Prasanna G. Alluri

Estrogen receptor mutations and their role in breast cancer progression.

Endocrine therapy is the mainstay of treatment in estrogen receptor-positive breast cancers and significantly reduces disease recurrence and breast cancer-related mortality. However, acquired resistance to therapy has been noted in nearly one-third of women treated with tamoxifen and other endocrine therapies. Mutations in the estrogen receptor have long been speculated to play a role in endocrine therapy resistance but have been rarely detected. However, recent studies utilizing next-generation sequencing on estrogen receptor-positive, metastatic clinical samples have revealed that recurrent ESR1 mutations are far more frequent than previously thought and may play an important role in acquired endocrine therapy resistance. Here we review recent advances in detection and characterization of ESR1 mutations in advanced, endocrine therapy-resistant breast cancers.

Basal-like and Triple Negative Breast Cancers: Searching For Positives Among Many Negatives

Triple-negative breast cancers (TNBC) are defined by their failure to express the estrogen receptor, progesterone receptor, and HER2/neu protein markers. This basic feature is clinically relevant because it indicates that these cancers cannot be managed with endocrine or anti-HER2 systemic therapies. Furthermore, most TNBC cases are also characterized as being of the genetically defined basal subtype, which is an inherently and biologically more aggressive pattern of disease. The two terms, however, are not synonymous, and some TNBC cases are prognostically more favorable. TNBC differs from non-TNBC in risk-factor profile, pattern, and rate of metastatic spread.

Simple reporter gene-based assays for hairpin poly(amide) conjugate permeability and DNA-binding activity in living cells

Hairpin poly(amide)s (HPs) are sequence specific DNA-binding compounds that have engendered considerable interest as potential pharmacological agents to manipulate the expression of specific genes. However, recent reports have indicated that the ability of HP conjugates to pass through cell membranes is sensitive to the cell type employed and the nature of the conjugate. Furthermore, while binding of HPs to DNA sequences in vitro is relatively well understood, packing of DNA into chromatin in living cells makes predicting the efficiency with which a given poly(amide) will bind its cognate site less certain. Previous methods to evaluate HP permeability and binding in vivo, while effective, are somewhat tedious and qualitative. We report here two related reporter gene-based assays that provide a more convenient and quantitative measure of poly(amide) permeability and DNA binding activity in living cells. We anticipate that these methods will complement existing tools and facilitate the development of HP conjugates with the desired biological activity.

BETs abet Tam-R in ER-positive breast cancer.

Epigenetic modifications such as histone acetylation play a central role in the transcriptional regulation of many oncogenic drivers. Accumulating evidence suggests that pharmacological modulation of certain key epigenetic reader proteins such as BRD2/3/4 may serve as an attractive strategy for treatment of many cancers, including tamoxifen-resistant breast cancer.

Androgen receptor as a mediator and biomarker of radioresistance in triple-negative breast cancer

Increased rates of locoregional recurrence have been observed in triple-negative breast cancer despite chemotherapy and radiation therapy. Thus, approaches that combine therapies for radiosensitization in triple-negative breast cancer are critically needed. We characterized the radiation therapy response of 21 breast cancer cell lines and paired this radiation response data with high-throughput drug screen data to identify androgen receptor as a top target for radiosensitization. Our radiosensitizer screen nominated bicalutamide as the drug most effective in treating radiation therapy-resistant breast cancer cell lines. We subsequently evaluated the expression of androgen receptor in >2100 human breast tumor samples and 51 breast cancer cell lines and found significant heterogeneity in androgen receptor expression with enrichment at the protein and RNA level in triple-negative breast cancer. There was a strong correlation between androgen receptor RNA and protein expression across all breast cancer subtypes (R2 = 0.72, p < 0.01). In patients with triple-negative breast cancer, expression of androgen receptor above the median was associated with increased risk of locoregional recurrence after radiation therapy (hazard ratio for locoregional recurrence 2.9–3.2)) in two independent data sets, but there was no difference in locoregional recurrence in triple-negative breast cancer patients not treated with radiation therapy when stratified by androgen receptor expression. In multivariable analysis, androgen receptor expression was most significantly associated with worse local recurrence-free survival after radiation therapy (hazard ratio of 3.58) suggesting that androgen receptor expression may be a biomarker of radiation response in triple-negative breast cancer. Inhibition of androgen receptor with MDV3100 (enzalutamide) induced radiation sensitivity (enhancement ratios of 1.22–1.60) in androgen receptor-positive triple-negative breast cancer lines, but did not affect androgen receptor-negative triple-negative breast cancer or estrogen-receptor-positive, androgen receptor-negative breast cancer cell lines. androgen receptor inhibition with MDV3100 significantly radiosensitized triple-negative breast cancer xenografts in mouse models and markedly delayed tumor doubling/tripling time and tumor weight. Radiosensitization was at least partially dependent on impaired dsDNA break repair mediated by DNA protein kinase catalytic subunit. Our results implicate androgen receptor as a mediator of radioresistance in breast cancer and identify androgen receptor inhibition as a potentially effective strategy for the treatment of androgen receptor-positive radioresistant tumors.Radiation: Drug target found for radioresistanceDrugs that block the androgen receptor (AR) protein can help make radiation-resistant breast tumors susceptible to ionizing therapy. Corey Speers from the University of Michigan, Ann Arbor, USA, and colleagues characterized the radiation sensitivity of 21 breast cancer cell lines and then paired the response data with the findings of a high-throughput drug screen to identify a medication called bicalutamide — an AR inhibitor — as one of the most potent agents for overcoming radiation resistance. The researchers then measured the expression of AR in more than 2100 human breast tumor samples and 51 breast cancer cell lines, and found that patients with triple-negative breast cancer, especially those who relapsed after radiation, had elevated levels of AR. Cell experiments with enzalutamide, a newer generation AR-blocking drug, confirmed that targeting AR helps reverse radiation resistance.

