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Supplementary Table 1 from PIK3CA and PIK3CB Inhibition Produce Synthetic Lethality when Combined with Estrogen Deprivation in Estrogen Receptor–Positive Breast Cancer

Supplementary Methods and Materials from PIK3CA and PIK3CB Inhibition Produce Synthetic Lethality when Combined with Estrogen Deprivation in Estrogen Receptor–Positive Breast Cancer

Several phosphoinositide 3-kinase (PI3K) catalytic subunit inhibitors are currently in clinical trial. We therefore sought to examine relationships between pharmacologic inhibition and somatic mutations in PI3K catalytic subunits in estrogen receptor (ER)–positive breast cancer, in which these mutations are particularly common. RNA interference (RNAi) was used to determine the effect of selective inhibition of PI3K catalytic subunits, p110α and p110β, in ER+ breast cancer cells harboring either mutation (PIK3CA) or gene amplification (PIK3CB). p110α RNAi inhibited growth and promoted apoptosis in all tested ER+ breast cancer cells under estrogen deprived-conditions, whereas p110β RNAi only affected cells harboring PIK3CB amplification. Moreover, dual p110α/p110β inhibition potentiated these effects. In addition, treatment with the clinical-grade PI3K catalytic subunit inhibitor BEZ235 also promoted apoptosis in ER+ breast cancer cells. Importantly, estradiol suppressed apoptosis induced by both gene knockdowns and BEZ235 treatment. Our results suggest that PI3K inhibitors should target both p110α and p110β catalytic subunits, whether wild-type or mutant, and be combined with endocrine therapy for maximal efficacy when treating ER+ breast cancer. [Cancer Res 2009;69(9):3955–62]

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.

Background: We have recently shown that acquired resistance to aromatase inhibitors (AI) can, in some instances, be reversed low-dose estradiol treatment until progression followed by repeat treatment with an aromatase inhibitor (Ellis, M. JAMA 302:774, 2009). We have also demonstrated that simultaneous inhibition of phosphoinositol-3-kinase (PI3K) and ER (though estrogen deprivation) activates cell death through apoptosis, which is reversed by estradiol, revealing a link between ER activation and the efficacy of PI3 kinase inhibition (Crowder, R. Cancer Res 69:3955, 2009). Consistent with this concept we have also shown that long term estrogen deprived (LTED) cells are co-resistant to both classes of agent (SABCS 2009 Abs#3131). We therefore studied ways to overcome resistance to PI3 kinase inhibitors in LTED cell lines using second line endocrine therapy strategies that could be offered to patients with AI resistant advanced disease. Methods: ER+ MCF7 and T47D (PIK3CA mutant) cells were cultured in phenol-red free media containing charcoal-stripped serum for 12 months to create long term estrogen deprivation (LTED) variants that mimicked clinical endocrine therapy resistance. A variant of the MCF7 LTED line that regained estrogen-dependent proliferation was created by re-exposure to estradiol for 4 months (MCF7 LTED-R). The effects of the PI3K isoform inhibitor BKM120, the mTOR inhibitor RAD001 and the dual PI3K/mTOR inhibitor BGT226 in MCF7 LTED and T47D LTED were assessed in combination with estrogen deprivation and fulvestrant. MCF7 LTED-R cells were treated with PI3 kinase pathway inhibitors after acute estradiol withdrawal. Apoptosis was measured using TUNEL flow cytometry. Results: MCF7 LTED were resistant to all three PI3 kinase inhibitors, but combination treatment with fulvestrant restored apoptosis to levels seen with parental MCF7 cells after acute estrogen-deprivation. Acute estrogen deprivation in MCF7 LTED-R cells was also effective in restoring an apoptotic response to BGT226. T47D LTED cells, which, unlike MCF7 cells, have markedly down-regulated ER expression, become more sensitive to PI3 kinase monotherapy than the parental T47D cell line, particularly in response to BKM120, and fulvestrant did not increase the apoptotic response. Conclusions: The MCF7 cell results suggest that PI3 kinase inhibitors will be effective in endocrine therapy resistant advanced breast cancer if these agents are administered with appropriate second line endocrine strategies. Potentially efficacious approaches include fulvestrant combinations and second-line aromatase inhibition, but only after restoration of AI sensitivity with estradiol. The T47D experiments suggest that in the setting of ER+ tumors that lose ER upon disease progression PI3 kinase inhibitors may be very effective. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P2-19-01.

