Amir Bahreini

Sensitive Detection of Mono- and Polyclonal ESR1 Mutations in Primary Tumors, Metastatic Lesions, and Cell-Free DNA of Breast Cancer Patients

Purpose: Given the clinical relevance of ESR1 mutations as potential drivers of resistance to endocrine therapy, this study used sensitive detection methods to determine the frequency of ESR1 mutations in primary and metastatic breast cancer, and in cell-free DNA (cfDNA). Experimental Design: Six ESR1 mutations (K303R, S463P, Y537C, Y537N, Y537S, D538G) were assessed by digital droplet PCR (ddPCR), with lower limits of detection of 0.05% to 0.16%, in primary tumors (n = 43), bone (n = 12) and brain metastases (n = 38), and cfDNA (n = 29). Correlations between ESR1 mutations in metastatic lesions and single (1 patient) or serial blood draws (4 patients) were assessed. Results: ESR1 mutations were detected for D538G (n = 13), Y537S (n = 3), and Y537C (n = 1), and not for K303R, S463P, or Y537N. Mutation rates were 7.0% (3/43 primary tumors), 9.1% (1/11 bone metastases), 12.5% (3/24 brain metastases), and 24.1% (7/29 cfDNA). Two patients showed polyclonal disease with more than one ESR1 mutation. Mutation allele frequencies were 0.07% to 0.2% in primary tumors, 1.4% in bone metastases, 34.3% to 44.9% in brain metastases, and 0.2% to 13.7% in cfDNA. In cases with both cfDNA and metastatic samples (n = 5), mutations were detected in both (n = 3) or in cfDNA only (n = 2). Treatment was associated with changes in ESR1 mutation detection and allele frequency. Conclusions: ESR1 mutations were detected at very low allele frequencies in some primary breast cancers, and at high allele frequency in metastases, suggesting that in some tumors rare ESR1-mutant clones are enriched by endocrine therapy. Further studies should address whether sensitive detection of ESR1 mutations in primary breast cancer and in serial blood draws may be predictive for development of resistant disease. Clin Cancer Res; 22(5); 1130–7. ©2015 AACR. See related commentary by Gu and Fuqua, p. 1034

Invasive Lobular Carcinoma Cell Lines Are Characterized by Unique Estrogen-Mediated Gene Expression Patterns and Altered Tamoxifen Response

Invasive lobular carcinoma (ILC) is a histologic subtype of breast cancer that is frequently associated with favorable outcomes, as approximately 90% of ILC express the estrogen receptor (ER). However, recent retrospective analyses suggest that patients with ILC receiving adjuvant endocrine therapy may not benefit as much as patients with invasive ductal carcinoma. On the basis of these observations, we characterized ER function and endocrine response in ILC models. The ER-positive ILC cell lines MDA MB 134VI (MM134) and SUM44PE were used to examine the ER-regulated transcriptome via gene expression microarray analyses and ER ChIP-Seq, and to examine response to endocrine therapy. In parallel, estrogen response was assessed in vivo in the patient-derived ILC xenograft HCI-013. We identified 915 genes that were uniquely E2 regulated in ILC cell lines versus other breast cancer cell lines, and a subset of these genes were also E2 regulated in vivo in HCI-013. MM134 cells were de novo tamoxifen resistant and were induced to grow by 4-hydroxytamoxifen, as well as other antiestrogens, as partial agonists. Growth was accompanied by agonist activity of tamoxifen on ER-mediated gene expression. Though tamoxifen induced cell growth, MM134 cells required fibroblast growth factor receptor (FGFR)-1 signaling to maintain viability and were sensitive to combined endocrine therapy and FGFR1 inhibition. Our observation that ER drives a unique program of gene expression in ILC cells correlates with the ability of tamoxifen to induce growth in these cells. Targeting growth factors using FGFR1 inhibitors may block survival pathways required by ILC and reverse tamoxifen resistance.

