Small Lung Cancer Cell Lines Research Articles

CADM1 promotes malignant features of small-cell lung cancer by recruiting 4.1R to the plasma membrane

Cell adhesion molecule 1 (CADM1), which mediates intercellular adhesion between epithelial cells, is shown to be highly expressed in small-cell lung cancer (SCLC) and to enhance tumorigenicity of SCLC cells in nude mice. Here, we investigated the molecular mechanism underlying the oncogenic role of CADM1 in SCLC. CADM1 promoted colony formation of SCLC cells in soft agar. Analysis of deletion and point mutants of the conserved protein-binding motifs in CADM1 revealed that the 4.1 protein-binding motif in the cytoplasmic domain is responsible for the promotion of colony formation. Among the actin-binding 4.1 proteins, 4.1R was the only protein whose localization to the plasma membrane is dependent on CADM1 expression in SCLC cells. Knockdown of 4.1R suppressed the colony formation enhanced by CADM1, suggesting that 4.1R is required for the oncogenic role of CADM1 in SCLC. In primary SCLC, CADM1 expression was correlated with membranous localization of 4.1R, as was observed in a SCLC cell line. Moreover, membranous co-localization of CADM1 and 4.1R was associated with more advanced tumor stage. These results suggest that the formation of CADM1–4.1R complex would promote malignant features of SCLC.

Effective ferroptotic small-cell lung cancer cell death from SLC7A11 inhibition by sulforaphane.

Small-cell lung cancer (SCLC) is a highly aggressive cancer with poor prognosis, due to a lack of therapeutic targets. Sulforaphane (SFN) is an isothiocyanate derived from cruciferous vegetables and has shown anticancer effects against numerous types of cancer. However, its anticancer effect against SCLC remains unclear. The present study aimed to demonstrate the anticancer effects of SFN in SCLC cells by investigating cell death (ferroptosis, necroptosis and caspase inhibition). The human SCLC cell lines NCI-H69, NCI-H69AR (H69AR) and NCI-H82 and the normal bronchial epithelial cell line, 16HBE14o- were used to determine cell growth and cytotoxicity, evaluate the levels of iron and glutathione, and quantify lipid peroxidation following treatment with SFN. mRNA expression levels of cystine/glutamate antiporter xCT (SLC7A11), a key component of the cysteine/glutamate antiporter, were measured using reverse transcription-quantitative PCR, while the levels of SLC7A11 protein were measured using western blot analysis. Following the addition of SFN to the cell culture, cell growth was significantly inhibited, and cell death was shown in SCLC and multidrug-resistant H69AR cells. The ferroptotic effects of SFN were confirmed following culture with the ferroptosis inhibitor, ferrostatin-1, and deferoxamine; iron levels were elevated, which resulted in the accumulation of lipid reactive oxygen species. The mRNA and protein expression levels of SLC7A11 were significantly lower in SFN-treated cells compared with that in the control cells (P<0.0001 and P=0.0006, respectively). These results indicated that the anticancer effects of SFN may be caused by ferroptosis in the SCLC cells, which was hypothesized to be triggered from the inhibition of mRNA and protein expression levels of SLC7A11. In conclusion, the present study demonstrated that SFN-induced cell death was mediated via ferroptosis and inhibition of the mRNA and protein expression levels of SLC7A11 in SCLC cells. The anticancer effects of SFN may provide novel options for SCLC treatment.

Combinations of proteasome inhibitors with obatoclax are effective for small cell lung cancer.

