SCLC Tumors Research Articles

Checkpoint Kinase 1 Inhibition Enhances Cisplatin Cytotoxicity and Overcomes Cisplatin Resistance in SCLC by Promoting Mitotic Cell Death

IntroductionPlatinum-based chemotherapy remains the standard treatment for patients with SCLC, but the benefit of the treatment is often hampered by rapid development of drug resistance. Thus far, there is no targeted therapy available for SCLC. More than 90% of SCLC tumors harbor mutations in the tumor suppressor gene tumor protein p53 (p53), an important DNA damage checkpoint regulator, and these tumor cells rely predominantly on the checkpoint kinases to control DNA damage response. MethodsWe examined whether and how inhibition of checkpoint kinase 1 (Chk1) affects cisplatin cytotoxicity in SCLC cells with and without p53 mutations, and evaluated the effect of Chk1 inhibitor and cisplatin combination in cisplatin-sensitive and -resistant preclinical models. ResultsInhibition of Chk1 synergized with cisplatin to induce mitotic cell death in the p53-deficeint SCLC cells. The effect was regulated in part through activation of caspase 2 and downregulation of E2F transcription factor 1 (E2F1). Furthermore, Chk1 inhibitors prexasertib and AZD7762 enhanced cisplatin antitumor activity and overcame cisplatin resistance in SCLC preclinical models in vitro an in vivo. We also observed that higher expression of Chk1 was associated with poorer overall survival of patients with SCLC. ConclusionsOur data account Chk1 as a potential therapeutic target in SCLC, and rationalize clinical development of Chk1 inhibitor and cisplatin combinational strategy for the treatment of SCLC.

Cells Lacking the RB1 Tumor Suppressor Gene Are Hyperdependent on Aurora B Kinase for Survival.

Small cell lung cancer (SCLC) accounts for 15% of lung cancers and is almost always linked to inactivating RB1 and TP53 mutations. SCLC frequently responds, albeit briefly, to chemotherapy. The canonical function of the RB1 gene product RB1 is to repress the E2F transcription factor family. RB1 also plays both E2F-dependent and E2F-independent mitotic roles. We performed a synthetic lethal CRISPR/Cas9 screen in an RB1 -/- SCLC cell line that conditionally expresses RB1 to identify dependencies that are caused by RB1 loss and discovered that RB1 -/- SCLC cell lines are hyperdependent on multiple proteins linked to chromosomal segregation, including Aurora B kinase. Moreover, we show that an Aurora B kinase inhibitor is efficacious in multiple preclinical SCLC models at concentrations that are well tolerated in mice. These results suggest that RB1 loss is a predictive biomarker for sensitivity to Aurora B kinase inhibitors in SCLC and perhaps other RB1 -/- cancers. SIGNIFICANCE: SCLC is rarely associated with actionable protooncogene mutations. We did a CRISPR/Cas9-based screen that showed that RB1 -/- SCLC are hyperdependent on AURKB, likely because both genes control mitotic fidelity, and confirmed that Aurora B kinase inhibitors are efficacious against RB1 -/- SCLC tumors in mice at nontoxic doses.See related commentary by Dick and Li, p. 169.This article is highlighted in the In This Issue feature, p. 151.

Abstract IA28: The genomic landscape of SCLC and other neuroendocrine lung tumors

Abstract Small cell lung cancer (SCLC) accounts for 15% of all lung cancers and belongs to the family of pulmonary neuroendocrine tumors. This tumor subgroup additionally includes large-cell neuroendocrine carcinomas (LCNEC) and pulmonary carcinoids (PCA), which occur in 3% and 2% of all lung cancer patients, respectively. Carcinoids are usually clinically benign, whereas SCLC and LCNEC are characterized as high-grade malignant tumors. Clinical and histologic studies have suggested similarities between SCLC and LCNECs: Both lung tumor subtypes are associated with heavy smoking and reveal an aggressive tumor growth resulting in a poor overall survival. We have performed comprehensive and multilevel genomic profiling on SCLC and LCNECs, including genome, exome, and transcriptome sequencing. SCLC and LCNEC tumors reveal high mutation rates with an average of 9.5 and 8.6 mutations per megabase, respectively. Our sequencing studies have shown that SCLC tumors are characterized by bi-allelic inactivation of TP53 and RB1 in almost 100% of the cases. LCNECs, on the other hand, are composed of two mutually exclusive subgroups, which we categorized as “type I LCNECs” and “type II LCNECs.” Type I LCNECs harbor TP53 and STK11 or KEAP1 alterations. Despite sharing genomic alterations with lung adenocarcinomas and squamous cell carcinomas, type I LCNECs exhibit a high neuroendocrine profile with close similarity to SCLC tumors. Type II LCNECs harbor loss of TP53 and RB1, thus showing genetic resemblance to SCLC. However, these tumors were revealed to be different from the majority of SCLC with reduced levels of neuroendocrine markers, an upregulated immune signaling profile, and high activity of the NOTCH pathway. Conclusively, our studies show marked biologic differences between high-grade neuroendocrine lung tumors and we emphasize the precise distinction of LCNEC subgroups from SCLC. Citation Format: Julie George. The genomic landscape of SCLC and other neuroendocrine lung tumors [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr IA28.

