Aisling O'Connor

An intact canonical NF-κB pathway is required for inflammatory gene expression in response to hypoxia.

Hypoxia is a feature of the microenvironment in a number of chronic inflammatory conditions due to increased metabolic activity and disrupted perfusion at the inflamed site. Hypoxia contributes to inflammation through the regulation of gene expression via key oxygen-sensitive transcriptional regulators including the hypoxia-inducible factor (HIF) and NF-κB. Recent studies have revealed a high degree of interdependence between HIF and NF-κB signaling; however, the relative contribution of each to hypoxia-induced inflammatory gene expression remains unclear. In this study, we use transgenic mice expressing luciferase under the control of NF-κB to demonstrate that hypoxia activates NF-κB in the heart and lungs of mice in vivo. Using small interfering RNA targeted to the p65 subunit of NF-κB, we confirm a unidirectional dependence of hypoxic HIF-1α accumulation upon an intact canonical NF-κB pathway in cultured cells. Cyclooxygenase-2 and other key proinflammatory genes are transcriptionally induced by hypoxia in a manner that is both HIF-1 and NF-κB dependent, and in mouse embryonic fibroblasts lacking an intact canonical NF-κB pathway, there is a loss of hypoxia-induced inflammatory gene expression. Finally, under conditions of hypoxia, HIF-1α and the p65 subunit of NF-κB directly bind to the cyclooxygenase-2 promoter. These results implicate an essential role for NF-κB signaling in inflammatory gene expression in response to hypoxia both through the regulation of HIF-1 and through direct effects upon target gene expression.

Vascular-targeted photodynamic therapy with BF2-chelated Tetraaryl-Azadipyrromethene agents: a multi-modality molecular imaging approach to therapeutic assessment.

Background:Photodynamic therapy (PDT) is a treatment modality for a range of diseases including cancer. The BF2-chelated tetraaryl-azadipyrromethenes (ADPMs) are an emerging class of non-porphyrin PDT agent, which have previously shown excellent photochemical and photophysical properties for therapeutic application. Herein, in vivo efficacy and mechanism of action studies have been completed for the lead agent, ADMP06.Methods:A multi-modality imaging approach was employed to assess efficacy of treatment, as well as probe the mechanism of action of ADPM06-mediated PDT.Results:Tumour ablation in 71% of animals bearing mammary tumours was achieved after delivery of 2 mg kg−1 of ADPM06 followed immediately by light irradiation with 150 J cm−2. The inherent fluorescence of ADPM06 was utilised to monitor organ biodistribution patterns, with fluorescence reaching baseline levels in all organs within 24 h. Mechanism of action studies were carried out using dynamic positron emission tomography and magnetic resonance imaging techniques, which, when taken together, indicated a decrease in tumour vascular perfusion and concomitant reduction in tumour metabolism over time after treatment.Conclusion:The encouraging treatment responses in vivo and vascular-targeting mechanism of action continue to indicate therapeutic benefit for this new class of photosensitiser.

The hydroxylase inhibitor dimethyloxallyl glycine attenuates endotoxic shock via alternative activation of macrophages and IL-10 production by B1 cells.

Localized tissue hypoxia is a feature of infection and inflammation, resulting in the upregulation of the transcription factors hypoxia-inducible factor 1&agr; and nuclear factor &kgr;B (NF-&kgr;B) via inhibition of oxygen sensing hydroxylase enzymes. Previous studies have demonstrated a beneficial role for the hydroxylase inhibitor dimethyloxallyl glycine (DMOG) in inflammatory conditions, including experimental colitis, by regulating the activity of hypoxia-inducible factor 1 and NF-&kgr;B. We have demonstrated in vivo that pretreatment with DMOG attenuates systemic LPS-induced activation of the NF-&kgr;B pathway. Furthermore, mice treated with DMOG had significantly increased survival in LPS-induced shock. Conversely, in models of polymicrobial sepsis, DMOG exacerbates disease severity. Dimethyloxallyl glycine treatment of mice promotes M2 polarization in macrophages within the peritoneal cavity, resulting in the downregulation of proinflammatory cytokines such as TNF-&agr;. In addition, in vivo DMOG treatment upregulates IL-10 expression, specifically in the peritoneal B1 cell population. This study demonstrates cell type-specific roles for hydroxylase inhibition in vivo and provides insight into the mechanism underlying the protection conveyed by DMOG in models of endotoxic shock.ABBREVIATIONS-MØ-macrophage; DMOG-dimethyloxallyl glycine