Proteasome inhibitors, bortezomib (BTZ), and carfilzomib (CFZ) are approved drugs for hematological malignancies, but lack anticancer activities against most solid tumors. Small cell lung cancer (SCLC) is a very aggressive neuroendocrine carcinoma of the lungs demanding effective therapy. In this study we investigated whether BTZ or CFZ combined with obatoclax (OBX), an antagonist for MCL-1 and a pan-BCL family inhibitor, could cause synergistic growth inhibition of SCLC cells. We showed that combined application of BTZ or CFZ with OBX caused synergistic growth inhibition of human SCLC cell lines (H82, H526, DMS79, H196, H1963, and H69) than single agent alone. Both BTZ-OBX and CFZ-OBX combinations displayed marked synergism on inducing apoptosis (~50% increase vs BTZ or CFZ alone). A comprehensive proteomics analysis revealed that BTZ preferentially induced the expression of MCL-1, an antiapoptotic protein, in SCLC cells. Thus, proteasome inhibitor-OBX combinations could specifically induce massive growth inhibition and apoptosis in SCLC cells. Subsequent proteome-wide profiling analysis of activated transcription factors suggested that BTZ- or CFZ-induced MCL-1 upregulation was transcriptionally driven by FOXM1. In nude mice bearing in SCLC H82 xenografts, both BTZ-OBX, and CFZ-OBX combinations exhibited remarkable antitumor activities against SCLC tumors evidenced by significant reduction of tumor size and the proliferation marker Ki-67 signals in tumor tissues as compared with single agent alone. Thus, proteasome inhibitor-OBX combinations are worth immediate assessments for SCLC in clinical settings.

Hypoxia Induces Resistance to EGFR Inhibitors in Lung Cancer Cells via Upregulation of FGFR1 and the MAPK Pathway.

Development of resistance remains the key obstacle to the clinical efficacy of EGFR tyrosine kinase inhibitors (TKI). Hypoxia is a key microenvironmental stress in solid tumors associated with acquired resistance to conventional therapy. Consistent with our previous studies, we show here that long-term, moderate hypoxia promotes resistance to the EGFR TKI osimertinib (AZD9291) in the non-small cell lung cancer (NSCLC) cell line H1975, which harbors two EGFR mutations including T790M. Hypoxia-induced resistance was associated with development of epithelial-mesenchymal transition (EMT) coordinated by increased expression of ZEB-1, an EMT activator. Hypoxia induced increased fibroblast growth factor receptor 1 (FGFR1) expression in NSCLC cell lines H1975, HCC827, and YLR086, and knockdown of FGFR1 attenuated hypoxia-induced EGFR TKI resistance in each line. Upregulated expression of FGFR1 by hypoxia was mediated through the MAPK pathway and attenuated induction of the proapoptotic factor BIM. Consistent with this, inhibition of FGFR1 function by the selective small-molecule inhibitor BGJ398 enhanced EGFR TKI sensitivity and promoted upregulation of BIM levels. Furthermore, inhibition of MEK activity by trametinib showed similar effects. In tumor xenografts in mice, treatment with either BGJ398 or trametinib enhanced response to AZD9291 and improved survival. These results suggest that hypoxia is a driving force for acquired resistance to EGFR TKIs through increased expression of FGFR1. The combination of EGFR TKI and FGFR1 or MEK inhibitors may offer an attractive therapeutic strategy for NSCLC. SIGNIFICANCE: Hypoxia-induced resistance to EGFR TKI is driven by overexpression of FGFR1 to sustain ERK signaling, where a subsequent combination of EGFR TKI with FGFR1 inhibitors or MEK inhibitors reverses this resistance. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/21/4655/F1.large.jpg.

Discovery of CC-90011: A Potent and Selective Reversible Inhibitor of Lysine Specific Demethylase 1 (LSD1).

Histone demethylase LSDl (KDMlA) belongs to the flavin adenine dinucleotide (FAD) dependent family of monoamine oxidases and is vital in regulation of mammalian biology. Dysregulation and overexpression of LSD1 are hallmarks of a number of human diseases, particularly cancers that are characterized as morphologically poorly differentiated. As such, inhibitors of LSD1 have potential to be beneficial as a cancer therapy. The most clinically advanced inhibitors of LSDl are covalent inhibitors derived from tranylcypromine (TCP). Herein, we report the discovery of a novel series of reversible and selective LSDl inhibitors. Exploration of structure-activity relationships (SARs) and optimization of ADME properties resulted in the identification of clinical candidate CC-90011. CC-90011 exhibits potent on-target induction of cellular differentiation in acute myeloid leukemia (AML) and small cell lung cancer (SCLC) cell lines, and antitumor efficacy in patient-derived xenograft (PDX) SCLC models. CC-90011 is currently in phase 2 trials in patients with first line, extensive stage SCLC (ClinicalTrials.gov identifier: NCT03850067).

A Bispecific DLL3/CD3 IgG-Like T-Cell Engaging Antibody Induces Antitumor Responses in Small Cell Lung Cancer.

