Chanpheng Phommaly

Endocrine-Therapy-Resistant ESR1 Variants Revealed by Genomic Characterization of Breast-Cancer-Derived Xenografts

To 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.

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, p110alpha and p110beta, in ER(+) breast cancer cells harboring either mutation (PIK3CA) or gene amplification (PIK3CB). p110alpha RNAi inhibited growth and promoted apoptosis in all tested ER(+) breast cancer cells under estrogen deprived-conditions, whereas p110beta RNAi only affected cells harboring PIK3CB amplification. Moreover, dual p110alpha/p110beta 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 p110alpha and p110beta catalytic subunits, whether wild-type or mutant, and be combined with endocrine therapy for maximal efficacy when treating ER(+) breast cancer.

Preclinical modeling of combined phosphatidylinositol-3-kinase inhibition with endocrine therapy for estrogen receptor-positive breast cancer

IntroductionInhibition 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.MethodsThe 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.ResultsDrug-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.ConclusionsEstrogen 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.

Abstract S3-05: Patient-derived xenograft study reveals endocrine therapy resistance of ER+ breast cancer caused by distinct ESR1 gene aberrations

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.

Functional Annotation of ESR1 Gene Fusions in Estrogen Receptor-Positive Breast Cancer.

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.

The Effect of the IGF1R Antibody R1507 on ER Positive Breast Cancer Cell Lines Growth and Survival.

Background: The insulin-like growth factor-1 receptor (IGF1R) mediates the biological actions of IGF-1 and IGF-2 and is expressed in most breast cancer cells, where receptor activation induces proliferative, cell-survival, and transforming activities. Because crosstalk exists between ER and IGF1R blockade of both pathways could increase antitumor activity. Further, IGF signaling may be enhanced in ER+ cells that have become refractory to long term estrogen deprivation (LTED) explaining resistance to aromatase inhibitor therapy. The effect of the selective IGF1R antibody (R1507) was therefore assessed in a series of breast cancer cell lines in both estrogen-dependent and estrogen-independent growth phases.Methods: The ER positive breast cancer cells lines MCF7, T47D and HCC712 were kept in phenol red-free medium containing 5% charcoal-stripped serum. Several doses of R1507 were used to evaluate effects on cell proliferation, signaling and apoptosis. Western blotting with phospho-specific antibodies was used to observe effects on cell signaling, cell growth was measured using resazurin reduction and apoptosis was quantified by flow cytometry using the TUNEL assay. STED (short-term estrogen deprived) cells were treated with R1507 between 2 and 3 weeks after estrogen depletion and LTED were treated with R1507 after 6+ months of estrogen depletion.Results: R1507 suppressed IGF1R expression and inhibited IGF-1 stimulated IGF1R and AKT phosphorylation in all cell lines. R1507 had anti-proliferative effects in MCF7 cells in both estrogen-dependent and independent growth phases and the magnitude of the effect was similar to fulvestrant. R1507 had a smaller effect on cell proliferation in HCC712 cells and no effects on proliferation were observed in T47D cells. Antibody treatment resulted in significant induction of apoptosis in MCF7 STED but MCF7 LTED cells were resistant to the apoptotic effects of IGF1R blockade.Conclusions: R1507 had an inhibitory effect on IGF1R signaling and an anti-proliferative effect in two of the three cell lines tested. In addition R1507 induced synthetic lethality in combination with acute estrogen deprivation in MCF7 cells, an effect we have recently reported for direct PI3 kinase inhibitors under the same circumstances (1). While the proapoptotic effects of R1507 were lost in the estrogen-independent growth phase of MCF7 cells, the anti-proliferative effect was not, indicating that R1507 might have efficacy in patients with endocrine therapy resistant tumors. In other cell lines the efficacy of R1507 was variable and synthetic lethality was not observed, suggesting that the development of predictive biomarkers for R1507 should be a priority. We are currently addressing the activity of R1507 in a clinical trial with two cohorts, one with acquired resistance to letrozole, where the antibody is added to continued letrozole treatment and a second group of patients with stable disease on letrozole to determine if R1507 can induce tumor regression under these circumstances.1. Crowder et al Cancer Research 69:3955-62, 2009 Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3126.

Abstract 1033: Estrogen receptor gene fusions drive endocrine therapy resistance in estrogen receptor positive breast cancer

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 PD2-03: Recurrent functionally diverse in-frameESR1gene fusions drive endocrine resistance in breast cancer

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 5478: Combination of fulvestrant with the allosteric Akt inhibitor MK-2206 promotes anti-tumor activity in estrogen deprivation-resistant estrogen receptor positive breast cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL 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