The anti-cancer activity of a cationic anti-microbial peptide derived from monomers of polyhydroxyalkanoate

The biodegradable polymer medium chain length polyhydroxyalkanoate (mclPHA), produced by Pseudomonas putida CA-3, was depolymerised and the predominant monomer (R)-3-hydroxydecanoic acid (R10) purified. R10 was conjugated to a d-peptide DP18 and its derivatives. All peptides conjugated with R10 exhibited greater anti-cancer activity compared to the unconjugated peptides. Unconjugated and conjugated peptides were cytocidal for cancer cells. Conjugation of R10 to peptides was essential for enhanced anti-proliferation activity, as unconjugated mixes did not result in enhancement of anti-cancer activity. The conjugation of R10 resulted in more rapid uptake of peptides into HeLa and MiaPaCa cells compared to unconjugated peptide. Both unconjugated and R10 conjugated peptides localized to the mitochondria of HeLa and MiaPaCa cells and induced apoptosis. Peptide conjugated with a terminally hydroxylated decanoic acid (ω-hydroxydecanoic acid) exhibited 3.3 and 6.3 fold higher IC(50) values compared to R10 conjugated peptide indicating a role for the position of the hydroxyl moiety in enhancement of anti-cancer activity. Conjugation of decanoic acid (C10) to peptides resulted in similar or higher IC(50) values compared to R10 conjugates but C10 conjugates did not exhibit any cancer selectivity. Combination studies showed that R10DP18L exhibited synergy with cisplatin, gemcitabine, and taxotere with IC(50) values in the nanomolar range.

Mechanism of cell death mediated by a BF2-chelated tetraaryl-azadipyrromethene photodynamic therapeutic: Dissection of the apoptotic pathway in vitro and in vivo

Photodynamic therapy (PDT) is an established treatment modality for cancer. ADPM06 is an emerging non‐porphyrin PDT agent which has been specifically designed for therapeutic application. Recently, we have demonstrated that ADPM06‐PDT is well tolerated in vivo and elicits impressive complete response rates in various models of cancer when a short drug‐light interval is applied. Herein, the mechanism of action of ADPM06‐PDT in vitro and in vivo is outlined. Using a drug and light combination that reduces the clonogenicity of MDA‐MB‐231 cells by >90%, we detected a well‐orchestrated apoptotic response accompanied by the activation of various caspases in vitro. The generation of reactive oxygen species (ROS) upon photosensitizer irradiation was found to be the key instigator in the observed apoptotic response, with the endoplasmic reticulum (ER) found to be the intracellular site of initial PDT damage, as determined by induction of a rapid ER stress response post‐PDT. PDT‐induced apoptosis was also found to be independent of p53 tumor suppressor status. A robust therapeutic response in vivo was demonstrated, with a substantial reduction in tumor proliferation observed, as well as a rapid induction of apoptosis and initiation of ER stress, mirroring numerous aspects of the mechanism of action of ADPM06 in vitro. Finally, using a combination of 18F‐labeled 3′‐deoxy‐3′‐fluorothymidine (18F‐FLT) nuclear and optical imaging, a considerable decrease in tumor proliferation over 24‐hr in two models of human cancer was observed. Taken together, this data clearly establishes ADPM06 as an exciting novel PDT agent with significant potential for further translational development.