SUMMARY RNA sequencing (RNA-seq) detects estrogen receptor alpha gene (ESR1) fusion transcripts in estrogen receptor-positive (ER+) breast cancer, but their role in disease pathogenesis remains unclear. We examined multiple ESR1 fusions and found that two, both identified in advanced endocrine treatment-resistant disease, encoded stable and functional fusion proteins. In both examples, ESR1-e6>YAP1 and ESR1-e6>PCDH11X, ESR1 exons 1–6 were fused in frame to C-terminal sequences from the partner gene. Functional properties include estrogen-independent growth, constitutive expression of ER target genes, and anti-estrogen resistance. Both fusions activate a metastasis-associated transcriptional program, induce cellular motility, and promote the development of lung metastasis. ESR1-e6>YAP1- and ESR1-e6>PCDH11X-induced growth remained sensitive to a CDK4/6 inhibitor, and a patient-derived xenograft (PDX) naturally expressing the ESR1-e6>YAP1 fusion was also responsive. Transcriptionally active ESR1 fusions therefore trigger both endocrine therapy resistance and metastatic progression, explaining the association with fatal disease progression, although CDK4/6 inhibitor treatment is predicted to be effective., In Brief Lei et al. show that transcriptionally active estrogen receptor gene (ESR1) fusions identified from late-stage, treatment-refractory estrogen receptor-positive (ER+) breast cancer drive pan-endocrine therapy resistance and metastatic progression. Growth of breast tumors driven by ESR1 fusions at primary and metastatic sties can be suppressed with a CDK4/6 inhibitor., Graphical Abstract

Background. Dysregulation of the 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 study compares functional, transcriptional, and pharmacological properties of additional ESR1 gene fusion events of both early stage (ESR1-NOP2) late stage (ESR1-YAP1 and ESR1-PCDH11x) breast cancers to gain a better understanding of therapeutic resistance and metastasis. Understanding the role of ESR1 fusions in inducing metastasis is critical, since the primary cause of death in breast cancer patients is through metastasis to distant sites. Methods. RNA-seq screens identified ESR1 fusions from early and late stage, endocrine therapy resistant breast tumor samples. Functional experiments were conducted using ER+ breast cancer cell lines, xenograft, and PDX models to test the ability of ESR1 fusions to induce therapeutic resistance and metastasis. ChIP-seq and RNA-seq were performed to examine transcriptional properties and differential gene expression induced by the fusions which directed subsequent pharmacological experiments with a CDK4/6 inhibitor. Results. ESR1-YAP1 and ESR1-PCDH11x promoted estrogen-independent and fulvestrant-resistant growth in vitro and induced greater tumor growth and increased metastatic capacity to the lungs of xenografted mice. In contrast, the ESR1-NOP2 fusion was sensitive to low estrogen conditions in vitro, and did not promote tumor growth. RNA-seq profiling revealed E2F targets pathway as the most highly enriched pathway induced by the ESR1 fusions. IHC revealed higher levels of pRb in ESR1-YAP1 and ESR1-PCDH11x xenograft tumors and subsequent CDK4/6 inhibition completely blocked tumor growth in an ESR1-YAP1 PDX model. Integrating RNA-seq with ChIP-seq data, we discovered a set of EMT and metastasis genes bound by all ESR1 fusions and WT-ER, but whose expression was strongly and uniquely up-regulated only by the ESR1-YAP1 and ESR1-PCDH11x fusions. These studies also revealed gained sites bound only by the ESR1-YAP1 and ESR1-PCDH11x fusions, not bound by WT-ER nor ESR1-NOP2. Genes mapping to these sites have a role in metastatic biology and were highly up-regulated by the YAP1 and PCDH11x fusions, potentially mediated by long range transcriptional activation. Conclusion. ESR1-YAP1 and ESR1-PCDH11x are driver fusions that occur in drug-resistant, advanced stage breast cancer and are a new class of recurrent somatic mutation that can cause acquired endocrine therapy resistance, yet can be treated with CDK4/6 inhibition. These driver fusions also confer increased metastatic ability through their ability to drive expression of genes that contribute to EMT and metastasis. In contrast, ESR1-NOP2 did not produce functional protein and appears to be a passenger event. These studies may provide pre-clinical rationale for targeting ESR1 translocated breast tumors, since the presence of an ESR1 driver fusion places a patient in a therapeutic category where none of the currently available endocrine therapies are likely to be effective. Citation Format: Lei JT, Shao J, Zhang J, Iglesia M, Chan DW, Cao J, Anurag M, Singh P, Haricharan S, Kavuri SM, Matsunuma R, Schmidt C, Kosaka Y, Crowder R, Hoog J, Phommaly C, Goncalves R, Ramalho S, Rodrigues-Peres RM, 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, Perou CM, Creighton CJ, Maher C, Ellis MJ. ESR1 gene fusions drive endocrine therapy resistance and metastasis in breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD8-03.

