Small Cell Lung Cancer Models Research Articles

Abstract 56: AZD0466, a nanomedicine of a potent dual Bcl-2/Bcl-xL inhibitor, exhibits anti-tumor activity in a range of hematological and solid tumor models

Abstract The induction of apoptosis in tumor cells represents a promising approach to the treatment of cancer. In tumor cells, the B cell lymphoma 2 (Bcl-2) protein family promotes cell survival through upregulation of anti-apoptotic Bcl-2 proteins, such as Bcl-2, Bcl-xL, Mcl-1 and Bcl-w. Clinical activity of the Bcl-2 inhibitor venetoclax has validated the approach of targeting this class of molecules, but additional value remains in jointly targeting Bcl-2 with other family members. AZD0466 is a novel drug-dendrimer conjugate, where the active moiety, AZD4320, is chemically conjugated to Starpharma's DEP® dendrimer platform, a 5-generation PEGylated poly-lysine dendrimer via a hydrolytically labile linker. AZD4320 is a potent dual Bcl-2/Bcl-xL inhibitor, with nanomolar affinity for both proteins1. AZD0466 has been optimized to maintain efficacy whilst mitigating anticipated on-target toxicities of AZD4320. The active moiety, AZD4320, was profiled in an unbiased 72 h cell proliferation screen of 764 cancer cell lines. The greatest degree of sensitivity to AZD4320 (IC50 value ≤0.1 µM) was observed in hematological and small cell lung cancer (SCLC) cell lines. AZD0466 demonstrated greater monotherapy activity than platinum/etoposide chemotherapy regimen or venetoclax monotherapy in 6 out of 11 SCLC PDX models. AZD0466 was also evaluated at different doses in the RS4;11 B-ALL xenograft model. Weekly intravenous dose of AZD0466 resulted in complete tumor regression at 34 and 103 mg/kg doses. Administration of a single dose of AZD0466 produced dose dependent induction of cleaved caspase 3 in tumors as measured by MSD ELISA, which was consistent with the concentrations of released AZD4320 measured in the tumor. All treatments were well tolerated. Anti-tumor activity of AZD0466 was also evaluated in the disseminated luciferase-tagged Ri-1-DLBCL tumor model. AZD0466 dosed weekly IV at 34 mg/kg showed approximately 82% inhibition of bioluminescence compared to vehicle treated animals, whereas 103 mg/kg and 340 mg/kg showed complete inhibition of bioluminescence. In the SUDHL-4 GCB DLBCL model, 103 mg/kg AZD0466 with 10 mg/kg Rituximab resulted in complete and durable regressions in 5/6 animals. Finally, combination of 103 mg/kg AZD0466 with 12.5 mg/kg BID Acalabrutinib, a Bruton's Tyrosine kinase inhibitor, was investigated in OCI-LY10 DLBCL model. While neither agent showed any demonstrable monotherapy activity the combination resulted in complete regressions in 8/8 mice in this model. These data show that AZD0466 has monotherapy activity and a differentiated response from Venetoclax in SCLC models. AZD0466 also has therapeutic potential as monotherapy and a combinatorial agent to increase the depth and duration of response to standard of care and BTK inhibitors in hematological tumors. 1Cidado, J; et al. AACR (2018) Citation Format: Srividya B. Balachander, Areya Tabatabai, Shenghua Wen, Francis D. Gibbons, Giulia Fabbri, Guangnong Sunny Zhang, Justin Cidado, Lorraine Graham, Marianne Ashford, Barry Davies. AZD0466, a nanomedicine of a potent dual Bcl-2/Bcl-xL inhibitor, exhibits anti-tumor activity in a range of hematological and solid tumor models [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 56.

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.

Abstract 420: Rlf-Mycl1 gene fusion as a novel oncogenic driver in a mouse model of small cell lung cancer