THERAPEUTIC RATIONALE TO TARGET HIGHLY EXPRESSED CDK7 CONFERRING POOR OUTCOMES IN TRIPLE-NEGATIVE BREAST CANCER

Triple-negative breast cancer (TNBC) patients commonly exhibit poor prognosis and high relapse after treatment, but there remains a lack of biomarkers and effective targeted therapies for this disease. Here, we report evidence highlighting the cell-cycle-related kinase CDK7 as a driver and candidate therapeutic target in TNBC. Using publicly available transcriptomic data from a collated set of TNBC patients (n = 383) and the METABRIC TNBC dataset (n = 217), we found CDK7 mRNA levels to be correlated with patient prognosis. High CDK7 protein expression was associated with poor prognosis within the RATHER TNBC cohort (n = 109) and the METABRIC TNBC cohort (n = 203). The highly specific CDK7 kinase inhibitors, BS-181 and THZ1, each downregulated CDK7-mediated phosphorylation of RNA polymerase II, indicative of transcriptional inhibition, with THZ1 exhibiting 500-fold greater potency than BS-181. Mechanistic investigations revealed that the survival of MDA-MB-231 TNBC cells relied heavily on the BCL-2/BCL-XL signaling axes in cells. Accordingly, we found that combining the BCL-2/BCL-XL inhibitors ABT-263/ABT199 with the CDK7 inhibitor THZ1 synergized in producing growth inhibition and apoptosis of human TNBC cells. Collectively, our results highlight elevated CDK7 expression as a candidate biomarker of poor prognosis in TNBC, and they offer a preclinical proof of concept for combining CDK7 and BCL-2/BCL-XL inhibitors as a mechanism-based therapeutic strategy to improve TNBC treatment. Cancer Res; 77(14); 3834-45. ©2017 AACR.

Abstract PD3-01: CDK7: A marker of poor prognosis and tractable therapeutic target in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is defined by absent expression of estrogen receptor (ER), progesterone receptor (PR) and non-overexpression of human epidermal growth factor receptor 2 (HER2), representing a heterogeneous subgroup of breast cancer with substantial genotypic and phenotypic diversity. TNBC patients commonly exhibit poor prognosis and high relapse rates at early stages after conventional treatments. Currently, there is a lack of biomarkers and targeted therapies for the management of TNBC. During tumour development and progression, alterations in cellular behaviour are frequently linked with kinase expression and activity. Here, we aimed to identify novel kinase targets that may play a pivotal role in the progression of TNBC and, thus, offer new therapeutic vantage points. We initially focused on identifying kinases correlated with differential outcome. Using publicly available transcriptomic data from a collated set of TNBC patients (n = 483), we identified 9 kinases that were significantly associated with survival at the mRNA level. From this in silico screen, CDK7 (cyclin-dependent kinase 7) was found to be correlated with poor recurrence-free survival. CDK79s trait as a marker of poor prognosis was further validated within another TNBC cohort (n=109) via assessment of a tissue microarray generated as part of the RATHER Consortium (www.ratherproject.com). At the protein level, high CDK7 expression was associated with poor breast cancer-specific, recurrence-free and distant recurrence-free survival. To evaluate CDK7 as a therapeutic target in TNBC, two TNBC cell lines (BT-549 and MDA-MB-231) were selected to evaluate phenotypic alterations post shRNA-mediated CDK7 knockdown. CDK7 silencing led to decreased cell proliferation, colony formation and migration in vitro. CDK7 down-regulation also increased TNBC cell sensitivity to doxorubicin. BS-181 and THZ1, two highly specific CDK7 inhibitors, attenuated TNBC tumour growth by inducing G2/M phase cell cycle arrest and apoptosis, as well as down-regulation of RNAPII phosphorylation, an indication of global RNA transcription inhibition. Moreover, the covalent CDK7 inhibitor THZ1 demonstrated 1000-fold higher potency than BS-181. Inhibition of global RNA transcription preferentially affects proteins with short half-lives. Accordingly, we detected a reduction in the expression of the anti-apoptotic protein MCL-1 in both cell lines. Next, we assessed anti-apoptotic dependence in MDA-MB-231 cells following treatment with THZ1 via BH3 profiling technology, and observed an increased response to the BAD and HRK peptides, inferring an elevated survival dependence on BCL-2/BCL-XL. We subsequently evaluated the combination of the BCL-2/BCL-XL inhibitor ABT-263 with THZ1 and discovered a synergistic inhibition of cell growth and apoptosis. Resulting combination index (CI) values demonstrated that synergistic cell death occurred following combined treatment with THZ1 and ABT-263/ABT-199 at various doses in both TNBC cell lines tested. Our data implicate high CDK7 expression as a promising biomarker of poor prognosis in TNBC. Moreover, these findings suggest that targeting CDK7, combined with the BCL-2/BCL-XL inhibitor ABT-263, may be a useful therapeutic strategy for TNBC. Citation Format: Gallagher WM, Li B, Ni Chonghaile T, Fan Y, Klinger R, O9Connor AE, Conroy E, Tarrant F, O9Hurley G, Mallya Udupi G, Gaber A, Chin S-F, Schouten PC, Dubois T, Linn S, Jirstrom K, Caldas C, Bernards R, O9Connor DP. CDK7: A marker of poor prognosis and tractable therapeutic target in triple-negative breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr PD3-01.