Abstract 3637: Expression of an immune checkpoint- Poliovirus Receptor (PVR) in small cell lung cancer

Abstract Background: Although immunotherapy has renewed hopes for the improved treatment of SCLC, the “classical” PD-1/PD-L1 pathway is not commonly expressed in SCLC. PVR (CD155) is expressed at low levels in a number of cell types of epithelial origin and is overexpressed in various carcinomas with epithelial and neurological origins, including colorectal carcinoma, breast carcinoma, neuroblastoma and glioblastoma. In this study, we evaluated the expression of PVR in SCLC cell lines and a cohort of patient tumors with association to clinical characteristics. Methods: Immunohistochemistry (IHC) was performed to evaluate PVR protein expression in 39 SCLC cell lines as well as a cohort of 77 resected tumors from SCLC patients with clinical data using a monoclonal PVR antibody (D8A5G,Cell Signaling, MA). Scoring data was generated using both the H-score and tumor proportion score (TPS) systems. PVR expression was correlated with characteristics of both the cell lines and the clinical cohort. Results: The SCLC cell lines were evaluated by the H-score system only. A total of 39 SCLC cell lines in a TMA were evaluated in which 37 cell lines (95%, 37/39) demonstrated PVR staining > 0 and 85% of the cell lines showed positive staining with an arbitrary H-score cutoff of 50. PVR expression was higher in cell lines established from pre-treated SCLC patients as compared to those established from treatment-naïve patients (P=0.037). There was no correlation between PVR expression and cell line growth as suspension versus adherent cells. In the SCLC patient cohort, PVR expression was found to be predominantly on the membrane of tumor cells, with minimal expression observed on immune cells in the tumor microenvironment. PVR expression in the SCLC patient cohort was 82% with H-score cutoff of ≥ 50. SCLC patients with positive PVR expression demonstrated a poorer prognosis, however the difference was not statistically significant (p=0.05). Positive PVR expression was also correlated with advanced stage (P=0.0073) and higher in male patients (P=0.04). With a TPS score system (cutoff of ≥ 50%) the prevalence of PVR expression is 79%. Using the TPS system, higher PVR expression was associated with poor prognosis and advanced stage (P=0.046, and P=0.045). PVR expression also as evaluated with the TPS score system was correlated with large tumor diameter (P=0.043) and older age of patients (P=0.046). Conclusion: PVR was broadly expressed in SCLC cell lines and tumor tissues. High PVR expression was associated with poor prognosis, advanced tumor stage, large tumor diameter, and advanced age of patients. We also found that PVR expression may correlate with chemotherapy treatment history in SCLC cell lines. These data suggest that the PVR-TIGIT pathway may be a promising target for immunotherapy in SCLC. Citation Format: Hui Yu, Camilla Koczara, Andrzej Badzio, Dexiang Gao, Christopher J. Rivard, Kim Ellison, Kenichi Suda, Shengxiang Ren, Charles Caldwell, Kristine A. Brovsky, Leslie Rozeboom, Fred R. Hirsch. Expression of an immune checkpoint- Poliovirus Receptor (PVR) in small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3637.

Abstract 2495: RUNX1T1 is amplified in combined small cell lung cancer

Abstract Small cell lung cancer (SCLC) is a highly aggressive malignancy that expresses neuroendocrine (NE) genes and represents approximately 15% of all lung cancers; the remaining histological subtype is non-small cell lung cancer (NSCLC). Clinically, there are two types of SCLC, termed ‘pure' and ‘combined'. Combined SCLC presents as a mixture of SCLC and NSCLC components within the same tumor mass. We analyzed 88 SCLC patient tumors by targeted exome sequencing and found that four SCLC patients demonstrated RUNX1T1 amplification. Interestingly, among these 88 SCLC patients there were two with combined SCLC and both demonstrated RUNX1T1 amplification only in the small cell component. Neither MYC (8p24) nor FGFR1 (8p11), two genes commonly amplified in SCLC that are nearby the RUNX1T1 locus (8p22), were co-amplified. RUNX1T1, also called ETO or MTG8, was first identified in the oncogenic fusion transcript AML1/ETO in acute myelogenous leukemia (AML) and was previously reported as a transcriptional co-repressor. Based on our observation we hypothesized that: 1) RUNX1T1 may be a specific marker for SCLC and may play a role in determining the SCLC phenotype, including NE expression, and 2) RUNX1T1 may play a role in transforming NSCLC to SCLC in the early stages of combined SCLC tumor formation. First we examined RUNX1T1 mRNA expression among cancer cell lines using the CCLE database and found that RUNX1T1 is highly expressed in SCLC cells compared to other cancer cells, especially NSCLC cells. This was also true in tumors using RNAseq data. Consistent with these mRNA profiles, our western blot results showed higher RUNX1T1 protein expression in SCLC compared to NSCLC cell lines. These findings validate that RUNX1T1 expression is specifically upregulated in SCLC compared to NSCLC. To investigate a role for RUNX1T1 in determining the SCLC phenotype, we overexpressed RUNX1T1 in SCLC cell lines by lentiviral transduction and observed marked growth suppression in H82, H2171, and H841 cells. Conversely, transient knock down of RUNX1T1 expression in the SCLC cell line H446 using siRNA decreased the expression of two NE proteins (NEUROD1 and TTF1). To explore a potential role of RUNX1T1 in NSCLC to SCLC transformation, we overexpressed RUNX1T1 in three NSCLC cell lines (PC-9, H1650 and A549) but observed no obvious phenotypic changes and no increase in NE gene expression. In conclusion, we have demonstrated that RUNX1T1 expression is upregulated in SCLC compared to NSCLC. Moreover, the results from RUNX1T1 overexpression and knockdown experiments in SCLC cell lines indicates a potential role of RUNX1T1 in regulating the SCLC phenotype and NE expression. Citation Format: Tian He, Karen McColl, Alyssa Savadelis, Yanwen Chen, Gary Wildey, Afshin Dowlati. RUNX1T1 is amplified in combined small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2495.