Recurrent hyperactive ESR1 fusion proteins in endocrine therapy-resistant breast cancer

Abstract Background Estrogen receptor-positive (ER-positive) metastatic breast cancer is often intractable due to endocrine therapy resistance. Although ESR1 promoter switching events have been associated with endocrine-therapy resistance, recurrent ESR1 fusion proteins have yet to be identified in advanced breast cancer. Patients and methods To identify genomic structural rearrangements (REs) including gene fusions in acquired resistance, we undertook a multimodal sequencing effort in three breast cancer patient cohorts: (i) mate-pair and/or RNAseq in 6 patient-matched primary-metastatic tumors and 51 metastases, (ii) high coverage (>500×) comprehensive genomic profiling of 287–395 cancer-related genes across 9542 solid tumors (5216 from metastatic disease), and (iii) ultra-high coverage (>5000×) genomic profiling of 62 cancer-related genes in 254 ctDNA samples. In addition to traditional gene fusion detection methods (i.e. discordant reads, split reads), ESR1 REs were detected from targeted sequencing data by applying a novel algorithm (copyshift) that identifies major copy number shifts at rearrangement hotspots. Results We identify 88 ESR1 REs across 83 unique patients with direct confirmation of 9 ESR1 fusion proteins (including 2 via immunoblot). ESR1 REs are highly enriched in ER-positive, metastatic disease and co-occur with known ESR1 missense alterations, suggestive of polyclonal resistance. Importantly, all fusions result from a breakpoint in or near ESR1 intron 6 and therefore lack an intact ligand binding domain (LBD). In vitro characterization of three fusions reveals ligand-independence and hyperactivity dependent upon the 3′ partner gene. Our lower-bound estimate of ESR1 fusions is at least 1% of metastatic solid breast cancers, the prevalence in ctDNA is at least 10× enriched. We postulate this enrichment may represent secondary resistance to more aggressive endocrine therapies applied to patients with ESR1 LBD missense alterations. Conclusions Collectively, these data indicate that N-terminal ESR1 fusions involving exons 6–7 are a recurrent driver of endocrine therapy resistance and are impervious to ER-targeted therapies.

Clinically Observed Estrogen Receptor Alpha Mutations within the Ligand-Binding Domain Confer Distinguishable Phenotypes

Objective: Twenty to fifty percent of estrogen receptor-positive (ER+) metastatic breast cancers express mutations within the ER ligand-binding domain. While most studies focused on the constitutive ER signaling activity commonly engendered by these mutations selected during estrogen deprivation therapy, our study was aimed at investigating distinctive phenotypes conferred by different mutations within this class. Methods: We examined the two most prevalent mutations, D538G and Y537S, employing corroborative genome-edited and lentiviral-transduced ER+ T47D cell models. We used a luciferase-based reporter and endogenous phospho-ER immunoblot analysis to characterize the estrogen response of ER mutants and determined their resistance to known ER antagonists. Results: Consistent with their selection during estrogen deprivation therapy, these mutants conferred constitutive ER activity. While Y537S mutants showed no estrogen dependence, D538G mutants demonstrated an enhanced estrogen-dependent response. Both mutations conferred resistance to ER antagonists that was overcome at higher doses acting specifically through their ER target. Conclusions: These observations provide a tenable hypothesis for how D538G ESR1-expressing clones can contribute to shorter progression-free survival observed in the exemestane arm of the BOLERO-2 study. Thus, in those patients with dominant D538G-expressing clones, longitudinal analysis for this mutation in circulating free DNA may prove beneficial for informing more optimal therapeutic regimens.

Upregulation of IRS1 Enhances IGF1 Response in Y537S and D538G ESR1 Mutant Breast Cancer Cells

Increased evidence suggests that somatic mutations in the ligand-binding domain of estrogen receptor [ER (ERα/ESR1)] are critical mediators of endocrine-resistant breast cancer progression. Insulinlike growth factor-1 (IGF1) is an essential regulator of breast development and tumorigenesis and also has a role in endocrine resistance. A recent study showed enhanced crosstalk between IGF1 and ERα in ESR1 mutant cells, but detailed mechanisms are incompletely understood. Using genome-edited MCF-7 and T47D cell lines harboring Y537S and D538G ESR1 mutations, we characterized altered IGF1 signaling. RNA sequencing revealed upregulation of multiple genes in the IGF1 pathway, including insulin receptor substrate-1 (IRS1), consistent in both Y537S and D538G ESR1 mutant cell line models. Higher IRS1 expression was confirmed by quantitative reverse transcription polymerase chain reaction and immunoblotting. ESR1 mutant cells also showed increased levels of IGF-regulated genes, reflected by activation of an IGF signature. IGF1 showed increased sensitivity and potency in growth stimulation of ESR1 mutant cells. Analysis of downstream signaling revealed the phosphoinositide 3-kinase (PI3K)-Akt axis as a major pathway mediating the enhanced IGF1 response in ESR1 mutant cells. Decreasing IRS1 expression by small interfering RNA diminished the increased sensitivity to IGF1. Combination treatment with inhibitors against IGF1 receptor (IGF1R; OSI-906) and ER (fulvestrant) showed synergistic growth inhibition in ESR1 mutant cells, particularly at lower effective concentrations. Our study supports a critical role of enhanced IGF1 signaling in ESR1 mutant cell lines, pointing toward a potential for cotargeting IGF1R and ERα in endocrine-resistant breast tumors with mutant ESR1.