Small cell lung cancer (SCLC) is the most lethal and aggressive subtype of lung carcinoma characterized by highly chemotherapy-resistant recurrence in the majority of patients. To effectively treat SCLC, we have developed a unique and novel IgG-like T-cell engaging bispecific antibody (ITE) that potently redirects T-cells to specifically lyse SCLC cells expressing Delta-like ligand 3 (DLL3), an antigen that is frequently expressed on the cell surface of SCLC cells, with no to very little detectable expression in normal tissues. The antitumor activity and mode of action of DLL3/CD3 ITE was evaluated in vitro using SCLC cell lines and primary human effector cells and in vivo in an SCLC xenograft model reconstituted with human CD3+ T-cells. Selective binding of DLL3/CD3 ITE to DLL3-positive tumor cells and T-cells induces formation of an immunological synapse resulting in tumor cell lysis and activation of T-cells. In a human T-cell engrafted xenograft model, the DLL3/CD3 ITE leads to an increase in infiltration of T-cells into the tumor tissue resulting in apoptosis of the tumor cells and tumor regression. Consistent with the mode of action, the DLL3/CD3 ITE treatment led to upregulation of PD-1, PD-L1, and LAG-3. This study highlights the ability of the DLL3/CD3 ITE to induce strictly DLL3-dependent T-cell redirected lysis of tumor cells and recruitment of T-cells into noninflamed tumor tissues leading to tumor regression in a preclinical in vivo model. These data support clinical testing of the DLL3/CD3 ITE in patients with SCLC.

SCLC-CellMiner: A Resource for Small Cell Lung Cancer Cell Line Genomics and Pharmacology Based on Genomic Signatures

SUMMARYCellMiner-SCLC (https://discover.nci.nih.gov/SclcCellMinerCDB/) integrates drug sensitivity and genomic data, including high-resolution methylome and transcriptome from 118 patient-derived small cell lung cancer (SCLC) cell lines, providing a resource for research into this “recalcitrant cancer.” We demonstrate the reproducibility and stability of data from multiple sources and validate the SCLC consensus nomenclature on the basis of expression of master transcription factors NEUROD1, ASCL1, POU2F3, and YAP1. Our analyses reveal transcription networks linking SCLC subtypes with MYC and its paralogs and the NOTCH and HIPPO pathways. SCLC subsets express specific surface markers, providing potential opportunities for antibody-based targeted therapies. YAP1-driven SCLCs are notable for differential expression of the NOTCH pathway, epithelial-mesenchymal transition (EMT), and antigen-presenting machinery (APM) genes and sensitivity to mTOR and AKT inhibitors. These analyses provide insights into SCLC biology and a framework for future investigations into subtype-specific SCLC vulnerabilities.

NRXN1 as a novel potential target of antibody-drug conjugates for small cell lung cancer.

Small cell lung cancer (SCLC) is a high-grade malignancy, and treatment strategies have not changed for decades. In this study, we searched for novel targets for antibody-drug conjugate (ADC) therapy for SCLC. We identified transmembrane proteins overexpressed specifically in SCLC with little or no expression in normal tissues and decided to focus on the cell adhesion molecule neurexin-1 (NRXN1). The cell surface overexpression of NRXN1 was confirmed using flow cytometry in SCLC cell lines (SHP77 and NCI-H526). The combination of a primary anti-NRXN1 monoclonal antibody and a secondary ADC exhibited anti-tumor activity in SCLC cell lines. Moreover, the knockout of NRXN1 in SHP77 cells resulted in a loss of the anti-tumor activity of NRXN1-mediated ADC therapy. Thus, NRXN1 could be a novel target for ADC therapy for the treatment of SCLC that is worth further research.

Exploring the mechanism of cisplatin resistance by transcriptome sequencing and reversing the chemoresistance by autophagy inhibition in small cell lung cancer

Cisplatin plays a key role in treating small cell lung cancer (SCLC); however, the rapid development of cisplatin resistance limits its treatment effect. The detailed mechanisms of cisplatin-resistance, particularly in SCLC, remain unclear. We analyzed the differentially expressed genes (DEGs) between cisplatin-resistant small cell lung cancer cell line H446/CDDP and its parental cell line H446, using the transcriptome sequencing technique. Gene ontology (GO) analysis and the subsequent tests demonstrated that the functions of protein ubiquitination and autophagy are more active in the H446/CDDP cells. Autophagy plays a protective role in the H446/CDDP cells by using the autophagy inhibitors, 3-methyladenine and bafilomycin A1. Moreover, antimalarial drugs that inhibit autophagy by increasing the pH of lysosomes can also enhance cisplatin-induced cell death.

