PELP1 Knockdown Research Articles

Abstract 3172: Significance of PELP1 signaling in liver cancer progression

Abstract Background: Hepatocellular carcinoma (HCC) is the fastest-rising cause of cancer-related mortality in the United States. Furthermore, advanced HCC has a very poor prognosis, with a median survival time of only about ten months. Effective new targeted therapies are urgently needed for the treatment of HCC. Proline, glutamic acid-, and leucine-rich protein 1 (PELP1) is a protooncogene. PELP1 signaling has been implicated in the development of several cancers, however, its role in the progression of HCC remains unclear. The goal of this project is to investigate the significance and therapeutic potential of targeting PELP1 in HCC. Methods: In this investigation, we have used six HCC cell lines (HUH7, HEP3B, SNU398, SNU475, SNU423, and SNU449). Using immunohistochemistry (IHC), the expression of PELP1 in HCC tissue microarrays was examined. PELP1shRNA lentiviral particles were used to create PELP1 knockdown (KD) cells. Using well-established in vitro tests for cell proliferation, colony formation, and apoptosis, the effects of PELP1 KD or PELP1 inhibitor (SMIP34) were investigated. RNA-seq, RT-qPCR, Western blotting, IHC, reporter gene assays, and signaling pathway analysis were used in mechanistic investigations. Preclinical evaluations were conducted using mice xenograft models. Results: TNM plot analysis showed that HCC samples had increased PELP1 expression relative to normal tissue. Immunohistochemistry analysis of tissue microarray confirmed that HCC specimens overexpressed PELP1 in comparison to normal specimens. PELP1 knockdown with shRNA dramatically decreased HCC cell invasion, clonogenicity, and proliferation. Similarly, PELP1 inhibitor SMIP34 treatment significantly reduced cell viability, invasiveness, colony forming capacity, and induced apoptosis of HCC cells. RNA-seq analyses using PELP1 KD cells confirmed that PELP1 modulates the ribosome and the eukaryotic translation elongation pathways. Mechanistic studies confirmed that SMIP34 treatment contributed to the disruption of the Rix complex, which is crucial for ribosomal biogenesis. Puromycin labelling studies confirmed that PELP1 KD or SMIP34 treatment contributes to the reduction of ribosomal biogenesis and new protein synthesis. Furthermore, PELP1 KD or treatment with SMIP34 dramatically slowed the progression of HCC tumors in xenograft models. IHC analysis revealed reduced levels of the proliferation marker Ki67 in PELP1 KD/SMIP34 treated HCC xenograft tumors compared to controls. Conclusions: Overall, the results of our study point to PELP1 as a potential therapeutic target for HCC intervention. Citation Format: Uday Pratap, Khaled Mohamed Nassar, Xue Yang, Adriana Baker, Rahul Gopalam, William C. Arnold, Timilehin Adeniran, Marian Helena Hernandez Fernandez, Gangadhara R. Sareddy, Suryavathi Viswanadhapalli, Luzhe Sun, Ratna K. Vadlamudi. Significance of PELP1 signaling in liver cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3172.

Abstract 440: The role of PELP1 inhibitor SMIP34 in the treatment of endometrial cancer

