Background:We had previously reported a novel small molecule, ERX-11, that directly interacts with ER and blocks the interaction between a subset of coregulators with both native and mutant forms of ER. ERX-11 effectively blocks ER oncogenic signaling and has potent anti-proliferative activity against therapy-sensitive and therapy-resistant human breast cancer cells. To enhance the clinical translation of ERX-11, we sought to pursue both lead optimization and evaluate combinations of ERX-11 with other approved agents in breast cancer. Methods: We designed, synthesized and tested 500 derivatives of ERX-11 in multiple models of ER+ breast cancer. We also tested combinations of ERX-11 with multiple agents, including other ER targeting agents, chemotherapies and CDK4/6 inhibitors. We tested the effect of combination therapy using breast cancer cells with acquired resistance (Tamoxifen, Letrozole, Ribociclib resistant) and engineered models that express ER mutations. In vitro activity was tested using Cell titer glo, MTT, and apoptosis assays. Mechanistic studies were conducted using Western blot, reporter gene assays and RNA-seq analysis. Xenograft, patient derived xenograft (PDX), patient derived explant (PDE) and xenograft derived explant (XDE) models were used for preclinical evaluation and toxicity. Result: Evaluation of 500 analogs of ERX-11 identified a number of leads with differential activity against ER+ and ER- breast cancer cells, identified several analogs including ERX-144, 208, 296, 315 with nanomolar potency against ER+ and therapy-resistant ER+ breast cancers. Validation of the mechanism of action of these analogs is ongoing. The combination of ERX-11 and palbociclib significantly blocked ER-mediated and ER-coregulators mediated oncogenic signaling and showed potent anti-proliferative activity against both endocrine therapy-sensitive and resistant breast cancer cells. In addition, ERX-11 inhibited ribociclib-resistant ER+ cell proliferation in a dose dependent manner. Mechanistic studies using IP-Mass spectrometry demonstrated that ERX-11 and palbociclib blocks the interaction between larger subset of coregulators with ER in therapy resistant breast cancer models. ERX-11 and palbociclib both exhibited potent anti-proliferative activity against therapy-sensitive and therapy-resistant ER+ve breast cancer cells, in xenograft models and in PDEs. Importantly, combination therapy of ERX-11 and palbociclib synergistically reduced the growth of tamoxifen and letrozole resistant xenograft tumors compared to either drug alone. Mass spec based DIA analyses and RNA-seq studies revealed that combinational treatment uniquely activated p53, unfolded response mediated apoptotic pathways, altered DNA damage response and suppressed E2F and Myc target genes. Biochemical studies confirmed combination therapy significantly altered E2F1, ER and DNA damage response pathways. Conclusion: We have successfully pursued two avenues to improving ERX-11 for clinical translation. We have developed ERX-11 analogs with higher potency against ER+ breast cancer. We have shown that combinational treatment with ERX-11 and palbociclib may overcome endocrine therapy resistance and CDK4/6 inhibitor (ribociclib) resistance. Citation Format: Viswanadhapalli S, Ma S, Lee T-K, Sareddy GR, Liu X, Ekoue D, Alluri A, Luo Y, Kassees K, Arteaga C, Alluri P, Weintraub SE, Tekmal RR, Ahn J-M, Raj GV, Vadlamudi RK. Enhancing the activity of a novel estrogen receptor coregulator binding modulator (ERX-11) against ER-positive therapy resistant breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-04-23.
#3022 Background: The estrogen receptor (ER) plays a central role in the progression of breast cancer. Current endocrine therapy for ER+ve breast cancer involves modulating ER-pathway using Tamoxifen, and blocking peripheral estrogen (E2) synthesis by Aromatase inhibitors. Despite the positive effects, de novo and/or acquired resistance to endocrine therapies frequently occur. Although mechanisms for hormonal therapy resistance remains elusive, most downstream events in these pathways converge upon modulation of cell cycle regulatory proteins including upregulation of Cyclin E and A, along with activation of Cyclin Dependent Kinase 2 (CDK2). ER signaling complexes are known to recruit various co-regulatory proteins and recent evidences suggest that deregulated expression, localization and activity of ER coregulators also plays vital role in hormonal resistance. In this study, we found that CDK signaling regulates ER coregulator PELP1 function via phosphorylation leading to hormonal resistance.