Abstract Background: Small cell lung cancer (SCLC) is the most aggressive subtype of lung cancer, accounting for about 15% of all lung cancer cases and approximately 200,000 deaths per year. SCLC genetic alterations are characterized by near-universal loss of function of the tumor suppressor genes TP53 and RB1. Several in-frame gene fusions have been identified in SCLC tumors and cell lines, with recurrent fusion events noted in up to 5% of cases. However, the biological and therapeutic implications of these fusions, including the recurrent in-frame RLF-MYCL1 gene fusion, are unknown. MYCL1 is frequently expressed in SCLC and differential expression of MYC family members has been associated with different SCLC subtypes. Here we investigate the functional significance of the RLF-MYCL1 gene fusion by developing a novel Rlf-Mycl1-driven genetically engineered mouse model (GEMM) of SCLC using CRISPR-Cas9 genome engineering. Methods: SgRNAs targeting intronic sequences of the Rlf and Mycl1 genes were cloned into a Cas9-expressing plasmid and co-transfected in NIH/3T3 cells. The presence of the Rlf-Mycl1 fusion was confirmed at the DNA and protein level. To test whether the RLF-MYCL1 gene fusion is important for the oncogenic switch during early SCLC development, we transduced Rb1Δ/Δ;Trp53Δ/Δ;Rbl2+/Δ precancerous neuroendocrine cells (preSCs), which were isolated from an early stage in tumorigenesis, with a lentiviral vector encoding Cas9 and with the dual sgRNAs targeting Rlf and Mycl1 genes (P-RM). Results: The P-RM transduced preSCs formed significantly more colonies in soft agar than those infected with non-targeting sgRNAs (P-NT) (p<0.02). To assess the effect of Rlf-Mycl1 gene fusion in vivo, we injected P-RM and P-NT cells into the flank of athymic nude mice (n=4/5). P-RM injected mice developed detectable tumors significantly earlier than P-NT injected mice (p<0.01). To induce the Rlf-Mycl1 rearrangement in vivo, we transduced a cohort (n=12) of adult GEMM Rb1fl/fl;Trp53fl/fl;Rbl2fl/fl;Rosa26LSL-Cas9-GFP mice by intratracheal administration of a lentiviral vector encoding sgRNAs targeting Rlf and Mycl1. Mice transduced with the Rlf-Mycl1 lentiviral vector had a significantly higher tumor burden than controls by quantitative monitoring with MRI of pulmonary tumors over 6 months. These GEMMs are currently being monitored for survival assessment. Summary: A major hurdle to SCLC clinical advancement is the absence of targetable driver mutations. Our findings indicate that the Rlf-Mycl1 gene fusion accelerates SCLC tumor growth. Defining mechanisms by which Rlf-Mycl1 accelerates tumorigenesis may identify therapeutic strategies to target SCLC harboring this fusion oncogene. Our study further supports a careful analysis of the involvement of other gene-fusions in SCLC. Citation Format: Metamia Ciampricotti, Triantafyllia Karakousi, Alvaro Quintanal Villalonga, Viola Allaj, Andrea Ventura, Triparna Sen, JT Poirier, Thales Papagiannakopoulos, Charles M. Rudin. Rlf-Mycl1 gene fusion as a novel oncogenic driver in a mouse model of small cell lung cancer [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 420.

Abstract 1060: Genetic and pharmacologic inhibition of Skp2, an E3 ubiquitin ligase and RB1-target, has antitumor activity in RB1-deficient human and mouse small cell lung cancer (SCLC)

Abstract The identification of driver mutations and their corresponding targeted drugs has led to significant improvements in the treatment of non-small cell lung cancer (NSCLC) and other solid tumors; however, similar advances have not been made in the treatment of small cell lung cancer (SCLC). Due to their aggressive growth, frequent metastases, and resistance to chemotherapy, the five-year overall survival of SCLC is less than 5%. While SCLC tumors can be sensitive to first-line therapy of cisplatin and etoposide, most patients relapse, often in less than 3 months after initial therapy. Dozens of drugs have been tested clinically in SCLC, including more than 40 agents that have failed in phase III trials. The near uniform bi-allelic inactivation of the tumor suppressor gene RB1 is a defining feature of SCLC. RB1 is mutated in highly aggressive tumors, including SCLC, where its functional loss, along with that of TP53, is both required and sufficient for tumorigenesis. While it is known that RB1 mutant cells fail to arrest at G1/S in response to checkpoint signals, this information has not led to effective strategies to treat RB1-deficient tumors, and it has been challenging to develop targeted drugs for tumors that are driven by the loss of gene function. Our group previously identified Skp2, a substrate recruiting subunit of the SCF-Skp2 E3 ubiquitin ligase, as an early repression target of pRb whose knockout blocked tumorigenesis in Rb1-deficient prostate and pituitary tumors. Here we used genetic mouse models to demonstrate that deletion of Skp2 completely blocked the formation of SCLC in Rb1/p53-knockout mice (RP mice). Skp2 KO caused an increased accumulation of the Skp2-degradation target p27, a cyclin-dependent kinase inhibitor, and we confirmed this was the mechanism of protection in the RP-Skp2 KO mice by using the knock-in of a mutant p27 that was unable to bind to Skp2. Building on the observed synthetic lethality between Rb1 and Skp2, we found that small molecules that bind to and/or inhibit Skp2 induced apoptosis and inhibited SCLC cell growth. In a panel of SCLC cell lines, growth inhibition by a Skp2 inhibitor was not correlated with sensitivity/resistance to etoposide. Targeting Skp2 also had in vivo antitumor activity in mouse tumors and human patient-derived xenograft models of SCLC. Using the genetic and pharmacologic approaches, antitumor activity was seen in vivo in established SCLC primary lung tumors, in liver metastases, and in chemotherapy-resistant tumors. The identification and validation of an actionable target downstream of RB1 could have a broad impact on treatment of SCLC and other advanced tumors with mutant RB1, for which there are currently no targeted therapies available. Citation Format: Hongling Zhao, Vineeth Sukrithan, Niloy Iqbal, Cari Nicholas, Yingjiao Xue, Joseph Locker, Juntao Zou, Liang Zhu, Edward L. Schwartz. Genetic and pharmacologic inhibition of Skp2, an E3 ubiquitin ligase and RB1-target, has antitumor activity in RB1-deficient human and mouse 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 1060.

Interferon-Induced Transmembrane Protein 1 (IFITM1) Promotes Distant Metastasis of Small Cell Lung Cancer.

