Solid Cancer Cell Lines Research Articles

Abstract 84: A discovery on splicing variant patterns of leucine-tRNA synthetase gene based on targeted RNA sequencing

Abstract A leucine-tRNA synthetase, which belongs to the class I aminoacyl-tRNA synthetase family, is encoded by LARS gene. LARS gene functions as an intracellular leucine sensor for mTORC1, mammalian target of rapamycin C1 signaling pathway. This cytosolic gene has a catalytic role of ligating leucine amino acid to its cognate tRNA, in ATP-dependent manner. Accordingly, the protein synthesis regulation, protein translation, cell size, and autophagy are known to be reported by this gene. Therefore, a comprehensive study on LARS gene is necessary for the sake of advanced drug research and precise personalized cancer therapy, in relation to mTOR pathway, which is the root cause of cancer genesis. We discovered the differently expressed splicing patterns in 16 transcripts in LARS gene. Alternative splicing variants of LARS gene were sequenced of 12 cancer cell lines using targeted RNA sequencing to verify their possibilities as a drug target; 12 cancer cell lines are consist of 8 hematologic cell lines and 4 solid cancer cell lines. Sequencing data of LARS gene is aligned by TopHat, and consequently transcript assembly and coverage percentage calculation are processed by Cufflinks, afterwards. Our data, observed from the integrated analysis of these values, shows outstanding features on three transcripts: known protein coding transcript LARS-001 (ENST00000394434, known processed transcript LARS-013 (ENST00000511505), and transcript LARS-015 (ENST00000508709), which is known to retain intron sequences. First of all, the coverage percentage value of transcript LARS-001 in a multiple myeloma cell line, KMS-12-BM, was substantially different from the other cell lines. Secondly, an exceptionally high coverage percent value of the transcript LARS-013 in HL-60, an acute promyelocytic leukemia cell line, was shown. Lastly, the transcript LARS-015 showed a considerably high coverage percent value in SK-MES-1, which is a lung squamous cell carcinoma cell line. Three distinct features of LARS transcripts, transcript LARS-001 in KMS-12-BM, transcript LARS-013 in HL-60, and transcript LARS-015 in SK-MES-1, were characterized. Interestingly, transcript LARS-001 is transcribed with no exon skipping and is only highly expressed in KMS-12-BM. On the other hand, the serial exon skipping from exon 14 to exon 26 in major transcript LARS-201 has shown commonly in 12 cell lines. This implies that LARS gene has unique patterns of alternative splicing variants, which can be consider of taking a significant role in carcinogenesis across diverse cancer types. Hence, additional analysis on exploring the biologically functional correlation between LARS gene and cancer, including cohort validation is to be demonstrated in the future research. Citation Format: HyoJin Song, Daeyoon Kim, Hyejoo Park, Hyun Sub Cheong, Sunghoon Kim, Youngil Koh, Sung-Soo Yoon. A discovery on splicing variant patterns of leucine-tRNA synthetase gene based on targeted RNA sequencing. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 84.

Abstract 3726: Predicting cellular response to small molecule Mcl-1 antagonists

Abstract Myeloid cell leukemia -1 (Mcl-1) is an anti-apoptotic member of the Bcl-2 protein family which many cancers overexpress and depend on for survival. Mcl-1 inhibits apoptosis by binding to Bax and Bak, inhibiting the effectors of apoptosis from oligomerizing at the outer membrane of the mitochondria to promote cell death. Due to its role in cancer and resistance development to conventional chemotherapies, efforts to potently and specifically target MCL-1 have been ongoing. We have discovered novel small molecule inhibitors that selectively disrupt Mcl-1-Bim binding in the sub-nanomolar range. Anti-proliferative activities of our leading Mcl-1 inhibitors were assessed in a panel of hematological and solid cancer cell lines. We found that our compound promoted apoptosis in a subset of cell lines tested. As there was a variable response, we used BH3 profiling, which is a functional assay that rapidly measures dependence on any of the anti-apoptotic Bcl-2 family proteins for an individual sample to predict in vitro sensitivity to our Mcl-1 antagonists. Using the MS1 peptide, a known potent and selective Mcl-1 binder as a positive control in the BH3 profiling assay, we found a correlation between the BH3 profiling measurement of anti-apoptotic dependence to Mcl-1 and increased cytotoxicity caused by our compounds. These studies demonstrate that our compounds promote apoptosis through a selective inhibition of Mcl-1 mechanism of action and supports further investigation of our compounds in in vivo models of cancer. Citation Format: Leah Hogdal, John Sensintaffar, Allison Arnold, Craig Goodwin, Zhiguo Bian, Subrata Shaw, Chris Tarr, Taekyu Lee, Edward Olejniczak, Stephen Fesik. Predicting cellular response to small molecule Mcl-1 antagonists. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3726.

