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

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.

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

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.

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.

Generation of mammaglobin-A-specific CD4 T cells and identification of candidate CD4 epitopes for breast cancer vaccine strategies.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Proteins associated with disease and clinical course in pancreas cancer: a proteomic analysis of plasma in surgical patients.

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.