Hoadley, Katherine A.

Abstract Dysregulation of estrogen receptor gene (ESR1) is an established mechanism of inducing endocrine therapy resistance. We previously discovered a chromosomal translocation event generating an estrogen receptor gene fused in-frame to C-terminal sequences of YAP1 (ESR1-YAP1) that contributed to endocrine therapy resistance in estrogen receptor positive (ER+) breast cancer models. This current study compares functional and pharmacological properties of additional ESR1 gene fusion events of both early stage (ESR1-NOP2) and advanced endocrine therapy resistant (ESR1-YAP1 and ESR1-PCDH11x) breast cancers. The YAP1 and PCDH11x fusions conferred estrogen-independent and fulvestrant-resistant growth in T47D, an ER+ breast cancer cell line in vitro and in vivo, in contrast to the NOP2 fusion which was sensitive to hormone deprivation. Immunohistochemical (IHC) staining of mouse lungs revealed significantly higher numbers of micrometastatic ER+ cells from the T47D tumors expressing the YAP1 and PCDH11x fusions than YFP control and NOP2 fusion. Estrogen response element (ERE) reporter and pull down assays revealed that although all ESR1 fusions studied bound EREs, only the YAP1 and PCDH11x caused ERE activation. Cell lines containing these “canonical” ESR1 fusions upregulated expression of ER responsive genes such as TFF1 and GREB1 in hormone deprived conditions. In contrast, the NOP2 fusion neither induced ERE activity nor upregulated TFF1 and GREB1 gene expression. The proliferative ability of canonical fusion-containing T47D cells was inhibited by palbociclib, a CDK4/6 inhibitor, in a dose-dependent manner. In vivo growth of patient-derived xenograft tumors naturally harboring the ESR1-YAP1 fusion (WHIM18) was significantly reduced in mice fed palbociclib-containing chow. Mice transplanted with WHIM18 also formed lung micrometastases, with an ER IHC staining pattern similar to lungs from YAP1 and PCDH11x fusion expressing T47D xenografts. In conclusion, in-frame ERE activating canonical fusions occur in end-stage, drug resistant, advanced breast cancer and can be added to ESR1 point mutations as a class of somatic mutation that may cause acquired resistance. Endocrine therapy resistant growth induced by these fusions can be treated with CDK4/6 inhibition, using an FDA approved drug, palbociclib, which could potentially improve outcomes in patients with ESR1 translocated tumors. Citation Format: Jonathan T. Lei, Jieya Shao, Jin Zhang, Michael Iglesia, Doug W. Chan, Ryoichi Matsunuma, Xiaping He, Purba Singh, Yoshimasa Kosaka, Robert Crowder, Svasti Haricharan, Shyam Kavuri, Jeremy Hoog, Chanpheng Phommaly, Rodrigo Goncalves, Susana Romalho, Wei-Chu Lai, Oliver Hampton, Anna Rogers, Ethan Tobias, Poojan Parikh, Sherri Davies, Cynthia Ma, Vera Suman, Kelly Hunt, Mark Watson, Katherine A. Hoadley, Aubrey Thompson, Charles Perou, Chad J. Creighton, Chris Maher, Matthew J. Ellis. Estrogen receptor gene fusions drive endocrine therapy resistance in estrogen receptor positive 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 1033. doi:10.1158/1538-7445.AM2017-1033