Abstract A35: WNT4 signaling mediates endocrine response and resistance in invasive lobular carcinoma cells

Invasive lobular carcinoma (ILC) is a histological subtype of breast cancer, affecting ~30,000 U.S. women annually. Over 90% of ILC are estrogen receptor (ER)-positive, however, endocrine therapy may have poorer efficacy in a subset of ILC patients compared to invasive ductal carcinoma (IDC) patients. Based on these observations, we assessed genome-wide ER-mediated gene expression and ER genomic binding in ILC cell lines MDA MB 134VI (MM134) and SUM44PE (44PE), to identify novel mediators of ER signaling and putative therapeutic targets specifically in ILC. Among ILC-specific estrogen-regulated genes, the most strongly induced was the Wnt ligand WNT4. In parallel, we identified an ILC-specific ER binding site (ERBS) at WNT4, suggesting that WNT4 is directly ER-controlled in ILC cells. We hypothesized that this would be an analog to progesterone-controlled WNT4 in mammary gland expansion, and assessed whether WNT4 is necessary for estrogen-induced growth in ILC cells. Using siRNAs, knockdown of WNT4 completely blocked estrogen-induced growth in ILC cells, but not IDC cells. Consistent with this, we found that the WNT4 ERBS is only occupied in ILC cells that strongly upregulate WNT4 in response to estrogen, whereas progesterone-regulated WNT4 expression in T47D cells was not associated with ER binding at the WNT4 ERBS. These data suggest that, via an ILC-specific ERBS at WNT4, ILC cells can drive estrogen-regulated proliferation by hijacking a developmental Wnt pathway. Canonical Wnt pathways activate β-catenin; however, we observed β-catenin dysfunction in ILC cells, and that WNT4 cannot activate β-catenin in cell lines. Thus, WNT4 regulates estrogen-induced growth in ILC cells via a novel non-canonical pathway. Using long-term estrogen-deprived (LTED) variants of MM134 and 44PE (4 and 2 lines, respectively), we assessed WNT4 in ILC endocrine resistance. WNT4 is over-expressed but uncoupled from ER in all MM134-LTED; expression is greatly reduced in 44PE-LTED, but weakly estrogen-regulated. Consistent with regulation, ER occupies the WNT4 ERBS only in 44PE-LTED cells. However, 44PE-LTED (low WNT4) are resistant to growth inhibition by WNT4 siRNA, while MM134-LTED (high WNT4) are growth-inhibited. Taken together, uncoupling and upregulating WNT4 may be necessary for LTED growth in MM134. Clinical observations suggest that ER regulates unique downstream pathways in ILC. We identified WNT4 as a putative downstream effector of endocrine signaling in ILC, having critical roles in both estrogen-induced growth and endocrine resistance. Targeting WNT4 signaling represents a novel approach to modulate endocrine response specifically for ILC patients. Citation Format: Matthew J. Sikora, Amir Bahreini, Caroline M. Alexander, Steffi Oesterreich. WNT4 signaling mediates endocrine response and resistance in invasive lobular carcinoma cells. [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 A35.

Non-coding single nucleotide variants affecting estrogen receptor binding and activity

BackgroundEstrogen receptor (ER) activity is critical for the development and progression of the majority of breast cancers. It is known that ER is differentially bound to DNA leading to transcriptomic and phenotypic changes in different breast cancer models. We investigated whether single nucleotide variants (SNVs) in ER binding sites (regSNVs) contribute to ER action through changes in the ER cistrome, thereby affecting disease progression. Here we developed a computational pipeline to identify SNVs in ER binding sites using chromatin immunoprecipitation sequencing (ChIP-seq) data from ER+ breast cancer models.MethodsER ChIP-seq data were downloaded from the Gene Expression Omnibus (GEO). GATK pipeline was used to identify SNVs and the MACS algorithm was employed to call DNA-binding sites. Determination of the potential effect of a given SNV in a binding site was inferred using reimplementation of the is-rSNP algorithm. The Cancer Genome Atlas (TCGA) data were integrated to correlate the regSNVs and gene expression in breast tumors. ChIP and luciferase assays were used to assess the allele-specific binding.ResultsAnalysis of ER ChIP-seq data from MCF7 cells identified an intronic SNV in the IGF1R gene, rs62022087, predicted to increase ER binding. Functional studies confirmed that ER binds preferentially to rs62022087 versus the wild-type allele. By integrating 43 ER ChIP-seq datasets, multi-omics, and clinical data, we identified 17 regSNVs associated with altered expression of adjacent genes in ER+ disease. Of these, the top candidate was in the promoter of the GSTM1 gene and was associated with higher expression of GSTM1 in breast tumors. Survival analysis of patients with ER+ tumors revealed that higher expression of GSTM1, responsible for detoxifying carcinogens, was correlated with better outcome.ConclusionsIn conclusion, we have developed a computational approach that is capable of identifying putative regSNVs in ER ChIP-binding sites. These non-coding variants could potentially regulate target genes and may contribute to clinical prognosis in breast cancer.