Resveratrol inhibits viability and induces apoptosis in the small‑cell lung cancer H446 cell line via the PI3K/Akt/c‑Myc pathway.

There have been no major breakthroughs in the treatment of small-cell lung cancer (SCLC) in recent decades. It is thus essential to explore new or adjuvant treatment options for SCLC. Resveratrol (Res) is a natural antioxidant revealed to influence the entire process of cancer development. Accordingly, the present study used the SCLC cell line H446 to explore the antitumor mechanism of Res. Cells were treated with 40 µg/ml Res with or without pretreatment with the antioxidant N-acetyl-L-cysteine (NAC). H446 cell viability and apoptosis were assessed with MTT and flow cytometry, and the expression of cytochrome c and the PI3K/Akt/c-Myc pathway and the nuclear translocation of apoptosis inducing factor (AIF) were assessed by western blotting. In addition, the changes in ROS content and mitochondrial membrane potential were determined. The results revealed that Res inhibited H446 cell viability and induced apoptosis, increased cytochrome c expression, inhibited the expression of PI3K/Akt/c-Myc signaling pathway components, and promoted the translocation of AIF from the cytoplasm to the nucleus in H446 cells. However, NAC pretreatment reversed these changes to various extents. The results of the present study indicated that Res may inhibit the viability and promote the apoptosis of human SCLC H446 cells through the PI3K/Akt/c-Myc pathway and that oxidative stress and mitochondrial membrane potential depolarization may be involved in the aforementioned processes.

Abstract 5328: mTOR inhibitor enhances anti-tumor effect of BET bromodomain inhibitor in small cell lung cancer

Abstract Introduction: Small cell lung cancer (SCLC) is one of the two recalcitrant cancers designated by NCI, and its 2-year survival rate has not improved over the past two decades. Novel therapy is urgently needed. BET bromodomain inhibitor (BETi), which targets transcriptional coactivator BET bromodomain proteins, has been reported to be effective in SCLC. However, given that SCLC is notorious for rapid development of drug resistance, it is necessary to identify drugs that can synergize with BETi to form a combinatorial drug regimen. Methods: A high-throughput drug combination matrix screen was performed to identify potential agents that can synergize with BETi. Synergy between the identified candidate drugs and BETi was further validated in various SCLC cell lines by using the CellTiter-Glo assay as well as in several SCLC PDX models by examining tumor growth suppression and survival of tumor-bearing mice. Cleavage of caspase 3 and poly (ADP-ribose) polymerase (PARP) was analyzed by IHC and western blot in SCLC PDX tumors and in vitro cell lines to assess apoptosis after drug treatment, respectively. Results: The high-throughput screen identified multiple inhibitors of the PI-3K/AKT/mTOR pathway to synergize with BETi by causing a significantly higher rate of apoptosis and cell growth inhibition. A comparison among the inhibitors that specifically target PI-3K, AKT or mTOR revealed that the combination of BETi and mTOR inhibitor (mTORi) have the highest level of synergy. The BETi/mTORi combo also showed synergy in several SCLC cell lines that are initially resistant to single agent BETi. The antitumor effect of the BETi/mTORi combinatorial treatment was further confirmed in several SCLC PDX mouse models. When compared to either single agent or vehicle, the BETi/mTORi combo treatment led to a greater degree of tumor growth suppression and a more prolonged survival of the tumor-bearing mice. This enhanced antitumor effect is connected with a significantly more apoptosis (cleaved caspase 3 or PARP) found in SCLC tumor tissues and cell lines treated with the BETi/mTORi combo. Conclusion: mTORi can enhance the antitumor effect of BETi in SCLC by causing more apoptosis. Citation Format: ANJU KUMARI, Lisa Gesumaria, Patricia Fetsch, Markku Miettinen, Keith Hughitt, Beverly Mock, Craig Thomas, David S. Schrump, Haobin Chen. mTOR inhibitor enhances anti-tumor effect of BET bromodomain inhibitor in 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 5328.

