Therapeutic Strategy For Small Cell Lung Cancer Research Articles

Cyclin A/B RxL Macrocyclic Inhibitors to Treat Cancers with High E2F Activity.

Cancer cell proliferation requires precise control of E2F1 activity; excess activity promotes apoptosis. Here, we developed cell-permeable and bioavailable macrocycles that selectively kill small cell lung cancer (SCLC) cells with inherent high E2F1 activity by blocking RxL-mediated interactions of cyclin A and cyclin B with select substrates. Genome-wide CRISPR/Cas9 knockout and random mutagenesis screens found that cyclin A/B RxL macrocyclic inhibitors (cyclin A/Bi) induced apoptosis paradoxically by cyclin B- and Cdk2-dependent spindle assembly checkpoint activation (SAC). Mechanistically, cyclin A/Bi hyperactivate E2F1 and cyclin B by blocking their RxL-interactions with cyclin A and Myt1, respectively, ultimately leading to SAC activation and mitotic cell death. Base editor screens identified cyclin B variants that confer cyclin A/Bi resistance including several variants that disrupted cyclin B:Cdk interactions. Unexpectedly but consistent with our base editor and knockout screens, cyclin A/Bi induced the formation of neo-morphic Cdk2-cyclin B complexes that promote SAC activation and apoptosis. Finally, orally-bioavailable cyclin A/Bi robustly inhibited tumor growth in chemotherapy-resistant patient-derived xenograft models of SCLC. This work uncovers gain-of-function mechanisms by which cyclin A/Bi induce apoptosis in cancers with high E2F activity, and suggests cyclin A/Bi as a therapeutic strategy for SCLC and other cancers driven by high E2F activity.

Single-Dose Cisplatin Pre-Treatment Enhances Efficacy of ROBO1-Targeted Radioimmunotherapy.

We previously reported that radioimmunotherapy (RIT) using 90Y-labeled anti-ROBO1 IgG (90Y-B5209B) achieved significant anti-tumor effects against small-cell lung cancer (SCLC) xenografts. However, subsequent tumor regrowth suggested the necessity for more effective therapy. Here, we evaluated the efficacy of combination 90Y-B5209B and cisplatin therapy in NCI-H69 SCLC xenograft mice. Mice were divided into four therapeutic groups: saline, cisplatin only, RIT only, or combination therapy. Either saline or cisplatin was administered by injection one day prior to the administration of either saline or 90Y-B5209B. Tumor volume, body weight, and blood cell counts were monitored. The pathological analysis was performed on day seven post injection of 90Y-B5209B. The survival duration of the combination therapy group was significantly longer than that of the group treated with RIT alone. No significant survival benefit was observed following the isolated administration of cisplatin (relative to saline). Pathological changes following combination therapy were more significant than those following the isolated administration of RIT. Although combination therapy was associated with an increase of several adverse effects such as weight loss and pancytopenia, these were transient. Thus, cisplatin pre-treatment can potentially enhance the efficacy of 90Y-B5209B, making it a promising therapeutic strategy for SCLC.

PARP inhibitors for small cell lung cancer and their potential for integration into current treatment approaches.

Small cell lung cancer (SCLC) is a very aggressive, highly lethal, neuroendocrine tumor that constitutes 15% of all lung cancer cases. It is characterized by its rapid disease progression and high relapse rate leading to poor survival for diagnosed patients. Recently, poly (ADP-ribose) polymerase inhibitors (PARPi) have emerged as a novel therapeutic strategy for SCLC. Preclinical studies have demonstrated that PARPi possesses cytotoxic activity as a single-agent and in combination with other anti-cancer agents. Predictive biomarkers of response to PARPi, such as SLFN11, have also been described in SCLC. This review aims to summarize the recent preclinical investigations and the relevant clinical trials that evaluate PARPi in SCLC. Here, we highlight the potential role of PARPi in a biomarker-selected manner and in combination with chemotherapy, targeted agents, radiotherapy and immunotherapy.

EphA2 inhibition suppresses proliferation of small-cell lung cancer cells through inducing cell cycle arrest

Small-cell lung cancer (SCLC) is characterized by one of neuroendocrine tumors, and is a clinically aggressive cancer due to its rapid growth, early dissemination, and rapid acquisition of multidrug resistance to chemotherapy. Moreover, the standard chemotherapeutic regimen in SCLC has not changed for three decades despite of the dramatic therapeutic improvement in non-SCLC. The development of a novel therapeutic strategy for SCLC has become a pressing issue. We found that expression of Eph receptor A2 (EphA2) is upregulated in three of 13 SCLC cell lines and five of 76 SCLC tumor samples. Genetic inhibition using siRNA of EphA2 significantly suppressed the cellular proliferation via induction of cell cycle arrest in SBC-5 cells. Furthermore, small molecule inhibitors of EphA2 (ALW–II–41-27 and dasatinib) also exclusively inhibited proliferation of EphA2-positive SCLC cells by the same mechanism. Collectively, EphA2 could be a promising candidate as a therapeutic target for SCLC.