Small cell lung cancer (SCLC) is a severe malignancy associated with early and widespread metastasis. To study SCLC metastasis, we previously developed an orthotopic transplantation model using the human SCLC cell line DMS273. In the model, metastatic foci were found in distant tissues such as bone and the adrenal gland, similarly as observed in patients with SCLC. In this study, we evaluated the differentially expressed genes between orthotopic and metastatic tumors in the model. We isolated tumor cells from orthotopic and metastatic sites, and the tumor cell RNA was analyzed using DNA microarray analysis. We found that 19 genes in metastatic tumors were upregulated by more than 4-fold compared with their expression in orthotopic tumors. One of these genes encodes a transmembrane protein, interferon (IFN)-induced transmembrane protein 1 (IFITM1), and immunohistochemical analysis confirmed the higher expression of the protein in metastatic sites than in orthotopic sites. IFITM1 was also detected in some SCLC cell lines and lung tumors from patients with SCLC. The overexpression of IFITM1 in DMS273 cells increased their metastatic formation in the orthotopic model and in an experimental metastasis model. Conversely, the silencing of IFITM1 suppressed metastatic formation by DMS273 cells. We also found that IFITM1 overexpression promoted the metastatic formation of NCI-H69 human SCLC cells. These results demonstrate that IFITM1 promotes distant metastasis in xenograft models of human SCLC.

Trp53 and Rb1 regulate autophagy and ligand-dependent Hedgehog signaling.

Ligand-dependent activation of Hedgehog (Hh) signaling in cancer occurs without mutations in canonical pathway genes. Consequently, the genetic basis of Hh pathway activation in adult solid tumors, such as small-cell lung cancer (SCLC), is unknown. Here we show that combined inactivation of Trp53 and Rb1, a defining genetic feature of SCLC, leads to hypersensitivity to Hh ligand in vitro, and during neural tube development in vivo. This response is associated with the aberrant formation of primary cilia, an organelle essential for canonical Hh signaling through smoothened, a transmembrane protein targeted by small-molecule Hh inhibitors. We further show that loss of both Trp53 and Rb1 disables transcription of genes in the autophagic machinery necessary for the degradation of primary cilia. In turn, we also demonstrate a requirement for Kif3a, a gene essential for the formation of primary cilia, in a mouse model of SCLC induced by conditional deletion of both Trp53 and Rb1 in the adult airway. Our results provide a mechanistic framework for therapeutic targeting of ligand-dependent Hh signaling in human cancers with somatic mutations in both TP53 and RB1.

Advances in Small-Cell Lung Cancer (SCLC) Translational Research.

Over the past several years, we have witnessed a resurgence of interest in the biology and therapeutic vulnerabilities of small-cell lung cancer (SCLC). This has been driven in part through the development of a more extensive array of representative models of disease, including a diverse variety of genetically engineered mouse models and human tumor xenografts. Herein, we review recent progress in SCLC model development, and consider some of the particularly active avenues of translational research in SCLC, including interrogation of intratumoral heterogeneity, insights into the cell of origin and oncogenic drivers, mechanisms of chemoresistance, and new therapeutic opportunities including biomarker-directed targeted therapies and immunotherapies. Whereas SCLC remains a highly lethal disease, these new avenues of translational research, bringing together mechanism-based preclinical and clinical research, offer new hope for patients with SCLC.

Abstract A38: A critical role of Wnt5a-Axin2 axis in small-cell lung cancer development

Abstract The paucity of readily actionable alterations in the small-cell lung cancer (SCLC) genome necessitates exploration of alternative targets for clinical intervention, including reactivated developmental signaling pathways. Despite the widespread implication of deregulated WNT signaling in cancer, its role in the pathogenesis of SCLC remains unknown. In this study, we observe selective induction of Axin2, a target of WNT pathway, and its genetic reporter in the lung tumors developed in genetically engineered mouse models (GEMM) of SCLC. Intriguingly, however, other transcription targets of b-catenin-dependent “canonical” pathway are downregulated in tumors relative to normal lung, and knockout of Ctnnb1 encoding b-catenin does not affect tumor development in the GEMM. On the other hand, Axin2 knockout, which enhances b-catenin level, suppresses the tumor development. These findings suggest not only the dispensability of b-catenin-dependent pathway but also the significant role of Axin2 in SCLC. Meanwhile, we observe that tumors and cell lines express significantly higher levels of ligands for b-catenin-independent “noncanonical” pathway, including Wnt5a, than precancerous cells (preSC) as well as whole lung. Ectopic Wnt5a in preSC enhances cell proliferation in culture and tumorigenic capacity in athymic nude mice while increasing Axin2 expression. Depletion of Wnt5a suppresses cell proliferation and tumor development. Taken together, these results suggest a critical role of the Wnt5a-Axin2 axis in SCLC development. Citation Format: Kee-Beom Kim, Dong-Wook Kim, Youngchul Kim, Jae-Il Park, Kwon-Sik Park. A critical role of Wnt5a-Axin2 axis in small-cell lung cancer development [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr A38.

Targeted Inhibition of the E3 Ligase SCFSkp2/Cks1 Has Antitumor Activity in RB1-Deficient Human and Mouse Small-Cell Lung Cancer.