Abstract Background: Endometrial cancer (ECa) is the fourth most prevalent type of cancer in women. Although ECa mostly affects older women, both the mortality and incidence rates are rapidly rising in women under the age of 40. Response rates and progression free survival are still low for recurrent ECa treatments such as hormone therapy, chemotherapy, and targeted therapies like Bevacizumab and Everolimus. There is an unmet need for the development of novel targeted therapeutics to support current ECa-directed treatments. The progression of many cancers, including ECa, is linked to proto-oncogenic PELP1 (proline-, glutamic acid-, and leucine-rich protein 1). In this investigation, we evaluated the efficacy of the PELP1 inhibitor, SMIP34, both alone and in combination with mTOR inhibitors in ECa. Methods: Using 13 distinct ECa cells, the effect of SMIP34 alone and in conjunction with mTOR inhibitors was assessed using the cell viability and colony formation assays. Using flow cytometry and Annexin-V/PI labeling, the ability of SMIP34 to promote apoptosis was evaluated. The effect of SMIP34 on cell cycle progression was evaluated using FACS analyses. RT-qPCR, Western blotting, and global RNA-seq were used for mechanistic studies. ECa patient-derived organoids (PDO), explants (PDEX) and xenograft model were used to assess the impact of SMIP34. TNM and Dependency Map (DepMap) databases were used to profile the status of PELP1 in ECa. The status of PELP1 in ECa tissues was analyzed using IHC analyses of ECa tumor tissue Microarrays. Results: Analysis of TNM plots showed that PELP1 expression was higher in ECa than in normal endometrium. Data from DepMap confirmed that PELP1 is required for the survival and expansion of ECa cells. SMIP34 treatment of established and primary patient-derived ECa cells significantly reduced cell viability and colony formation with an IC50 of 2-10µM. PELP1 knockdown significantly reduced the effect of SMIP34. Further, SMIP34 treatment induced apoptosis and promoted S-phase arrest in ECa cells. RNA-seq analyses showed that SMIP34 regulated genes positively correlated with p53 and apoptosis pathways, while negatively correlated with ribosome and eukaryotic translation elongation pathways. Western blot analyses confirmed that SMIP34 treatment reduced the levels of PELP1 and its complex of proteins including TEX10, LAS1L, SENP3. Mechanistic studies demonstrated that SMIP34 treatment attenuated the activation of mTOR signaling in ECa cells. Accordingly, SMIP34 enhanced the effect of mTOR inhibitors on ECa cells. SMIP34 is effective in reducing cell viability in PDO and limited the cells proliferation of PDEX. Importantly, SMIP34 treatment significantly reduced of the growth of ECa cell line-derived xenografts. Conclusions: Collectively, our results suggest that SMIP34 inhibits ECa growth in vitro, in vivo, and ex vivo and combination of SMIP34 with mTOR inhibitor represents a novel therapeutic strategy for ECa. Citation Format: Xue Yang, Zexuan Liu, Weiwei Tang, Uday P. Pratap, Alexia B. Collier, Kristin A. Alwegg, Nicole Spencer, Xiaonan Li, Philip T. Valente, Edward R. Kost, Suryavathi Viswanadhapalli, Ratna K. Vadlamudi. The role of PELP1 inhibitor SMIP34 in the treatment of endometrial cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 440.

SETDB1 interactions with PELP1 contributes to breast cancer endocrine therapy resistance

BackgroundMethyltransferase SETDB1 is highly expressed in breast cancer (BC), however, the mechanisms by which SETDB1 promotes BC progression to endocrine therapy resistance remains elusive. In this study, we examined the mechanisms by which SETDB1 contribute to BC endocrine therapy resistance.MethodsWe utilized therapy sensitive (MCF7 and ZR75), therapy resistant (MCF7-TamR, MCF7-FR, MCF7-PELP1cyto, MCF7-SETDB1) estrogen receptor alpha positive (ER+)BC models and conducted in vitro cell viability, colony formation, 3-dimensional cell growth assays to investigate the role of SETDB1 in endocrine resistance. RNA-seq of parental and SETDB1 knock down ER+ BC cells was used to identify unique pathways. SETDB1 interaction with PELP1 was identified by yeast-two hybrid screen and confirmed by immunoprecipitation and GST-pull down assays. Mechanistic studies were conducted using Western blotting, reporter gene assays, RT-qPCR, and in vitro methylation assays. Xenograft assays were used to establish the role of PELP1 in SETDB1 mediated BC progression.ResultsRNA-seq analyses showed that SETDB1 regulates expression of a subset of estrogen receptor (ER) and Akt target genes that contribute to endocrine therapy resistance. Importantly, using yeast-two hybrid screen, we identified ER coregulator PELP1 as a novel interacting protein of SETDB1. Biochemical analyses confirmed SETDB1 and PELP1 interactions in multiple BC cells. Mechanistic studies confirmed that PELP1 is necessary for SETDB1 mediated Akt methylation and phosphorylation. Further, SETDB1 overexpression promotes tamoxifen resistance in BC cells, and PELP1 knockdown abolished these effects. Using xenograft model, we provided genetic evidence that PELP1 is essential for SETDB1 mediated BC progression in vivo. Analyses of TCGA datasets revealed SETDB1 expression is positively correlated with PELP1 expression in ER+ BC patients.ConclusionsThis study suggests that the PELP1/SETDB1 axis play an important role in aberrant Akt activation and serves as a novel target for treating endocrine therapy resistance in breast cancer.