Abstract 1473: CXCL12 inhibition with NOX-A12 (olaptesed pegol) increases T-cell infiltration in tumor-stroma spheroids and synergizes with PD-1 immune checkpoint blockade

Abstract Immune checkpoint inhibition promotes T cell-mediated killing of cancer cells and can induce striking responses, but objective control of tumor growth is observed in only 10-30% of patients with cancer types that generally respond to this treatment (Fearon 2014, Cancer Immunol Res 2:187). A possible cause for this limitation of checkpoint inhibition may be an immune-privileged tumor microenvironment (TME) which excludes the cytotoxic T cells from the vicinity of cancer cells. The chemokine CXCL12 has recently been described as an important T cell exclusion factor in the TME-driven immune suppression. In this study we aimed to investigate whether CXCL12 inhibition by the clinical stage L-aptamer (Spiegelmer®) NOX-A12 (olaptesed pegol) is able to enhance T cell infiltration in 3D tumor-stroma spheroids, thereby facilitating effective immunotherapy. We established 3D multicellular microtissues that mimic a solid tumor with a CXCL12-abundant TME. For this purpose, CXCL12-expressing murine stromal MS-5 cells were co-cultured with solid human cancer cell lines in ultra-low attachment plates for three days. Primary human T cells isolated from healthy donors were added to the spheroids in the presence of various concentrations of NOX-A12. The next day, spheroids were washed and dissociated for T cell quantification by flow cytometry. T cell localization in the 3D microtissues was assessed by immunohistochemistry (IHC). In order to examine T cell activation in the spheroids, a bioluminescent reporter-based PD-1/PD-L1 blockade bioassay (Promega) was adapted to the 3D format: Jurkat-PD-1/Luc T cells were incubated with anti-PD-1 and added to NOX-A12-treated spheroids (CHO-PD-L1 + MS-5). We found that NOX-A12 increases the amount of T cells in tumor-stroma spheroids in a dose-dependent manner; flow cytometry analyses revealed a 2-3 fold increase in spheroid T cell infiltration at 10 nM NOX-A12 in all examined 3D co-culture types. Enhanced T cell infiltration in the presence of NOX-A12 was corroborated by IHC. In line with this, we found increased infiltration and activation of Jurkat-PD-1/luc T cells in the MS-5/CHO-PD-L1 spheroids treated with NOX-A12. Importantly, NOX-A12 synergized with anti-PD1-induced T cell activation. Taken together, in heterotypic 3D models that mimic the complexity of the TME, the CXCL12 antagonist NOX-A12 improved T cell-based tumor immunotherapy by increasing T cell infiltration. By modulating the CXCL12 gradients within the complex 3D structure, NOX-A12 appears to break the immune-privilege of the TME, thereby paving the way for T cell migration into the tumor. These data provide a rationale for the combination of NOX-A12 with checkpoint inhibitors as well as other T cell-based therapies in patients with solid cancer. Citation Format: Dirk Zboralski, Lisa Bauer, Dirk Eulberg, Axel Vater. CXCL12 inhibition with NOX-A12 (olaptesed pegol) increases T-cell infiltration in tumor-stroma spheroids and synergizes with PD-1 immune checkpoint blockade. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1473.

Overcoming resistance to TRAIL-induced apoptosis in solid tumor cells by simultaneously targeting death receptors, c-FLIP and IAPs

The discovery of the TRAIL protein and its death receptors DR4/5 changed the horizon of cancer research because TRAIL specifically kills cancer cells. However, the validity of TRAIL-based cancer therapies has yet to be established, as most cancer cells are TRAIL-resistant. In this report, we demonstrate that TRAIL-resistance of many cancer cell lines can be overcome after siRNA- or rocaglamide-mediated downregulation of c-FLIP expression and simultaneous inhibition of IAPs activity using AT406, a pan-antagonist of IAPs. Combined triple actions of the TRAIL, the IAPs inhibitor, AT406, and the c-FLIP expression inhibitor, rocaglamide (ART), markedly improve TRAIL-induced apoptotic effects in most solid cancer cell lines through the activation of an extrinsic apoptosis pathway. Furthermore, this ART combination does not harm normal cells. Among the 18 TRAIL-resistant cancer cell lines used, 15 cell lines become sensitive or highly sensitive to ART, and two out of three glioma cell lines exhibit high resistance to ART treatment due to very low levels of procaspase-8. This study provides a rationale for the development of TRAIL-induced apoptosis-based cancer therapies.