Abstract Background. We previously reported an alternative ESR1 somatic gain-of-function chromosomal translocation event in a patient presenting with aggressive, endocrine therapy resistant estrogen receptor (ER) positive disease, producing an in-frame fusion gene consisting of N-terminal ESR1 and the C-terminus of the Hippo pathway coactivator YAP1 (ESR1-YAP1). We recently identified another ESR1 fusion through RNA sequencing (RNA-seq) in advanced stage ER+ disease from a chest wall recurrence in a male patient that was refractory to multiple lines of treatment. Two examples of fusions discovered in primary breast cancer samples include ESR1 fused in-frame to C-terminal sequences from NOP2 (ESR1-NOP2), identified in a resistant cohort from a RNA-seq screen focused on 81 primary breast cancers from aromatase inhibitor clinical trials, and a second ESR1 fusion, fused in-frame to the entire coding sequence of POLH (ESR1-POLH), that was identified from RNA-seq analysis of 728 Cancer Genome Atlas breast samples. This current study extends our previous characterization of ESR1-YAP1 by comparing functional and pharmacological properties of these three additional ESR1 gene fusion events of both early stage and advanced breast cancers. Methods. In vitro and in vivo experiments were conducted to test ESR1 fusions to induce therapeutic resistance, and metastasis. The transcriptional and binding properties of each fusion was also examined. Pharmacological inhibition with Palbociclib, a cyclin-dependent kinase 4/6 inhibitor, was utilized to assess drug sensitivity in ESR1 fusion containing breast cancer cells and in a patient derived xenograft (PDX) model expressing ESR1-YAP1 (WHIM18). Results. The YAP1 and PCDH11x fusions conferred estrogen-independent and fulvestrant-resistant growth. Immunohistochemistry revealed significantly higher numbers of ER+ cells in lungs of mice xenografted with T47D cells expressing the YAP1 and PCDH11x fusions compared to YFP control, NOP2 and POLH fusions. Results from ChIP-seq and microarray studies suggest that these two fusions promote proliferation and metastasis through genomic action by binding estrogen response elements (ERE) and subsequent gene activation. We thereby define these fusions as “canonical” fusions compared to “non-canonical” NOP2 and POLH fusions, which demonstrated dramatically decreased genomic binding ability. The non-canonical fusions induced genes associated with basal-like breast cancer and promoted HER2, EGFR, and MAPK gene expression signatures in contrast to genes associated with cell cycle/proliferation induced by canonical fusions. The proliferative ability of canonical fusion-containing ER+ cells was inhibited by Palbociclib in a dose-dependent manner. In vivo WHIM18 tumors in mice fed with Palbociclib-containing chow demonstrated significantly reduced tumor volume, growth rate, and weight compared to tumors in mice on control chow. Conclusions. In-frame ERE activating canonical fusions occur in end-stage drug resistant advanced breast cancer and can be added to ESR1 point mutations as a class of recurrent somatic mutation that may cause acquired resistance. Growth induced by these fusions can be antagonized by Palbociclib and is potentially clinically helpful. Citation Format: Lei JT, Shao J, Zhang J, Iglesia M, Cao J, Chan DW, He X, Kosaka Y, Schmidt C, Matsunuma R, Haricharan S, Crowder R, Hoog J, Phommaly C, Goncalves R, Ramalho S, Lai W-C, Hampton O, Rogers A, Tobias E, Parikh P, Davies S, Ma C, Suman V, Hunt K, Watson M, Hoadley KA, Thompson A, Chen X, Perou CM, Creighton CJ, Maher C, Ellis MJ. Recurrent functionally diverse in-frame ESR1 gene fusions drive endocrine resistance in breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr PD2-03.

Abstract Fusion transcript 24analysis by five groups, each using "in house" analytical workflow to analyze RNA-seq data from 813 breast tumors from The Cancer Genome Atlas project detected 2514 high confidence RNA fusions (2 or more junction spanning reads detected by 2 or more groups). A subset of tumors with fusions were evaluated for chromosomal rearrangement using whole genome sequence data (WGS), and about half of the high confidence fusions could be validated, with the remainder likely subclonal and therefore below the limits of detection by WGS at the relatively low depth of coverage (30X) available. The majority of tumors contained one or more fusion transcript (range 0-24 fusions/tumor), but recurrence of specific chimaeric transcripts was low. Only ESR1->CCDC170, C20orf3->ACSS1, and USP22->MYH10 fusions were detected in 10 or more tumors. Additional ESR1 fusions were detected with AKAP12, BNC2, C6orf211, GNAS, POLH, USP25, and UTRN, within 19 tumors, primarily of the Luminal B subtype. RARA was the second most common 5' fusion partner with 17 tumors, predominately of the HER2-enriched subtype, expressing out of frame RARA fusions to 19 different 3' fusion partners. Other common 5' fusion partners included FBXL20, USP22, USP32, BCAS3, MED1, BPTF, ERBB2, MED24, and PPF1A1 (expressed in 10 or more tumors each). The most common 3' fusion partners were ERBB2, CCDC170, PITPNC1, ACSS1, MYH10, IKZF3, and RAB6A (10 or more tumors each). HER2-enriched tumors expressed significantly more fusions than other subtypes (4.9 fusions/tumor vs 3.3 for Basal-like, 1.3 for LumA, and 3.2 for LumB). Almost all HER2-enriched tumors expressed fusions (97%), whereas 85% of Basal-like tumors and 79% of Luminal B tumors expressed one or more fusions. Among Luminal A tumors, only 48% expressed one or more fusion transcript. Those Luminal A tumors with multiple fusion transcripts contained significantly more copy number aberrations and were enriched for expression of mitotic cell cycle genes (p=3.07E-18), characteristic of the recently described high risk CNH subclass of Luminal A tumors. We conclude that, consistent with other observations, breast tumors generally exhibit a high level of genomic instability, associated with multiple copy number alterations and multiple fusion transcripts. However, recurrence of specific fusion transcripts is low. About half of Luminal A tumors express fusion transcripts; and these tumors exhibit multiple copy number events, consistent with high level genomic instability, as well as enriched expression of mitotic cell cycle genes. These tumors appear to be related to the relatively high risk Luminal A CNH subclass, and express high levels of aurora kinases and polo-like kinases, which might be considered as therapeutic targets. Given the increasing emphasis on transcript profiling of clinical samples and the clinical significance of high risk Luminal A tumors, it may be timely to consider inclusion of fusion transcripts in such analyses as surrogate markers of genomic instability and potential therapeutic and/or prognostic indicators. Citation Format: Thompson EA, Asmann YW, Su X, Ellis MJ, Shao J, Hu Y, White KP, Cherniack AD, Hoadley KA, Serie DJ, Perez EA, Perou CM. A comprehensive analysis of fusion transcripts in breast cancer reveals associations between number of fusion transcripts, copy number events, gene expression profiles, and potentially clinical outcome. [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 S4-02.