver tissue. AMG 757 antitumor activity was associated with increased CD8+ T-cell infiltration as assessed by flow cytometry and IHC and upregulation of the T cell surface activation markers CD25, CD69, PD-1, and 4-1BB as assessed by flow cytometry. Together, these preclinical data demonstrate that AMG 757 can recruit and engage T cells to tumors, consistent with the BiTE® mechanism of action, and that AMG 757-mediated redirected T-cell lysis can drive significant antitumor activity in established SCLC tumor models. Citation Format: Keegan Cooke, Juan Estrada, Jinghui Zhan, Jonathan Werner, Sean Caenepeel, Mike Giffin, Julie M. Bailis, Angela Coxon, Paul E. Hughes, Jude Canon. Antitumor activity of AMG757, a half-life extended (HLE) bispecific T-cell engager (BiTE®) immune therapy targeting DLL3, in human PDX and orthotopic mouse models of small cell lung cancer (SCLC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4558.

CRISPR-mediated modeling and functional validation of candidate tumor suppressor genes in small cell lung cancer

Small cell lung cancer (SCLC) is a highly aggressive subtype of lung cancer that remains among the most lethal of solid tumor malignancies. Recent genomic sequencing studies have identified many recurrently mutated genes in human SCLC tumors. However, the functional roles of most of these genes remain to be validated. Here, we have adapted the CRISPR-Cas9 system to a well-established murine model of SCLC to rapidly model loss-of-function mutations in candidate genes identified from SCLC sequencing studies. We show that loss of the gene p107 significantly accelerates tumor progression. Notably, compared with loss of the closely related gene p130, loss of p107 results in fewer but larger tumors as well as earlier metastatic spread. In addition, we observe differences in proliferation and apoptosis as well as altered distribution of initiated tumors in the lung, resulting from loss of p107 or p130 Collectively, these data demonstrate the feasibility of using the CRISPR-Cas9 system to model loss of candidate tumor suppressor genes in SCLC, and we anticipate that this approach will facilitate efforts to investigate mechanisms driving tumor progression in this deadly disease.

Oncolytic virotherapy for small-cell lung cancer induces immune infiltration and prolongs survival.

Oncolytic virotherapy has been proposed as an ablative and immunostimulatory treatment strategy for solid tumors that are resistant to immunotherapy alone; however, there is a need to optimize host immune activation using preclinical immunocompetent models in previously untested common adult tumors. We studied a modified oncolytic myxoma virus (MYXV) that shows high efficiency for tumor-specific cytotoxicity in small-cell lung cancer (SCLC), a neuroendocrine carcinoma with high mortality and modest response rates to immune checkpoint inhibitors. Using an immunocompetent SCLC mouse model, we demonstrated the safety of intrapulmonary MYXV delivery with efficient tumor-specific viral replication and cytotoxicity associated with induction of immune cell infiltration. We observed increased SCLC survival following intrapulmonary MYXV that was enhanced by combined low-dose cisplatin. We also tested intratumoral MYXV delivery and observed immune cell infiltration associated with tumor necrosis and growth inhibition in syngeneic murine allograft tumors. Freshly collected primary human SCLC tumor cells were permissive to MYXV and intratumoral delivery into patient-derived xenografts resulted in extensive tumor necrosis. We confirmed MYXV cytotoxicity in classic and variant SCLC subtypes as well as cisplatin-resistant cells. Data from 26 SCLC human patients showed negligible immune cell infiltration, supporting testing MYXV as an ablative and immune-enhancing therapy.

Small cell lung cancer tumors and preclinical models display heterogeneity of neuroendocrine phenotypes.

Small cell lung cancer (SCLC) is a deadly, high grade neuroendocrine (NE) tumor without recognized morphologic heterogeneity. However, over 30 years ago we described a SCLC subtype with "variant" morphology which did not express some NE markers and exhibited more aggressive growth. To quantitate NE properties of SCLCs, we developed a 50-gene expression-based NE score that could be applied to human SCLC tumors and cell lines, and genetically engineered mouse (GEM) models. We identified high and low NE subtypes of SCLC in all of our sample types, and characterized their properties. We found that 16% of human SCLC tumors and 10% of SCLC cell lines were of the low NE subtype, as well as cell lines from the GEM model. High NE SCLC lines grew as non-adherent floating aggregates or spheroids while Low NE lines had morphologic features of the variant subtype and grew as loosely attached cells. While the high NE subtype expressed one of the NE lineage master transcription factors ASCL1 or NEUROD1, together with NKX2-1, the entire range of NE markers, and lacked expression of the neuronal and NE repressor REST, the low NE subtype had lost expression of most NE markers, ASCL1, NEUROD1 and NKX2-1 and expressed REST. The low NE subtype had undergone epithelial mesenchymal transition (EMT) and had activated the Notch, Hippo and TGFβ pathways and MYC oncogene . Importantly, the high and low NE group of SCLC lines had similar gene expression profiles as their SCLC tumor counterparts. SCLC tumors and cell lines can exhibit distinct inter-tumor heterogeneity with respect to expression of NE features. Loss of NE expression results in major alterations in morphology, growth characteristics, and molecular properties. These findings have major clinical implications as the two subtypes are predicted to have very different responses to targeted therapies.