402. Bioengineering of Peripheral Blood Derived Gamma Delta T Cells in a Serum-Free Expansion Medium

To date the main source of effector cells in preclinical and clinical immunotherapy trials has been αβ T cells. Although cellular products utilizing these cells have shown some promise, there is a need for better control of antitumor immunogenicity, especially in the context of chimeric antigen receptor technologies. Therefore, as an alternative, we are investigating the use of a T cell subset, γΔ T cells. These are a potentially attractive cellular therapeutic as they can be expanded to clinically useful levels and have potent MHC independent intrinsic anti-tumorigenicity driven by their recognition of stress antigens on tumor cells. Our efforts have focused on development of a clinically reliable expansion and manufacturing process of genetically engineered γΔ T cells. In the current study we describe the optimization of culture conditions in serum free media (SFM) that can support robust expansion followed by genetic modification of the expanded cells. Peripheral blood mononuclear cells (PBMC) isolated from healthy donors were cultured in several SFMs including: OpTmizer, X-Vivo, SCGM, and AIM V as well as commonly used media containing serum, RPMI1640 supplemented with either 10% FBS or human serum. Complete growth media also included 2 mM L-glutamine, zoledronic acid (5µM added only at the start of culture), and IL-2 (either 100 or 1000 lU/ml added twice weekly). Of the SFM cultures, robust γΔ T cell expansion was only observed in OpTmizer supplemented with high-dose IL-2, which resulted in expansion and γΔ T cell percentages to levels sufficient for clinical use. Specifically, in OpTmizer, the percentage of γΔ T cells increased from a mean of 2.4% (0.8%-3.6%) in the starting product to 66.4% (35%-94.5%, 6 of 9 cultures were >70% γΔ T cell), resulting in 80-fold expansion at 14 days. The SFM-expanded γΔ T cells were evaluated for their anti-tumor cytotoxicity and shown to be equally effective at killing both hematologic and solid cancer cell lines as γΔ T cells expanded in serum containing media. Transduction efficiency was tested using GFP encoding self-inactivating lentiviral vectors (LV). These studies demonstrated that SIV and HIV transduce expanded γΔ T cells equally, and that compared to EF1α, cassettes employing an MSCV promoter provide greater transgene expression. The timing of LV addition was optimized with respect to i) γΔ T cell expansion, ii) LDL Receptor (LDL-R) expression, and iii) LV dose (3-4 consecutive transductions over 48 hours). The greatest transduction efficiencies were achieved in cultures with the greatest expansion. As expected a range of transduction efficiencies was observed for various donors with a mean of approximately 20%, which is similar to LV transduction levels observed for αβ T cell transductions with similar multiplicity of infection and rates of expansion. Therefore, an optimized strategy to expand and transduce γΔ T cells with demonstrated anti-cancer properties was developed. The utilization of SFM to expand these cells to clinically useful doses and redirection toward tumor cells via bioengineering can result in a clinically safe, widely applicable, and potentially more efficacious cellular immunotherapy.

Cytotoxic macrophage-released tumour necrosis factor-alpha (TNF-α) as a killing mechanism for cancer cell death after cold plasma activation

The present study aims at studying the anticancer role of cold plasma-activated immune cells. The direct anti-cancer activity of plasma-activated immune cells against human solid cancers has not been described so far. Hence, we assessed the effect of plasma-treated RAW264.7 macrophages on cancer cell growth after co-culture. In particular, flow cytometer analysis revealed that plasma did not induce any cell death in RAW264.7 macrophages. Interestingly, immunofluorescence and western blot analysis confirmed that TNF-α released from plasma-activated macrophages acts as a tumour cell death inducer. In support of these findings, activated macrophages down-regulated the cell growth in solid cancer cell lines and induced cell death in vitro. Together our findings suggest plasma-induced reactive species recruit cytotoxic macrophages to release TNF-α, which blocks cancer cell growth and can have the potential to contribute to reducing tumour growth in vivo in the near future.

Using Human Cancer Cell Lines as In vitro Model for Testing the Efficacy of CDBPA; a New Anticancer Drug

The aim of the present study was to test the efficacy of cis-coordinated complexes of platinum (II) with the polymer of benzene-poly-carboxylic acids derived from lignin (CDBPA) (laboratory code BP-C1), an innovative anticancer compound, on the growth of several solid human cancer cell lines: bladder cancer, chondrosarcoma, colonic cancer, head and neck cancer, hepatic cancer, ovary cancer, pancreatic cancer and prostatic cancer. Furthermore, the effect of CDBPA on non-Hodgkin lymphoma cell lines was also tested. The effect of CDBPA on cell viability was detected by XTT assay and toxicity was detected by measuring the leakage of Lactate dehydrogenase from the cells to the media. The present study has demonstrated that CDBPA is not toxic and able to reduce cell viability substantially and significantly in various human cancer cell lines. When comparison of viability in percentage of the controls at the maximum given dose of CDBPA for each type of cancer cell line, it was found that the largest impact on the viability was on sarcoma, and then decreases via breast, prostatic, head and neck-, pancreatic, colonic cancer and finally ovarian cancer. In addition, the effect of CDBPA on non-Hodgkin lymphoma cell lines was similar to that found in sarcoma cells. We conclude that the effect of CDBPA on cell viability is different and may be dependent on genotype of the cancer cell type. This may indicate different mechanisms of action in the different cancer types. The results obtained from the in vitro studies are important for designing future in vivo studies using animal models and to predict the clinical outcome in human cancer.