NK Cells Mediate Synergistic Antitumor Effects of Combined Inhibition of HDAC6 and BET in a SCLC Preclinical Model.

Small-cell lung cancer (SCLC) has the highest malignancy among all lung cancers, exhibiting aggressive growth and early metastasis to distant sites. For 30 years, treatment options for SCLC have been limited to chemotherapy, warranting the need for more effective treatments. Frequent inactivation of TP53 and RB1 as well as histone dysmodifications in SCLC suggest that transcriptional and epigenetic regulations play a major role in SCLC disease evolution. Here we performed a synthetic lethal screen using the BET inhibitor JQ1 and an shRNA library targeting 550 epigenetic genes in treatment-refractory SCLC xenograft models and identified HDAC6 as a synthetic lethal target in combination with JQ1. Combined treatment of human and mouse SCLC cell line-derived xenograft tumors with the HDAC6 inhibitor ricolinostat (ACY-1215) and JQ1 demonstrated significant inhibition of tumor growth; this effect was abolished upon depletion of NK cells, suggesting that these innate immune lymphoid cells play a role in SCLC tumor treatment response. Collectively, these findings suggest a potential new treatment for recurrent SCLC.Significance: These findings identify a novel therapeutic strategy for SCLC using a combination of HDAC6 and BET inhibitors. Cancer Res; 78(13); 3709-17. ©2018 AACR.

Abstract A12: CRISPR-mediated modeling and functional validation of candidate tumor-suppressor genes in small cell lung cancer

Abstract 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 a large number of recurrently mutated genes in human SCLC tumors. However, apart from a few notable examples, the functional roles of most of these genes remain to be validated. The CRISPR-Cas9 system has been utilized in genetically engineered mouse models of cancer to generate somatic genetic alterations in tumor cells in vivo. This has greatly accelerated efforts to study the roles of candidate genes of interest in various cancers. In this study, we adapted the CRISPR-Cas9 system to murine models of SCLC (mSCLC) to enable rapid modeling and functional validation of candidate tumor-suppressor genes in SCLC. We generated a Cre-activated allele of Cas9 and crossed it into both the Trp53flox/flox; Rb1flox/flox and the Trp53flox/flox; Rb1flox/flox; p130flox/flox models of mSCLC. By infecting these animals with adenoviral vectors coexpressing Cre recombinase and a target sgRNA, we can initiate SCLC tumors harboring loss-of-function mutations in candidate genes of interest. To validate this system, we infected animals with adenoviral vectors expressing sgRNAs targeting either p107 (Rbl1) or p130 (Rbl2), two genes that are recurrently mutated in a subset of human SCLC tumors. SCLC tumors that developed in these animals harbored loss-of-function mutations at the respective sgRNA target sites in the genome. Loss of p107 or p130 expression in these tumors was confirmed by immunohistochemical staining. As expected, CRISPR-mediated loss of p130 in Trp53flox/flox; Rb1flox/flox animals resulted in acceleration of tumor progression compared with animals infected with a control sgRNA, consistent with the phenotype previously observed in Trp53flox/flox; Rb1flox/flox; p130flox/flox animals. Loss of p107 in Trp53flox/flox; Rb1flox/flox animals also resulted in acceleration of tumor progression, as seen by an increase in tumor burden and a decrease in overall survival compared with control animals. Such an acceleration was not observed with loss of p107 in Trp53flox/flox; Rb1flox/flox; p130flox/flox animals, suggesting potential functional redundancy between p107 and p130 in SCLC. In summary, we have demonstrated the feasibility of using the CRISPR-Cas9 system to rapidly model loss-of-function mutations in candidate tumor-suppressor genes in mSCLC, and have used this approach to validate the role of p107 as a tumor suppressor in SCLC. Such an approach will greatly accelerate efforts to investigate the molecular mechanisms of tumor progression in SCLC. Citation Format: Sheng Rong Ng, William M. Rideout, III, Bethany L. Wagner, Tyler Jacks. CRISPR-mediated modeling and functional validation of candidate tumor-suppressor genes in small cell lung cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr A12.

