Sarah Maguire

SF3B1 mutations constitute a novel therapeutic target in breast cancer

Mutations in genes encoding proteins involved in RNA splicing have been found to occur at relatively high frequencies in several tumour types including myelodysplastic syndromes, chronic lymphocytic leukaemia, uveal melanoma, and pancreatic cancer, and at lower frequencies in breast cancer. To investigate whether dysfunction in RNA splicing is implicated in the pathogenesis of breast cancer, we performed a re‐analysis of published exome and whole genome sequencing data. This analysis revealed that mutations in spliceosomal component genes occurred in 5.6% of unselected breast cancers, including hotspot mutations in the SF3B1 gene, which were found in 1.8% of unselected breast cancers. SF3B1 mutations were significantly associated with ER‐positive disease, AKT1 mutations, and distinct copy number alterations. Additional profiling of hotspot mutations in a panel of special histological subtypes of breast cancer showed that 16% and 6% of papillary and mucinous carcinomas of the breast harboured the SF3B1 K700E mutation. RNA sequencing identified differentially spliced events expressed in tumours with SF3B1 mutations including the protein coding genes TMEM14C, RPL31, DYNL11, UQCC, and ABCC5, and the long non‐coding RNA CRNDE. Moreover, SF3B1 mutant cell lines were found to be sensitive to the SF3b complex inhibitor spliceostatin A and treatment resulted in perturbation of the splicing signature. Albeit rare, SF3B1 mutations result in alternative splicing events, and may constitute drivers and a novel therapeutic target in a subset of breast cancers.

Three-dimensional modelling identifies novel genetic dependencies associated with breast cancer progression in the isogenic MCF10 model

The initiation and progression of breast cancer from the transformation of the normal epithelium to ductal carcinoma in situ (DCIS) and invasive disease is a complex process involving the acquisition of genetic alterations and changes in gene expression, alongside microenvironmental and recognized histological alterations. Here, we sought to comprehensively characterise the genomic and transcriptomic features of the MCF10 isogenic model of breast cancer progression, and to functionally validate potential driver alterations in three‐dimensional (3D) spheroids that may provide insights into breast cancer progression, and identify targetable alterations in conditions more similar to those encountered in vivo. We performed whole genome, exome and RNA sequencing of the MCF10 progression series to catalogue the copy number and mutational and transcriptomic landscapes associated with progression. We identified a number of predicted driver mutations (including PIK3CA and TP53) that were acquired during transformation of non‐malignant MCF10A cells to their malignant counterparts that are also present in analysed primary breast cancers from The Cancer Genome Atlas (TCGA). Acquisition of genomic alterations identified MYC amplification and previously undescribed RAB3GAP1–HRAS and UBA2–PDCD2L expressed in‐frame fusion genes in malignant cells. Comparison of pathway aberrations associated with progression showed that, when cells are grown as 3D spheroids, they show perturbations of cancer‐relevant pathways. Functional interrogation of the dependency on predicted driver events identified alterations in HRAS, PIK3CA and TP53 that selectively decreased cell growth and were associated with progression from preinvasive to invasive disease only when cells were grown as spheroids. Our results have identified changes in the genomic repertoire in cell lines representative of the stages of breast cancer progression, and demonstrate that genetic dependencies can be uncovered when cells are grown in conditions more like those in vivo. The MCF10 progression series therefore represents a good model with which to dissect potential biomarkers and to evaluate therapeutic targets involved in the progression of breast cancer.

Evaluation of CDK12 Protein Expression as a Potential Novel Biomarker for DNA Damage Response Targeted Therapies in Breast Cancer

Disruption of Cyclin-Dependent Kinase 12 (CDK12) is known to lead to defects in DNA repair and sensitivity to platinum salts and PARP1/2 inhibitors. However, CDK12 has also been proposed as an oncogene in breast cancer. We therefore aimed to assess the frequency and distribution of CDK12 protein expression by IHC in independent cohorts of breast cancer and correlate this with outcome and genomic status. We found that 21% of primary unselected breast cancers were CDK12 high, and 10.5% were absent, by IHC. CDK12 positivity correlated with HER2 positivity but was not an independent predictor of breast cancer–specific survival taking HER2 status into account; however, absent CDK12 protein expression significantly correlated with a triple-negative phenotype. Interestingly, CDK12 protein absence was associated with reduced expression of a number of DDR proteins including ATR, Ku70/Ku80, PARP1, DNA-PK, and γH2AX, suggesting a novel mechanism of CDK12-associated DDR dysregulation in breast cancer. Our data suggest that diagnostic IHC quantification of CDK12 in breast cancer is feasible, with CDK12 absence possibly signifying defective DDR function. This may have important therapeutic implications, particularly for triple-negative breast cancers. Mol Cancer Ther; 17(1); 306–15. ©2017 AACR.