Several phosphoinositide 3-kinase (PI3K) catalytic subunit inhibitors are currently in clinical trial. We therefore sought to examine relationships between pharmacologic inhibition and somatic mutations in PI3K catalytic subunits in estrogen receptor (ER)–positive breast cancer, in which these mutations are particularly common. RNA interference (RNAi) was used to determine the effect of selective inhibition of PI3K catalytic subunits, p110α and p110β, in ER+ breast cancer cells harboring either mutation (PIK3CA) or gene amplification (PIK3CB). p110α RNAi inhibited growth and promoted apoptosis in all tested ER+ breast cancer cells under estrogen deprived-conditions, whereas p110β RNAi only affected cells harboring PIK3CB amplification. Moreover, dual p110α/p110β inhibition potentiated these effects. In addition, treatment with the clinical-grade PI3K catalytic subunit inhibitor BEZ235 also promoted apoptosis in ER+ breast cancer cells. Importantly, estradiol suppressed apoptosis induced by both gene knockdowns and BEZ235 treatment. Our results suggest that PI3K inhibitors should target both p110α and p110β catalytic subunits, whether wild-type or mutant, and be combined with endocrine therapy for maximal efficacy when treating ER+ breast cancer. [Cancer Res 2009;69(9):3955–62]

New biomarkers for pancreas cancer are needed to improve its detection and management. We surveyed the plasma of patients undergoing surgical resection to identify proteins which change in abundance after complete resection of tumor. Using longitudinally collected specimens from surgical patients, we control for normal inter-individual variation which can confound cross-sectional analysis. Recent refinements in two-dimensional gel electrophoresis allowed us to quantify changes in low abundance plasma proteins with precision. To circumvent the traditional limitations of image analysis in comparing two-dimensional gels, we used fluorometric two-dimensional difference gel electrophoresis to resolve the proteins from pre- and post-surgical plasma from each patient on one physical gel. Furthermore, we increased the ability of our assay to detect low-abundance proteins by depleting the plasma of 12 high-abundance proteins with a multi-affinity column. Informative protein spots from 20 plasma samples across 10 patients were submitted for identification with mass-spectrometry. We identified a group of proteins which change consistently in plasma following complete resection of pancreas tumor. Furthermore, we identified proteins which correlate with post-surgical rapid recurrence of disease. With further identification and validation, the candidate biomarkers which we identify in this study may prove to be useful in the diagnosis, management and prognostication of patients with pancreas cancer.

Endocrine therapy resistance occurs in 50% of estrogen receptor positive (ER+) luminal breast cancers but the underlying mechanisms are poorly understood. To gain insight, we have taken advantage of whole-genome and RNA sequencing data of five late-stage hormone-resistant luminal breast tumors all of which have been successfully established as patient-derived mouse xenograft (PDX) models. Here we describe genetic alterations in ESR1-the gene coding for ERa-in three of these five tumors. They include an ESR1 point mutation (Y537S), a gene translocation causing an in-frame fusion between N-terminal ER and C-terminal Yes-associated protein 1 (YAP1), and ESR1 gene amplification. Functional characterization of the ESR1(Y537S) mutant and ESR1-YAP1 fusion in ER+ cell lines indicated that they both possess constitutive transcriptional activity and drive hormone-independent cell proliferation, mirroring the endocrine resistance of the originating tumors and the estradiol-independent growth of the PDX tumors. ESR1 (Y537S), a known gain-of-function experimental mutation in the ligand-binding domain of ER is not seen in primary breast cancer (TCGA data), suggesting it is a mutation associated with acquired resistance. Regarding the ESR1-YAP1 fusion gene, the truncated N-terminal ER fragment lacks the hormone-dependent transactivation domain (AF2) and the ligand binding domain but retains the hormone-independent transactivation domain (AF1) and therefore drives resistance to all endocrine approaches. As for ESR1 gene amplification, the tumor of origin, though resistant to aromatase inhibition, paradoxically responded to estradiol treatment, and this was recapitulated in the PDX model. Interestingly, acquired ESR1 gene amplification also occurred in long-term estrogen-deprived breast cancer MCF-7 cells which similarly regress upon estradiol exposure. Thus, ESR1 amplification may be a biomarker for paradoxical therapy with estradiol. Together, our focused study of advanced endocrine resistant luminal breast tumors revealed three distinct mutational mechanisms affecting the ESR1 gene that drive endocrine therapy resistance. Prevalence studies using RNAseq are underway to determine the frequency of somatic changes in the ESR1 gene in advanced breast cancer samples and additional ER+ PDX models. These additional data will be presented at the meeting. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr S3-05.