Abstract 421: Comprehensive genomic analysis of metastatic breast cancers revealsESR1fusions as a recurrent mechanism of endocrine therapy resistance

Metastatic breast cancer is often intractable due to its inherent ability to overcome current therapies. Genomic alterations are frequently responsible for therapeutic resistance. To better understand genomic mechanisms of acquired resistance in breast cancer we undertook a detailed characterization of single nucleotide variation (SNV) and structural variation (SV) in paired primary-metastasis metachronous tumors from 6 breast cancer patients (median time to recurrence 7.3 years). In ER-positive recurrent tumors treated with endocrine therapies, we identified multiple metastatic-acquired variants in ESR1 including a novel constitutively active, ligand-independent ESR1-DAB2 gene fusion. Importantly, this fusion resulted from a breakpoint in intron 4, retaining the DNA-binding domain but eliminating the ligand-binding domain (LBD), concordant to a similar fusion reported previously in a xenograft model. Hybrid capture based genomic profiling from >7,800 breast cancers identified similar exon/intron 4 fusions in 5 tumors with direct paired-read evidence. Using a novel copy number shift detection strategy, 58 additional tumors showed indirect evidence of a rearrangement at exon 4 based on a novel copy number shift detection strategy. ESR1 fusion and copy number shift positive tumors are strongly enriched in metastatic disease (78%; p Citation Format: Ryan J. Hartmaier, Nolan Priedigkeit, Laurie Gay, Michael E. Goldberg, James Suh, Siraj Ali, Jeffery Ross, Michaela Tsai, Barbara Haley, Julio Peguero, Rena D. Callahan, Irina Sachelarie, John Cho, Amir Bahreini, Shannon L. Puhalla, Steffi Oesterreich, Aju Mathew, Peter C. Lucas, Nancy E. Davidson, Adam M. Brufsky, Philip J. Stephens, Juliann Chmielecki, Adrian V. Lee. Comprehensive genomic analysis of metastatic breast cancers reveals ESR1 fusions as a recurrent mechanism of endocrine therapy resistance [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 421. doi:10.1158/1538-7445.AM2017-421

Abstract P3-04-05: Invasive lobular carcinoma cell lines utilize WNT4 signaling to mediate estrogen-induced growth