Abstract IA12: Intra- and intertumoral heterogeneity and response to therapy in mouse models of SCLC

Abstract Small cell lung cancer (SCLC) is a high-grade neuroendocrine carcinoma that accounts for ~15% of all lung cancers and causes over 200,000 deaths worldwide each year. The majority of SCLC patients have metastatic disease at the time of diagnosis; for these patients, the median survival, even with treatment, is 7-12 months, and the 5-year overall survival is <1%. SCLC is managed with first-line chemotherapy–often cisplatin or carboplatin with etoposide. However, the majority of SCLC patients suffer relapse within months of completing initial therapy due to the persistence of a subpopulation of chemotherapy-resistant cells. Treatment options for SCLC have remained virtually unchanged for the past 30 years, and there are no approved targeted therapies in SCLC. To dissect the molecular changes that regulate SCLC progression, it is critical to analyze purified SCLC cells from tumors at known stages of tumorigenesis. This has not been possible previously using either mouse models or human specimens due to the 1) limited access to lung cancers at defined stages of malignant progression, 2) inability to unambiguously isolate SCLC cells away from stromal cells, and 3) difficulty in performing genome-wide analyses on the few cells that can be isolated from metastases in mouse models or patient biopsies. To overcome these obstacles, we have developed mouse models of SCLC including Cre/lox-mediated deletion of key tumor suppressors (p53, Rb, and the Rb family member p130) as well as activation of fluorescent reporters. This triple-knockout (TKO) model recapitulates the genetics, histology, and metastatic ability of human SCLC. Using this mouse model, we have recently identified new levels of intratumoral heterogeneity in SCLC tumors, including the presence of tumor-propagating cells (TPCs) as well as Notch-active non-neuroendocrine tumor cell population that promotes the growth of TPCs in a non-cell autonomous but tumor-intrinsic manner. Using TKO mice, we have also uncovered distinct mechanisms of intertumoral heterogeneity during tumor progression; in particular, we have shown that one group of tumors gains metastatic potential by upregulation of the Nfib transcription factor and extensive changes in their chromatin state, while others become metastatic in absence of Nfib-driven chromatin dynamics. These observations will be discussed in the context of how SCLC tumors respond to chemotherapy and candidate targeted therapies. Citation Format: Jing Shan Lim, Dian Yang, Yan Ting Shue, Monte Winslow, Julien Sage. Intra- and intertumoral heterogeneity and response to therapy in mouse models of SCLC [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr IA12.

Targeting CREB Pathway Suppresses Small Cell Lung Cancer.

Small cell lung cancer (SCLC) is the most deadly subtype of lung cancer due to its dismal prognosis. We have developed a lentiviral vector-mediated SCLC mouse model and have explored the role of both the NF-κB and CREB families of transcription factors in this model. Surprisingly, induction of NF-κB activity, which promotes tumor progression in many cancer types including non-small cell lung carcinoma (NSCLC), is dispensable in SCLC. Instead, suppression of NF-κB activity in SCLC tumors moderately accelerated tumor development. Examination of gene expression signatures of both mouse and human SCLC tumors revealed overall low NF-κB but high CREB activity. Blocking CREB activation by a dominant-negative form of PKA (dnPKA) completely abolished the development of SCLC. Similarly, expression of dnPKA or treatment with PKA inhibitor H89 greatly reduced the growth of SCLC tumors in syngeneic transplantation models. Altogether, our results strongly suggest that targeting CREB is a promising therapeutic strategy against SCLC.Implications: Activity of the transcription factor CREB is elevated in SCLC tumors, which helps to maintain its neuroendocrine signature and cell proliferation. Our results highlight the importance of targeting the CREB pathway to develop new therapeutics to combat SCLC. Mol Cancer Res; 16(5); 825-32. ©2018 AACR.

Antitumor activity of CD56-chimeric antigen receptor T cells in neuroblastoma and SCLC models

The CD56 antigen (NCAM-1) is highly expressed on several malignancies with neuronal or neuroendocrine differentiation, including small-cell lung cancer and neuroblastoma, tumor types for which new therapeutic options are needed. We hypothesized that CD56-specific chimeric antigen receptor (CAR) T cells could target and eliminate CD56-positive malignancies. Sleeping Beauty transposon-generated CD56R-CAR T cells exhibited αβT-cell receptors, released antitumor cytokines upon co-culture with CD56+ tumor targets, demonstrated a lack of fratricide, and expression of cytolytic function in the presence of CD56+ stimulation. The CD56R-CAR+ T cells are capable of killing CD56+ neuroblastoma, glioma, and SCLC tumor cells in in vitro co-cultures and when tested against CD56+ human xenograft neuroblastoma models and SCLC models, CD56R-CAR+ T cells were able to inhibit tumor growth in vivo. These results indicate that CD56-CARs merit further investigation as a potential treatment for CD56+ malignancies.

Integrative genomic profiling of large-cell neuroendocrine carcinomas reveals distinct subtypes of high-grade neuroendocrine lung tumors

Pulmonary large-cell neuroendocrine carcinomas (LCNECs) have similarities with other lung cancers, but their precise relationship has remained unclear. Here we perform a comprehensive genomic (n = 60) and transcriptomic (n = 69) analysis of 75 LCNECs and identify two molecular subgroups: “type I LCNECs” with bi-allelic TP53 and STK11/KEAP1 alterations (37%), and “type II LCNECs” enriched for bi-allelic inactivation of TP53 and RB1 (42%). Despite sharing genomic alterations with adenocarcinomas and squamous cell carcinomas, no transcriptional relationship was found; instead LCNECs form distinct transcriptional subgroups with closest similarity to SCLC. While type I LCNECs and SCLCs exhibit a neuroendocrine profile with ASCL1high/DLL3high/NOTCHlow, type II LCNECs bear TP53 and RB1 alterations and differ from most SCLC tumors with reduced neuroendocrine markers, a pattern of ASCL1low/DLL3low/NOTCHhigh, and an upregulation of immune-related pathways. In conclusion, LCNECs comprise two molecularly defined subgroups, and distinguishing them from SCLC may allow stratified targeted treatment of high-grade neuroendocrine lung tumors.