Abstract P6-06-05: RadiotypeDx: Identification and validation of a radiation sensitivity signature in human breast cancer

Purpose: An unmet clinical need in breast cancer (BC) management is the identification of which patients will respond to radiation therapy (RT). We hypothesized that the integration of post-RT clonogenic survival data with gene expression data across a large spectrum of BC cell lines would generate a BC-specific RT sensitivity signature predictive for RT response in BC patients and allow identification of patients with tumors refractive to conventional therapy. Methods: Using clonogenic survival assays, we identified the range of surviving fraction (SF) after 2 Gy of RT across 21 BC cell lines. Using SF as a continuous variable, the RT sensitivity score (RSS) was correlated to gene expression using a Spearman correlation method on an individual gene basis. Genes were selected for the signature based on positive or negative correlation with a p-value Results: Clonogenic survival identifies a range of radiation sensitivity in human BCC lines (SF 77%-17%) with no significant correlation (r value Conclusion: In this study, we derive a human BC-specific RT sensitivity signature (RadiotypeDx) with biologic relevance from preclinical studies and validate this signature for prediction of recurrence in an independent clinical dataset. The signature is not correlated to the intrinsic subtypes of human breast cancer and thus provides useful information beyond traditional breast cancer subtyping. By identifying patients with tumors refractory to standard RT, this signature has the potential to allow for personalization of radiotherapy, particularly in patients for whom treatment intensification is needed. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-06-05.

Abstract 2156: Targeting estrogen receptor mutations for treatment of endocrine therapy resistance in breast cancer

Approximately 70% of all breast cancers express the Estrogen Receptor (ER) and inhibition of the ER signaling remains the mainstay of systemic therapy in such cancers. The advent of endocrine therapies (ET) have contributed significantly to reduction in the incidence, recurrence and mortality associated with breast cancer. However, approximately one-third of patients with ER-positive breast cancer do not benefit from adjuvant ET, and nearly all patients with ER-positive metastatic breast cancer (MBC) ultimately become refractory to all known ETs. While several mechanisms of resistance to ET have been proposed, there are currently no clinically effective treatments for ET resistance. Thus, ET resistance remains a major obstacle to the effective treatment of a significant subset of ER-positive breast cancers. We have recently reported recurrent somatic mutations in the ligand binding domain (LBD) of the Estrogen Receptor gene (ESR1) in metastatic breast cancer patients with a history of prior treatment with ET. The ESR1 mutations result in constitutive activation of the ER and confer ET resistance when ectopically expressed in ER-positive cell lines. In an attempt to develop novel therapeutic strategies to treat ET-resistant breast cancer, we have evaluated OTX015, a clinical bromodomain and extraterminal (BET) domain inhibitor, in pre-clinical models of ET resistance due to ESR1 mutations. Our studies suggest that BET inhibition may serve as an attractive strategy for treatment of ET resistance due to ESR1 mutations. Citation Format: Prasanna G. Alluri, Jose Larios, Rohit Malik, James Rae, Arul M. Chiinaiyan. Targeting estrogen receptor mutations for treatment of endocrine therapy resistance in breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2156.

