The discovery that circulating nucleic acid-containing complexes in the serum of autoimmune lupus patients can stimulate B cells and plasmacytoid dendritic cells via Toll-like receptors 7 and 9 ...suggested that agents that block these receptors might be useful therapeutics. We identified two compounds, AT791 {3-4-(6-(3-(dimethylamino)propoxy)benzodoxazol-2-yl)phenoxy-N,N-dimethylpropan-1-amine} and E6446 {6-3-(pyrrolidin-1-yl)propoxy)-2-(4-(3-(pyrrolidin-1-yl)propoxy)phenylbenzodoxazole}, that inhibit Toll-like receptor (TLR)7 and 9 signaling in a variety of human and mouse cell types and inhibit DNA-TLR9 interaction in vitro. When administered to mice, these compounds suppress responses to challenge doses of cytidine-phosphate-guanidine (CpG)-containing DNA, which stimulates TLR9. When given chronically in spontaneous mouse lupus models, E6446 slowed development of circulating antinuclear antibodies and had a modest effect on anti-double-stranded DNA titers but showed no observable impact on proteinuria or mortality. We discovered that the ability of AT791 and E6446 to inhibit TLR7 and 9 signaling depends on two properties: weak interaction with nucleic acids and high accumulation in the intracellular acidic compartments where TLR7 and 9 reside. Binding of the compounds to DNA prevents DNA-TLR9 interaction in vitro and modulates signaling in vivo. Our data also confirm an earlier report that this same mechanism may explain inhibition of TLR7 and 9 signaling by hydroxychloroquine (Plaquenil; Sanofi-Aventis, Bridgewater, NJ), a drug commonly prescribed to treat lupus. Thus, very different structural classes of molecules can inhibit endosomal TLRs by essentially identical mechanisms of action, suggesting a general mechanism for targeting this group of TLRs.
Inhibition of Poly(ADP-ribose) Polymerase1 (PARP1) impairs DNA damage repair, and early generation PARP1/2 inhibitors (olaparib, niraparib, etc.) have demonstrated clinical proof of concept for ...cancer treatment. Here, we describe the development of the novel PARP inhibitor E7449, a potent PARP1/2 inhibitor that also inhibits PARP5a/5b, otherwise known as tankyrase1 and 2 (TNKS1 and 2), important regulators of canonical Wnt/β-catenin signaling. E7449 inhibits PARP enzymatic activity and additionally traps PARP1 onto damaged DNA; a mechanism previously shown to augment cytotoxicity. Cells deficient in DNA repair pathways beyond homologous recombination were sensitive to E7449 treatment. Chemotherapy was potentiated by E7449 and single agent had significant antitumor activity in BRCA-deficient xenografts. Additionally, E7449 inhibited Wnt/β-catenin signaling in colon cancer cell lines, likely through TNKS inhibition. Consistent with this possibility, E7449 stabilized axin and TNKS proteins resulting in β-catenin de-stabilization and significantly altered expression of Wnt target genes. Notably, hair growth mediated by Wnt signaling was inhibited by E7449. A pharmacodynamic effect of E7449 on Wnt target genes was observed in tumors, although E7449 lacked single agent antitumor activity in vivo, a finding typical for selective TNKS inhibitors. E7449 antitumor activity was increased through combination with MEK inhibition. Particularly noteworthy was the lack of toxicity, most significantly the lack of intestinal toxicity reported for other TNKS inhibitors. E7449 represents a novel dual PARP1/2 and TNKS1/2 inhibitor which has the advantage of targeting Wnt/β-catenin signaling addicted tumors. E7449 is currently in early clinical development.
Chlorotoxin (Cltx) isolated from scorpion venom is an established tumor targeting and antiangiogenic peptide. Radiolabeled Cltx therapeutic (
I-TM601) yielded promising results in human glioma ...clinical studies, and the imaging agent tozuleristide, is under investigation in CNS cancer studies. Several binding targets have previously been proposed for Cltx but none effectively explain its pleiotropic effects; its true target remains ambiguous and is the focus of this study.
A peptide-drug conjugate (ER-472) composed of Cltx linked to cryptophycin as warhead was developed as a tool to probe the molecular target and mechanism of action of Cltx, using multiple xenograft models.