 Methods: Significance of CDK2 axis in the therapy resistance was tested using breast cancer models cells that acquired resistance to endocrine therapy and by using chemical inhibitors that block CDK2 activity. Immunoprecipitation, and confocal analysis was used to confirm protein-protein interactions. In vitro kinase and ortho-phosphate labeling assays were used to test CDK2 phosphorylation of PELP1. Utilizing Breast cancer model cells that express PELP1 mutants that cannot be phosphorylated by CDK2, we examined the significance of PELP1 phosphorylation in cell cycle progression. Using PELP1siRNA nanoparticles, we tested the effect of PELP1 knockdown in hormone therapy resistance.
 Results: ER coregulator PELP1 interacts with CDK2 upon E2 stimulation. In vitro kinase assays using both purified CDK2/CyclinE and CDK2/CyclinA complexes showed that full length PELP1 is a potential substrate of CDK2. PELP1 exhibited phosphorylation at the time points that corresponds to CDK2 activation in MCF7 cells. PELP1 overexpression increases E2F luciferase activity while PELP1 mutants that lack CDK2 sites failed to enhance the E2F activity. CDK2 mediated phosphorylation of PELP1 is important for PELP1 regulation of E2F and ER target genes. Combination therapies using PELP1 siRNA nano particles or Roscovitine along with tamoxifen or letrozole, sensitized the therapy resistance cells for endocrine therapy.
 Conclusions: We have identified ER coregulator PELP1 as a novel substrate of CDK2. Because CDK2 activity is deregulated in breast tumors and implicated in therapy resistance, our findings suggests that CDK2-PELP1 axis deregulation may contribute therapy resistance. Combinatorial therapeutic strategy using Roscovitine along with PELP1 siRNA nanoparticles will provide new therapeutic opportunity to increase the sensitivity of hormone resistant cells to Tamoxifen and Letrozole therapy. These studies were supported by DOD breast cancer grant BC083207. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3022.
#5036 Background: Estrogen stimulates breast tissue to increase cell divisions (mitosis) and is implicated in breast cancer progression. ER action is complex and requires functional interactions with coregulators. Proline-, glutamic acid-, and leucine-rich protein (PELP)-1, also known as modulator of nongenomic actions of estrogen receptor (MNAR), is a novel nuclear receptor (ER) coregulator with multitude of functions. Emerging evidence suggest that PELP1 expression is deregulated in breast cancer and serves as a scaffolding protein that couples various signaling complexes with estrogen receptor. In this study we found that ER coregulator PELP1 plays a novel role in mitosis.
 Material and Methods: To understand the mechanism by which ER coregulator PELP1 contribute to breast cancer progression, we have utilized small RNA interference methodology and established breast cancer model cells that stably express PELP1-shRNA (MCF7-PELP1shRNA). FACS analysis was used to determine the cell cycle status of the model cells. Using confocal microscopy, immunoprecipitation, in vitro kinase assays, site directed mutagenesis and Western analysis using phospho-antibodies we studied the mechanism and significance of PELP1 signaling in mitotic progression. We also developed PELP1 siRNA nanoparticles and used them as well as CDK1 inhibitors in cell proliferation studies.