The RB1 tumor suppressor gene is mutated in highly aggressive tumors including small-cell lung cancer (SCLC), where its loss, along with TP53, is required and sufficient for tumorigenesis. While RB1-mutant cells fail to arrest at G1-S in response to cell-cycle restriction point signals, this information has not led to effective strategies to treat RB1-deficient tumors, as it is challenging to develop targeted drugs for tumors that are driven by the loss of gene function. Our group previously identified Skp2, a substrate recruiting subunit of the SCF-Skp2 E3 ubiquitin ligase, as an early repression target of pRb whose knockout blocked tumorigenesis in Rb1-deficient prostate and pituitary tumors. Here we used genetic mouse models to demonstrate that deletion of Skp2 completely blocked the formation of SCLC in Rb1/Trp53-knockout mice (RP mice). Skp2 KO caused an increased accumulation of the Skp2-degradation target p27, a cyclin-dependent kinase inhibitor, which was confirmed as the mechanism of protection by using knock-in of a mutant p27 that was unable to bind to Skp2. Building on the observed synthetic lethality between Rb1 and Skp2, we found that small molecules that bind/inhibit Skp2 have in vivo antitumor activity in mouse tumors and human patient-derived xenograft models of SCLC. Using genetic and pharmacologic approaches, antitumor activity was seen with Skp2 loss or inhibition in established SCLC primary lung tumors, in liver metastases, and in chemotherapy-resistant tumors. Our data highlight a downstream actionable target in RB1-deficient cancers, for which there are currently no targeted therapies available. SIGNIFICANCE: There are no effective therapies for SCLC. The identification of an actionable target downstream of RB1, inactivated in SCLC and other advanced tumors, could have a broad impact on its treatment.

MAX Functions as a Tumor Suppressor and Rewires Metabolism in Small Cell Lung Cancer

Small cell lung cancer (SCLC) is a highly aggressive and lethal neoplasm. To identify candidate tumor suppressors we applied CRISPR/Cas9 gene inactivation screens to a cellular model of early-stage SCLC. Among the top hits was MAX, the obligate heterodimerization partner for MYC family proteins that is mutated in human SCLC. Max deletion increases growth and transformation in cells and dramatically accelerates SCLC progression in an Rb1/Trp53-deleted mouse model. In contrast, deletion of Max abrogates tumorigenesis in MYCL-overexpressing SCLC. Max deletion in SCLC resulted in derepression of metabolic genes involved in serine and one-carbon metabolism. By increasing serine biosynthesis, Max-deleted cells exhibit resistance to serine depletion. Thus, Max loss results in metabolic rewiring and context-specific tumor suppression.

Preclinical evaluation of SC0191, a small molecule inhibitor of Wee1 kinase.

e15637 Background: TP53 mutation is common in cancer cells, especially in small cell lung cancer (SCLC) and pancreatic cancer (PC). The G1 checkpoint function is lost in these cancer cells, which more rely on G2 checkpoint to repair damaged DNA. Wee1 is a serine/threonine kinase, which phosphorylates the Tyr15 locus of CDC2, inhibits the activity of CDC2, and stays in the G2 phase. Wee1 inhibition can promote the cells with DNA damage into the M phase, thus driving the TP53 mutant cancer cells to apoptosis. The purpose of this study was to investigate the in vitro and in vivo antitumor activity of SC0191, a small molecule inhibitor of Wee1 kinase, in preclinical models of SCLC and PC with TP53 mutation. Methods: The kinase inhibiting activity of SC0191 was determined using the Wee1 kinase assays. The cellular anti-proliferative activity was evaluated with TP53 mutant NCI-H446 SCLC and BxPC-3 PC cell lines. The in vivo antitumor activity of SC0191 was evaluated in the NCI-H446 and BxPC-3 cells-derived xenograft (CDX) mouse models, respectively. Results: SC0191 displayed potent kinase inhibiting activity for Wee1 with IC50 22.3 nM. SC0191 significantly inhibited tumor cell proliferation in TP53 mutant NCI-H446 cells with IC50 231.1 nM and BxPC-3 cells with IC50 223.8 nM. Moreover, SC0191 oral administration showed significant antitumor efficacy which was better than AZD1775, in the NCI-H446 and BxPC-3 CDX mouse models, respectively. Conclusions: A novel Wee1 inhibitor, SC0191, has demonstrated excellent antitumor efficacy in TP53 mutant solid cancer preclinical studies, and represents a promising clinical candidate for treating solid tumors, such as SCLC and PC.

Abstract B15: The oral Chk1 inhibitor, SRA737, synergizes with immune checkpoint blockade in small-cell lung cancer (SCLC)