3D modelling identifies novel genetic dependencies associated with breast cancer progression in the isogenic MCF10 model.

The initiation and progression of breast cancer from the transformation of the normal epithelium to ductal carcinoma in situ (DCIS) and invasive disease is a complex process involving the acquisition of genetic alterations and changes in gene expression, alongside microenvironmental and recognized histological alterations. Here, we sought to comprehensively characterise the genomic and transcriptomic features of the MCF10 isogenic model of breast cancer progression, and to functionally validate potential driver alterations in three‐dimensional (3D) spheroids that may provide insights into breast cancer progression, and identify targetable alterations in conditions more similar to those encountered in vivo. We performed whole genome, exome and RNA sequencing of the MCF10 progression series to catalogue the copy number and mutational and transcriptomic landscapes associated with progression. We identified a number of predicted driver mutations (including PIK3CA and TP53) that were acquired during transformation of non‐malignant MCF10A cells to their malignant counterparts that are also present in analysed primary breast cancers from The Cancer Genome Atlas (TCGA). Acquisition of genomic alterations identified MYC amplification and previously undescribed RAB3GAP1–HRAS and UBA2–PDCD2L expressed in‐frame fusion genes in malignant cells. Comparison of pathway aberrations associated with progression showed that, when cells are grown as 3D spheroids, they show perturbations of cancer‐relevant pathways. Functional interrogation of the dependency on predicted driver events identified alterations in HRAS, PIK3CA and TP53 that selectively decreased cell growth and were associated with progression from preinvasive to invasive disease only when cells were grown as spheroids. Our results have identified changes in the genomic repertoire in cell lines representative of the stages of breast cancer progression, and demonstrate that genetic dependencies can be uncovered when cells are grown in conditions more like those in vivo. The MCF10 progression series therefore represents a good model with which to dissect potential biomarkers and to evaluate therapeutic targets involved in the progression of breast cancer.

Capture Hi-C identifies putative target genes at 33 breast cancer risk loci

Genome-wide association studies (GWAS) have identified approximately 100 breast cancer risk loci. Translating these findings into a greater understanding of the mechanisms that influence disease risk requires identification of the genes or non-coding RNAs that mediate these associations. Here, we use Capture Hi-C (CHi-C) to annotate 63 loci; we identify 110 putative target genes at 33 loci. To assess the support for these target genes in other data sources we test for associations between levels of expression and SNP genotype (eQTLs), disease-specific survival (DSS), and compare them with somatically mutated cancer genes. 22 putative target genes are eQTLs, 32 are associated with DSS and 14 are somatically mutated in breast, or other, cancers. Identifying the target genes at GWAS risk loci will lead to a greater understanding of the mechanisms that influence breast cancer risk and prognosis.Risk loci for breast cancer have been identified by genome-wide association studies. Here, the authors use Capture Hi-C to identify 110 putative target genes at 33 loci and assessed associations of gene expression, SNP genotype, and survival, providing evidence of mechanisms that may influence the prognosis and risk of breast cancer.