Targeting PELP1 Attenuates Angiogenesis and Enhances Chemotherapy Efficiency in Colorectal Cancer

Simple SummaryExcessive angiogenesis is a distinct feature of colorectal cancer (CRC) and plays a pivotal role in tumor development and metastasis. Therefore, it is essential to clarify the underlying mechanism of angiogenesis. In this study, we found that the level of proline-, glutamic acid, and leucine-rich protein 1 (PELP1) was positively correlated with microvessel density (MVD). In vitro and in vivo assays further showed PELP1 regulated angiogenesis via the Signal transducer and activator of transcription 3 (STAT3)/Vascular endothelial growth factor (VEGFA). Notably, we found that inhibition of PELP1 enhanced the efficacy of chemotherapy due to vascular normalization. Thus, targeting of PELP1 may be a potentially therapeutic strategy for CRC.Abnormal angiogenesis is one of the important hallmarks of colorectal cancer as well as other solid tumors. Optimally, anti-angiogenesis therapy could restrain malignant angiogenesis to control tumor expansion. PELP1 is as a scaffolding oncogenic protein in a variety of cancer types, but its involvement in angiogenesis is unknown. In this study, PELP1 was found to be abnormally upregulated and highly coincidental with increased MVD in CRC. Further, treatment with conditioned medium (CM) from PELP1 knockdown CRC cells remarkably arrested the function of human umbilical vein endothelial cells (HUVECs) compared to those treated with CM from wildtype cells. Mechanistically, the STAT3/VEGFA axis was found to mediate PELP1-induced angiogenetic phenotypes of HUVECs. Moreover, suppression of PELP1 reduced tumor growth and angiogenesis in vivo accompanied by inactivation of STAT3/VEGFA pathway. Notably, in vivo, PELP1 suppression could enhance the efficacy of chemotherapy, which is caused by the normalization of vessels. Collectively, our findings provide a preclinical proof of concept that targeting PELP1 to decrease STAT3/VEGFA-mediated angiogenesis and improve responses to chemotherapy due to normalization of vessels. Given the newly defined contribution to angiogenesis of PELP1, targeting PELP1 may be a potentially ideal therapeutic strategy for CRC as well as other solid tumors.

Abstract 733: Significance of PELP1/SETDB1 axis in endocrine therapy resistance

Abstract BACKGROUND: PELP1 is commonly overexpressed in breast cancer (BCa) and is implicated in endocrine therapy resistance. However, the mechanism by which PELP1 contributes to therapy resistance remains elusive. Our ongoing studies using yeast two hybrid screen identified histone methyltransferase SETDB1 as a novel interactor of PELP1. The objective of this study is to determine the significance of PELP1 interaction with SETDB1 in BCa endocrine therapy resistance. METHODS: We used two ER+ BCa cell lines (MCF7 and ZR75) and three endocrine therapy resistant cell lines (Tamoxifen resistant MCF7-TamR, Fulvestrant resistant MCF7-FR, Tamoxifen resistant MCF7-PELP1-cyto). Additional BCa models with stable overexpression and under expression of SETDB1 and PELP1 (MCF7-SETDB1-PELP1shRNA, ZR75-SETDB1-PELP1shRNA, ZR75-SETDB1, MCF7-SETDB1, MCF7-TamR-SETDB1shRNA, MCF7-FR-SETDB1shRNA) were generated using lentiviral transduction. Functional significance of cross-talk was tested using MTT, soft agar, and colony formation assays. Mechanistic studies were conducted using immunoprecipitation, Western blotting, and RT-qPCR. RESULTS: Analyses of TCGA databases showed that SETDB1 expression is positively correlated with PELP1 expression in ER+ BCa (r=0.30, p<0.0001). Immunoprecipitation assays using multiple ER+ BCa cell lysates confirmed the interaction of SETDB1 with PELP1. Using ER+ BCa models with overexpression of SETDB1 or knockdown, we provided evidence that SETDB1 plays an important role in the proliferation of BCa cells. PELP1 knockdown attenuated SETDB1 mediated BCa cells proliferation. SETDB1 upregulation contributed to tamoxifen resistance, while PELP1 knockdown re-sensitized cells to tamoxifen therapy. Further, endocrine therapy resistant model cells (MCF7-TamR, MCF7-FR, MCF7-PELP1-cyto) showed increased expression of SETDB1, and its knockdown sensitized them to endocrine therapy. Mechanistic studies revealed that PELP1 is needed for SETDB1 mediated Akt phosphorylation and activation of its downstream targets such as S6, and Cyclin D1. CONCLUSIONS: The results of study suggest that the PELP1/SETDB1 interactions contribute to endocrine therapy resistance via aberrant activation of Akt signaling. Targeting PELP1/SETDB1 axis could represent a new therapeutic avenue to mitigate endocrine resistance. Supported by VA grant I01BX004545 (R.K. Vadlamudi) Citation Format: Zexuan Liu, Junhao Liu, Weiwei Tang, Uday P. Pratap, Kristin A. Altwegg, Behnam Ebrahimi, Xiaonan Li, Gangadhara R. Sareddy, Suryavathi Viswanadhapalli, Ratna K. Vadlamudi. Significance of PELP1/SETDB1 axis in endocrine therapy resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 733.