Abstract 4922: NEUROD1 is highly expressed in extensive small cell lung cancer by promoting migration

Abstract Introduction:Small cell lung cancer (SCLC) exhibits high-grade neuroendocrine features and recent genomic analyses revealed that aberrant expression of bacic helix loop helix transcription factors such as neurogenic differentiation 1 (NEUROD1) as well as achaete-scute homolog 1(ASCL1) have also been observed in SCLC. NEUROD1 plays important role for the development, maturation in several nerve system and formation of distal lung of neuroendocrine morphology. Previous report showed that mRNA expression as well as target genes of NEUROD1 are largely distinct from those of ASCL1 in human SCLC cell lines, however, protein level and expression patterns of these transcription factors in SCLC clinical tumor samples as well as its relation to clinicopathological characteristics have not been fully elucidated. Method: We retrospectively analyzed specimens from 95 SCLC patients between June 1988 and December 2017, and immunohistochemical (IHC) staining of NEUROD1 as well as ASCL1 was performed. Furthermore, we examined the effect of gain or loss of NEUROD1 function on proliferation and migration by transfection in SCLC cell lines. Result:Dual high staining score of ASCL1 and NEUROD1 was present in SCLC samples (32/95: 34%) and IHC score of NEUROD1 was higher in extensive disease (ED) samples than those in limited disease (LD) samples (median score 160 vs 80 ; P = 0.0389).Overall survival did not significantly differ between SCLC patients with high or low score of NEUROD1 or ASCL1. Next, our vitro data showed that upregulation of NEUROD1 in SCLC cell lines promoted transwell migration whereas downregulation of NEUROD1 reduced cell migration. Conclusion: Our results from clinical and in vitro experimental data collectively indicate that NEUROD1 expression increases in extensive SCLC by promoting migration and metastasis of SCLC cells. Citation Format: Yuki Ikematsu, Kentaro Tanaka, Nobuhisa Ando, Kayo Ijichi, Yasuto Yoneshima, Hiroyuki Inoue, Goji Toyokawa, Tetsuzo Tagawa, Yoichi Nakanishi, Isamu Okamoto. NEUROD1 is highly expressed in extensive small cell lung cancer by promoting migration [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 4922.

Abstract 4124: The combination of PARP inhibitor talazoparib with low-dose temozolomide results in increased cell lethality in BRCA1/2 wild-type melanoma, small cell lung cancer, ovarian, and colon cancer cell lines and mouse xenografts via the formation of DNA double-strand breaks during S-phase