PO-054 Common genetic variants at the breast cancer risk region 2Q35 map to putative IGFbp5 enhancers

Introduction Genome-wide association studies (GWAS) have identified over 100 independently-associated regions of the genome with germline variants that modulate risk of developing breast cancer. Most signals map to non-coding regions of the genome and are thought to act at the level of gene regulation. Whilst GWAS are instrumental in highlighting regions of interest, it is important to identify the causal variant(s) at each locus and the mechanisms by which they influence risk. Material and methods We investigated the 2q35 breast cancer risk locus using two complementary methods. The first method involved selecting potentially causal variants based primarily on breast relevant functional data (Capture Hi-C, ChIP-seq, DNase-seq, GRO-seq) The second method involved conventional fine-mapping of case-control samples, to generate a short list of potentially causal variants that cannot be excluded on statistical grounds alone followed by intersection of the same breast-relevant functional data. Results and discussions The two methods identify distinct subsets of putative causal SNPs mapping to ERα/FOXA1 enhancer-like elements targeting the IGFBP5 gene promoter, a gene known to play a role in breast development. We confirmed enhancer status by performing luciferase reporter assays containing each of the regions of interest; furthermore, by introducing minor SNP alleles we identified a subset of SNPs that modulate strength of enhancer activity. ChIP-seq in heterozygous cell lines also confirms an allelic preference in enhancer binding. We are now using CRISPR/Cas9-based systems to investigate the regions of interest through perturbation studies, and generating isogenic cell lines to further investigate SNP effects on transcription factor binding, chromosome conformation and gene expression. Conclusion The 2q35 breast cancer risk locus consists of three independent risk-associated signals. Statistical and functional approaches to variant prioritisation are complementary, identifying largely overlapping but distinct sets of candidate causal variants in putative regulatory elements (PREs). Subsequent functional investigation suggests these genetic variants may modulate the strength of these PREs; however, further investigation in isogenic cell lines is required.

Abstract 816: Molecular and genomic characterization of a newly established male breast cancer cell line

Although the majority of breast cancers affect women, approximately 1% of cases occur in men. At present comparatively little is known about the molecular mechanisms that influence male breast cancer predisposition and tumorigenicity. This is largely due to the current unavailability of an established cell line model in which to study male breast cancer. Development of in vitro models of the disease is therefore warranted, not least because growing evidence indicates that it is sufficiently different from female breast cancer such that extrapolating knowledge from one to the other may be misleading. Here we report the establishment and characterisation of a male breast cancer cell line derived from a primary tumor arising in a 61-year old male patient. We used 3T3-J2 mouse fibroblast co-culture in the presence of a ROCK inhibitor to conditionally reprogram epithelial cells from freshly resected tumor tissue. Once established, the cell line showed a rapid proliferation rate and growth in full conditioned media without the support of mouse fibroblasts. Immunohistochemical profiling demonstrated expression of epithelium-specific antigens AE1/AE3, breast epithelial marker CK7 and confirmed estrogen receptor positivity in concordance with the primary tumor. Germline sequencing detected no pathogenic germline predisposition mutations in either BRCA1 or BRCA2. There was no evidence of somatic mutation in any of the established female breast cancer driver genes. Somatic whole genome sequencing of early and late passages indicated a paucity of structural aberration and little evidence for obvious accumulation of gross genetic alterations. In conclusion, we have established a novel in vitro model for studying male breast cancer. That the cell line genome displays minimal evidence of significant acquired genetic changes arising due to reprogramming suggests that it will be of value in future studies of the biology of male breast cancer. Citation Format: Tereza Vaclova, Sarah Maguire, Matthew Pugh, Peter Barry, Nick Orr. Molecular and genomic characterization of a newly established male breast cancer cell line [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 816. doi:10.1158/1538-7445.AM2017-816

Abstract A28: Evaluation of CDK12 protein expression as a novel biomarker for PARP1/2 inhibitor sensitivity in breast cancer