Abstract Background: Small-cell lung cancer (SCLC) is the most aggressive form of lung cancer. Despite the recent success of immunotherapy in other indications, only a minority of SCLC patients respond to immune checkpoint blockade (ICB) targeting programmed cell death protein 1 (PD-1) or programmed death ligand 1 (PD-L1) either as a monotherapy or combination. Therefore, there is a strong need to develop strategies to enhance the efficacy of immunotherapy in SCLC. Our group previously discovered that SCLC exhibits high expression of checkpoint kinase 1 (Chk1) and showed that preclinical in vivo models of SCLC respond to Chk1 inhibition. Based on data from others and our group, we hypothesized that targeting Chk1 can enhance antitumor immunity and synergize with ICB. Results: SRA737 treatment decreased cell viability with a range of potencies in a panel of SCLC cell lines in vitro. This was accompanied by an induction of double-strand breaks in sensitive cell lines as demonstrated by increased γ-H2AX. Intriguingly, SRA737 also led to an increase in micronuclei formation and STING activation in cells in vitro. Cell surface and total PD-L1 protein were increased following SRA737 treatment in vitro, further supporting a potential benefit of combining the drug with immune checkpoint blockade therapy in vivo. As hypothesized, SRA737 showed strong synergy with anti-PD-L1 antibody in an immunocompetent xenograft SCLC model. Triple-knockout SCLC cells generated from a GEMM mouse model with conditional deletion of Trp53, Rb1 and p130 were implanted into the flank of B6129F1 mice. The mice were treated for three weeks with either IgG (control), SRA737 (100mg/kg, either 3/7 or 5/7 days), anti-PD-L1 (300ug, 1/7 days) or the combination. While anti-PD1 antibody treatment was largely ineffective, SRA737 significantly delayed tumor growth (at Day 21: T/C=0.30 for 3/7 days and T/C=0.28 for 5/7 days). Combination treatment with SRA737 and anti-PD-L1 demonstrated remarkable antitumor efficacy, resulting in stable disease following SRA737 schedule of 3/7 days (T/C=0.12) and tumor regressions following SRA737 schedule of 5/7 days (T/C=0.1). These effects were sustained after treatment cessation and the long-term survival benefit is being assessed. Discussion: The intrinsic antitumor activity of the Chk1 inhibitor, SRA737, was significantly enhanced by addition of an anti-PD-L1 antibody, leading to tumor regressions in an immunocompetent SCLC model. Preliminary evidence suggests SRA737 induces micronuclei formation, STING activation and PD-L1 expression in tumor cells. Further studies to elucidate the mechanism of Chk1 inhibition-induced antitumor immunity in SCLC are ongoing. SRA737 is currently being tested in clinical trials both as a monotherapy and in combination with other agents. Given that the anti-PD-L1 antibody opdivo is now approved for SCLC, our data suggest intriguing possibilities for therapeutic synergy between the oral Chk1 inhibitor, SRA737, and ICB therapy that warrant further clinical investigation. Citation Format: Triparna Sen, Snezana Milutinovic, Robert J. Cardnell, Lixia Diao, Youhong Fan, Ryan J. Hansen, Bryan Strouse, Michael P. Hedrick, Christian Hassig, Jing Wang, Lauren A. Byers. The oral Chk1 inhibitor, SRA737, synergizes with immune checkpoint blockade in small-cell lung cancer (SCLC) [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B15.

MiR-665 Promotes the Biological Behavior of Small Cell Lung Cancer by Targeting LLGL1

背景与目的MicroRNAs（miRNAs）是一种广泛存在于真核生物体中的非编码小分子RNA，尽管一些miRNA在肿瘤中作用已被发现，但是miR-665对小细胞肺癌的中的表达及影响还尚不清楚。本研究旨在分析miR-665对肺癌细胞增殖、周期、侵袭和迁移的影响，探讨miR-665在小细胞肺癌中发挥的作用及其工作机制。方法qRT-PCR检测miR-665在肺癌组织和癌旁正常组织中的表达水平；TargetScan预测miR-665的潜在靶基因并用双荧光素酶报告基因实验、qRT-PCR和Western blot进行验证；免疫组化、qRT-PCR和Western blot检测LLGL1在肺癌组织和癌旁正常组织中的表达水平；CCK8法、流式细胞法、Transwell和细胞划痕实验检测miR-665和LLGL1对肺癌细胞NCI-H446、NCI-H1688增殖、侵袭、迁移以及S期细胞比值的影响；构建肺癌裸鼠移植瘤模型并观察miR-665对小鼠肿瘤生长的影响。结果miR-665在肺癌组织中的表达水平明显高于癌旁正常组织；miR-665能靶向作用于LLGL1的3’-UTR并抑制其表达；相比于癌旁正常组织，LLGL1在肺癌组织中的表达水平明显降低；抑制miR-665的表达可以抑制肺癌NCI-H446细胞的增殖、S期细胞比值、侵袭和迁移能力，而干扰LLGL1能逆转这种抑制效果；上调miR-665则促进肺癌NCI-H1688的增殖、S期细胞比值、侵袭和迁移能力，但这种促进效果同样被LLGL1的过表达逆转；在肺癌裸鼠移植瘤模型中，抑制miR-665能上调LLGL1蛋白的表达并抑制肿瘤的生长，而上调miR-665的表达则可以产生相反的结果。结论miR-665表达水平的变化与肺癌密切相关，miR-665可以通过抑制其靶基因LLGL1的表达促进肺癌细胞的生物学行为，在小细胞肺癌中发挥促癌基因的作用。

IA23 Developing New Therapies in Small-Cell Lung Cancer Using Parallel Clinical and Laboratory-Based Studies