Invasive lobular carcinoma (ILC) is a histological subtype of breast cancer representing 10-15% of newly diagnosed breast tumors. Over 90% of ILC are ER-positive, however, endocrine response and estrogen signaling are not well described in ILC. Retrospective analyses suggest that ILC patients treated with endocrine therapy have poorer outcomes than similar invasive ductal carcinoma (IDC) patients, and that ILC patients may not benefit from adjuvant tamoxifen. Additionally, we recently identified ILC-specific ER-target genes and de novo tamoxifen resistance driven by ER in ILC model systems. Based on these observations, we hypothesize that ILC-specific signaling pathways driven by ER mediate growth and endocrine resistance in ILC cells. Among ILC-specific estrogen-regulated genes in the ILC cell lines MDA MB 134VI (MM134) and SUM44PE (SUM44), Wnt signaling genes were highly differentially expressed. The secreted ligand WNT4 was the most strongly estrogen-induced gene in ILC cells. The frizzled receptor FZD7 is also strongly induced in ILC cells, but only transiently induced in the ER-positive IDC cell line MCF-7. Among IDC cell lines, either WNT4 or FZD7 is over-expressed in ER-positive or ER-negative cells, respectively. Conversely, MM134 and SUM44 over-express both WNT4 and FZD7. Also, we identified an ILC-specific ER binding site at WNT4; located in intron 1, this site contains a predicted estrogen response element. Direct WNT4 regulation and parallel regulation of pathway genes suggests that ER controls a WNT4 signaling pathway in ILC cells. In samples from the Cancer Genome Atlas, WNT4 and FZD7 are each over-expressed in ER-positive ILC versus IDC; co-expression is also enriched only in ILC. These observations suggest that a WNT4 signaling pathway may be specifically active in ILC tumors. To assess whether WNT4 is necessary for estrogen-induced growth, we used siRNA to knock down WNT4. Using either of two siRNAs, WNT4 knockdown completely blocks estrogen-induced growth in ILC cells, but not IDC cells. Consistent with this, WNT4 knockdown abrogated estrogen-regulation of a subset of ER-target genes in MM134 cells; induction or repression was inhibited by WNT4 knockdown prior to estrogen treatment. Thus, a subset of estrogen-induced gene expression changes is mediated by WNT4 signaling. Though Wnt signaling typically acts via the canonical, β-catenin-dependent pathway, we observed that β-catenin signaling is dysfunctional in ILC cells. Additionally, WNT4 over-expression or recombinant protein cannot activate canonical Wnt signaling in breast cancer cell lines. This suggests that WNT4 signaling mediates estrogen-induced growth in ILC cells via a novel non-canonical signaling pathway. Wnt signaling pathway genes including WNT4 are uniquely regulated in ILC cell lines, and are over-expressed in ILC tumors, suggesting that a WNT4-driven pathway may be active specifically in ILC. WNT4 is necessary for estrogen-mediated growth in ILC cells, and likely signaling via a novel non-canonical signaling pathway. Targeting WNT4 signaling represents a novel approach to modulate endocrine response specifically for ILC patients. Future studies will focus on identifying the signaling pathway controlled by WNT4 in order to identify novel therapeutic targets. Citation Format: Matthew J Sikora, Amir Bahreini, Caroline M Alexander, Steffi Oesterreich. Invasive lobular carcinoma cell lines utilize WNT4 signaling to mediate estrogen-induced growth [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 P3-04-05.

Abstract A1-14: Discovery of a functional SNP in an estrogen receptor binding site in the IGF1R gene

Estrogen receptor (ER) positive luminal breast cancer represents approx 70% of all newly diagnosed breast cancers. ER is known to drive breast cancer development and progression, and targeting ER has been one of the most successful targeted therapies in oncology. Given the paucity of functional DNA sequence variants (DSVs) within the ER in primary breast tumors, we asked whether DSVs in ER binding sites could contribute to altered hormone response through changes in ER recruitment to DNA. We developed a pipeline to extract DSVs in ER binding sites, and to assess their impact on ER binding in a genome-wide manner, using ChIP-seq data from hormone responsive breast cancer cells. We utilized MACS for peak calling and GATK for calling DSVs including SNVs and short indels. Using 22,143 ER binding sites which overlapped in at least six data sets, we detected 1,409,406 DSVs from 441,855,039 reads. Position weight matrix (PWM) was used to scan the genome for potential ERE motifs, and identified 126 potential DSVs which were predicted to change ER binding. Of those, 29 DSV-containing EREs were in proximity or within estrogen regulated genes. Intriguingly, the top hit was a previously reported SNP within intron 2 of the IGF1R gene (rs62022087), which our data predicted to increase the affinity for ER binding. The ERE lies in a regulatory region characterized by active histone marks such as H3K29ac and H3k4Me1, and recruitment of a number of transcription factors including c-myc, FoxA1, GATA-1, and finally DNase I hypersensitive sites. Further analysis of published ChIP-seq studies on 6 ER+ cell lines and 15 primary tumors revealed that the presence of rs62022087 was associated with the enrichment of reads over the ER binding site, suggesting that the DSV indeed increases binding affinity of ER. Our further in-vitro functional studies confirmed that ER is more preferentially binding to mutant allele vs wild-type allele. In summary, we have shown that our pipeline is capable of predicting potential regulatory DSVs in ER binding sites. Studies are ongoing to understand functional and clinical relevance of rs62022087, especially with respect to crosstalk between estrogen and IGF1 signaling. Citation Format: Amir Bahreini, Amir Bahreini, Lucas Santanas, Peilu Wang, Panayiotis V. Benos, Adrian V. Lee, Adrian V. Lee, Steffi Oesterreich, Steff Oesterreich. Discovery of a functional SNP in an estrogen receptor binding site in the IGF1R gene. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A1-14.