Abstract 3935: Dissecting small cell lung carcinoma heterogeneity and chemotherapy resistance with mass cytometry

Abstract Introduction: Small cell lung cancer (SCLC) is a high grade neuroendocrine carcinoma of the lung responsible for up to 25% of lung cancer deaths. Treatment in SCLC has not changed significantly in the last 20 years. SCLC initially responds well to chemotherapy, but inevitably recurs. Characterization of tumor heterogeneity and changes in SCLC cell signaling and phenotypes after chemotherapy could yield new insights and therapeutic options. Mass cytometry uses metal labeled antibodies to profile expression and phosphorylation of more than 40 proteins in single cells and offers the opportunity to identify new subpopulations including potential cancer stem cell populations as well as targets for novel therapies in SCLC. Methods: Nude mice with SCLC patient derived xenografts (PDXs) were treated with one cycle of carboplatin/etoposide or saline injection. Tumors were harvested at ~2000mm3, disaggregated, and cryopreserved. PDX samples were stained with a 22 marker panel and an intercalator dye to identify nucleated cells. This panel measured phospho-signaling, neuroendocrine, immune, and mesenchymal cell markers, and functional markers including ki67 and cleaved caspase 3. ViSNE analysis and biaxial gating were used to identify major subpopulations of interest. Results: PDX tumors released viable tumor and stromal cells suitable for cryopreservation and mass cytometry. ACK buffer and enzymatic dissociation yielded the best quality cells by depleting red blood cells. Mouse cells, including leukocytes, were excluded using mouse MHC1 gating and iridium intercalator was used to identify nucleated cells. Single cell protein expression and phosphorylation was analyzed using viSNE and yielded at least 9 distinct subpopulations based on density islands with neuroendocrine (CD56+) and non-neuroendocrine (CD56-) populations. Chemotherapy treated cells had dramatic changes in subpopulation distribution compared to matched mock treated tumor. This included 2-3 fold expansion of SOX2+, CD117+, and pSTAT3+ populations with chemotherapy treatment. A small CD44+ tumor subpopulation identified in the chemotherapy treated cells was not present in the matched mock treated tumor suggesting a potential chemotherapy resistant/ stem- like subpopulation. Kinase activity showed stable p-AKT overall, but increased p-S6 in the chemotherapy treated cells. Conclusions: Mass cytometry was able to identify multiple neuroendocrine and non-neuroendocrine cell populations from SCLC PDXs and characterize their signaling. Chemotherapy treated PDX had differential subpopulation distribution with enrichment of multiple stem-like signaling factors. This work demonstrates the utility of mass cytometry and viSNE as novel techniques to identify subpopulations associated with chemotherapy resistance for future targeting and demonstrates the feasibility of this technique for characterizing signaling heterogeneity in human SCLC tumors. Citation Format: Jonathan M. Lehman, Nalin Leelatian, Bradford Harris, Megan Hoeksema, Zou Yong, Deon B. Doxie, Jonathan M. Irish, Pierre P. Massion. Dissecting small cell lung carcinoma heterogeneity and chemotherapy resistance with mass cytometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3935. doi:10.1158/1538-7445.AM2017-3935

Capsaicin synergizes with camptothecin to induce increased apoptosis in human small cell lung cancers via the calpain pathway

Small cell lung cancer (SCLC) is characterized by excellent initial response to chemotherapy and radiation therapy with a majority of the patients showing tumor shrinkage and even remission. However, the challenge with SCLC therapy is that patients inevitably relapse and subsequently do not respond to the first line treatment. Recent clinical studies have investigated the possibility of camptothecin-based combination therapy as first line treatment for SCLC patients. Conventionally, camptothecin is used for recurrent SCLC and has poor survival outcomes. Therefore, drugs which can improve the therapeutic index of camptothecin should be valuable for SCLC therapy. Extensive evidence shows that nutritional compounds like capsaicin (the spicy compound of chili peppers) can improve the anti-cancer activity of chemotherapeutic drugs in both cell lines and animal models. Statistical analysis shows that capsaicin synergizes with camptothecin to enhance apoptosis of human SCLC cells. The synergistic activity of camptothecin and capsaicin is observed in both classical and variant SCLC cell lines and, in vivo, in human SCLC tumors xenotransplanted on chicken chorioallantoic membrane (CAM) models. The synergistic activity of capsaicin and camptothecin are mediated by elevation of intracellular calcium and the calpain pathway. Our data foster hope for novel nutrition based combination therapies in SCLC.