Small-cell lung cancer (SCLC) is an aggressive high-grade neuroendocrine malignancy with high metastatic potential and poor clinical outcomes. My translational research program utilizes both preclinical studies and clinical trial strategies to develop improved treatments for patients with SCLC. Preclinical and clinical studies have demonstrated activity of poly (ADP-ribose) polymerase (PARP) inhibitors in SCLC, though overall the activity of PARP inhibitor monotherapy has been quite modest. Combinations with other DNA-damaging agents have shown greater potential in trials. We conducted a phase I/II trial of combination olaparib tablets and temozolomide in previously treated SCLC. We established a recommended phase 2 dose (RP2D) of olaparib 200 mg PO BID with temozolomide 75 mg/m2 daily, both on days 1-7 of a 21-day cycle, and expanded to a total of 50 patients. The confirmed overall response rate (ORR) was 41.7% (20/48 evaluable), median progression-free survival (mPFS) was 4.2 months (95% CI 2.8-5.7), and median overall survival (mOS) was 8.5 months (95% CI 5.1-11.3) after a median follow-up of 7.1 months (Farago et al., Cancer Discovery 2019, PMID 31416802). Overall, these results indicate promising activity of combination olaparib and temozolomide in SCLC. In parallel, we have generated a panel of patient-derived xenograft (PDX) models of SCLC, using both tissue biopsies and circulating tumor cells (Drapkin et al., Cancer Discovery 2018, PMID 29483136). This panel includes 6 PDX models derived from patients enrolled to the olaparib/temozolomide trial. The responses of these in vivo tumor models to olaparib/temozolomide recapitulated the clinical responses of the corresponding patients. This enabled a coclinical trial in 32 PDX models, which we then utilized to identify putative biomarkers of response and resistance to olaparib/temozolomide. Using paired-end transcriptome sequencing, we identified a correlation between low basal expression of inflammatory response genes and cross-resistance to both olaparib/temozolomide and standard first-line chemotherapy, etoposide/platinum. We are now exploring mechanisms of acquired resistance to olaparib/temozolomide using serially derived PDX models from patients before and after treatment with this regimen. Updated data will be presented at the meeting.

SR9009 induces a REV-ERB dependent anti-small-cell lung cancer effect through inhibition of autophagy.

Rationale: The circadian clock coordinates cell proliferation and metabolism and impacts the progression of some diseases, particularly cancer. Pharmacological modulation of the circadian machinery may be an effective therapeutic approach for treating cancer. SR9009 is a specific synthetic agonist of the REV-ERBs, essential circadian clock components. However, the potential efficacy and antitumor mechanism of this drug in small-cell lung cancer (SCLC) remains poorly understood.Methods: Here, we used chemosensitive cells (H69 and H446) and the corresponding chemoresistant cells (H69AR and H446DDP) to assess the efficacy of the REV-ERB agonist SR9009 for the treatment of SCLC in vitro and further validated the antitumor effect in subcutaneous tumor models of SCLC. Then, we determined whether REV-ERBα was correlated with the anti-SCLC effect of SR9009. Chromatin immunoprecipitation (ChIP) sequencing assays were conducted to identify potential DNA sequences directly regulated by REV-ERBα. Autophagy regulation by REV-ERBα and its possible mechanism in SR9009-based SCLC therapy were analyzed.Results: Here, we showed that the REV-ERB agonist SR9009 is specifically lethal to both chemosensitive and chemoresistant SCLC cells. REV-ERBα was involved in the antitumor effect of SR9009 in SCLC. The core autophagy gene Atg5 was identified as a direct downstream target of REV-ERBα and was suppressed by the REV-ERB agonist SR9009 in SCLC. Furthermore, the interaction of REV-ERBα with this autophagy gene impaired autophagy activity, leading to SR9009 cytotoxicity in SCLC cells.Principal conclusions: Our study provided a novel viewpoint indicating that the REV-ERB agonist SR9009 could be a novel and promising therapeutic strategy in first- or second-line SCLC treatment. The anti-SCLC effect of SR9009 is mediated by REV-ERB dependent suppression of autophagy via direct repression of the autophagy gene Atg5.

CDK7 Inhibition Potentiates Genome Instability Triggering Anti-tumor Immunity in Small Cell Lung Cancer

Cyclin-dependent kinase 7 (CDK7) is a central regulator of the cell cycle and gene transcription. However, little is known about its impact on genomic instability and cancer immunity. Using a selective CDK7 inhibitor, YKL-5-124, we demonstrated that CDK7 inhibition predominately disrupts cell-cycle progression and induces DNA replication stress and genome instability in small cell lung cancer (SCLC) while simultaneously triggering immune-response signaling. These tumor-intrinsic events provoke a robust immune surveillance program elicited by Tcells, which is further enhanced by the addition of immune-checkpoint blockade. Combining YKL-5-124 with anti-PD-1 offers significant survival benefit in multiple highly aggressive murine models of SCLC, providing a rationale for new combination regimens consisting of CDK7 inhibitors and immunotherapies.

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.