Neuropilin-1 (NRP1), an endocytic receptor on tumor and endothelial cells, was identified as a novel Cltx target, and NRP1 binding by Cltx increased drug uptake into tumor. Metabolism of Cltx to peptide bearing free C-terminal arginine, a prerequisite for NRP1 binding, took place in the tumor microenvironment, while native scorpion Cltx with amidated C-terminal arginine did not bind NRP1, and instead acts as a cryptic peptide. Antitumor activity of ER-472 in xenografts correlated to tumor NRP1 expression. Potency was significantly reduced by treatment with NRP1 blocking antibodies or knockout in tumor cells, confirming a role for NRP1-binding in ER-472 activity. Higher cryptophycin metabolite levels were measured in NRP1-expressing tumors, evidence of NRP1-mediated enhanced drug uptake and presumably responsible for the superior antitumor efficacy.
NRP1 was identified as a novel Cltx target which enhances tumor drug uptake. This finding should facilitate tumor selection for chlorotoxin-based therapeutics and diagnostics.
Folate receptor alpha (FOLR1/FRA) is reported to be overexpressed in epithelial ovarian cancers (EOC), especially the serous histotype. Further, while dysregulation of the folate-dependent 1-carbon ...cycle has been implicated in tumorogenesis, little is known relative to the potential mechanism of action of FOLR1 expression in these processes. We therefore investigated the expression of FOLR1, other folate receptors, and genes within the 1-carbon cycle in samples of EOC, normal ovary and fallopian tube on a custom TaqMan Low Density Array. Also included on this array were known markers of EOC such as MSLN, MUC16 and HE4. While few differences were observed in the expression profiles of genes in the 1-carbon cycle, genes previously considered to be overexpressed in EOC (e.g., FOLR1, MSLN, MUC16 and HE4) showed significantly increased expression when comparing EOC to normal ovary. However, when the comparator was changed to normal fallopian tube, these differences were abolished, supporting the hypothesis that EOC derives from fallopian fimbriae and, further, that markers previously considered to be upregulated or overexpressed in EOC are most likely not of ovarian origin, but fallopian in derivation. Our findings therefore support the hypothesis that the cell of origin of EOC is tubal epithelium.
Ulcerative colitis (UC) and Crohn's disease (CD) are common inflammatory bowel diseases producing intestinal inflammation and tissue damage. Although emerging evidence suggests these diseases are ...distinct, ∼10% of patients remain classified as indeterminate inflammatory bowel disease even after invasive colonoscopy intended for diagnosis. A molecular diagnostic assay using a clinically accessible tissue would greatly assist in the classification of these diseases. In the present study we assessed transcriptional profiles in peripheral blood mononuclear cells from 42 healthy individuals, 59 CD patients, and 26 UC patients by hybridization to microarrays interrogating more than 22,000 sequences. Supervised analysis identified a set of 12 genes that distinguished UC and CD patient samples with high accuracy. The alterations in transcript levels observed by microarray were verified by real-time polymerase chain reaction. The results suggest that a peripheral blood mononuclear cell-based gene expression signature can provide a molecular biomarker that can complement the standard dia-gnosis of UC and CD.
Purpose: Given their accessibility, surrogate tissues, such as peripheral blood mononuclear cells (PBMC), may provide potential predictive
biomarkers in clinical pharmacogenomic studies. In leukemias ...and lymphomas, the prognostic value of peripheral blast expression
profiles is clear; however, it is unclear whether circulating mononuclear cells of patients with solid tumors might yield
profiles with similar prognostic associations.
Experimental Design: In this study, we evaluated the association of expression profiles in PBMCs with clinical outcomes in patients with advanced
renal cell cancer. Transcriptional patterns in PBMCs of 45 renal cell cancer patients were compared with clinical outcome
data at the conclusion of a phase II study of the mTOR kinase inhibitor CCI-779 to determine whether pretreatment transcriptional
patterns in PBMCs were correlated with eventual patient outcomes.
Results: Unsupervised hierarchical clustering of the PBMC profiles using all expressed genes identified clusters of patients with
significant differences in survival. Cox proportional hazards modeling showed that the expression levels of many PBMC transcripts
were predictors for the patient outcomes of time to progression and overall survival (time to death). Supervised class prediction
approaches identified multivariate expression patterns in PBMCs capable of assigning favorable outcomes of time to death and
time to progression in a test set of renal cancer patients, with overall performance accuracies of 72% and 85%, respectively.