Abstract PS17-15: Targeting the PELP1 axis for treating ESR1 mutant driven breast cancer

Abstract Background: Mutations in ESR1 genes (30-40% frequency) play an important role in acquired endocrine therapy resistance and metastases. The most commonly observed are two ESR1 LBD mutations, D538G andY537S. These mutant ERα (MT) proteins have high constitutive transcriptional activity leading to therapy resistance. Furthermore, the ability of the constitutively active mutants to interact with coregulators is associated with the promotion of tumor growth. Proline, glutamic acid-, and leucine-rich protein 1 (PELP1), an oncogenic coregulator of ERα, plays a critical role in ERα signaling, and its dysregulated expression is a prognostic indicator for poorer breast cancer (BCa) survival. The objective of this study was to test the utility of Small Molecule Inhibitor of PELP1 (SMIP34) for treating ESR1 mutant (MT-ER) driven BCa. Methods: Four BCa models that express either ESR1 mutation D538G or Y537S and their respective wild-typeERα (WT-ER) controls were used to test the utility of targeting the PELP1 axis using PELP1-specific shRNA orSMIP34. Celltiter Glo, MTT, colony formation, and Boyden chamber invasion assays were used to test the efficacy of SMIP34. Western blot, RNA-Seq, and reporter gene assays were utilized to uncover the mechanistic insights. Pre-clinical evaluation was performed using MT-ER expressing xenograft explant (XDEX) and patient-derived explant (PDEX) assays. Results: BCa model cells expressing MT-ER showed increased cell proliferation, whilst PELP1 knock-down significantly reduced their proliferation. Immunoprecipitation results confirmed that PELP1 constitutively associates with MT-ER. PELP1 knock-down or treatment with PELP1 inhibitor SMIP34 significantly reduced proliferation of the four MT-ER models with an IC50 of 3-5μM. PELP1 knock-down or SMIP34 treatment significantly reduced the constitutive ERE reporter activity observed in MT-ER models. RTqPCR assays confirmed the downregulation of MT-ER target genes in PELP1 knock-down and SMIP34 treated cells. Furthermore, PELP1 knock-down or SMIP34 treatment significantly reduced the invasiveness and colony formation of MT-ER BCa models. Mechanistic studies using Western blot revealed that SMIP34 contributes to PELP1 degradation by its direct binding to PELP1. SMIP34 significantly decreased proliferation of MT-ER BCa cells in XDEX andPDEX assays, as measured by Ki67 staining. Conclusion: Our results suggest that PELP1 associates with MT-ER and targeting the PELP1 axis with SMIP34will have therapeutic utility in treating MT-ER driven BCa. Supported by CPRIT Predoctoral Fellowship CPRIT RTA; RP170345 (K.A. Altwegg) and VA grant I01BX004545(R.K.V) Citation Format: Kristin A Altwegg, Suryavathi Viswanadhapalli, Junhao Liu, Zexuan Liu, Uday P. Pratap, Benham Ebrahimi, Hariprasad Vankayalapati, Ratna K. Vadlamudi. Targeting the PELP1 axis for treating ESR1 mutant driven breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-15.