Abstract B171: Anti-oncogenic effects of RP10107, a novel and potent glutaminase inhibitor, in solid cancer cell lines

Abstract Background: Cancer cells preferentially channel glucose towards lactate production even when oxygen is plentiful, a process termed “aerobic glycolysis” or the Warburg effect. The entire metabolic machinery is reprogrammed in cancer cells wherein glutamine utilization is increased via elevation of glutaminase activity thereby generating the necessary substrates required for eventual ATP synthesis and energy production. Targeting glutaminase therefore represents a potential therapeutic strategy to curb malignant transformation of cells and combat cancer progression. Herein, we describe the biological and pharmacokinetic properties of RP10107, a novel small molecule glutaminase inhibitor, with scope to be further developed as a clinical candidate for solid tumors. Methods: Inhibition of glutaminase (GLS1) activity was determined in rodent brain lysates and recombinant human enzyme preparations using a colorimetric or fluorometric procedure. Selectivity over liver glutaminase (GLS2) was confirmed using mouse liver mitochondrial lysates. Viability was assessed using the colorimetric MTT reagent after incubation of RP10107 with cell lines representative of breast, lung, and colorectal cancer. Glutamate concentrations in cell lines following treatment with RP10107 was estimated by LC-MS/MS. Expression of GLS-1, cMyc, pS6, and p-mTOR were estimated by Western Blotting and bands were quantified using ImageJ after normalization with Actin. Pharmacokinetic profile of RP10107 in plasma after single dose oral administration (10 mg/kg) or IV injection (1 mg/kg) was determined in mice. Results: RP10107 inhibited glutaminase (GLS1) activity in mouse brain lysate, rat brain lysate, and recombinant human enzyme with IC50 of 21.2, 18.2, and 26.4 nM, respectively. In contrast, activity in liver mitochondrial preparations (GLS2) was minimal (<10% inhibition) even at 10 μM concentration. RP10107 caused a dose-dependent inhibition in proliferation of breast, lung, and colorectal cell lines. Response was the highest in MDA-MB-231 (GI50 = 27 nM) with half-maximal growth inhibition for ZR.75.1, MCF-7, A549, H441, HCT-8, and HCT-116 observed at <2 μM RP10107. Reduction in cell viability was accompanied by a dose-dependent increase in the ratio of glutamine to glutamate with >50% response noticed at 100 nM in MDA-MB-231 cells. While p-mTOR expression levels were not affected, addition of RP10107 caused a reduction in cMyc and pS6 (>50% at 100-1000 nM) in lung and breast cancer cell lines. Pharmacokinetic studies of RP10107 in mice indicated high oral bioavailability (62%) with maximum plasma concentrations (Cmax) exceeding 10 μM. Conclusions: RP10107 is a potent and selective GLS1 inhibitor that inhibited growth of solid tumor cell lines with a concomitant increase in glutamine to glutamate ratio. Reduction in glutaminase activity resulted in downregulation of cMyc and pS6 in breast and lung cancer cells. Based on the in vitro efficacy and excellent pharmacokinetic profile, RP10107 is current being tested in various mouse xenograft models. Citation Format: Srikant Viswanadha, Prashant Bhavar, Gayatriswaroop Merikapudi, Babu Govindrajulu, Satyanarayana Eleswarapu, Sridhar Veeraraghavan, Swaroop Vakkalanka. Anti-oncogenic effects of RP10107, a novel and potent glutaminase inhibitor, in solid cancer cell lines. [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 B171.

Abstract 5472: Selinexor, a selective inhibitor of nuclear export (SINE), acts through NF-κB deactivation and combines with proteasome inhibitors to synergistically induce tumor cell death