Identification and Targeting of Long-Term Tumor-Propagating Cells in Small Cell Lung Cancer

Small cell lung cancer (SCLC) is a neuroendocrine lung cancer characterized by fast growth, early dissemination, and rapid resistance to chemotherapy. We identified a population of long-term tumor-propagating cells (TPCs) in a mouse model of SCLC. This population, marked by high levels of EpCAM and CD24, is also prevalent in human primary SCLC tumors. Murine SCLC TPCs are numerous and highly proliferative but not intrinsically chemoresistant, indicating that not all clinical features of SCLCare linked to TPCs. SCLC TPCs possess a distinct transcriptional profile compared to non-TPCs, including elevated MYC activity. Genetic and pharmacological inhibition of MYC in SCLC cells to non-TPC levels inhibits long-term propagation but not short-term growth. These studies identify a highly tumorigenic population of SCLC cells in mouse models, cell lines, and patient tumors and a means to target them in this most fatal form of lung cancer.

Abstract 4610: A drug repositioning approach identifies tricyclic antidepressants as inhibitors of small cell lung cancer and other neuroendocrine tumors

Abstract Lung cancer is the leading cause of cancer deaths worldwide, with more than 1.3 million deaths annually. SCLC is a subtype of lung cancer representing ∼15% of all lung cancer cases, and is characterized by the rapid expansion and metastasis of small cells with neuroendocrine features. Patients are typically treated with 4-6 rounds of etoposide and a platinum-based agent, to which there is a 30-40% complete response rate. However, patients almost invariably relapse with disease that is resistant to their primary therapy, and rapidly succumb to their malignancy due to this chemoresistance. No targeted therapy has ever been approved despite numerous attempts. Consequently, 5-year survival for patients has hovered near 5% for the last 30 years. Drug repositioning, which is the discovery of new indications for existing drugs, is an increasingly attractive mode of therapeutic discovery. Recent advances in computing, concomitant with the increasing availability of large datasets, have enabled the development of in silico approaches to drug discovery. Here we applied a systematic drug-repositioning bioinformatics approach querying a large compendium of gene expression profiles to identify candidate FDA-approved drugs to treat small cell lung cancer (SCLC). We found that tricyclic antidepressants (TCAs) and related molecules, such as imipramine and promethazine, potently induce apoptosis in both chemonaïve and chemoresistant SCLC cells in culture, in mouse and human SCLC tumors transplanted into immunocompromised mice, and in endogenous tumors from a mouse model for human SCLC. The candidate drugs activate stress pathways and induce cell death in SCLC cells, at least in part by disrupting autocrine/paracrine survival signals involving neurotransmitters and their receptors. In particular, the mechanism of action of these drugs in inhibiting SCLC tumor growth is through antagonizing G- protein coupled receptors (GPCRs) and blocking signaling downstream, leading to a decrease in cAMP levels and PKA activity, subsequently resulting in activation of the c-Jun/JNK pathway. Moreover, TCAs induce rapid cell death in other neuroendocrine tumors, including carcinoid tumors and Merkel cell carcinoma. These experiments identify novel targeted strategies that can be rapidly evaluated in patients with neuroendocrine tumors through the repurposing of approved drugs. Since these drugs have already been vetted in terms of safety, dosage and toxicity, they can rapidly enter clinical trials. Citation Format: Nadine S. Jahchan, Joel T. Dudley, Pawel K. Mazur, Natasha Flores, Dian Yang, Alec Palmerton, Anne-Flore Zmoos, Dedeepya Vaka, Kim Q.t. Tran, Margaret Zhou, Karolina Krasinska, Jonathan W. Riess, Joel W. Neal, Purvesh Khatri, Kwon S. Park, Atul J. Butte, Julien Sage. A drug repositioning approach identifies tricyclic antidepressants as inhibitors of small cell lung cancer and other neuroendocrine tumors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4610. doi:10.1158/1538-7445.AM2014-4610