Abstract C091: Preclinical evaluation of PK, PD, and antitumor activity of the oral, non-covalent, potent and highly selective CDK7 inhibitor, SY-5609, provides rationale for clinical development in multiple solid tumor indications

Abstract Introduction: CDK7 is a key regulator of transcription and cell cycle progression and has been implicated in multiple tumor types driven by aberrant transcriptional control (e.g. MYC-, ESR1-activation) and/or aberrant cell cycle control (e.g. RB1, CCNE1, CDKN2A alterations). SY-5609 is an oral, potent, and highly selective CDK7 inhibitor that is advancing through IND-enabling studies to support initiation of a planned Phase 1 oncology trial in early 2020. Here we report on the relationship between pharmacokinetics (PK), pharmacodynamics (PD), and tumor growth inhibition (TGI) in xenograft models of tumor types with transcriptional and/or cell cycle aberrations including high grade serous ovarian cancer (HGSOC), small cell lung cancer (SCLC), triple negative breast cancer (TNBC), and estrogen receptor positive breast cancer (ER+BC). Methods: The relationship between SY-5609 PK, PD, and TGI was evaluated in xenograft models of HGSOC (OVCAR3) and TNBC (HCC70) using once daily (QD) or twice daily (BID) dosing via oral gavage. SY-5609 TGI was evaluated as a single agent (SA) QD in patient-derived xenograft (PDX) models of HGSOC (n=3), SCLC (n=4), TNBC (n=4), and in combination with once weekly fulvestrant in ER+BC PDX models selected in vivo for resistance to the CDK4/6 inhibitor palbociclib (n=1) or resistance to both palbociclib and fulvestrant (n=1). Results: SY-5609 plasma exposure was dose proportional and did not accumulate after repeated therapeutic doses. Dose-dependent transcriptional responses in xenograft tissue were observed within 4 hours of SY-5609 dosing and were sustained for 24 hours. TGI was dose-dependent, with tumor regressions observed at doses significantly below the maximum tolerated dose (MTD). Similar TGI was seen when the same daily dose was administered either QD or BID suggesting that the effect was driven by overall exposure or minimum concentration. In HGSOC, SCLC, and TNBC PDX models, SA SY-5609 induced >50% TGI in all models tested (11/11), with 7/11 (64%) demonstrating robust anti-tumor activity (≥90% TGI or regression): 3/3 HGSOC, 2/4 SCLC, and 2/4 TNBC. In a palbociclib-resistant ER+BC PDX model, the combination of SY-5609 and fulvestrant induced significant TGI (89%), with no evident tumor regrowth up to 21 days after dosing cessation, distinguishing the observed effects from SY-5609 SA or fulvestrant SA. In a palbociclib and fulvestrant double-resistant ER+ BC PDX model, SY-5609 SA resulted in 33% TGI and fulvestrant SA had no activity. In contrast, the combination of SY-5609 and fulvestrant demonstrated significant TGI (68%; p<0.0001 vs fulvestrant SA), suggesting re-sensitization to fulvestrant. Conclusions: We have characterized PK, PD, and anti-tumor activity of the CDK7 inhibitor SY-5609 in a series of xenograft models. Exposure in plasma is dose-proportional and does not accumulate at therapeutic dose levels. SY-5609 induces dose-dependent transcriptional responses in tumor xenograft tissue and shows robust TGI, including regressions, in PDX models derived from multiple solid tumor indications. These results highlight the broad potential for SY-5609 across a variety of solid tumor types, including treatment resistant ER+BC, and support the development of SY-5609 in early phase clinical trials. Citation Format: Liv H Johannessen, Shanhu Hu, Nan Ke, Anthony D'Ippolito, Nisha Rajagopal, Jason Marineau, Anneli Savinainen, William Zamboni, Graeme Hodgson. Preclinical evaluation of PK, PD, and antitumor activity of the oral, non-covalent, potent and highly selective CDK7 inhibitor, SY-5609, provides rationale for clinical development in multiple solid tumor indications [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C091. doi:10.1158/1535-7163.TARG-19-C091

Abstract B064: The Aurora kinase A-inhibitor, alisertib, is a potential candidate for combination with immunotherapy in small cell lung cancer

Abstract Background: Small cell lung cancer (SCLC) is characterized by a loss of TP53 and RB1 and by amplification of MYC in about 25% of patients. Our group and others have shown that MYC-driven SCLC cell lines are vulnerable to Aurora Kinase A (AURKA) inhibition. Alisertib, a selective AURKA inhibitor, has also shown clinical efficacy in MYC-positive relapsed SCLC patients in combination with paclitaxel in a phase II trial (NCT02038647). AURKA regulates DNA chromosome alignment during cell division and thus its inhibition results in mitotic stress and subsequently DNA damage. We have previously demonstrated that DNA damage response (DDR) inhibitors synergize with immune checkpoint inhibition in SCLC in vivo models by activating a STING-mediated innate immune response. Based on these findings, we hypothesized that AURKA inhibition may activate the STING pathway and induce PD-L1 expression in SCLC. Methods: We compared AURKA gene expression among a panel of SCLC and NSCLC cell lines and, similarly, in tumor versus normal tissue in SCLC clinical dataset (Sato et al.). In parallel, we explored the landscape of STING and immune genes expression in two SCLC clinical datasets (George et al. and Sato et al.).We tested human and GEMM-derived SCLC cell lines for changes in immune and DDR-related protein expression by Western blot analysis after treatment with the AURKA-i alisertib. Results: AURKA gene expression was higher in SCLC cell lines as compared to NSCLC cell lines (p=0.023) and in SCLC tumors compared to normal tissue (FC=1.6; p<0.001), thus making AURKA an attractive target in SCLC. Furthermore, in SCLC clinical datasets, we found that CXCL10 and CCL5, the two downstream chemokines of STING pathway, are highly expressed in about 30% SCLC tumors and their expression is significantly associated with CMYC (Spearman Rho>0.45, p<0.001), possibly due to high baseline levels of replication stress and also to CD274, CD8 and other markers and chemokines suggestive of an inflamed immune microenvironment. We observed that in vitro treatment with alisertib (1µM for 72 hours) further increases the level of phospho-H2AX protein, a marker of DNA damage, along with PD-L1. Furthermore, alisertib treatment resulted in STING pathway activation, as indicated by increased phospho-STING_S366 and phospho-TBK1_S172 protein levels, in both human and murine SCLC cells. Conclusions: Our results demonstrate the potential of alisertib, the selective AURKA inhibitor, in modulation of PD-L1 and STING pathway activation in SCLC cells, thus supporting the rationale for in vivo testing with immunotherapy drugs. In particular, evaluation of this strategy in MYC-positive SCLC models could open new therapeutic investigations in this highly resistant SCLC subgroup. Citation Format: Carminia M. Della Corte, Liz Lauren Ajpacaja, Robert J Cardnell, Carl M Gay, Lixia Diao, Qi Wang, Kavya Ramkumar, Allison C. Stewart, Rebecca B. Kow, Hannah E Stumpf, You-Hong Fan, Jing Wang, John V Heymach, Lauren A. Byers. The Aurora kinase A-inhibitor, alisertib, is a potential candidate for combination with immunotherapy in small cell lung cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B064. doi:10.1158/1535-7163.TARG-19-B064