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

Mammaglobin-A (MGB) is a breast cancer-associated antigen that is an attractive target for immune intervention. MGB has been shown to induce a specific CD8 T cell response in breast cancer patients, but little is known about a possible MGB-specific CD4 T cell response.Peripheral blood-derived CD4(+)CD25(-) T cells were stimulated in vitro with MGB-pulsed antigen-presenting cells (APC). The MGB and human leukocyte antigen (HLA) class II specificity of the CD4 T cell lines was confirmed by cytokine release following restimulation with autologous and allogenic APC pulsed with MGB from different sources. Candidate HLA class II-restricted epitopes were identified by computer algorithm and validated in cytokine release assays.MGB-specific CD4 T cells were successfully generated in cultures from six of seven donors. Restimulation of MGB-specific CD4 T cells with MGB-pulsed APC induced significantly higher levels of interferon (IFN)-gamma release than APC pulsed with an irrelevant protein (P = 0.0004). Cultures from five of seven donors showed a pure Th1 type response as evidenced by the absence of interleukin (IL)-4. MGB-specific CD4 T cells recognized both recombinant and naturally processed MGB presented by APC. This recognition was HLA class II-restricted, as HLA-DR mismatched APC were not recognized. MGB-specific CD4 T cells from three of four donors recognized MGB-derived, HLA class II-restricted peptides pulsed onto APC.We have successfully generated MGB-specific CD4 T cell cultures and identified candidate MGB HLA class II epitopes. These studies should facilitate study of the CD4 T cell response to MGB, and the development and monitoring of vaccine strategies targeting this unique antigen.

Background. We recently identified an in-frame ESR1 translocation in a tumor and PDX pair from a patient with endocrine therapy resistant advanced disease. The fusion gene preserved the N-terminal 365aa of ESR1 containing the intact activation function 1 (AF1), DNA-binding domain (DBD) and hinge, followed by the C-terminal transactivation domain of YAP1. The ESR1-365>YAP1 fusion drove estrogen-independent and anti-estrogen-resistant tumor cell growth (Cell Reports 4:1116, 2013). We therefore sought to further investigate the role of ESR1 fusion genes in breast cancer. Methods. 711 cases from TCGA, 82 ER+ cases from neoadjuvant endocrine therapy trials and 25 ER+ systemic relapse samples were screened for fusion genes using RNAseq. Fusion genes involving ESR1 were subsequently assayed for estrogen response element (ERE)-mediated transcriptional activity and ability to drive estradiol independent breast cancer cell growth. Results. Multiple ESR1 fusions were identified in mostly luminal B cancers. ESR1 fusions resulted most frequently from rearrangements involving another gene on Chromosome (Chr) 6 but also fusions with genes on other chromosomes. The fusions involving partners on Chr6 that retained an intact N terminal AF1 and DNA binding domain (DBD) of ESR1 (≥1-253aa) included an Out-of-Frame (OF) fusion event with CCDC170 (a gene immediately centromeric to ESR1), POLH (in-frame or IF), AKAP12 (IF), PCMT1 (OF), SYNE1 (OF) and GPR126 (OF). Also identified were inter-chromosomal translocations involving Chr12p-NOP2 (IF) and ChrX-PCDH11X (IF) and Chr7q-AKR1D1 (OF). The ESR1 fusions with transcriptional potential (because of a retained DNA binding domain) included variable 5’ ESR1 exons that preserved 253aa, 365aa, 412aa or 458aa of ESR1 sequence: in all cases disrupting ligand binding though loss of C terminal sequence. The effect of the IF and OF 3’ sequences on the transcriptional activity of the relevant ESR1 fragment was highly variable. The most transcriptionally active fusion (more active than the relevant ESR1 fragment alone) was the ESR1-365>YAP1 as well as ESR1-365>PCDH11X and ESR1-253>CCDC170. This is remarkable since PCDH11X is not considered a transcription factor and the CCDC170 sequence was OF – in both cases suggesting the activity of the fusion was due to "neomorphic" properties. Two fusions, NOP2 and POLH were inactive in the ERE reporter assay, yet both stimulated estradiol independent growth. In these cases we suggest that the ESR1 locus can function as a "promoter trap" which allows the identification of genes with previously unknown functions in endocrine therapy resistance. Conclusions: In luminal-type breast cancer the ESR1 gene was fused to multiple 3’ partners with remarkably heterogeneous functions. The two most transcriptionally active, ESR1-365>YAP1 and ESR1-365>PCDH11X, were both identified in endocrine therapy refractory advanced disease – suggesting a role in fatal disease progression. Citation Format: Jieya Shao, Jin Zhang, Robert J Crowder, Rodrigo Goncalves, Chanpheng Phommaly, Breast AWG and Network, Charles M Perou, Christopher A Maher, E Aubrey Thompson, Matthew J Ellis. ESR1 gene fusions implicated in endocrine therapy resistance of ER+ 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 PD6-4.