Progress in the Management of Advanced Thoracic Malignancies in 2017.

The treatment paradigm of NSCLC underwent a major revolution during the course of 2017. Immune checkpoint inhibitors (ICIs) brought remarkable improvements in response and overall survival both in unselected pretreated patients and in untreated patients with programmed deathligand 1 expression of 50% or more. Furthermore, compelling preliminary results were reported for new combinations of anti-programmed cell death 1/programmed death ligand 1 agents with chemotherapy or anti-cytotoxic T-lymphocyte associated protein 4 inhibitors. The success of the ICIs appeared to extend to patients with SCLC, mesothelioma, or thymic tumors. Furthermore, in SCLC, encouraging activity was reported for an experimental target therapy (rovalpituzumab teserine) and a new chemotherapeutic agent (lurbinectedin). For oncogene-addicted NSCLC, next-generation tyrosine kinase inhibitors (TKIs) (such as osimertinib or alectinib) have demonstrated increased response rates and progression-free survival compared with first-generation TKIs in patients with both EGFR-mutated and ALK receptor tyrosine kinase gene (ALK)-rearranged NSCLC. However, because of the lack of mature overall survival data and considering the high efficacy of these drugs in patients with NSCLC previously exposed to first- or second-generation TKIs, definitive conclusions concerning the best treatment sequence cannot yet be drawn. In addition, new oncogenes such as mutant BRAF, tyrosine-protein kinase met gene (MET) and erb-b2 receptor tyrosine kinase 2 gene (HER2), and ret proto-oncogene (RET) rearrangements have joined the list of potential targetable drivers. In conclusion, the field of thoracic oncology is on the verge of a breakthrough that will open up many promising new therapeutic options for physicians and patients. The characterization of biomarkers predictive of sensitivity or resistance to immunotherapy and the identification of the optimal therapeutic combinations (for ICIs) and treatment sequence (for oncogene-addicted NSCLC) represent the toughest upcoming challenges in the domain of thoracic oncology.

Abstract 966: Phase II clinical trial patient responses to the macrophage activating agent RRx-001 correlate to TGF- β pathway activation and markers for fibrosis

Abstract Introduction: RRx-001 is a novel systemically non-toxic small molecule macrophage stimulating agent with promising activity in an ongoing clinical trial in small cell and non-small cell lung cancer and in neuroendocrine tumors. In preclinical studies, RRx-001 activated normally immune-suppressive M2-like Tumor Associated Macrophages (TAMs) to express a series of pro-inflammatory cytokines and chemokines such as transforming growth factor-β1 (TGF-β1), tumor necrosis factor-α TNF-α), inducible nitric oxide synthase (iNOS), and interleukins-10 and -12, a profile that resembles the M1 activated macrophage state. Due to its chemotactic activity for macrophages, TGF-β1 was hypothesized to be involved in the antitumor mechanism of RRx-001. We therefore compared expression patterns of TGF-β1 and TGF-βRI in patient-derived biopsy samples obtained at screening and post RRx-001. As TGF-β1 is closely associated with the induction of fibrosis, we also examined key fibrosis markers. Methods: Tumor biopsies before and after treatment with RRx-001 were obtained from consented patients with NSCLC, SCLC, and neuroendocrine tumors in the QUADRUPLE THREAT Phase II clinical trial (NCT02489903). Post treatment biopsies were obtained following 6 weeks of once-weekly RRx-001 treatment co-incidental with the first on-study CT scan. Tumor samples were evaluated immune-histochemically with putative markers for TGFβ pathway activation (TGF-β1, TGF-βR1), fibrosis (alpha-smooth muscle actin [α-SMA], Matrix metallopeptidase-9 [MMP-9], and Collagen III deposition), and macrophage activation. Patients were subsequently followed for tumor progression. Results: Positive immune-histochemical staining for TGF-β1 and TGF-βR1 was seen in all responding patients, and in none of the non-responding patients to date. In responders we also found that MMP-9 and α-SMA activity was down regulated post RRx-001. The down-regulation of these fibrosis markers is suggestive of less invasive malignancy. The number of tumor associated macrophages and their activation was related to the activity of RRx-001. Conclusions: Our pre and post dose patient derived data to date indicate a correlation between TGF-β signaling activation and a response to RRx-001 that may also correlate with increased numbers of macrophages, and their activation status, in the vicinity of the tumor. These data suggest that activation of the TGF-β1 pathway, as evident by expression of TGF-β1 and TGF-βR1 by tumor cells, could be a predictive biomarker for RRx-001 treatment. Citation Format: Saheli Jha, Thomas A. Summers, Karen Zeman, Christine Brzezniak, Corey Carter, Lindsey Ferry, Jan Scicinski, Bryan Oronsky, Scot Caroen, Jane B. Trepel, Pedro Cabrales, Regina Day. Phase II clinical trial patient responses to the macrophage activating agent RRx-001 correlate to TGF- β pathway activation and markers for fibrosis [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 966. doi:10.1158/1538-7445.AM2017-966

Abstract 944: Chemokine receptor signaling as a new tool to improve lung cancer diagnostics and therapy