Abstract 1542: Comprehensive genome and transcriptome analyses on small cell lung cancer

Abstract Small cell lung cancer (SCLC) is a malignant neuroendocrine tumor of the lung accounting for 15% of all lung cancer cases. SCLC is a highly aggressive disease characterized by early metastasis and a high mortality rate of 95%. The standard of care is chemotherapy, which remains largely ineffective due to quick relapse of chemo-resistant tumors. To date, targeted therapies have not been identified for the treatment of SCLC. In order to comprehensively characterize the genomic alterations in this cancer type, we sought for a worldwide collaboration to collect rare surgical specimen of SCLC patients. Thus, we were able to study copy number alterations in 130 cases, and performed whole genome sequencing on over 78 tumor-normal pairs and transcriptome sequencing of at least 62 primary tumors and 16 SCLC cell lines. In a complementary approach we analyzed tumors derived from a murine SCLC model that was established by the conditional inactivation of Rb1 and Trp53 in lung epithelial cells (Meuwissen et al., 2003). We collected copy number alteration data for 20 mouse SCLC cases and performed exome and genome sequencing of at least 8 tumors derived from SCLC mice which additionally harbored a conditional knockout of Rbl2 (Schaffer et al., 2010). Human SCLC tumors comprise large areas of copy number alterations, revealing focal amplifications of MYC transcription factors. In line with this finding recurrent MYCL1 amplifications were also detected in murine SCLC. Human primary tumors were additionally found to harbor focal homozygous deletions of the CDKN2A locus. Furthermore, low frequent amplifications of oncogenes were identified among which therapeutic tractable FGFR1 amplifications were observed in approximately 6% of the cases. The genomic data allowed for the comprehensive characterization of the TP53 and RB1 locus at higher resolution, aiding in the identification of mutations, hemizygous and homozygous deletions, and larger genomic rearrangements. Consequently, lesions of TP53 and RB1 were found to affect both alleles in almost 100% of the cases analyzed. Other TP53 and RB1 family members were also altered in human tumor specimen thus supporting the mouse models for SCLC. In line with previous findings PTEN inactivation was found in 14% of the cases. Furthermore, recurrent mutations were detected in neuroendocrine specific genes and in chromatin and histone modifiers, among which the inactivation of the histone-acyl-transferases CREBBP and EP300 occurred at a frequency of 24%. In summary, as a continuation of our initial pilot study on 29 SCLC exomes (Peifer et al., 2012) this comprehensive large scale genomic analysis provides further insight into the complex genomic architecture of SCLC tumors and substantially improves the resolution to detect genomic alterations. This larger SCLC tumor cohort allows for the identification of low frequent alterations that might play a decisive role in the development and progression of this disease. Citation Format: Julie George, Martin Peifer, Lynnette Fernandez-Cuesta, Roman Thomas. Comprehensive genome and transcriptome analyses on small cell lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1542. doi:10.1158/1538-7445.AM2014-1542

Pharmacogenomic approach to identify drug sensitivity in small-cell lung cancer.

There are currently no molecular targeted approaches to treat small-cell lung cancer (SCLC) similar to those used successfully against non-small-cell lung cancer. This failure is attributable to our inability to identify clinically-relevant subtypes of this disease. Thus, a more systematic approach to drug discovery for SCLC is needed. In this regard, two comprehensive studies recently published in Nature, the Cancer Cell Line Encyclopedia and the Cancer Genome Project, provide a wealth of data regarding the drug sensitivity and genomic profiles of many different types of cancer cells. In the present study we have mined these two studies for new therapeutic agents for SCLC and identified heat shock proteins, cyclin-dependent kinases and polo-like kinases (PLK) as attractive molecular targets with little current clinical trial activity in SCLC. Remarkably, our analyses demonstrated that most SCLC cell lines clustered into a single, predominant subgroup by either gene expression or CNV analyses, leading us to take a pharmacogenomic approach to identify subgroups of drug-sensitive SCLC cells. Using PLK inhibitors as an example, we identified and validated a gene signature for drug sensitivity in SCLC cell lines. This gene signature could distinguish subpopulations among human SCLC tumors, suggesting its potential clinical utility. Finally, circos plots were constructed to yield a comprehensive view of how transcriptional, copy number and mutational elements affect PLK sensitivity in SCLC cell lines. Taken together, this study outlines an approach to predict drug sensitivity in SCLC to novel targeted therapeutics.