 Results: Down regulation of PELP1 expression resulted in decreased estrogen mediated cell proliferation, delayed mitotic progression and induced accumulation of mitotic cells. Interestingly, PELP1 depleted cells also exhibited multinucleation. Western analysis of various markers of mitotic progression revealed a delay in the kinetics of G2M initiation and progression. Confocal analysis revealed colocalization of CDK1 and PELP1 in G2M. Immunoprecipitation assays demonstrate that endogenous CDK1 form functional complex with PELP1 and Src kinase during mitosis. Using deletion and mutagenesis approach, we have mapped the putative CDK1 phosphorylation sites on PELP1. Down regulation of PELP1 or overexpression of PELP1 mutants (that cannot be phosphorylated by CDK1), reduces the magnitude of Src activation, which is an essential driving force for timely progression of M phase. PELP1 siRNA nanoparticles alone or in combination with CDK1 inhibitors have shown to significantly reduce the proliferation of breast cancer cells and showed increased response in tamoxifen resistant breast cancer cells.
 Discussion: These results suggest that ER coregulator PELP1 play a novel role in G2M progression. Since PELP1 expression is deregulated in breast cancer, PELP1 ability to regulate mitosis could contribute to the progression of cancer by causing genomic instability through the deregulation of mitosis. Taken together our findings suggest that estrogen can promote neoplasia using coregulators by two distinct mechanisms (1) Coregulator induction of target genes and (2) Coregulator mediated actions in mitosis. ER coregulator PELP1 play multiple roles in Estrogen mediated neoplasia, and thus represent a target for novel therapeutic breast cancer strategies by forming the “next generation” of antimitotic drugs. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5036.
#17 The majority of breast cancer patients are postmenopausal women with estrogen receptor-positive (ER+) tumors. Approximately 66% of breast carcinomas contain aromatase. Aromatase inhibitors are proving to be more effective than tamoxifen in the treatment of postmenopausal women with ER+ breast cancer. However, the development of resistance to treatment is a concern. The transition of the ER+ tumors from a responsive to unresponsive state is associated with different molecular pathways. We have therefore investigated the properties of resistance that develops in response to the aromatase inhibitor letrozole in the preclinical MCF-7-aromatase xenograft animal model following long-term treatment with letrozole by using the derived letrozole-resistant MCF-7-aromatase LTLTca breast cancer cells. The combination treatment of letrozole and the ERβ agonist diarryl propionitrile (DPN) restored sensitivity to letrozole in the letrozole-resistant LTLTca cells. Studies from our group using this postmenopausal breast cancer preclinical model clearly showed the therapeutic advantage of the ERβ agonist in overcoming unresponsiveness to letrozole. A 67% reduction in tumour size was observed in the group of animals bearing the letrozole-resistant LTLTca tumors that received the combination of letrozole and DPN. A novel delivery method using letrozole-DPN nanoparticles showed enhanced sensitivity (∼80%) to the combination nanoparticles. Letrozole was found to be relatively ineffective in these animals with only a 36% reduction in tumor size with tumors having a mean tumor weight of 0.95g by the end of four weeks of treatment. Biochemical analysis showed a reduction in the tumor aromatase enzyme levels in mice treated with letrozole + DPN when compared to letrozole alone. QRT-PCR as well as protein analysis studies of the letrozole-resistant LTLTca tumors showed a decrease in P27 levels and elevated expression levels of HER-2. Expression levels of cyclin D1 and the ERα to ERb ratio was higher in the untreated letrozole-resistant tumors compared to tumors that responded to the combination therapy. These findings provide further support for the development and testing of novel therapeutic approaches for selective regulation of ER-dependent (ERα and β) actions. Combination treatments involving agents that modulate ERβ activity may provide therapeutic advantage in the treatment, for overcoming resistance to estrogen-ablation and prevention of breast cancer. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 17.