Abstract Background: The tumor microenvironment plays a critical role in cancer development, progression, and control. We demonstrated in vitro and in vivo that a bidirectional crosstalk between tumor-associated macrophages and cancer cells via CCR2-CCL2 and CX3CR1-CXCL1 signaling is fundamental for lung cancer growth and metastasis offers new treatment concepts. Aim: Our working hypothesis is to develop a new platform for specific chemokine receptor (CCR2) PET imaging and a range of theranostic applications in lung cancer. For this purpose, we planned to define the CCR2 expression in NSCLC as well as in SCLC, tumor cells as well as immune/inflammatory cells. Material and Methods: This is a multicenter trial with patients from Zentralklinik Bad Berka GmbH (n =100 NSCLC, n=100 SCLC). All patients gave informed consent according to the declaration of Helsinki. FFPE material was available in all cases. CCR2 CXCR4 and CX3CR1 expression was determined by immunohistochemistry with the avidin-biotin-peroxidase-complex. Results: Analyzing 96 NSCLC (47 adenocarcinoma, 49 squamous cell carcinoma) and 45 SCLC tumor samples, we found CXCR4 and CCR2 chemokine receptor expression in tumor, immune and inflammatory cells. In NSCLC, co-expression between CCR2 and CXCR4 staining was very high concerning their expression on tumor cells rising with higher tumor stages, proliferation rate and grading. CCR2 expression in immune cells was restricted to lower stages; the same pattern could be observed in 45 SCLC tumor samples where CCR2 expression on immune cells was found especially in limited disease rather than in metastatic disease; CXCR4 expression in tumor cells was found to be high in all stages of SCLC. To evaluate the therapeutic efficacy of CCR2 antagonism in vivo, we injected LLC1 s.c. into WT mice treated with CCR2 antagonist (RS504393) or vehicle. Notably, tumor growth was significantly inhibited in CCR2 antagonist-treated mice as compared with vehicle-treated mice. Discussion: Our preliminary results show a significant portion of CCR2 positive tumors - NSCLC as well as SCLC which would qualify for a CCR2-based imaging as well as - demonstrated in our mice models - CCR2 inhibitory strategies. This should be evaluated in further preclinical trials. Citation Format: Katalin Nagy-Major, Jörg Sänger, Harshad R. Kulkarni, Peter Fix, Almut Kunze, Amelie Lupp, Reiner Bonnet, Werner Seeger, Richard P. Baum, Patricia Grabowski, Rajkumar Savai. Chemokine receptor signaling as a new tool to improve lung cancer diagnostics and therapy [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 944. doi:10.1158/1538-7445.AM2017-944

Abstract 3632: BiTE® antibody constructs for the treatment of SCLC

Abstract Small cell lung cancer (SCLC) is a highly aggressive neuroendocrine tumor with a poor prognosis and limited therapeutic options. Most patients present with extensive stage disease, where recent advances in immunotherapies, including bi-specific T cell engager (BiTE®) antibody constructs, represent a promising new therapeutic approach. BiTE® molecules have demonstrated durable complete responses in the clinic in hematological malignancies. Similar to hematological malignancies, SCLC is also a widely-disseminated malignancy that shows very high response rate to first line therapies with high rates of disease recurrence, features which may support efficacy of the BiTE® modality. Next generation sequencing (NGS) identified Delta-like Ligand 3 (DLL3) as a highly specific tumor associated antigen for SCLC, with consistent expression in tumors and very low expression in normal tissues. Tumor expression of DLL3 protein was confirmed by IHC, with 30 of 35 SCLC tumors staining positive for DLL3. DLL3 BiTE® antibody constructs showed low pM potency in vitro and also demonstrated significant inhibition of tumor growth in vivo in an orthotopic model of SCLC. A half-life extended (HLE) BiTE® targeting DLL3 demonstrated antibody-like pharmacokinetic properties in single-dose studies in non-human primates (NHP), with a half-life of 11 days. This is predicted to support every other week dosing in humans. The combination of high potency and excellent PK properties suggests that HLE BiTE® molecules may provide a useful tool for targeting residual disease in SCLC patients whose tumors express DLL3. Citation Format: Michael J. Giffin, Ed K. Lobenhofer, Keegan Cooke, Tobias Raum, Jennitte Stevens, Pedro J. Beltran, Angela Coxon, Paul E. Hughes. BiTE® antibody constructs for the treatment of SCLC [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 3632. doi:10.1158/1538-7445.AM2017-3632

P2.03a-048 The CDK4/6 Inhibitor G1T28 Protects Immune Cells from Cisplatin-Induced Toxicity in vivo and Inhibits SCLC Tumor Growth: Topic: Clinical Trials

P2.03a-048 The CDK4/6 Inhibitor G1T28 Protects Immune Cells from Cisplatin-Induced Toxicity in vivo and Inhibits SCLC Tumor Growth: Topic: Clinical Trials

Small Cell Lung Cancer Exhibits Frequent Inactivating Mutations in the Histone Methyltransferase KMT2D/MLL2: CALGB 151111 (Alliance)