Deeper understanding the mechanisms by which luminal-type breast cancer develops resistance to endocrine therapy and development of novel strategies to treat these patients requires model systems recapitulate human breast cancer as accurately as possible. An increasing body of work suggests patient derived xenografts (PDX) may represent an informative model for development of novel therapeutics. We therefore established seven xenograft tumor lines from late-stage breast cancer patients with estrogen positive (ER+) disease. To date five ER+ PDX lines have been tested for responses to estradiol treatment in overiectomized NOD/SCID mice. Three showed estradiol independent-growth, one estrogen-stimulated growth and in one estradiol-induced a regression. These patterns mimicked the clinical phenotypes of each patient, tracking survival and responses to serial endocrine treatments. To define new mechanisms for resistance, whole genome DNA sequencing, RNA sequencing and Reverse Phase Protein Assay analysis was conducted. These studies identified an ESR1/YAP1 balanced translocation in a PDX model and tumor of origin showing low levels of ER, paradoxical high level expression form luminal genes and extreme ET resistance. The ESR1 YAP1 fusion maintained the N terminal DNA binding motif of ESR1, but the hormone binding and AF2 motifs were replaced with the C terminal transactivation domain of YAP1. Expression ESR1 YAP1 in ER+ breast cancer models down-regulated ER and induced estrogen independent growth. PDX endocrine phenotypes parallel tumor of origin responses to endocrine therapy and revel novel mechanism for endocrine therapy resistance. Citation Format: Matthew J. Ellis, Shunqiang Li, Dong Shen, Li Ding, Robert Crowder, Jeiya Shao, Rodrigo Goncalves, Yu Tao, Jingqin Luo, Aleix Prat, Wenbin Liu, Ana Maria Gonzalez-Angulo, Shuying Liu, Joshua F. McMichael, Chris Miller, Dave Larson, Robert S. Fulton, Tom Mooney, Jeremy Hoog, Li Lin, Therese Giuntoli, Caroline Bumb, Crystal Cooper, Rebecca Aft, Robert T. Kitchens, Stephen N. Johnson, Chanpheng Phommaly, Megha Shiyam Kavuri, Katherine DeSchryver, Austin Lin, YiYu Dong, Cynthia X. Ma, Timothy Pluard, Michael Naughton, Ron Bose, Rama Suresh, Reida G. McDowell, Loren Michel, Richard Wilson, Shaomeng Wang, Christopher Maher, Gordon B. Mills, Charles Perou, Elaine R. Mardis. Patient-derived xenografts from advanced luminal-type breast cancer: insights into endocrine therapy resistance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-265. doi:10.1158/1538-7445.AM2013-LB-265