Abstract B09: Evaluating transcription factor networks as targets for the treatment of small cell lung cancer

Abstract Achaete-scute homolog 1 (ASCL1) is a transcription factor that is highly expressed in most small cell lung cancer (SCLC) tumors and cell lines and is critical for their proliferation. Inhibiting ASCL1 transcriptional activity may thus be a valid therapeutic strategy for SCLC. However, transcription factors (TFs) have been historically difficult to target. Given that TFs belong to complex regulatory networks, we propose to identify and target multiple components within an ASCL1-transcriptional network rather than ASCL1 alone. We have previously detected ASCL1-bound genomic regions associated with the active chromatin epigenetic mark, H3K27Ac in SCLC cells. Here we report the identification of an ASCL1-containing transcriptional network in SCLC cells and evaluate its targeting by the transcriptional inhibitor, mithramycin. We conducted chromatin immunoprecipitation (ChIP) by immunoprecipitating the H3K27Ac chromatin mark from NCI-H2107 and NCI-H69 SCLCs (5X107 cells) nuclear lysates with10 μg H3K27Ac antibodies (Abcam-Ab4729), and sequenced ChIP libraries on an Illumina High-Seq2000. Further characterization using siRNA transfections, immunoblotting and MTS-cell proliferation assays were conducted following standard procedures. We found 82 overlapping TF genes, including lineage-specific TF genes for ASCL1, Forkhead box protein A2 (FOXA2), and nuclear factor 1 B-type (NFIB), associated with the active chromatin epigenetic marks H3K27Ac in SCLC cell lines. This suggests that ASCL1, FOXA2, and NFIB may belong to the same transcriptional network. siRNA-mediated knockdown of ASCL1 leads to decreased FOXA2 levels, but it has no effect on NFIB levels, implicating ASCL1 as a transcriptional regulator of FOXA2 but not NFIB. Mithramycin stops cell proliferation and reduces ASCL1, FOXA2 but not NFIB protein levels. In conclusion, our studies identify the beginning of a transcriptional network necessary for SCLC proliferation in which ASCL1 regulates FOXA2. Targeting multiple TFs within a network with mithramycin can stop cell proliferation and as such suggests a therapeutic strategy for SCLC. Because of the importance of NFIB in mediating metastatic behavior of SCLC, these data indicate that approaches other than targeting ASCL1 and use of mithramycin will be needed to target NFIB. Citation Format: Karine Pozo, Rahul K. Kollipara, John D. Minna, Adi F. Gazdar, Jane E. Johnson. Evaluating transcription factor networks as targets for the treatment of small cell lung cancer [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 B09.

Abstract B07: Characterization of branched-chain aminotransferase 1 as a novel therapeutic target for small cell lung cancer

Abstract Few studies have explored tumor metabolism to target small cell lung cancer (SCLC), for which treatment options are limited and eventually fail with resistance. The objective of this study is to identify novel biomarkers and therapeutic targets among molecular changes specific to the cancer cells relative to precancerous precursor cells. In our previous study, comparative profiling of precancerous cells with SCLC cells revealed branched-chain aminotransferase1 (Bcat1) as one of the most upregulated genes in the cancer cells relative to their precursors as well as normal lung. Based on this result, we hypothesize that the elevated Bcat1 plays a role in promoting SCLC. Here, we confirmed elevated Bcat1 protein expression in mouse SCLC cells relative to precancerous cells and in human SCLC lines. Targeting Bcat1 in mouse and human SCLC cells with shRNA or small-molecule inhibitors significantly reduced colony expansion in soft agar and formation of subcutaneous tumors in athymic nude mice. Inhibition of Bcat1 also reduced protein synthesis rate as measured by puromycin incorporation into protein. Additionally, we found Bcat1 plays a significant role in leucine catabolism in mouse SCLC using a coupled enzymatic assay. Taken together, these novel findings suggest that elevated Bcat1 promotes SCLC growth by supporting protein synthesis, which is necessary for proliferating cells. Given the proven safety profile of existing inhibitors of Bcat1, our findings are significant in supporting the concept of targeting Bcat1 as a novel and particularly valuable therapeutic strategy for SCLC. Citation Format: Napoleon B. Butler, Keebeom Kim, Dong-wook Kim, Kwon Park. Characterization of branched-chain aminotransferase 1 as a novel therapeutic target for small cell lung cancer [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 B07.