Capsaicin causes apoptosis of human small cell lung cancers cells via the TRPV6/calpain pathway (644.8)

Small cell lung cancer (SCLC) is characterized by aggressive clinical course and a dismal survival rate. The present study examines the apoptotic activity of capsaicin (the pungent ingredient of chili peppers) in human SCLC. We observed that capsaicin induced apoptosis in a panel of human SCLC in a concentration‐dependent and time‐dependent manner. Most importantly, capsaicin caused robust apoptosis in a panel of human SCLC cell lines, but did not affect normal human lung epithelial cells. The dietary administration of capsaicin decreased the growth H69 and DMS53 human SCLC tumors in nude mice and in chicken chorioallantoic membrane (CAM) models. The apoptotic activity of capsaicin was reversed with the generalized TRPV antagonist ruthenium red, indicating that TRPV receptors are involved in capsaicin induced apoptosis. However, antagonists to TRPV1, namely capsazepine and SB‐366791, did not affect the apoptotic activity of capsaicin, suggesting the involvement of a TRPV1‐independent mechanism. In contrast, depletion of TRPV6 by siRNA ablated the apoptotic activity of capsaicin. Capsaicin‐induced apoptosis was also inhibited by calpeptin, a chemical inhibitor of the calpain pathway. Our findings suggest that capsaicin may have potential applications as a novel therapeutic agent for SCLCs.Grant Funding Source: Supported by an NIH R15‐Area Grant, a FAMRI YCSA Grant, and WV INBRE

Abstract C128: TAS-116, an orally available HSP90α/β selective inhibitor, has potent antitumor activity in small cell lung cancer as a single agent and in combination with an anthracycline derivative, amrubicin.

Abstract Background: The molecular chaperone HSP90 has been considered a promising target for cancer therapy. We have previously reported the discovery of an orally available HSP90α/β selective inhibitor, TAS-116. TAS-116 led to tumor shrinkage in various human tumor xenograft models, while not inducing retinal toxicity in rats including at doses given above the maximum tolerated dose. Extensive small cell lung cancer (SCLC) is one of the most aggressive types of cancer, and has an unmet medical need for more effective treatments. An anthracycline, amrubicin (AMR), has been used to treat platinum resistant SCLC in Japan. Here we report on the possible application of TAS-116 to treat SCLC both as a single agent and in combination with AMR. Materials and Methods: In vitro drug combination effects were evaluated with calculation of the combination index and cell cycle analysis by fluorescence-activated cell sorting. To clarify the mechanism involved in TAS-116’s enhancement of anthracycline-induced apoptosis the extent of apoptosis was measured in combination with several kinase inhibitors including SB 218078 (CHK1) and MK-2206 (AKT). Antitumor activities of TAS-116 alone and in combination with AMR were evaluated in human SCLC xenograft models. Results: TAS-116 induced cell growth arrest and apoptosis in SCLC cell lines by inhibiting both PI3K/AKT and MAPK signaling through downregulation of HSP90 clients such as AKT and RAF1. TAS-116 also showed antitumor activity against human SCLC tumor xenograft models. In in vitro explorative combination studies in SCLC cell lines, structurally-related anthracyclines, AMR and doxorubicin (DOX), both showed synergistic effect upon apoptosis induction in combination with TAS-116. Moreover, TAS-116 enhanced the antitumor activity of AMR in human SCLC tumor xenograft models. Since anthracyclines exert antitumor activity based on their DNA damaging potential through topoisomerase II inhibition, we evaluated whether DNA damage signaling proteins and apoptosis-related proteins were affected by TAS-116 when given in combination with DOX. TAS-116 significantly reduced phosphorylation of CHK1, AKT and ERK1/2. In combination with these kinase inhibitors, SB 218078 significantly and MK-2206 moderately enhanced DOX-mediated apoptosis. Comparatively, TAS-116 enhanced apoptosis to a greater extent than SB 218078. Conclusion: TAS-116 showed antitumor activity against human SCLC tumor xenograft models alone and in combination with AMR. Our data suggest that TAS-116 enhanced the antitumor activity of anthracyclines through the combined actions of abrogation of the DNA damage-induced G2/M checkpoint and the inhibition of a survival signal by CHK1 and AKT downregulation, respectively. These results suggest that TAS-116 could be a promising agent to treat extensive SCLC. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C128. Citation Format: Hiromi Muraoka, Chihoko Yoshimura, Akihiro Hashimoto, Kenjiro Ito, Makoto Kitade, Kazuhiko Yonekura, Shuichi Ohkubo, Teruhiro Utsugi. TAS-116, an orally available HSP90α/β selective inhibitor, has potent antitumor activity in small cell lung cancer as a single agent and in combination with an anthracycline derivative, amrubicin. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C128.