Introduction: Activation of the phosphatidylinositol-3-kinase (PI3K)/Akt pathway through gene mutation, most frequently in PIK3CA, is common in estrogen receptor-positive (ER+) breast cancer (BC) and deregulation of PI3K signaling contributes to endocrine resistance. These data suggest that proteins within the PI3K/Akt cascade are important therapeutic targets for patients who experience relapse despite adjuvant endocrine treatment. We have demonstrated previously that simultaneous inhibition of PI3K catalytic subunits and ER (through estrogen deprivation) activates cell death in estrogen-dependent ER+ BC cells (Cancer Res 69:3955, 2009). In order to develop rationales for combination therapies in the advanced disease setting we tested a clinical grade Akt inhibitor, MK-2206 in both estrogen-dependent BC cells and in BC cells that had lost the requirement for estrogen due to long-term estrogen deprivation (LTED). Methods: The effects of MK-2206 on cell signaling and apoptosis were determined in a panel of growth-arrested ER+ BC cells following short-term estrogen deprivation (STED) and in estrogen-independent MCF7 and T47D cells established following LTED. The impact of estrogen deprivation and treatment with the ER down-regulator fulvestrant on MK-2206 treatment-induced apoptosis was assessed in STED and LTED cells. We also determined the activity of MK-2206 with and without fulvestrant in vivo in MCF7 LTED xenografts, a model for aromatase inhibitor-resistant ER+ breast cancer. Results: MK-2206-induced apoptosis was most marked in STED cells with PIK3CA mutation and PTEN loss, indicating that ER+ BC with PI3K pathway mutations will be most sensitive to MK-2206 treatment. Estradiol antagonized MK2206-induced apoptosis following STED, emphasizing a role for estrogen-deprivation therapy in promoting Akt inhibitor activity. MCF7 LTED cells exhibited increased ER expression and resistance to MK-2206 which was reversed by treatment with fulvestrant. In contrast, T47D LTED cells exhibited ER loss and resistance to MK-2206 that was not overcome by fulvestrant. In MCF7 LTED xenografts the combination MK-2206 with fulvestrant showed a synergistic effect on inhibiting tumor cell proliferation and tumor growth. Conclusions: In estrogen-dependent ER+ BC cells with PIK3CA mutation or PTEN loss, acute estrogen deprivation increases the apoptotic effects of MK-2206 providing a rationale for Akt/aromatase inhibitor combinations in the first line setting. In ER+ BC cells that have lost the requirement for estrogen following LTED, fulvestrant antagonizes ER function and promotes Akt inhibitor activity suggesting that this combination would be appropriate for testing as a second line approach. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5478. doi:10.1158/1538-7445.AM2011-5478

e13646 Background: We have recently shown that acquired resistance to aromatase inhibitors can, in some instances, be reversed by the therapeutic use of lower dose estradiol suggesting a cyclical a...

Abstract #3063 Background: Endocrine therapy with estrogen deprivation (ED) induces cell cycle arrest in sensitive estrogen receptor positive (ER+) breast cancers, but typically not apoptosis, thus relapse is common despite ED due to residual surviving tumor cells. The phosphoinositide 3-kinase (PI3K) pathway, which promotes cell survival, is hyperactivated in ER+ breast cancer due to mutations of genes in the PI3K signaling cascade, most commonly PIK3CA. We therefore investigated the role of p110α and p110β, the gene products of PIK3CA and PIK3CB, in promoting cell survival in ER+ breast cancer. Materials and Methods: RNA interference (RNAi) was used to selectively inhibit p110α and p110β expression in the ER+, PIK3CA mutant MCF-7 and T47D cell lines; the ER+, PIK3CA wild-type but PIK3CB gene amplified HCC712 cell line; and the ER-, PIK3CA and PIK3CB wild-type MDA-MB-231 cell line. Western blotting was used to confirm p110α and p110β knockdown efficiencies and to determine the effects of specific knockdowns on PI3K pathway signaling. Cell line growth was measured using a resazurin reduction assay. Apoptosis was quantified using TUNEL/Hoechst staining. Gene copy number for PIK3CB was assessed by FISH in cell lines and tumor samples. Results: PIK3CA RNAi inhibited growth and activated apoptosis in all tested ER+ cell lines under ED, whereas the addition of estrogen reversed PIK3CA RNAi-induced apoptosis. PIK3CB RNAi inhibited proliferation and promoted apoptosis in the absence of estrogen only in the PIK3CB-gene amplified HCC712 cell line. In combination, PIK3CA and PIK3CB gene knockdowns produced a synergistic apoptotic effect on T47D and HCC712 cell lines, but not in MCF-7 cells which contain low levels of p110β protein. In contrast PIK3CA and PIK3CB RNAi did not promote apoptosis in the ER- MDA-MB-231 cell line. Finally, since PIK3CB gene amplification has not been described previously we conducted a screen by FISH and array CGH and detected low-level PIK3CB copy number gain or amplification in ∼5% of clinical samples from a series of breast cancer patients representing diverse clinical phenotypes (ER+, ER-, ERBB2+). Conclusions: Inhibition of the PI3K pathway through suppression of PI3K catalytic subunit expression produces a synthetic lethal effect in the absence of estrogen specifically in ER+ breast cancer cells. These data establish a preclinical rationale for combining endocrine therapy with PI3K pharmacological inhibitors in ER+ breast cancer, but suggest that both PIK3CA and PIK3CB are therapeutic targets and gene mutation or amplification in both catalytic subunits may have to be taken into account when identiying a sensitive population for PI3 kinase-directed therapy. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3063.