Abstract Background: The primary nuclear export protein, Exportin 1 (XPO1/CRM1) is overexpressed in most cancers, which frequently correlates with poor prognosis. SINE compounds are a family of small-molecule bioavailable drugs that bind covalently to XPO1 leading to nuclear retention of major tumor suppressor proteins such as p53 and IκB, resulting in selective tumor cell death. Selinexor is the most advanced SINE with >500 hematological and solid tumor cancer patients treated to date in Phase I/II clinical trials. Here we investigated the role of NF-κB transcriptional deactivation in SINE induced anti tumor activity by nuclear retention of IκB. We report the results of combining selinexor or related SINE compounds with proteasome inhibitors, for which inhibition of NF-κB has also been shown to be an important mechanism for cancer cell killing. Methods: Whole protein cell lysates from solid and hematological cancer cell lines treated with selinexor with or without proteasome inhibitors were analyzed by immunoblots. IκB localization was evaluated by immunofluorescence. Cytotoxic effects of SINE compounds were evaluated in the presence or absence of proteasome inhibitors. NF-κB transcriptional activity was analyzed using an ELISA assay. H929 multiple myeloma xenografts in NOD-SCID mice were treated with selinexor alone or in combination with carfilzomib to determine effects on tumor growth. Results: NF-κB transcriptional activity was upregulated in SINE resistant sarcoma cell lines HT1080R (IC50 = 2.4 μM versus HT1080 parental IC50 = 0.03 μM) and ASPS-KY (IC50>10μM). In those resistant lines, combination of the proteasome inhibitors bortezomib or carfilzomib with SINE compound showed dramatic synergistic cytotoxic potency of more than 10 folds. Also, the combination of bortezomib with selinexor led to synergistic nuclear retention of IκB. Furthermore, IκB silencing by siRNA led to reduced selinexor cytotoxic potency, increasing IC50 from 58 nM to 830 nM (9-fold) in IM-9 multiple myeloma cells and from 27 nM to 1.9 μM (66-fold) in U2OS osteosarcoma cells. Finally, the combination of selinexor with carfilzomib was synergistic in reducing tumor growth in the H929 multiple myeloma xenograft model. Conclusion: Inhibition of NF-κB transcriptional activity through forced nuclear retention of IκB appears to be an important mechanism in the selective tumor cell cytotoxicity of selinexor and related SINE compounds. Furthermore, the combination of selinexor and proteasome inhibitors, which are also known to act at least in part through inhibition of NF-κB, leads to synergistic activity in vitro and in vivo, suggesting that such combinations may provide clinically more effective than the single agents. A Phase 1 trial to study the safety and efficacy of selinexor in combination with carfilzomib in multiple myeloma is ongoing (NCT02199665). Citation Format: Yosef Landesman, Trinayan Kashyap, Marsha Crochiere, Boris Klebanov, Sharon Friedlander, William Senapedis, Robert Carlson, Michael Kauffman, Sharon Shacham. Selinexor, a selective inhibitor of nuclear export (SINE), acts through NF-κB deactivation and combines with proteasome inhibitors to synergistically induce tumor cell death. [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 5472. doi:10.1158/1538-7445.AM2015-5472

Bcl-xl and Mcl-1 are the major determinants of the apoptotic response to dual PI3K and MEK blockage

The dual targeting of PI3K-AKT-mTOR and Ras-Raf-MEK-ERK pathways is a potential anticancer therapy, but unfortunately, the response rate has been low in early phase clinical trials. Pre-clinical models have suggested that an apoptotic response to dual PI3K and MEK targeting is relatively rare and understanding apoptotic avoidance could lead to increased clinical efficiency. This study investigated solid cancer cell lines, which are known to be sensitive to dual PI3K and MEK inhibition but to have a limited apoptotic response. The cells were exposed to dual PI3K and MEK blockage in combination with a panel of additional pharmacological agents and cytotoxicity and apoptosis were analyzed. Our results indicated that the BH3 mimetic ABT-263, the HDAC inhibitor entinostat and the multikinase inhibitor dasatinib increased the cytotoxicity and apoptotic response of dual PI3K and MEK targeting. Furthermore, ABT-263 and entinostat was able to induce apoptosis in combination with single agent PI3K and MEK inhibitors. Protein expression, immunoprecipitation and siRNA knockdown models suggested that Bcl-xl and Mcl-1 were the most important factors circumventing PI3K and/or MEK inhibition-mediated apoptosis. The results suggest that the cytotoxicity of PI3K and/or MEK inhibitor treatments can be augmented by combinatory approaches targeting anti-apoptotic mediators Bcl-xl and Mcl-1.

Circulating microRNAs as Candidate Biomarkers for the Surveillance of Melanoma Patients

Circulating microRNAs as Candidate Biomarkers for the Surveillance of Melanoma Patients

Synthesis and antiproliferative activity of α-branched α,β-unsaturated ketones in human hematological and solid cancer cell lines

A series of α-branched α,β-unsaturated ketones were prepared via boron trifluoride etherate mediated reaction between arylalkynes and carboxaldehydes. The evaluation of the antiproliferative activity over hematological (NB4) and solid cancer (A549, MCF-7) cell lines provided a structure-activity relationship. 5-Parameter QSAR equations were built which were able to explain 80%–92% of the variance in activity. The resulting selective lead compound showed IC50 value 0.6 μM against the hematological cell line and did not cause apoptosis, but blocked cell cycle in G0/G1. Moreover, it was demonstrated that this compound enhances and accelerates retinoic acid induced granulocytic differentiation.