MS05.03 Notch Signalling

The Notch signaling pathway is a highly conserved pathway that has a vital role in embryonic development and post-embryonic functions such as hematopoiesis, neural cell development and angiogenesis (1). The signaling pathway is activated by the interaction of 4 Notch receptors and their interactions with 5 different ligands. Signaling is characterized by juxtacrine interaction between ligands and receptors on neighboring cells or within the same cell. Alterations in this pathway have been detected in several tumors both as tumor promoter and tumor suppressor. Notch signaling does appear to have relevance in non-small cell lung cancers (NSCLC) (2). In approximately 30% of NSCLCs loss of Numb, a negative regulator of Notch1, leads to increased Notch activity and about 10% of NSCLCs have gain of function mutations in Notch1. A meta-analysis demonstrated that higher expression of Notch1 was correlated with more advanced tumors (3). In addition, higher expressions of both Notch1 and Notch3 were associated with poor prognosis (4,5). In pre-clinical studies inhibition of Notch3 signaling has reduced growth of lung cancers. In addition, it has been demonstrated that there is cross talk between Notch3 and EGFR pathways and inhibition of both pathways can induce expression of anti-apoptotic protein BIM. Finally, Notch signaling may have a role in induction of the epithelial to mesenchymal phenotype (EMT) in cancers (6). EMT is known to be associated with resistance to both cytotoxic agents and targeted agents and inhibition of Notch signaling can not only reverse EMT but also can enhance the anti-tumor activity of cytotoxic agents such as cisplatin and targeted agents such as erlotinib. The EMT phenotype is frequently observed in a sub-population of cancer cells with self-renewal capacity or cancer stem cells. Notch signaling may be crucial to survival of cancer stem cells and persistence of this population may contribute to resistance to therapeutic agents. In squamous cell lung cancers Notch signaling may have tumor suppressive properties (7). Loss of function mutations in Notch family of genes, predominantly in Notch receptors, are frequently identified in several squamous cell cancers including squamous cell cancer of the lung. Similarly, loss of function mutations in Notch genes, particularly Notch1 have been identified in small cell lung cancer (SCLC). Expression of Notch receptor in a mouse model of SCLC reduced tumor burden, suggesting its tumor suppressive properties. The expression of DLL3, one of the Notch ligands is induced in SCLC by a key transcription factor ASCL1.DLL3 is shown to downregulate Notch signaling in SCLCs and enhance the carcinogenic phenotype. All the above data suggest that Notch signaling is highly contextual. In some tumors this pathway may have tumor suppressive properties but in others tumor promoting properties. Defining the role of this pathway in tumor types may guide development of therapeutic strategies targeting the Notch signaling pathway. Bigas A, Espinosa L. The multiple usages of Notch signaling in development, cell differentiation and cancer. Curr Opin Cell Biol 2018;55:1-7. Westhoff B, Colaluca IN, D’Ario G, et al. Alterations of the Notch pathway in lung cancer. Proc Natl Acad Sci 2009;106:22293-8. Yuan X, Wu H, Xu H, et al. Meta-analysis reveals the correlation of Notch signaling with non-small cell lung cancer progression and prognosis. Sci Rep 2015;5:10338. Donnem T, Andersen S, Al-shibili K, et al. Prognostic impact of Notch ligands and receptors in nonsmall cell lung cancer: coexpression of Notch-1 and vascular endothelial growth factor-A predicts poor survival. Cancer 2010:116:5674-85. Hassan WA, Yoshida R, Kudoh S, et al. Evaluation of role of Notch3 signaling pathway in human lung cancer cells. J Cancer Res Clin Oncol 2016;142:981-93. Yuan X, Wu H, Han N, et al. Notch signaling and EMT in non-small cell lung cancer: biological significance and therapeutic application. J Hematol Oncol 2014;7:87. Nowell CS, Radtke F. Notch as a tumor suppressor. Nat Rev Cancer 2017;17:145-159. squamous, Tumor suppressor, NOTCH