Conclusions: The present study provides the first example of gene expression profiling in peripheral blood, a clinically accessible surrogate
tissue, for identifying patterns of gene expression associated with higher likelihoods of positive outcome in patients with
a solid tumor.
Expression profiling has demonstrated that transcriptomes of primary malignancies differ from those in normal tissue. It is unknown, however, whether there exist "surrogate" transcriptional markers ...in peripheral blood mononuclear cells (PBMCs) of patients with solid tumors. We identified transcripts expressed differentially between PBMCs from renal cell carcinoma patients and normal subjects, some of which appear to reflect specific immune responses of circulating cells. We also identified small sets of predictor genes distinguishing PBMCs from renal cell carcinoma patients and normal volunteers with high accuracy. The present findings have important implications for diagnosis and future clinical pharmacogenomic studies of antitumor therapies.
Abstract
Ewing's sarcoma affects mostly adolescents and young adults with tumors that predominate in bone, characterized by the presence of fusion proteins created by chromosomal translocations ...(EWS-FLI1, EWS-ERG etc.). Recent studies demonstrated an interaction between EWS fusion proteins and PARP1; increased DNA damage and elevated PARP levels upon fusion protein expression as well as sensitivity to PARP inhibition have been reported. E7449, a potent, orally available PARP inhibitor was evaluated in various xenograft models of Ewing's sarcoma as a single agent and in combination with TMZ or irinotecan, chemotherapies often used in relapsed patients.
In an RD-ES (ATCC® HTB166™) Ewing's sarcoma (EWS-FLI1) s.c. xenograft model, treatment with single agent E7449 or TMZ resulted in no antitumor activity. However, when combined exquisite synergy that resulted in tumor regression and tumor-free mice was observed, even at the low E7449 combination dose of 1 mg/kg. E7449 dose responsive re-growth of tumors was observed and 8/8 mice remained tumor-free in the highest dose group (30 mg/kg) at study termination. Enhanced toxicity as measured by body weight loss was observed in combination treatment groups but mice generally recovered well. Significant antitumor activity was observed for irinotecan alone in the RD-ES model; combination with E7449 enhanced that activity. Combining TMZ and irinotecan was also efficacious in the RD-ES model. Antitumor activity was further enhanced by the addition of E7449 which also potentiated combination toxicity. Studies are ongoing to optimize dosing of E7449 and chemotherapy and to identify a treatment schedule that maximizes activity and tolerability of the various combinations.
Additionally, the activity of E7449 alone and in combination was evaluated in 2 Ewing's sarcoma patient-derived xenograft (PDx) models. In one model (CTG-0143) neither TMZ nor E7449 as single agents had significant antitumor activity. However, the combination of E7449 + TMZ resulted in striking synergy and tumor regression. Although tumors were large when treatment started, significant antitumor activity was also observed with single agent irinotecan and combination treatment. The second model (CTG-0142) was sensitive to E7449 alone at high dose and to both chemotherapies (TMZ and irinotecan) as single agents. Addition of E7449 to either TMZ or irinotecan resulted in enhanced anti-tumor activity as measured by significantly delayed tumor re-growth following regression. Biomarker analysis is ongoing, including gene expression profiling and assessment of the DNA damage response in tumors from RD-ES and PDx models following drug treatments.
In conclusion, the combination of E7449 with TMZ and irinotecan resulted in highly potent anti-cancer activity against cell line and PDx models of Ewing's sarcoma. These data support the assessment of E7449 as a combination therapy for Ewing's sarcoma in clinical trials.