Abstract Background: Previous studies of PARP inhibitors have shown efficacy in tumors with defects in homologous recombination repair, particularly in persons with ovarian and breast cancers with germline deleterious BRCA1/2 mutations (gBRCA1/2+). There is much interest in expanding the use of PARP inhibitors beyond these populations, with one strategy being the creation of DNA damage with cytotoxic chemotherapy with concomitant inhibition of its repair with a PARP inhibitor. Murai, et al, have also shown that PARP is trapped on the DNA at the site of damage in the presence of a PARP inhibitor. In April 2016, my colleague Dr Zev Wainberg presented Phase I safety and efficacy data at AACR (abstract CT011) with PARP inhibitor talazoparib in combination with alkylating agent temozolomide in cancer patients without gBRCA1/2+, demonstrating this to be a promising combination. Methods: We evaluated the molecular mechanisms underlying cancer cell lethality with talazoparib plus temozolomide in melanoma, small cell lung, ovarian, and colorectal cancer cell lines and mouse xenografts. We determined growth-adjusted IC50s for talazoparib in multiple histologies to choose cell lines of low and high sensitivity to the PARP inhibitor as monotherapy. We studied percent growth inhibition of talazoparib, temozolomide, and combination therapy at increasing doses. We investigated cell cycle dynamics at 24 hours and 48 hours; PARP inhibitors have been shown to activate the G2/M cell cycle checkpoint, which could confound growth arrest and DNA repair studies. We performed western blot analysis to identify phosphorylated histone H2AX as a marker of DNA double-strand breaks. We used FACS to determine apoptotic fractions and to correlate phospho-H2AX formation with cell cycle phase. We used confocal microscopy to correlate phospho-H2AX with nuclear PARP. We used mouse xenografts to demonstrate sensitivity of tumors to temozolomide, talazoparib, and combination therapy. Results: The combination of temozolomide with talazoparib results in the formation of DNA double-strand breaks during S-phase, which results in increased cell lethality in BRCA1/2 wild-type cell lines and mouse xenografts of melanoma, colorectal, ovarian, and small cell lung histologies even when those cell lines are not sensitive to either talazoparib or temozolomide alone. We were unable to demonstrate colocalization of PARP and phospho-H2AX due to PARP's high expression in the nucleus during S-phase. We were also not able to demonstrate PARP-trapping by western blot at doses utilized in human patients, but this is likely due to lack of assay sensitivity. Conclusions: Taking into consideration both the molecular data demonstrating efficacy of talazoparib plus temozolomide therapy in BRCA1/2 wild-type cell lines and mouse xenografts and the Phase I trial data showing safety and efficacy with this combination, PARP inhibitors likely have utility beyond treatment of gBRCA1/2+ patients. Use of the alkylating agent might be particularly important, as it is recognized during S-phase and trapping of PARP on the DNA could result in collapse of a stalled replication fork into a DNA double-strand break. Citation Format: Kelly Elizabeth McCann, Erika von Euw, Neil O'Brien, Dennis Slamon. The combination of PARP inhibitor talazoparib with low-dose temozolomide results in increased cell lethality in BRCA1/2 wild-type melanoma, small cell lung cancer, ovarian, and colon cancer cell lines and mouse xenografts via the formation of DNA double-strand breaks during S-phase [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 4124.

Abstract 5260: Preclinical studies with ONC201 in combination with clinically approved chemotherapy as a novel treatment strategy against Small Cell Lung Cancer (SCLC)

Abstract According to the American Cancer Society, about 10-15 percent of all lung cancer diagnoses are small cell lung cancer (SCLC). The median survival rate of limited-stage small cell lung cancer is about 12 to 16 months. Several clinically approved chemotherapy drugs have been used to treat SCLC, with several more currently under clinical trials. However, a novel combination is still needed to increase patient life expectancy. ONC201 is an impridone, currently in clinical trials, that has proven to have proapoptotic effects in multiple tumor types. We hypothesized that treating SCLC cell lines with ONC201 in conjunction with clinically approved chemotherapy drugs, such as etoposide and carboplatin, will have a synergistic effect and serve as a potential novel combination treatment plan. SCLC cell lines H1048 and H1105 were treated for 72 hours with ONC201 and etoposide, or ONC201 and carboplatin. CellTiter-Glo cell viability assay and Western blotting of PARP cleavage and ATF4 were used to check synergy in the combination treatments. The combination of ONC201 and clinically approved chemotherapy drugs, etoposide and carboplatin, revealed that there was an upregulation in cleaved PARP and a synergistic effect on the overall cell samples. When looking at the combination index of the cell proliferation assays, the best index for each combination was 0.867 with an ONC201 dosage of 4 μM and an etoposide dosage of 0.05 μg/mL in H1048, 0.43191 at ONC201 dosage 0.3125 μM and etoposide dosage 2.5 μg/mL in H1105, and 0.20278 at ONC201 dosage 2.5 μM and carboplatin dosage 3.125 μg/mL in H1105. We are currently testing drug combinations including a triple drug combination of ONC201 and two clinically approved drugs and studying the mechanism underlying the synergies, with the potential to translate this novel combination treatment plan of ONC201 and clinically approved drugs into a clinical regimen to improve the outcome of SCLC patients. Citation Format: Young S. Lee, Lanlan Zhou, Christopher Azzoli, Wafik S. El-Deiry. Preclinical studies with ONC201 in combination with clinically approved chemotherapy as a novel treatment strategy against 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 5260.