Abstract PS17-34: Proto-oncogene PELP1 interactions with SETDB1 contribute to aberrant activation of AKT1 in breast cancer

Abstract BACKGROUND: Hyperactivation of PI3K/Akt signaling is implicated in breast cancer (BCa) progression. SETDB1, a methyltransferase, is also implicated in BCa, however, the mechanism remains elusive. Recent studies have shown SETDB1 can methylate non-histone substrates such as Akt1 and contribute to its aberrant activation, thus leading to tumor progression. PELP1 is a proto-oncogene which is overexpressed in BCa and participates in both the nuclear and extra nuclear functions of various nuclear receptors. In a subset of BCa, PELP1 uniquely localizes in the cytoplasm and contributes to endocrine therapy resistance. The objective of this study is to characterize the significance of PELP1 in SETDB1 mediated Akt signaling in BCa progression. METHODS: Using yeast two-hybrid screening, we identified PELP1 as a novel interacting protein of SETDB1. Functional significance of cross-talk was tested using MTT, proliferation, stemness and colony formation assays. Mechanistic studies were conducted using immunoprecipitation, RNA-seq, shRNA, overexpression, Western blotting, and RT-qPCR. Biological significance of PELP1 and SETDB1 in endocrine therapy resistance was also examined. RESULTS: Analyses of TCGA databases showed that SETDB1 is highly expressed in BCa and associated with poor clinical outcome. Further, SETDB1 expression is positively correlated with PELP1 expression in BCa (r=0.30, p<0.0001). Immunoprecipitation assays using multiple BCa cell lysates confirmed the interaction of SETDB1 with PELP1. Using two different shRNAs targeting SETDB1 and multiple BCa model cells, we provided evidence that SETDB1 plays an important role in the proliferation of BCa cells. SETDB1 upregulation is sufficient to accelerate proliferation and PELP1 knockdown attenuated SETDB1 oncogenic functions. SETDB1 overexpression contributed to resistance to tamoxifen treatment, while PELP1 knockdown re-sensitized cells to therapy. RNA-seq identified Akt signaling pathways were activated by SETDB1 in BCa. Mechanistic studies showed that SETDB1 overexpression increased Akt phosphorylation and its downstream signaling, while PELP1 knock down attenuated SETDB1 mediated Akt activation in both MCF7 and ZR75 models. Furthermore, in BCa model cells that uniquely express PELP1 in cytoplasm, knockdown of SETDB1 reduced activation of Akt and its downstream pathways. CONCLUSIONS: Our study results suggest that the PELP1/SETDB1 interactome plays an important role in aberrant Akt activation. Drugs that target PELP1/SETDB1 axis may be useful in modulating aberrant Akt signaling. Supported by VA grant I01BX004545. Citation Format: Zexuan Liu, Junhao Liu, Weiwei Tang, Uday P. Pratap, Kristin A. Altwegg, Xiaonan Li, Suryavathi Viswanadhapalli, Ratna Vadlamudi. Proto-oncogene PELP1 interactions with SETDB1 contribute to aberrant activation of AKT1 in breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-34.

PELP1 promotes the expression of RUNX2 via the ERK pathway during the osteogenic differentiation of human periodontal ligament stem cells

ObjectiveThe aim of this study was to determine the physiological function and mechanism of proline-, glutamic acid-, and leucine-rich protein 1 (PELP1) in the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) at the molecular level in vitro. DesignDuring the osteogenic differentiation of hPDLSCs, the change of PELP1 and the osteogenic commitment markers runt-related transcription factor 2(RUNX2), alkaline phosphatase (ALP) and osteocalcin (OCN) were monitored by quantitative real-time PCR (qRT-PCR) and western blots. To elucidate how PELP1 regulates RUNX2, the expression of RUNX2, the phosphorylation of extracellular regulated protein kinases (ERK) and subcellular location of PELP1 were detected under conditions that PELP1 was either knockdown by specific siRNA or overexpressed. A pharmacological inhibitor of ERK, U0126 was used while PELP1 was overexpressed, and the expression of RUNX2 was monitored by qRT-PCR. ResultsPELP1 was upregulated during the osteogenic differentiation of hPDLSCs. Knockdown of PELP1 suppressed the expression of RUNX2, whereas overexpression of PELP1 increased RUNX2 expression. Moreover, PELP1 knockdown resulted in reduced ERK phosphorylation and RUNX2 expression, and PELP1 overexpression induced RUNX2 expression was inhibited by U0126 in the hPDLSCs. ConclusionsPELP1 regulates the expression of RUNX2 during the osteogenic differentiation of hPDLSCs and that the ERK pathway is involved in this process.