Abstract P6-03-08: Androgen receptor (AR): A novel target for radiosensitization and treatment in triple-negative breast cancers (TNBC)

Background: Increased rates of locoregional recurrence have been observed in TNBC despite the use of chemotherapy and radiation (RT). Thus, approaches that result in radiosensitizaton of TNBC are critically needed. We have previously characterized the radiation response of 21 breast cancer cell (BCC) lines using clonogenic survival assays. We now pair this data with high-throughput drug screen data available through cancer cell line encyclopedia studies to identify AR as a top target for radiosensitization and assess AR inhibition as a radiosensitization strategy for TNBC. Methods: Clonogenic survival assays were performed to determine the intrinsic RT sensitivity of 21 BCC lines (0-8 Gy RT). IC50 values were determined for 130 clinically available compounds and correlation coefficients were calculated using IC50 values (for drug sensitivity) and SF-2Gy (for radiation sensitivity). Gene expression was measured using Affymetrix microarrays and protein expression was measured using reverse-phase protein lysate arrays (RPPA) of human tumor samples (n=2,061) and BCC lines (n=51). AR function was assessed using siRNA knockdown or inhibition with MDV3100 (enzalutamide). Kaplan-Meier analysis was performed to determine the clinical impact of AR expression on local control and survival. A Cox proportional hazards model was constructed to identify potential factors of survival, and multivariate analysis was used to determine variables most significantly associated with LRF survival. Results: Our radiosensitizer screen nominated bicalutamide as one of the most effective drugs in treating radioresistant BCC lines (R2= 0.46, p-value 2000 human breast tumor samples and found signifi[not]cant heterogeneity in AR expression with an increase in TNBC (35% of tumors) compared to non-TNBC (28% of tumors). This same heterogeneity was also identified in human BCC lines. There was a strong correlation between AR RNA expression and protein expression (R2= 0.72, p Conclusion: Our results implicate AR as a mediator of radioresistance in breast cancer and support the rationale for developing clinical strategies to inhibit AR as a novel radiosensitizing target in TNBC. Citation Format: Corey Speers, Shuang G Zhao, Meilan Liu, Joseph Evans, Prasanna Alluri, Daniel F Hayes, Felix Y Feng, Lori J Pierce. Androgen receptor (AR): A novel target for radiosensitization and treatment in triple-negative breast cancers (TNBC) [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 P6-03-08.

Abstract P6-04-04: Maternal embryonic leucine zipper kinase (MELK) is a novel radiosensitizing and therapeutic target and is independently prognostic in triple-negative breast cancer

Background: While effective targeted therapies exist for estrogen receptor (ER)-positive and HER2/neu-positive breast cancer, no such effective therapies exist for ER-negative, PR-negative, and HER2-negative (“triple negative”) cancers. Given the lack of targeted agents for triple negative (TN) disease and their relative radiation insensitivity, it is clear that additional targets for treatment are critically needed. Our previous work identified one such novel molecular target as maternal embryonic leucine zipper kinase (MELK), and we sought to investigate the impact of MELK expression on radiation response and patient outcomes. Methods: Using gene expression arrays, we interrogated the expression of MELK in 2,061 breast tumor samples as well as a panel of 51 breast cancer cell lines. We measured protein expression in TN cancers with western blotting and used clonogenic survival assays to quantitate radiosensitivity of BCC lines at baseline and after MELK inhibition. Multiple datasets were used to evaluate the prognostic import of MELK. Kaplan-Meier analysis using local control and survival data was performed. Chi squared scores were calculated to determine significance and hazard ratios (HR) and 95% confidence intervals (CI) were calculated. A Cox proportional hazards model was constructed to identify potential factors of survival. Results: We demonstrate that MELK expression is significantly elevated in human TN breast cancers, including chemoradiation resistant tumors (305 tumors compared to 1756 non-TN breast tumors; p-value 7.5 e-21). MELK protein and RNA expression is induced by ionizing radiation (5.6-7.5 fold at 72 hours, p-value Conclusion: Here, we identify MELK as a potential biomarker of radioresistance and target for radiosensitization in triple negative breast cancers. MELK overexpression was associated with local failure across multiple data sets. MVA identified MELK as the strongest factor associated with poor local control. Our results support the rationale for developing clinical strategies to inhibit MELK as a novel target in triple-negative breast cancer. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-04-04.