Citation Format: Sharon McGonigle, Zhihong Chen, Jingzang Tao Miu, Kuan-Chun Huang, Donna Kolber-Simonds, Nanding Zhao, Natalie C. Twine, Qiongfang Cao, Galina Kuznetsov, Shanqin Xu, Kenichi Nomoto. Antitumor activity of the PARP inhibitor E7449 in Ewing's sarcoma. 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 2733. doi:10.1158/1538-7445.AM2014-2733
Abstract
Introduction: In a small neoadjuvant study in patients with triple negative breast cancer (TNBC) the combination of eribulin plus carboplatin was effective, with a pathologic complete ...response rate of 43% following 4 cycles of treatment. Significant numbers of sporadic TNBC tumors are deficient in DNA repair capacity and share clinical and pathological features with hereditary BRCA1 mutant disease. PARP inhibitors have demonstrated synthetic lethality in cancer cells with defective DNA repair and have therapeutic potential for TNBC. In this study we describe the combination of PARP inhibitor E7449 with eribulin +/- carboplatin in preclinical models of TNBC.
Methods: E7449, an orally available PARP inhibitor, was administered in combination with eribulin +/- carboplatin to 4 s.c. xenograft models of TNBC: MDA-MB-436 (BRCA1 mutant, PTEN deficient), MDA-MB-468 (BRCA wild type, PTEN deficient), HCC1806 and MDA-MB-231 (BRCA and PTEN wild type).
Results and Discussion: Addition of E7449 to eribulin significantly delayed tumor progression in PTEN deficient MDA-MB-468 xenografts. In the BRCA1 mutant and PTEN deficient MDA-MB-436 xenograft model, combination of E7449 with eribulin enhanced antitumor activity versus eribulin alone. Similar potentiation was observed for carboplatin upon combination with E7449. Treatment of MDA-MB-436 xenografts with the triple combination of E7449 + eribulin + carboplatin was more efficacious than any double combination and was well tolerated at the doses examined. In contrast, no significant combination activity was observed for E7449 plus eribulin in the BRCA and PTEN wild type xenografts HCC1806 and MDA-MB-231, and similarly no potentiation of carboplatin was observed in an MDA-MB-231 xenograft. Notably, combination activity was observed in the BRCA1 mutant (MDA-MB-436) and PTEN deficient (MDA-MB-436 and MDA-MB-468) xenografts and not in the BRCA and PTEN wild-type models (HCC1806 and MDA-MB-231). Data from ongoing studies to evaluate the combination activity of E7449 + eribulin in patient-derived xenograft (PDx) models of TNBC will be presented at the meeting.
Potential biomarkers of sensitivity to the combination are under investigation in both cell line xenograft and PDx models and will be described.
Conclusion: The addition of E7449 to eribulin +/- carboplatin increased antitumor activity in a subset of TNBC models. Biomarker studies aimed at a better understanding of the underlying cause of sensitivity are underway. The preclinical data support assessment of E7449 + eribulin + carboplatin combination therapy in the current phase I/II clinical trial.
Citation Format: Sharon McGonigle, Jiayi Wu, Donna Kolber-Simonds, Natalie C Twine, Jue-lon Shie, Noel Taylor, Sergei Agoulnik, Zoltan Dezso, Shannon McGrath, Mark Matijevic, Shanqin Xu, Galina Kuznetsov, Mary Woodall-Jappe, Kenichi Nomoto. Combination of the PARP inhibitor E7449 with eribulin +/- carboplatin in preclinical models of triple negative breast cancer abstract. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-06-03.
Alzheimer's disease (AD) is a complex genetic disease, and variants identified through genome-wide association studies (GWAS) explain only part of its heritability. Epistasis has been proposed as a ...major contributor to this 'missing heritability', however, many current methods are limited to only modelling additive effects. We use VariantSpark, a machine learning approach to GWAS, and BitEpi, a tool for epistasis detection, to identify AD associated variants and interactions across two independent cohorts, ADNI and UK Biobank. By incorporating significant epistatic interactions, we captured 10.41% more phenotypic variance than logistic regression (LR). We validate the well-established AD loci, APOE, and identify two novel genome-wide significant AD associated loci in both cohorts, SH3BP4 and SASH1, which are also in significant epistatic interactions with APOE. We show that the SH3BP4 SNP has a modulating effect on the known pathogenic APOE SNP, demonstrating a possible protective mechanism against AD. SASH1 is involved in a triplet interaction with pathogenic APOE SNP and ACOT11, where the SASH1 SNP lowered the pathogenic interaction effect between ACOT11 and APOE. Finally, we demonstrate that VariantSpark detects disease associations with 80% fewer controls than LR, unlocking discoveries in well annotated but smaller cohorts.
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