The CycK/CDK12 complex is known to protect normal cells from genomic instability by regulating the DNA damage response (DDR) and has been postulated as a tumor suppressor gene in high-grade serous ovarian cancer. We have previously shown that in breast cancer, CDK12 is recurrently targeted by both DNA rearrangements (13% of HER2-amplified breast cancers) and recurrent point mutations (2.6% of unselected breast cancers). Furthermore, we have shown that loss of CDK12 confers sensitivity to PARP1/2 inhibitors, and therefore may constitute a biomarker of response. The aims of this study were i) to determine the frequency of CDK12 protein loss in unselected breast cancers, and any potential correlations with prognosis and/or molecular subtype; ii) assess the frequency of CDK12 protein loss in HER2-positive breast cancer, given the high frequency of CDK12 breakpoints in HER2-amplified tumors and correlate this with response to anti-HER2 therapy and iii) to identify additional genomic mechanisms of CDK12 disruption. We stained two tissue microarrays (TMA) with a validated antibody against CDK12 (Clone 57311, Abcam). The first cohort comprised 794 unselected primary breast cancers and the second comprised 121 HER2-positive breast cancers that had been treated with Herceptin. CDK12 expression was quantified using a modified Allred score, with a score of 0 being considered negative and a score of 7 or 8, positive. Correlation statistics were calculated using SPSS with a Pearson9s Chi Square or Fisher9s Exact Test p-value of In the TMA tumor cohort, 73 tumors were CDK12 negative (9%), and 146 were CDK12 positive (18%). In both these groups, a significant correlation with HER2 expression was seen, with 96% of CDK12 negative tumors being HER2 negative (p = 0.006) and only 39% of CDK12 positive tumors being HER2 positive (p In conclusion, our results highlight that CDK12 is disrupted via multiple genomic mechanisms and is a potential tumor suppressor gene in breast cancer. Loss of CDK12 protein expression is seen in 9% of unselected breast cancers and 18% of triple negative breast cancers, and may be a worthwhile treatment option for triple negative patients. Routine diagnostic quantification of CDK12 protein in tumors using immunohistochemistry is feasible, making it a viable biomarker for response to PARP1/2 inhibitor therapy. Citation Format: Kalnisha Naidoo, Patty Wai, Sarah Maguire, Frances Daley, Yinyin Yuan, Emad A. Rakha, Ian O. Ellis, Christopher J. Lord, Andrew R. Green, Rachael C. Natrajan. Evaluation of CDK12 protein expression as a novel biomarker for PARP1/2 inhibitor sensitivity in breast cancer. [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 A28.

Abstract PR06: Modeling the tumor microenvironment to identify novel loss of function mutations in breast cancer progression

Recent next generation sequencing studies have comprehensively mapped the genetic landscape of breast cancer and revealed that only a small number of genes are recurrently mutated in more than 10% of unselected tumors (i.e. TP53, PIK3CA and GATA3), and that the vast majority of recurrent mutations occur at low frequencies. Although some have been shown to be drivers (i.e. confer a selective advantage), such as oncogenic ERBB2 mutations, there is a myriad of significantly altered lower frequency mutations whose functional impact is unknown. We utilized a functional genomics approach silencing the 200 most frequently mutated genes in breast cancer in 3D spheroid cultures that more accurately recapitulate in vivo like conditions, using the MCF10A progression series cell line panel to identify novel loss of function mutations that affect breast cancer progression from non-malignant to highly invasive disease. Genes whose silencing significantly altered spheroid growth were integrated with comprehensive copy number and mutation data in order to analyze the impact of these genes in concert with additional driver alterations in genes such as TP53 and PIK3CA mutations. We identified 11 genes whose silencing with siRNA had a significant effect on growth in two or more cell lines in 3D, including FMN2, FOXA1, NIPBL and CREBBP. Silencing of FMN2 increased spheroid growth in the invasive cell lines only, suggesting loss of function mutations are a later event in breast cancer progression. A second targeted validation screen showed that silencing of a cohort of these genes had limited effect under traditional 2D culture conditions, for example, silencing of maltase-glucoamylase (MGAM) resulted in increased growth in AT1 and DCIS.com cells in 3D while having no effect in 2D; an effect that was recapitulated by treating cells with an established MGAM inhibitor. Furthermore, loss of NIPBL significantly increased spheroid growth in cells harboring TP53 nuclear accumulation, and was significantly co-mutated in TP53 mutant primary tumors, suggestive of epistasis. Integrating genes that when silenced decreased spheroid growth with mutation status in the cell lines identified 3D specific oncogenic dependencies with PIK3CA and novel SZT2 mutations. Using a functional genomic approach in 3D models we have identified recurrently mutated genes whose loss or gain of function contribute to breast cancer progression and furthermore may be epistatic or cooperate with established driver mutations in breast cancer. Citation Format: Barrie Peck, Sarah Maguire, Eamonn Morrison, Patty Wai, Rachael Natrajan. Modeling the tumor microenvironment to identify novel loss of function mutations in breast cancer progression. [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 PR06.