Finbarr Tarrant

Integration of genomic, transcriptomic and proteomic data identifies two biologically distinct subtypes of invasive lobular breast cancer

Invasive lobular carcinoma (ILC) is the second most frequently occurring histological breast cancer subtype after invasive ductal carcinoma (IDC), accounting for around 10% of all breast cancers. The molecular processes that drive the development of ILC are still largely unknown. We have performed a comprehensive genomic, transcriptomic and proteomic analysis of a large ILC patient cohort and present here an integrated molecular portrait of ILC. Mutations in CDH1 and in the PI3K pathway are the most frequent molecular alterations in ILC. We identified two main subtypes of ILCs: (i) an immune related subtype with mRNA up-regulation of PD-L1, PD-1 and CTLA-4 and greater sensitivity to DNA-damaging agents in representative cell line models; (ii) a hormone related subtype, associated with Epithelial to Mesenchymal Transition (EMT), and gain of chromosomes 1q and 8q and loss of chromosome 11q. Using the somatic mutation rate and eIF4B protein level, we identified three groups with different clinical outcomes, including a group with extremely good prognosis. We provide a comprehensive overview of the molecular alterations driving ILC and have explored links with therapy response. This molecular characterization may help to tailor treatment of ILC through the application of specific targeted, chemo- and/or immune-therapies.

Gene Expression Profiling Identifies Interferon Signalling Molecules and IGFBP3 in Human Degenerative Annulus Fibrosus

Low back pain is a major cause of disability especially for people between 20 and 50 years of age. As a costly healthcare problem, it imposes a serious socio-economic burden. Current surgical therapies fail to replace the normal disc in facilitating spinal movements and absorbing load. The focus of regenerative medicine is on identifying biomarkers and signalling pathways to improve our understanding about cascades of disc degeneration and allow for the design of specific therapies. We hypothesized that comparing microarray profiles from degenerative and non-degenerative discs will lead to the identification of dysregulated signalling and pathophysiological targets. Microarray data sets were generated from human annulus fibrosus cells and analysed using IPA ingenuity pathway analysis. Gene expression values were validated by qRT-PCR, and respective proteins were identified by immunohistochemistry. Microarray analysis revealed 238 differentially expressed genes in the degenerative annulus fibrosus. Seventeen of the dysregulated molecular markers showed log2-fold changes greater than ±1.5. Various dysregulated cellular functions, including cell proliferation and inflammatory response, were identified. The most significant canonical pathway induced in degenerative annulus fibrosus was found to be the interferon pathway. This study indicates interferon-alpha signalling pathway activation with IFIT3 and IGFBP3 up-regulation, which may affect cellular function in human degenerative disc.

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 4674: Epigenetic modulators for the treatment of invasive lobular carcinoma breast cancer

Invasive lobular carcinoma (ILC) is a breast cancer subtype comprising 10% of breast tumors. The majority of ILC (90%) are estrogen receptor (ER)-positive and therefore candidates for endocrine therapy. Unfortunately, de novo resistant to endocrine therapies occurs in 33% of women and a further 40% will relapse on treatment. Therefore, novel therapeutic targets are required for ILC. Deregulated transcription is a recurring theme in cancer, which can be due to epigenetic events. The bromdomain & extra-terminal domain (BET) family of proteins (BRD2, BRD3, BRD4, BRDT) function as chromatin readers that bind acetylated lysine residues on histones and regulate transcription. We performed RNA-Sequencing analysis on 61 primary ILC samples and found that high expression of BRD3 is associated with poor survival in ILC (log rank test, p=0.037). We validated this finding, that high expression of BRD3 is associated with poor survival, in a second cohort of 99 ILC primary samples from the METABRIC dataset (log rank test, p=0.0157). Next, we tested if ILC cell lines were sensitive to BET inhibition using the small molecule inhibitor JQ1, which inhibits all BET family proteins. JQ1 downregulates growth and survival genes in ILC cell lines including MYC, ER and BCL-XL. Pathway analysis following RNA sequencing revealed that JQ1 targets the apoptotic and Wnt signalling pathways in ILC cell lines. Interestingly, JQ1 inhibited the cell growth in all ILC cell lines tested, however apoptosis was only induced in two ILC cell lines. Furthermore, ILC cell lines which were relatively resistant to JQ1-induced apoptosis expressed both the BCL-2 and BCL-XL anti-apoptotic proteins. This led us to assess the combination of JQ1 and the BH3 mimetics, ABT-199 and ABT-263. ABT-199 is a selective small molecule inhibitor of BCL-2, whereas ABT-263 is an inhibitor of BCL-2, BCL-XL and BCL-W proteins. We found the combination of JQ1 and ABT-263, but not the combination of JQ1 & ABT-199, to be synergistic and enhance apoptosis in ILC cell lines. This is in accordance with BH3 profiling of ILC cell lines which indicated that ILC cell lines are dependent on BCL-2/ BCL-XL proteins for cell survival. As JQ1 is a pan-BET family inhibitor, we also wish to determine which BET protein is responsible for sensitivity to JQ1. Following knockdown of each BET protein in the CAMA-1 cell line we found that BRD3 and BRD4 were responsible for loss of cell viability. Future work will include determining the specific role of BRD3 in ILC and the effectiveness of the JQ1 and ABT-263 combination in vivo. Our work suggests that inhibition of BET proteins in combination with BH3 mimetics may be a rational therapeutic combination for ILC. Citation Format: Louise Walsh, Bruce Moran, Sudipto Das, Finbarr Tarrant, Philip Schouten, Suet-Feung Chin, Rene Bernards, Carlos Caldas, William Gallagher, Triona Ni Chonghaile*, Darran O9Connor*. Epigenetic modulators for the treatment of invasive lobular carcinoma breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4674. doi:10.1158/1538-7445.AM2017-4674