IntroductionSCLC is a lethal neuroendocrine tumor type that is highly prone to metastasis. There is an urgency to understand the mutated genes that promote SCLC, as there are no approved targeted therapies yet available. SCLC is rarely resected, limiting the number of samples available for genomic analyses of somatic mutations. MethodsTo identify potential driver mutations in human SCLC we sequenced the whole exomes of 18 primary SCLCs and seven cell lines along with matched normal controls. We extended these data by resequencing a panel of genes across 40 primary SCLCs and 48 cell lines. ResultsWe report frequent mutations in the lysine methyltransferase 2D gene (KMT2D) (also known as MLL2), a key regulator of transcriptional enhancer function. KMT2D exhibited truncating nonsense/frameshift/splice site mutations in 8% of SCLC tumors and 17% of SCLC cell lines. We found that KMT2D mutation in human SCLC cell lines was associated with reduced lysine methyltransferase 2D protein levels and reduced monomethylation of histone H3 lysine 4, a mark associated with transcriptional enhancers. We also found mutations in other genes associated with transcriptional enhancer control, including CREB binding protein gene (CREBBP), E1A binding protein p300 gene (EP300), and chromodomain helicase DNA binding protein 7 gene (CHD7), and we report mutations in additional chromatin remodeling genes such as polybromo 1 gene (PBRM1). ConclusionsThese data indicate that KMT2D is one of the major mutated genes in SCLC, and they point to perturbation of transcriptional enhancer control as potentially contributing to SCLC.

Abstract A41: Use of patient-derived tumor xenografts (PDX) for discovery and development of an anti-Notch2/3 monoclonal antibody targeting cancer stem cells

Abstract Accumulating evidence has suggested that tumors may arise and grow as a result of the formation of a subset cell population termed cancer stem cells (CSC) or tumor initiating cells. Several research reports have indicated that CSCs are relatively resistant to conventional therapies. Thus, therapeutic strategies that specifically target cancer stem cells could have a major impact on cancer patient survival. Patient-derived tumor xenografts (PDX) have played a major role in the development of new cancer therapies. The advantage of PDX over standard cell-line xenograft models is that PDX retain much of the molecular, genetic, and histological heterogeneity of the original tumor and are minimally passed. In addition, self-renewal and lineage differentiation, the hallmarks of cancer stem cells, can be demonstrated through serial transplantation in immunodeficient mice. Our therapeutic approach in cancer stem cell drug discovery has been to target key developmental pathways that have been strongly implicated in cancer including the Notch pathway. We carried out tumorigenicity studies in three SCLC PDX models utilizing naïve cells positively enriched for either Notch2 or Notch3 expression. In two of the three models, Notch2+ and/or Notch3+ cell populations resulted in enhanced tumor formation. In a SCLC tumor that expressed low levels of Notch3, the Notch2+ population resulted in 70% tumor formation compared to CD44+ (10%) or CD133+ (45%) enriched populations. We have developed a monoclonal antibody, tarextumab (OMP-59R5), which selectively inhibits the function of both Notch2 and Notch3. Our preclinical data in PDX models demonstrate that tarextumab was efficacious in inhibiting the growth of various indications with minimal intestinal toxicity. Notably, the sensitivity of tarextumab in combination with gemcitabine or gemcitabine plus nab-paclitaxel in pancreatic tumors was associated with higher levels of Notch3 gene expression. Interference with Notch2/3 signaling by tarextumab delays tumor recurrence, decreases cancer stem cell frequency (as determined by in vivo LDA studies) and modulates the function of tumor vasculature. Our ALPINE Phase 1b clinical trial indicates that tarextumab is generally well-tolerated and shows signs of anti-tumor efficacy and modulation of Notch pathway signaling in the clinic. Furthermore, we observe a higher response rate and longer survival in patients with Notch3 high tumors receiving gemcitabine/nab-paclitaxel/tarextumab combination therapy. Analysis of pre- and post-treatment tumor biopsies showed an inhibition of Notch pathway and CSC gene signatures. Ongoing Phase 2 clinical trials evaluate first-line treatment with tarextumab in metastatic pancreatic cancer (ALPINE) and small cell lung cancer (PINNACLE). Collectively, these results demonstrate the utility of PDX models for discovery and development of anti-cancer stem cell therapeutics, identifying pharmacodynamic endpoints of drug actions, identifying predictive biomarkers for patient stratification, and translating these preclinical findings into clinical trials. Citation Format: Marcus M. Fischer, Wan-Ching Yen, Fumiko Axelrod, Christopher Bond, Jennifer Cain, Belinda Cancilla, Randall Henner, Rene Meisner, Aaron Sato, Jalpa Shaw, Tracy Tang, Breanna Wallace, Min Wang, Chun Zhang, Ann Kapoun, Lei Zhou, Jakob Dupont, John Lewicki, Austin Gurney, Tim Hoey. Use of patient-derived tumor xenografts (PDX) for discovery and development of an anti-Notch2/3 monoclonal antibody targeting cancer stem cells. [abstract]. In: Proceedings of the AACR Special Conference: Patient-Derived Cancer Models: Present and Future Applications from Basic Science to the Clinic; Feb 11-14, 2016; New Orleans, LA. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(16_Suppl):Abstract nr A41.