SummaryTo characterize patient-derived xenografts (PDXs) for functional studies, we made whole-genome comparisons with originating breast cancers representative of the major intrinsic subtypes. Structural and copy number aberrations were found to be retained with high fidelity. However, at the single-nucleotide level, variable numbers of PDX-specific somatic events were documented, although they were only rarely functionally significant. Variant allele frequencies were often preserved in the PDXs, demonstrating that clonal representation can be transplantable. Estrogen-receptor-positive PDXs were associated with ESR1 ligand-binding-domain mutations, gene amplification, or an ESR1/YAP1 translocation. These events produced different endocrine-therapy-response phenotypes in human, cell line, and PDX endocrine-response studies. Hence, deeply sequenced PDX models are an important resource for the search for genome-forward treatment options and capture endocrine-drug-resistance etiologies that are not observed in standard cell lines. The originating tumor genome provides a benchmark for assessing genetic drift and clonal representation after transplantation.

Introduction Inhibition of phosphatidylinositol-3-kinase (PI3K) induces apoptosis when combined with estrogen deprivation in estrogen receptor (ER)-positive breast cancer. The aims of the present study were to identify effective PI3K pathway inhibitor and endocrine therapy combinations, to evaluate the effect of PI3K pathway mutations and estrogen dependency on tumor response, and to determine the relevance of PIK3CA mutation in recurrent disease. Methods The PI3K catalytic subunit inhibitor BKM120, the mammalian target of rapamycin (mTOR) inhibitor RAD001 and the dual PI3K/mTOR inhibitor BGT226 were tested against ER-positive breast cancer cell lines before and after long-term estrogen deprivation (LTED). The impact of estradiol deprivation and the ER downregulator fulvestrant on PI3K pathway inhibitor-induced apoptosis was assessed. PIK3CA hotspot mutation analysis was performed in 51 recurrent or metastatic breast cancers and correlated with ER status and survival. Results Drug-induced apoptosis was most marked in short-term estrogen-deprived cells with PIK3CA mutation and phosphatase and tensin homolog loss. Apoptosis was most highly induced by BGT226, followed by BKM120, and then RAD001. Estradiol antagonized PI3K inhibitor-induced apoptosis following short-term estrogen deprivation, emphasizing a role for estrogen-deprivation therapy in promoting PI3K inhibitor activity in the first-line setting. ER-positive MCF7 LTED cells exhibited relative resistance to PI3K pathway inhibition that was reversed by fulvestrant. In contrast, T47D LTED cells exhibited ER loss and ER-independent PI3K agent sensitivity. PIK3CA mutation was prevalent in relapsed ER-positive disease (48%) and was associated with persistent ER positivity and a late relapse pattern. Conclusions Estrogen deprivation increased the apoptotic effects of PI3K and dual PI3K/mTOR inhibitors in ER-positive disease, providing a rationale for PI3K/aromatase inhibitor combinations as first-line therapy. In LTED cells, differential effects on ER expression may be a relevant consideration. When ER was persistently expressed, fulvestrant strongly promoted PI3K drug activity. When ER was lost, PI3K inhibitor monotherapy was sufficient to induce high-level apoptosis. Although tumors with PIK3CA mutation had a late recurrence pattern, these mutations were common in metastatic disease and were most often associated with persistent ER expression. Targeting PIK3CA mutant tumors with a PI3K pathway inhibitor and fulvestrant is therefore a feasible strategy for aromatase-inhibitor-resistant ER-positive relapsed breast cancer.