Abstract A30: RATHER: High-resolution molecular profiling of invasive lobular breast cancers

Introduction: RATHER (Rational Therapy for Breast Cancer) is an international multi-site collaborative effort that aims to use high resolution molecular profiling techniques to identify novel kinase targets for two subtypes of breast cancer, invasive lobular cancers (ILC) and triple negatives (TN) where no targeted therapies are available at present. Experiments: DNA, RNA and protein were extracted from 137 ILC and 155 TN samples with an average of 5 years clinical follow-up. A variety of high resolution molecular profiling methods were used such as copy number analysis (Affymetrix SNP6), gene expression profiling (Agilent 4x44K gene arrays), targeted sequencing (Agilent customized kinome panel & Illumina Nextera Custom Enrichment), whole transcriptomic sequencing and reverse phase protein lysate array (RPPA) analysis. Results: Combining copy number and gene expression data, we have classified the ILC tumors into the intergrative Cluster (IntClust) subgroups that we have previously identified from our large-scale breast cancer study, METABRIC (Molecular Taxonomy of Breast Cancer International Consortium). The ILC tumors were predominantly in IntClust3 (37.2%) and IntClust8 (21.2%). Only two genes were found to be frequently mutated (>10%) ie. CDH1 (40.8%) and PIK3CA (35%ILC). The PI3K pathway has been found to be frequently altered in ILCs by either mutations (PIK3CA and AKT1) or copy number alterations (PTEN). Integrating with transcriptomic and proteomic data, two main subtypes of ILCs were identified: (i) an immune responsive subtype with mRNA up-regulation of PDL1, PD1 and CTLA4 and greater sensitivity to DNA-damaging agents in representative cell line models; (ii) a hormone receptor signalling subtype, associated with Epithelial to Mesenchymal Transition (EMT), and gain of chromosomes 1q and 8q and loss of chromosome 11q. Using somatic mutation rate and eIF4B protein level, we identified three groups with different clinical outcomes, including a group with extremely good prognosis. Conclusion: We provide a comprehensive overview of the molecular alterations driving ILC and have explored links with therapy response. This molecular characterization will help to tailor treatment of ILC through the application of specific targeted, chemo- and/or immune-therapies. Citation Format: Suet-Feung Chin, Magali Michault, Ian Majewski, Tesa M. Severson, Tycho Bismeijer, Leanne De Korning, Justine Peeters, Phillip Schouten, Oscar M. Rueda, Astrid Bosma, Finbarr Tarrant, Yue Fan, BeiLei He, Bernard Pereira, Helen A. Bardwell, Elena Provenzano, Darran P. O9Connor, Sabine Linn, Thierry Dubois, Iris Simon, William Gallagher, Lodewyk Wessels, Rene Bernards, Carlos Caldas. RATHER: High-resolution molecular profiling of invasive lobular breast cancers. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A30.

Abstract P6-04-02: Defining clinically relevant molecular subtypes in lobular breast cancer within the RATHER consortium

Background: The Rational Therapy for Breast Cancer (RATHER) Consortium aims to identify novel kinase targets for therapy in poor-prognosis subtypes of breast cancer for which there are currently no targeted therapies available. In this project, the focus is on invasive lobular carcinomas (ILC), which represent 10% of breast tumors. The main strength of the RATHER project is the unique combination of comprehensive molecular data together with detailed clinical information, which enables translation of state-of-the art genomics analyses to the clinic. Our main goal is to identify and validate novel kinase targets for breast cancer therapy in a comprehensive way using large-scale and complementary genomics data (DNA and RNA sequence, copy number variation, gene expression and protein expression). Integrated analysis of these molecular data is performed to define molecular subtypes of ILC with differential clinical outcome. Methods: One hundred and fifty ILC samples (fresh frozen) with >5 years follow-up were collected from two institutes (Cambridge, Netherlands Cancer Institute). All samples were processed following one standard operating protocol to isolate RNA, DNA and protein of high quality. We used a five-pronged approach to identify and validate novel kinase targets for therapy in ILC, namely i) direct re-sequencing of the kinome of 150 ILC breast tumors, ii) determination of abundance and activation status of kinases in these tumors by reverse phase protein lysate array (RPPA) technology, iii) determination of copy number variation (CNV) by genome-wide SNP arrays, iv) mRNA quantitation of both genome and kinome using DNA microarrays and v) RNAseq of a subset of ILC tumors. ILC are compared to triple negative, which allows us to highlight differences and/or similarities between these subtypes and provide clues for therapeutic targets. Results: Data from these independent genome-scale technologies were integrated, yielding a prioritized list of potential kinase targets for therapy in ILC breast cancer. Deep sequencing of the kinome has revealed somatic mutations characteristic of ILC, which are currently being validated via mass spectrometry-based genotyping technology and their possible effects confirmed with gene expression, protein expression and phosphorylation changes. In addition, on a subset of the ILC samples, RNA sequencing was performed to confirm expression of particular mutants. Known gene mutations in ILC such as loss of CDH1 were confirmed. Moreover, the PI3K pathway is found to be frequently altered (50% of the samples). Gene expression analysis, as well as integrative analysis of CNV and gene expression data revealed subsets of ILCs that significantly regulate alternate biological processes and show differential clinical outcome. Such biological subsets are currently being validated with clinical and follow-up data. Updated results will be presented at the meeting. Conclusion: The RATHER project aims to deliver proof-of-concept for novel therapeutic interventions, together with companion molecular diagnostic assays for patient stratification, for up to 10% of breast cancer patients, where current treatment options are unsatisfactory. Ongoing validation of a number of potential targets proves to be promising. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-04-02.