Abstract 122: Comparative analysis of small cell lung cancer and other pulmonary neuroendocrine tumors

Abstract Small cell lung cancer (SCLC) is accounting for 15% of all lung cancer cases and belongs to the family of pulmonary neuroendocrine tumors. This tumor entity additionally includes large-cell neuroendocrine carcinomas (LCNEC) and pulmonary carcinoids (PCA), which occur in 3% and 2% of all lung cancer patients, respectively. Whereas lung carcinoids are clinically benign, SCLC and LCNEC are characterized as high-grade malignant tumors, which are associated with heavy smoking and a poor 5-year survival rate of less than 5%. We aimed to characterize the genomic alterations in neuroendocrine lung tumors and performed whole genome, whole exome and transcriptome sequencing on up to 110 SCLC cases, over 40 pulmonary carcinoids and over 50 LCNEC tumors. SCLC and LCNEC tumors reveal high mutation rates with an average of 9.5 and 8.6 mutations per megabase, respectively. Our sequencing studies have shown that SCLC tumors are characterized by bi-allelic inactivation of TP53 and RB1 in almost 100% of the cases. Recurrent significant genomic alterations affected among others TP73 (13%) and NOTCH family genes (25%). Further studies in a pre-clinical mouse model confirmed NOTCH as tumor suppressor and as a regulator of neuroendocrine differentiation in SCLC (George et al., 2015). LCNEC tumors showed TP53 and RB1 alterations in up to 20% of the cases. Additionally, LCNECs were found with STK11 and KEAP1 mutations, which occurred mutually exclusive to RB1 alterations (P<0.05). In light of these distinct mutational characteristics, we analyzed the transcriptome sequencing data of neuroendocrine lung tumors in comparison to lung adenocarcinomas (LUAD) and lung squamous cell carcinomas (LUSQ). An unsupervised clustering approach led to the identification of five expression clusters: LUAD, LUSQ and PCA formed distinct transcriptional classes; the majority of SCLC and LCNEC tumors clustered into two subgroups. While a few LCNEC tumors clustered with LUAD, LUSQ and PCA, RB1-mutated LCNEC tumors predominantly clustered with SCLC samples (P <0.05). KEAP1 and STK11 mutated LCNECs did not segregate with a particular histological tumor type. Neuroendocrine lung tumors confirmed high expression levels of neuroendocrine markers. A detailed comparison of LCNEC and SCLC tumors revealed that the majority of the SCLC and a few LCNEC tumors were found in a transcriptional group that showed high expression of the neuroendocrine lineage transcription factor ASCL1, gastrin releasing peptide (GRP) and DLL3, which was less pronounced in transcriptional subsets formed by the majority of LCNEC tumors. Patient tumors that clustered to the transcriptional class marked by higher ASCL1 expression were identified to have a worse overall survival (P<0.05). In summary, these findings point to transcriptional differences in SCLC and LCNEC tumors and emphasize the precise analysis of these histological tumor types with respect to their molecular biology and therapeutic treatment option. Citation Format: Julie George, Lynnette Fernandez-Cuesta, Vonn Walter, Neil Hayes, Roman Thomas. Comparative analysis of small cell lung cancer and other pulmonary neuroendocrine tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 122.

Abstract 2531: Marker free isolation and expansion of cancer stem cells from small cell lung cancer

Abstract Small cell lung cancer (SCLC) accounts for approximately 15% of all lung cancer cases and, contrary to the advances in diagnostics and therapeutic options for non small cell lung cancer, SCLC long term survival has failed to materially improve over the last 3 decades. Early dissemination with hematogenous metastasis, advanced stages at diagnosis, dramatic response to chemotherapy with early and aggressive relapses are the clinical hallmarks. Limited surgical samples, early palliation and limited second line treatment options negating the benefit of repeat biopsies have limited tissue available for research to understand the biology of this relatively rare tumor. Recent data has suggested the presence of rare populations of cells within the primary SCLC tumors which have stem cell-like properties. We hypothesize that novel in vitro culture platforms and methods can be used to isolate and expand cancer stem cells (CSCs) within SCLC tumors and that the expanded CSCs will yield a renewable cell source for research applications and for predictive drug response profiling. Many groups have relied upon cell surface markers such as CD133 and CD24 to identify the CSC population. Our approach is unique in that we have employed a marker-free approach utilizing a combination of chemical and functional isolation. We then use a combination of growth factors, extracellular matrix proteins, and oxygen tension in a 3D perfusion culture system to further isolate, purify and expand isolated cells with the goal of having quantities of functional SCLC stem cells for molecular, proteomic and functional in vitro and in vivo assays. Initial cell sources will include immortalized cell lines and patient derived xenograft lines, with eventual adaptation to primary patient samples. Using numerous techniques, we have qualified these cells based upon marker expression (flow cytometry, RT-PCR), dye exclusion, drug response, and limited dilution assays. Future experiments will confirm stemness with limited dilution tumorigenicity assays in vivo. Initial data suggest poor correlation of canonical CSC marker expression with clonogenic growth in cell lines, supporting the need for unbiased and functional isolation methods. These expanded populations of CSCs may be used to identify novel CSC markers and/or targets, mechanisms of resistance and screen and develop novel therapeutics Our ultimate goals are to be able to use our novel culture platforms and methods to expand CSCs for real time precision medicine applications to identify novel therapies in real time that can eradicate the CSC population and improve outcomes of patients with SCLC. Citation Format: Tessa M. DesRochers, Melissa Millard, Alina Lotstein, Lillia Holmes, Hal E. Crosswell. Marker free isolation and expansion of cancer stem cells from small cell lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2531.