BACKGROUND: Recently,CDK4/6 inhibitors in combination with endocrine therapy (AE/AI/SERDs) is approved for the treatment of ER+ advanced breastcancer (BCa). However, not all patients benefit from CDK4/6 inhibitors therapy. Emerging studies indicate many therapy-resistant tumors retainER signaling, via interaction with critical oncogenic coregulatorproteins. Considering complex signaling interplay of ER and CDK4/6 axis, combination therapy of CDK inhibitor with other potent ER-targeted agents that block ER coregulatory signaling may extend the efficacy and may prevent the development of resistance to the CDK4/6 inhibitors. We recently developed a small organic molecule, ER coregulator binding modulator ERX-11 (EtiraRx-11). The objective of this study is to test the utility of novel combination therapy of ERX-11 with CDK4/6 inhibitor palbociclib in treating therapy resistant advanced BCa. METHODS: We have utilized multiple therapy sensitive and therapy-resistant BCa models with various genetic backgrounds. We tested efficacy using both acquired resistance and engineered models that express ER mutations or oncogenes. Efficacy of combination therapy was tested using established in vitro assays including, MTT, colony formation, apoptosis, and cell cycle progression. Mechanistic studies were conducted using reporter gene assays, gene expression, RNA-seq analysis and signaling alterations. Patient-derived BCa explant and Xenograft studies were used to determine the in vivo efficacy of the combination therapy. RESULTS: ERX-11 effectively blocked ER-mediated and ER-coregulator mediated oncogenic signaling and has potent anti-proliferative activity against both endocrine therapy-sensitive and therapy-resistant BCa cells. Mechanistic studies using IP-Mass spectrometry showed that ERX-11 blocks the interaction between a subset of coregulators with ER in resistant BCa models. ERX-11 exhibited potent anti-proliferative activity against therapy-sensitive and therapy-resistant ER-driven BCa cells in vitro, in xenograft models in vivo and in patient-derived breast tumor explants ex vivo. Co-treatment of ERX-11 with palbociclib synergistically reduced cell viability and induced apoptosis of therapy sensitive and resistant BCa model cells. Importantly, combination therapy of ERX-11 and the palbociclib synergistically reduced the growth and induced apoptosis of tamoxifen and letrozole resistant xenograft tumors compared to either drug alone. RNA-seq studies revealed that combinational treatment with ERX-11 and palbociclib uniquely activated p53 and unfolded response mediated apoptotic pathways and suppressed E2F and Myc target genes. Biochemical studies confirmed combination therapy significantly altered E2F1 and ER signaling pathways and promoted apoptosis. CONCLUSIONS: Our data support a critical role of blocking ER coregulator signaling in treating therapy resistance in advanced ER+ BCa. Combinational treatment with ERX-11 and palbociclib may overcome/delay endocrine therapy resistance. Citation Format: Viswanadhapalli S, Sareddy GR, Zhou M, Ali E, Li X, Ma S-H, Lee T-K, Tekmal RR, Ahn J-M, Raj GV, Vadlamudi RK. Blocking ER coregulator signaling enhances CDK4/6 inhibitor palbociclib therapy in ER-positive advanced 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 P1-09-06.
#601 Back ground: Estradiol (E2) and estrogen receptor (ER) signaling play a key role in development and progression of breast cancer. ER signaling is complex, involves coregulatory proteins and the status of ER coregulators in tumor cells plays an important role in hormonal responsiveness and tumor progression. In addition, ER also participates in non-genomic signaling events in the cytoplasm, however the significance of non-genomic signaling in mammary tumorigenesis remain unknown. PELP1/MNAR is novel ER coregulator that participates in ER genomic and non-genomic actions. PELP1 expression is deregulated in breast tumors and in a subset of tumors PELP1 is predominantly localized in the cytoplasm. Since PELP1 cytoplamsic localization promotes excessive activation of Src and AKT pathways, we hypothesized that PELP1 mediated excessive activation of ER-nongenomic functions may play a role tumorigenesis. To test this, we have generated MMTV-PELP1cyto TG model that uniquely express PELP1 in the cytoplasm of mammary glands that mimics the pathological situation of PELP1 localization seen breast cancer.
 Methods: As a means of targeting the expression of the PELP1 transgene to the mammary gland, we placed the PELP1cyto cDNA under the control of the MMTV promoter. PELP1 transgene integration was verified by PCR and expression levels by Western and IHC in each founder line. Whole-mount preparations and IHC analysis was performed using Tg and age controlled wild type littermates from different developmental stages. Total protein extracts of mammary gland were used for western blot analysis of nongenomic signaling components.