Synergistic killing of human small cell lung cancer cells by the Bcl-2-inositol 1,4,5-trisphosphate receptor disruptor BIRD-2 and the BH3-mimetic ABT-263.

Small cell lung cancer (SCLC) has an annual mortality approaching that of breast and prostate cancer. Although sensitive to initial chemotherapy, SCLC rapidly develops resistance, leading to less effective second-line therapies. SCLC cells often overexpress Bcl-2, which protects cells from apoptosis both by sequestering pro-apoptotic family members and by modulating inositol 1,4,5-trisphosphate receptor (IP3R)-mediated calcium signaling. BH3-mimetic agents such as ABT-263 disrupt the former activity but have limited activity in SCLC patients. Here we report for the first time that Bcl-2-IP3 receptor disruptor-2 (BIRD-2), a decoy peptide that binds to the BH4 domain of Bcl-2 and prevents Bcl-2 interaction with IP3Rs, induces cell death in a wide range of SCLC lines, including ABT-263-resistant lines. BIRD-2-induced death of SCLC cells appears to be a form of caspase-independent apoptosis mediated by calpain activation. By targeting different regions of the Bcl-2 protein and different mechanisms of action, BIRD-2 and ABT-263 induce cell death synergistically. Based on these findings, we propose that targeting the Bcl-2–IP3R interaction be pursued as a novel therapeutic strategy for SCLC, either by developing BIRD-2 itself as a therapeutic agent or by developing small-molecule inhibitors that mimic BIRD-2.

Abstract C153: Investigation of Chk1 as a novel therapeutic target for small cell lung cancer (SCLC)

Abstract Background: Small cell lung cancer (SCLC) is the most lethal form of lung cancer, accounting for around 15% of lung cancers in the United States, currently having no targeted therapies with established efficacy. Using a high-throughput proteomic screen, we demonstrated overexpression of checkpoint kinase 1 (Chk1) in SCLC. Chk1 being the master regulator of the cell cycle in absence of p53, we hypothesize that Chk1 targeting may be effective SCLC because of near ubiquitous loss of p53. In the present study, we tested the effect of Chk1 targeting by shRNA-mediated knockdown and pharmacologic inhibition in an extensive panel of SCLC cell lines and in animal models. Experimental procedures: shRNA-mediated knockdown of Chk1 was done in select human SCLC lines and confirmed at the mRNA and protein level. The effect of Chk1 inhibition by LY2606368 alone and in combination with cisplatin or the PARP1/2 inhibitor talazoparib was tested in 39 SCLC cell lines. Proliferation was assessed by cell titer glo analysis and effect on cell cycle was tested by flow cytometry. Drug sensitivity (IC50) was then correlated with baseline expression level of >190 total or phosphorylated proteins measured by reverse phase protein array (RPPA) to identify potential predictive biomarkers. In vivo effect of the Chk1 inhibitor with or without cisplatin or talazoparib was tested in syngeneic and xenograft model of SCLC. Results: shRNA-mediated targeted Chk1 knockdown significantly decreased the proliferation of SCLC lines confirming Chk1 as a viable target (p<0.01). Targeted Chk1 knockdown also increased the expression of pγH2Ax indicating double-stranded DNA damage upon Chk1 inhibition. Chk1 inhibition by LY2606368 had striking single-agent activity in a majority of SCLC lines (median IC50 2-50nM) and the combination of LY2606368 with either cisplatin or talazoparib was additive in a majority of cell lines. Proteomic analysis revealed association between Chk1 inhibitor sensitivity and elevated basal expression of total and phospho cMyc (p = 0.01), 14-3-3 protein (p = 0.003), pro-apoptotic protein Bax (p<0.05); in contrast, resistance to LY2606368 at multiple doses was correlated to with higher levels of PARP, SGK and p90RSK (p<0.05). In syngeneic in vivo model, single agent LY2606368 was well tolerated and resulted in near-total regression of tumor (>90% reduction in volume) which was durable (ongoing response >50 days in 7/9 animals). The combination with cisplatin or talazoparib also resulted in tumor regression and was significantly more effective than either cisplatin or talazoparib alone. Tumor regression following LY2606368 treatment was also observed in cis-resistant H69 xenograft model of SCLC. Conclusion: The study established checkpoint kinase 1 (Chk1) targeting as a novel and active therapeutic strategy for SCLC, a disease for which the standard of care has remained unchanged for >30 years. Chk1 knockdown was sufficient to inhibit proliferation in SCLC cell lines and LY2606368, a Chk1 inhibitor in current clinical trials, showed activity as a single agent and in combination with cisplatin and PARP inhibition in vitro and in vivo. TP53-mutant cells (hallmark of SCLC) rely on Chk1 for cell cycle arrest; hence, inhibition of Chk1 in SCLC likely works by driving cells to mitotic catastrophe and apoptosis. These findings support the further investigation of Chk1 targeting and Chk1 predictive biomarkers in SCLC. Citation Format: Triparna Sen, Pan Tong, C. Allison Stewart, You Hong-Fan, Ying Feng, Jing Wang, Lauren A. Byers. Investigation of Chk1 as a novel therapeutic target for small cell lung cancer (SCLC). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C153.