Pan-cancer stratification of solid human epithelial tumors and cancer cell lines reveals commonalities and tissue-specific features of the CpG island methylator phenotype

BackgroundThe term CpG island methylator phenotype (CIMP) has been used to describe widespread DNA hypermethylation at CpG-rich genomic regions affecting clinically distinct subsets of cancer patients. Even though there have been numerous studies of CIMP in individual cancer types, a uniform analysis across tissues is still lacking.ResultsWe analyze genome-wide patterns of CpG island hypermethylation in 5,253 solid epithelial tumors from 15 cancer types from TCGA and 23 cancer cell lines from ENCODE. We identify differentially methylated loci that define CIMP+ and CIMP− samples, and we use unsupervised clustering to provide a robust molecular stratification of tumor methylomes for 12 cancer types and all cancer cell lines. With a minimal set of 89 discriminative loci, we demonstrate accurate pan-cancer separation of the 12 CIMP+/− subpopulations, based on their average levels of methylation. Tumor samples in different CIMP subclasses show distinctive correlations with gene expression profiles and recurrence of somatic mutations, copy number variations, and epigenetic silencing. Enrichment analyses indicate shared canonical pathways and upstream regulators for CIMP-targeted regions across cancer types. Furthermore, genomic alterations showing consistent associations with CIMP+/− status include genes involved in DNA repair, chromatin remodeling genes, and several histone methyltransferases. Associations of CIMP status with specific clinical features, including overall survival in several cancer types, highlight the importance of the CIMP+/− designation for individual tumor evaluation and personalized medicine.ConclusionsWe present a comprehensive computational study of CIMP that reveals pan-cancer commonalities and tissue-specific differences underlying concurrent hypermethylation of CpG islands across tumors. Our stratification of solid tumors and cancer cell lines based on CIMP status is data-driven and agnostic to tumor type by design, which protects against known biases that have hindered classic methods previously used to define CIMP. The results that we provide can be used to refine existing molecular subtypes of cancer into more homogeneously behaving subgroups, potentially leading to more uniform responses in clinical trials.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-015-0007-7) contains supplementary material, which is available to authorized users.

Absolute configuration and anti-tumor activity of torrubiellin B

The dimeric anthracene derivative torrubiellin B (1) was isolated from the endophytic fungus Acremonium sp. that had been obtained from leaves of the Mangrove plant Sonneratia caseolaris. The absolute configuration of 1 was established as (5′R,10′S,10a′R) for the first time on the basis of its electronic circular dichroism (ECD) spectra aided with TDDFT-ECD calculations. Torrubiellin B (1) exhibited strong anti-tumor activity when tested in vitro against several solid cancer cell lines including cells that are resistant against the widely used cytostatic drug cisplatin. The IC50 values of 1 against cisplatin sensitive and cisplatin resistant cells were in the range of 0.2–2.6μM depending on cell line investigated.

CUB-domain-containing protein 1 overexpression in solid cancers promotes cancer cell growth by activating Src family kinases.

The transmembrane glycoprotein, CUB (complement C1r/C1s, Uegf, Bmp1) domain-containing protein 1 (CDCP1) is overexpressed in several cancer types and is a predictor of poor prognosis for patients on standard of care therapies. Phosphorylation of CDCP1 tyrosine sites is induced upon loss of cell adhesion and is thought to be linked to metastatic potential of tumor cells. Using a tyrosine-phosphoproteomics screening approach, we characterized the phosphorylation state of CDCP1 across a panel of breast cancer cell lines. We focused on two phospho-tyrosine pTyr peptides of CDCP1, containing Tyr707 and Tyr806, which were identified in all six lines, with the human epidermal growth factor 2-positive HCC1954 cells showing a particularly high phosphorylation level. Pharmacological modulation of tyrosine phosphorylation indicated that, the Src family kinases (SFKs) were found to phosphorylate CDCP1 at Tyr707 and Tyr806 and play a critical role in CDCP1 activity. We demonstrated that CDCP1 overexpression in HEK293 cells increases global phosphotyrosine content, promotes anchorage-independent cell growth and activates several SFK members. Conversely, CDCP1 downregulation in multiple solid cancer cell lines decreased both cell growth and SFK activation. Analysis of primary human tumor samples demonstrated a correlation between CDCP1 expression, SFK and protein kinase C (PKC) activity. Taken together, our results suggest that CDCP1 overexpression could be an interesting therapeutic target in multiple solid cancers and a good biomarker to stratify patients who could benefit from an anti-SFK-targeted therapy. Our data also show that multiple tyrosine phosphorylation sites of CDCP1 are important for the functional regulation of SFKs in several tumor types.

Mcl-1 antagonism is a potential therapeutic strategy in a subset of solid cancers

Cancer cell survival is frequently dependent on the elevated levels of members of the Bcl-2 family of prosurvival proteins that bind to and inactivate BH3-domain pro-apoptotic cellular proteins. Small molecules that inhibit the protein–protein interactions between prosurvival and proapoptotic Bcl-2 family members (so-called “BH3 mimetics”) have a potential therapeutic value, as indicated by clinical findings obtained with ABT-263 (navitoclax), a Bcl-2/Bcl-xL antagonist, and more recently with GDC-0199/ABT-199, a more selective antagonist of Bcl-2. Here, we report study results of the functional role of the prosurvival protein Mcl-1 against a panel of solid cancer cell lines representative of different tumor types. We observed silencing of Mcl-1 expression by small interfering RNAs (siRNAs) significantly reduced viability and induced apoptosis in almost 30% of cell lines tested, including lung and breast adenocarcinoma, as well as glioblastoma derived lines. Most importantly, we provide a mechanistic basis for this sensitivity by showing antagonism of Mcl-1 function with specific BH3 peptides against isolated mitochondria induces Bak oligomerization and cytochrome c release, therefore demonstrating that mitochondria from Mcl-1-sensitive cells depend on Mcl-1 for their integrity and that antagonizing Mcl-1 function is sufficient to induce apoptosis. Thus, our results lend further support for considering Mcl-1 as a therapeutic target in a number of solid cancers and support the rationale for development of small molecule BH3-mimetics antagonists of this protein.