 Results: Preliminary analysis of mammary gland from PELP-cyto mice showed hyperplasia, increased proliferation as analyzed by PCNA staining. Mammary tumors were observed as early as 32 weeks. No spontaneous mammary tumors were found in the wild type cohort. Pathological analysis revealed that these tumor masses represent full blown mammary adenocarcinomas. Mammary tumors showed excessive activation of nongenomic signaling including activation of Src and AKT pathways. A clear induction of aromatase expression was found in PELP1 tumors compared with the wild-type that showed no aromatase expression in the mammary gland.
 Discussion: We have established and characterized a transgenic mouse model that mimics deregulated ER-nongenomic signaling. Our results suggest that PELP1 is a proto-oncogene and demonstrates its in vivo tumorigenic potential. PELP1 driven tumors are ER+ve, express aromatase, thus provide an interesting in vivo model for studying ER-mediated tumorigenesis and to study effect of local E2 on ER-mediated tumorigenesis. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 601.
BACKGROUND: Cancer is caused by abnormalities of the genome and epigenome including the frequently occurring epigenetic changes of histone modifications. Specifically, estrogen-induced breast carcinogenesis is characterized by alterations in histone modifications. Reader proteins that recognize these modifications facilitate modulation of genes and their resulting biological actions. In addition to regulation by several coregulators, estrogen receptor alpha (ER) transcriptional activation or repression depends on the modulation of histone methylation at target promoters. The ER coregulator PELP1 plays an important role in ER signaling, is a proto-oncogene with aberrant expression in hormone- related cancers and a prognostic indicator of decreased survival in breast cancer patients. The molecular mechanisms by which PELP1 promotes oncogenesis remain unknown; however, previous studies from our lab have shown that PELP1 has the potential to function as a reader of chromatin modifications. MATERIALS AND METHODS: A histone peptide array (Active Motif) that contains 384 unique combinations of histone modifications was used to explore the PELP1 epigenetic interactome using the manufacturer's protocol. Purified full length and various deletions of epitope tagged PELP1 were used for the screening. Using MCF7 and ZR75 model cells with stable expression of PELP1 or PELP1 shRNA, we examined the significance and role of PELP1 on the histone arginine methylation using ERE reporter gene assays, confocal microscopy and ChIP assays. Pre-clinical nude mice based models were used to validate the PELP1 effects on arginine methylation in vivo and PELP1-siRNA liposomes were used to downregulate PELP1 expression in vivo. IHC studies were performed to examine the status of histone arginine methylation in murine breast tumors. RESULTS: Our results show that PELP1 uniquely recognizes histones modified by arginine dimethylation, arginine citrullination and lysine dimethylation. Phosphorylation of residues adjacent to a methyl modification affects the ability of PELP1 to recognize histone methylation. Using various deletions of PELP1 peptides, we have found that PELP1 acts as a module for recognition of a specific histone modification through the carboxyl-terminal glutamic acid rich region. Reporter gene assays showed that PELP1 functionally interacts with arginine methyltransferases including CARM1 and PRMT6, both shown to be dysregulated in human cancers, and synergistically enhances ER-transactivation. Chromatin immunoprecipitation assays revealed that PELP1 has the potential to alter histone H3 arginine methylation status at ER target gene promoters. PELP1 knockdown via PELP1-sRNA liposomes using xenograft based assays resulted in decreased arginine dimethylation with concomitant reduction in tumor volume. Conclusions: Our findings suggest that PELP1 is a reader of histone methyl modifications and deregulation of PELP1 may have implications on tumor proliferation via epigenetic alterations at ER target promoters. Targeting these epigenetic alterations through inhibition of PELP1 and the arginine methyltransferases could be a promising cancer therapeutic. This study was funded by CPRIT pre-doctoral fellowship grant and Komen KG090447. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-05-01.
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