Abstract P6-03-02: Functional genomic screening identifies USP11 as a novel therapeutic target in breast cancer

The estrogen receptor (ER) is the principal driver of growth and differentiation in breast cells and de-regulated receptor function is a key feature of almost 75% of breast cancers. Here, we investigated the role of de-ubiquitinating enzymes (DUBs), which act to remove ubiquitin moieties from proteins, in regulating the transcriptional activity of ER in breast cancer. To identify DUBs involved in the regulation of ER transcriptional activity, we performed an RNAi loss-of-function screen using a library of shRNA vectors targeting all known human DUB genes (108 genes/432 shRNAs in total). We found that suppression of a number of DUBs markedly repressed or enhanced the activity of an estrogen-response-element (ERE) luciferase reporter to estradiol (E2). In particular, suppression of the BRCA2-associated DUB, USP11, was found to down-regulate ERα transcriptional activity (both in the presence and absence of E2), as demonstrated by a pronounced decrease in estrogen-response element (ERE) luciferase reporter activity. Subsequent validation of the screen using multiple individual hairpins and ZR-75-1 stable USP11 knockdown cells confirmed the suppression of ERE-reporter activity and further revealed a notable reduction in expression of the endogenous ER target genes TFF1 and PgR following USP11 knockdown. In vitro phenotypic analysis also revealed a global decrease in cell survival, decreased ERK and AKT phosphorylation and increased sensitivity to DNA-damaging agents in USP11 knockdown cell lines compared to non-targeting controls. In silico analysis of publically available breast cancer gene expression datasets revealed a highly significant association between high USP11 mRNA levels and poor prognosis. We observed a highly significant correlation between high expression of USP11 mRNA in ER positive patients and poor overall survival (OS)(HR 1.51, CI 1.07-2.14, p=0.018) and distant metastasis-free survival (DMFS)(HR 1.35, CI 1.04-1.73, p=0.023). This correlation was also significant in ER positive patients who had received endocrine therapy (OS, HR 3.4, CI 1.2-9.81, p=0.014), DMFS, HR 2.16, CI 1.23-3.8, p=0.0083). These results suggest a role for USP11 in driving cellular growth and identify USP11 as a rationale and novel therapeutic target in breast cancer. Citation Format: Darran P O9Connor, Lisa Dwane, Aisling E O9Connor, Annette M Dirac, Rene Bernards, William M Gallagher. Functional genomic screening identifies USP11 as a novel therapeutic target in 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 P6-03-02.