Interaction of transcription factor AP-2 gamma with proto-oncogene PELP1 promotes tumorigenesis by enhancing RET signaling.

A significant proportion of estrogen receptor-positive (ER+) breast cancer (BC) initially responds to endocrine therapy but eventually evolves into therapy-resistant BC. Transcription factor AP-2 gamma (TFAP2C) is a known regulator of ER activity, and high expression of TFAP2C is associated with a decreased response to endocrine therapies. PELP1 is a nuclear receptor coregulator, commonly overexpressed in BC, and its levels are correlated with poorer survival. In this study, we identified PELP1 as a novel interacting protein of TFAP2C. RNA-seq analysis of PELP1 knockdown BC cells followed by transcription factor motif prediction pointed to TFAP2C being enriched in PELP1-regulated genes. Gene set enrichment analysis (GSEA) revealed that the TFAP2C-PELP1 axis induced a subset of common genes. Reporter gene assays confirmed PELP1 functions as a coactivator of TFAP2C. Mechanistic studies showed that PELP1-mediated changes in histone methylation contributed to increased expression of the TFAP2C target gene RET. Furthermore, the TFAP2C-PELP1 axis promoted the activation of the RET signaling pathway, which contributed to downstream activation of AKT and ERK pathways in ER+ BC cells. Concomitantly, knockdown of PELP1 attenuated these effects mediated by TFAP2C. Overexpression of TFAP2C contributed to increased cell proliferation and therapy resistance in ER+ BC models, while knockdown of PELP1 mitigated these effects. Utilizing ZR75-TFAP2C xenografts with or without PELP1 knockdown, we provided genetic evidence that endogenous PELP1 is essential for TFAP2C-driven BC progression invivo. Collectively, our studies demonstrated that PELP1 plays a critical role in TFAP2C transcriptional and tumorigenic functions in BC and blocking the PELP1-TFAP2C axis could have utility for treating therapy resistance.

Abstract 4371: PELP1-TFAP2C crosstalk promotes endocrine resistance in breast cancer cells

Abstract Background: A significant proportion of estrogen receptor positive (ER+) breast cancer (BC) initially respond to antiestrogens or aromatase inhibitors but become therapy resistant-BC. Development of effective therapies for endocrine therapy resistant BC represent the highest unmet need. PELP1 is a nuclear receptor coregulator, commonly overexpressed in BC, contributes to therapy resistance and correlate with poor survival. TFAP2C (transcription factor AP-2 gamma) is a known regulator of ER activity and high expression of TFAP2C is associated with a decreased response to Fulvestrant. The objective of this study is to examine the significance of PELP1-TFAP2C crosstalk in the development of breast cancer therapy resistance. Methods: We used yeast two-hybrid screen to identify PELP1 interacting transcription factors. Interaction of PELP1 and TFAP2C was confirmed by immunoprecipitation and GST pull down assays. Functional significance of the cross talk was tested using CellTiter-Glo, MTT and colony formation assays in the presence or absence of endocrine therapy. Mechanistic studies were conducted using shRNA, overexpression, Western blotting, reporter gene assays, RT-qPCR, ChIP-seq and RNA-seq analysis. Biological significance of PELP1 and TFAP2C in endocrine therapy resistance was examined using overexpression and under expression models of PELP1 and TFAP2C in multiple BC models including MCF7 and ZR75. Results: Yeast based screening of a mammary gland cDNA expression library using PELP1 as the bait identified TFAP2C as a novel interacting protein of PELP1. Immunoprecipitation assays using multiple BC cell lysates confirmed the interaction of PELP1 with TFAP2C. Using various deletions of PELP1, and by using GST pull down assays, we identified N-terminal 400-600aa region of PELP1 as the major interaction site for TFAP2C. Using RNA-seq of PELP1 knockdown BC model cells, we predicted TFAP2C as an enriched transcription factor in PELP1 regulated genes. The GSEA results from RNA-seq showed TFAP2C and PELP1 induce a subset of common genes. Reporter gene assays confirmed that PELP1 functions as a coactivator of TFAP2C. Mechanistic studies showed that TFAP2C activates both AKT and ERK pathways in ER+ cell lines, while knock down of PELP1 attenuated these effects. Overexpression of TFAP2C contributed to increased cell proliferation and endocrine therapy resistance in MCF7 and ZR75 models, while knock down of PELP1 attenuated these effects. Utilizing ZR75-TFAP2C xenograft models with WT PELP1 or PELP1 knock down, we provided genetic evidence that endogenous PELP1 is essential for TFAP2C drive n breast cancer progression in vivo. Conclusions: Collectively, our studies demonstrated that PELP1 functions as a coactivator of TFAP2C in modulating a set of ER target genes, TFAP2C functions as a transcription factor of PELP1 regulated genes and blocking the PELP1-TFAP2C axis could have therapeutic utility for treating therapy resistance. Supported by VA grant I01BX004545 (R.K. Vadlamudi) Citation Format: Junhao Liu, Suryavathi Viswanadhapalli, Mengxing Li, Uday P. Pratap, Weiwei Tang, Zexuan Liu, Yiliao Luo, Kristin A. Altwegg, Xiaonan Li, Gangadhara R. Sareddy, Rajeshwar R. Tekmal, Ratna K. Vadlamudi. PELP1-TFAP2C crosstalk promotes endocrine resistance in breast cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4371.