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&amp;lt;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&amp;lt;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 705: A cell-based screening assay to identify agents interfering protein-protein interaction of MAPKAPK2 and OCT4

Abstract c-MYC overexpression plays an important role in tumorigenesis as well as in the progression of tumors. Historically, direct targeting strategies have been challenging due to c-MYC being a transcription factor. Our observation demonstrated that MAPKAPK2 as one of the two major protein kinases involved in binding and phosphorylating OCT4 at serine 111 residue to induce c-MYC activation in progressive disease neuroblastoma. In targeting this novel pathway of c-MYC activation, we considered two options: direct inhibition of MAPKAPK2 or inhibition of MAPKAPK2-OCT4 protein-protein interaction. Given the limitations of direct targeting kinases, namely resistance and off-target effect, we pursued the inhibition of the protein-protein interaction. The purpose of this study is to introduce a novel cell-based assay for compound screening and validation steps to identify compounds that inhibit OCT4-MAPKAPK2 interaction. First, OCT4 (NTD and POUs: the domains critical for c-MYC activation) and MAPKAPK2 were exogenously expressed by a single vector with CMV promoter using a lentiviral vector system in NCI-H82, a small cell lung cancer cell line. Upon the optimization of the assay, we screened approximately 100,000 compounds to identify “hits” by statistically analyzing the reduction in luminescence. Seventy-six compounds were found to inhibit the interaction of MAPKAPK2-OCT4. Subsequently, we have used a two-step hit validation for the 76 compounds: 1) the decrease in pOCT4S111 expression along with the reduction in c-MYC expression by immunoblotting: 2) and the inhibition of binding between MAPKAPK2 and OCT4 by co-immunoprecipitation. The validation step further narrowed down the hits to three compounds that reduced the phosphorylation of OCT4 at S111 and inhibited the MAPKAPK2-OCT4 interaction. To verify the compounds interfering the MAPKAPK2-OCT4 interaction, we used in vitro assay using recombinant human MAPKAPK2 kinase and OCT4 and all three compounds inhibited phosphorylation of OCT4S111. These all compounds also tested minimally affected ATP-dependent MAPKAPK2 kinase relative to the known MAPKAPK2 inhibitor (PF3644022). In conclusion, our screening assay and validation experiments was successfully implemented in identifying hits and the validation step confirmed three novel compounds that demonstrated: 1) impairment of MAPKAPK2-mediated phosphorylation of OCT4; and 2) inhibition of the MAPKAPK2-OCT4 kinase-substrate interaction. These compounds may be further optimized to minimize systemic toxicities and improve the anti-cancer activity for the treatment of neuroblastoma or small cell lung cancer with high c-MYC expression. Citation Format: Inhyoung Yang, Sung-Jen Wei, Ismail Mohiuddin, Gloria M. Martinez, Min H. Kang. A cell-based screening assay to identify agents interfering protein-protein interaction of MAPKAPK2 and OCT4 [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 705.

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 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.

A novel multi-target tyrosine kinase inhibitor anlotinib combined with irinotecan has in-vitro anti-tumor activity against human small-cell lung cancer.

Anlotinib is a multi-target tyrosine kinase inhibitor developed independently in China. Its biological effects remain unclear in small-cell lung cancer (SCLC). The current study aimed to evaluate the effects of anlotinib in combination with irinotecan on H446 and H2227 SCLC cell lines and provide new treatment strategy for SCLC. Cell growth of two cell lines was inhibited by anlotinib, irinotecan and the combination in a dose-dependent manner. After 72 h incubation, the inhibition rate was greater in the combination group than all single drug group. A similar result was found when apoptosis was assessed after 12 h, but not after 6 h of treatment. Compared with single drug, combination drug suppressed the migration and invasion abilities in two cell lines; however, there was no difference between individual anlotinib or irinotecan. The colony formation rate was obviously lower in the combination group. Vascular endothelial growth factor receptor, fibroblast growth factor receptor (FGFR) and platelet-derived growth factor receptor were expressed in two cell lines after treatment regardless single or combination, but FGFR was expressed more after combination treatment than anlotinib. The expression of phosphorylated (p) ERK was decreased with anlotinib alone or combination treatment and pAKT expression was impaired with combination treatment, but not with anlotinib or irinotecan alone. The biological function of anlotinib and irinotecan may be mediated through the AKT/ERK signaling pathway. Additional investigations on biomarker-guided patient-stratification and elucidating individualized targets in patients anlotinib are urgently needed.

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.