Abstract 1374: Identification of a transcription factor driving BET dependency in SCLC

Abstract The regulation, and subsequent recognition, of covalent chromatin modifications by an array of cellular proteins are key determinants of gene expression. For instance, the binding of bromodomain and extra terminal domain (BET) proteins to acetylated histone tails has been shown to regulate c-MYC transcription in some cell types. Consequently, pharmacological inhibition of BET proteins leads to an anti-proliferative activity in cancers dependent on c-MYC activity such as multiple myeloma and acute myelogenous leukemia. Since c-MYC has been implicated as a driver of cell proliferation in many cancer types, we investigated if BET inhibitors may have anti-proliferative activity in solid tumor cancer cell lines as well. Strikingly, in a panel of over 100 mixed histology lung cancer cell lines we identified selective growth inhibition of lines derived from small cell lung cancer (SCLC) histology. To better understand the mechanism of this dependency we performed 12-point dose response microarray analyses using the BET inhibitor, JQ1, in order to identify genes that are regulated by BET proteins in SCLC. In contrast to what has been observed in myeloma and leukemia, BET inhibition is not always accompanied by inhibition of c-MYC expression in the SCLC cell lines. From this analysis a lineage-specific transcription factor was found to be highly regulated in a dose-dependent manner. Previous studies have reported this transcription factor to be essential in maintaining the differentiated neuroendocrine phenotype characteristic of human SCLC. It has also been shown that loss of this transcription factor results in growth inhibition in SCLC tumor models. We have confirmed the growth dependency of SCLC to BET via dysregulation of this transcription factor using RNAi and a small molecule inhibitor. Furthermore, chromatin immunoprecipitation followed by deep-sequencing confirmed binding of BRD4, a BET family member, and RNA polymerase II, a marker of active gene transcription, to the enhancer region of this target gene in two sensitive SCLC cell lines. This binding is disrupted in the presence of BET compound treatment. SCLC is a disease with high unmet medical need, and the results of our studies have identified BET inhibition as a potential novel therapeutic opportunity for this subtype of lung cancer. Our results have also provided a mechanistic basis for the sensitivity of SCLC to BET inhibition, and a rationale for the clinical development of BET inhibitors beyond hematologic cancers. Citation Format: Susan Wee, Tai Wong, BMS. Identification of a transcription factor driving BET dependency in SCLC. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1374. doi:10.1158/1538-7445.AM2014-1374

Abstract 4235: Anti-proliferative effect of (19Z)-halichondramide from the sponge Chondrosia corticata via G2/M cell cycle arrest and suppression of mTOR signaling in human lung cancer cells

Abstract Five oxazole-containing macrolides isolated from the marine sponge Chondrosia corticata were evaluated for their anti-proliferative activity in a panel of human solid cancer cell lines. (19Z)-Halichondramide ((19Z)-HCA), a novel trisoxazole-containing macrolide, exhibited the highest potency among the macrolides, with IC50 values in the submicro-molar ranges. Prompted by the high potency of growth inhibition of cancer cells, we investigated the mechanism of action of the anti-proliferative activity of (19Z)-HCA in human A549 lung cancer cells. (19Z)-HCA induced cell cycle arrest in the G2/M phase, and this event was highly correlated with the expression of checkpoint proteins, including the up-regulation of p53 and GADD45α and the down-regulation of cyclin B1, cyclin A, CDC2, and CDC25C. In addition, the growth inhibition by (19Z)-HCA was associated with the suppression of mTOR and its downstream effector molecules 4EBP1 and p70S6K. The modulation of mTOR signaling by (19Z)-HCA was found to be mediated by the regulation of upstream proteins, including the down-regulation of Akt and p38 MAPK and the up-regulation of AMPK. These data suggest the potential of (19Z)-HCA to serve as a candidate for cancer chemotherapeutic agents derived from marine organisms by virtue of arresting the cell cycle in the G2/M phase and the modulation of mTOR/AMPK signaling pathways. [Acknowledgements: This work was supported by the MarineBio Research Program (NRF-2013045101) of the National Research Foundation of Korea (NRF) Grant funded by the Korean Government (MEST).] Citation Format: Song Yi Bae, Jayoung Song, Yoonho Shin, Won Kyung Kim, Jedo Oh, Tae Joon Choi, Eun Ju Jeong, So Hyun Park, Eun Jeong Jang, Ji In Kang, Hyen Joo Park, Ji-Young Hong, Gi Dae Kim, Ju-eun Jeon, Jongheon Shin, Sang Kook Lee. Anti-proliferative effect of (19Z)-halichondramide from the sponge Chondrosia corticata via G2/M cell cycle arrest and suppression of mTOR signaling in human lung cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4235. doi:10.1158/1538-7445.AM2014-4235