Abstract 5316: Check point kinase 1 (Chk1) targeting as a novel therapeutic strategy in small cell lung cancer (SCLC)

Abstract Background: Small cell lung cancer (SCLC) is the most aggressive form of lung cancer, accounting for 14% of lung cancers. It is associated with poor outcomes and few effective treatments. Identification of novel therapeutic targets is imperative for improving treatment outcomes. In our previous work we identified several DNA repair proteins including check point kinase 1 (Chk1) and poly (ADP-ribose) polymerase 1 (PARP1) that are overexpressed in SCLC and are high priority candidate targets. TP53-mutant cells (a hallmark of SCLC) rely on Chk1 to arrest the S and G2 phases of the cell cycle. Hence, inhibition of Chk1 in p53 mutant cells abrogates the S and G2 checkpoints driving such cells to mitotic catastrophe and apoptosis. In this study we tested the in vitro efficacy of Chk1 inhibitors as single agents and in combination with a PARP1 inhibitor and cisplatin. Experimental design: SCLC cell lines [human cell lines plus those from a patient derived xenograft (PDX) and genetically engineered mouse models (GEMMs)] were treated with Chk1 inhibitors (SCH900776, LY2603618, LY2606368) alone and in combination with olaparib (PARP inhibitor) or cisplatin. Cell proliferation was measured by Cell Titer Glo assay. Drug sensitivity (IC50) was correlated with baseline expression levels of >190 total or phosphorylated proteins measured by reverse phase protein array (RPPA) to identify potential predictive markers. Result: Single-agent Chk1 suppression decreased proliferation of SCLC cells in a dose-dependent manner. SCLC lines exhibited varied levels of sensitivity to the Chk1 inhibitors, with greatest single agent activity observed with LY2606368 (IC50 2-30nM). LY2606368 also showed appreciable single agent activity in PDX and GEMM-derived cell lines (IC50 2-7nM). Single agent activity was also observed with the other Chk1 inhibitors (SCH900776 IC50 0.3-10uM; LY2603618 IC50 0.2-10uM). Combination of LY2606368 with olaparib and cisplatin revealed an additive effect. Proteomic analysis revealed an association between Chk1 inhibitor sensitivity and elevated basal expression of pro-apoptotic proteins (cleaved PARP, Bax, and Caspase 3; p <0.05). In contrast, Chk1 resistance was associated with higher levels of PI3K pathway proteins (pAkt, pGSK, p <0.05). Conclusion: Identification and characterization of novel therapeutic targets is essential to improve efficacy of SCLC therapy. In this preliminary study we tested Chk1 inhibitors as potential therapies in SCLC and provide evidence that targeting Chk1 could be a promising therapeutic strategy for SCLC. Further preclinical and clinical investigation is needed to confirm the molecular mechanism and in vivo outcome of Chk1 targeting. Acknowledgement: 1. 2013 NCI Cancer Clinical Investigator Team Leadership Award, P30 CA016672 (Byers) 2. UT MD Anderson Small Cell Lung Cancer Working Group and Abell Hangar Foundation Distinguished Professor Endowment (Glisson). Citation Format: Aly A. Valliani, Triparna Sen, Fatehmeh Masrorpour, Lixia Diao, Robert J. Cardnell, Jing Wang, Bonnie S. Glisson, Helen Piwnica-Worms, Don L. Gibbons, Lauren A. Byers. Check point kinase 1 (Chk1) targeting as a novel therapeutic strategy in small cell lung cancer (SCLC). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5316. doi:10.1158/1538-7445.AM2015-5316