Abstract 1801: APR-246, a clinical-stage mutant p53-reactivating compound, resensitizes ovarian cancer cells to platinum compounds and doxorubicin

Abstract Background: Platinum-based drugs are since decades used as first-line treatment for many solid tumors. Patients with ovarian cancer often respond well to platinum compounds but a majority of patients rapidly develop resistance and die of chemotherapy refractory disease. The mechanisms underlying resistance are multifactorial, but two of the main causes are mutations in the tumor suppressor p53 and elevated intracellular glutathione (GSH) levels. Mutations in p53 occur in about 60% of ovarian tumors. APR-246 (PRIMA-1MET) is the first clinical-stage compound that reactivates mutant p53. APR-246 is a prodrug that accumulates in cancer cells and is converted to the active form MQ, a Michael acceptor that binds to mutant p53, refolds it to wild type conformation and triggers apoptosis (Lambert et al. Cancer Cell 15, 2009). APR-246 has been tested in a Phase I/IIa clinical trial with promising results (Lehmann et al. J Clin Oncol 30, 2012), and a Phase Ib/II study with platinum-based combination therapy in recurrent p53 mutant ovarian cancer is underway. Methods: Cell viability was assessed with WST-1, MTT or FMCA assay. p53 gene status was determined by Sanger sequencing and single strand conformation analysis, and p53 protein expression by Western blotting. Intracellular GSH levels were assessed with GSH kit (Cayman). Results: We have previously shown outstanding synergistic anticancer effects with APR-246 in combination with platinum compounds in p53 mutant solid cancer cell lines, including cisplatin resistant ovarian cancer cells. Synergistic effects were also observed ex vivo as well as in vivo in mice carrying human tumor xenografts. Here we show that APR-246 not only reactivates mutant p53 but also decreases intracellular GSH levels in a dose-dependent manner, presumably via adduct formation between MQ and GSH. APR-246 resensitized cisplatin resistant p53 mutant ovarian A2780-CP20 carcinoma cells to cisplatin, as shown by a decrease in the IC50 value from 52 to 2.9 µM, similar to the IC50 in parental A2780 cells. APR-246 also restored the sensitivity of resistant A2780ADR cells to doxorubicin. A2780-CP20 and A2780ADR were developed from the parental A2780 line with wild type p53 by chronic exposure to the respective drug. Moreover, APR-246 resensitized the p53 mutant OVCAR-3 cell line, established from a drug resistant patient, to cisplatin. Conclusions: Our results show that APR-246 not only reactivates mutant p53 but also decreases intracellular glutathione levels. We propose that this unique dual mechanism of action accounts for the resensitization and strong synergistic effects with APR-246 and platinum drugs. Our results provide strong rationale for the planned clinical study in ovarian cancer and suggest that combination treatment with APR-246 and DNA damaging drugs could have broad applicability in the treatment of drug resistant p53 mutant human tumors. Citation Format: Nina Mohell, Jessica Alfredsson, Åsa Fransson, Vladimir Bykov, Mikael von Euler, Klas Wiman, Ulf Björklund. APR-246, a clinical-stage mutant p53-reactivating compound, resensitizes ovarian cancer cells to platinum compounds and doxorubicin. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1801. doi:10.1158/1538-7445.AM2014-1801

From the covalent linkage of drugs to novel inhibitors of ribonucleotide reductase: Synthesis and biological evaluation of valproic esters of 3′-C-methyladenosine

We synthesized a series of serum-stable covalently linked drugs derived from 3′-C-methyladenosine (3′-Me-Ado) and valproic acid (VPA), which are ribonucleotide reductase (RR) and histone deacetylase (HDAC) inhibitors, respectively. While the combination of free VPA and 3′-Me-Ado resulted in a clear synergistic apoptotic effect, the conjugates had lost their HDAC inhibitory effect as well as the corresponding apoptotic activity. Two of the analogs, 2′,5′-bis-O-valproyl-3′-C-methyladenosine (A160) and 5′-O-valproyl-3′-C-methyladenosine (A167), showed promising cytotoxic activities against human hematological and solid cancer cell lines. A167 was less potent than A160 but had interesting features as an RR inhibitor. It inhibited RR activity by competing with ATP as an allosteric effector and concomitantly reduced the intracellular deoxyribonucleoside triphosphate (dNTP) pools. A167 represents a novel lead compound, which in contrast to previously used RR nucleoside analogs does not require intracellular kinases for its activity and therefore holds promise against drug resistant tumors with downregulated nucleoside kinases.