Abstract P3-10-01: Development and characterization of a first-in-class small molecule inhibitor of PELP1

Abstract Background: Breast cancer (BCa) is the most commonly diagnosed cancer and the second leading cause of cancer death in women. BCa is composed of distinct molecular subtypes, such as ER positive BCa (ER+ BCa) and triple negative BCa (TNBC). Development of novel effective therapies for patients with therapy resistant breast cancer (TR-BC) and TNBC remains the highest unmet need in patient treatment and survivorship. Proline-, glutamic acid-, and leucine-rich protein 1 (PELP1) plays a critical role in multiple nuclear receptor functions leading to TR-BC and TNBC progression. PELP1 expression is dysregulated in BCa, is a prognostic indicator of poorer BCa survival, and its deregulation contributes to BCa therapy resistance. The objective of this study is development and characterization of a small molecule inhibitor of PELP1 (SMIP) as novel therapeutic for treating BCa. Methods: Using yeast two-hybrid screening, we identified PELP1 Inhibitory Peptide (PIP1) from a library of peptides. PIP1 binds PELP1 with high affinity and functions to inhibit PELP1 oncogenic activity. Direct binding of PIP1 to PELP1 was confirmed using biotin pull-down assays and inhibition of BCa proliferation confirmed using MTT assays. We used the Hit-Ligand interaction site with PIP1 hot spot residues based on 3D alignment and morphology to generate a library of peptidomimetics (small chemical molecules). In vitro activity was assessed using Celltiter Glo, MTT, and matrigel invasion chamber assays in multiple BCa models. Mechanistic studies were conducted using Western blot, reporter gene assays, and peptide competition assays. Xenograft and patient derived explant (PDEX) assays were used for preclinical evaluation and preliminary toxicity analysis. Results: Bioactivity screens revealed PELP1 Inhibitory Peptide (PIP1) significantly attenuates PELP1-mediated proliferation with an IC50 of 10µM across multiple BCa cell lines. We confirmed PIP1 binding to PELP1 using peptide pull-down assays with nuclear lysates from BCa cells. Using Hit-Ligand-Based interaction site with the PIP1 hot spot residues, we identified 61 potential hits using a 10,000 Diverse Set. Screening of these 61 potential hits using the MTT assays lead to the selection of SMIP34 (tetrahydropyrazolo [1,5a) pyrazole) as lead inhibitor of PELP1. SMIP34 treatment reduced proliferation at an IC50 of 3-10µM in ER+ BCa (ZR-75, MCF-7, and T- 47D); TR-BC (MCF-7-TamR, MCF-7-LTLT, ZR-75-MT-ER537S, and ZR-75-MT-ER538G); and TNBC (MDA- MB-231, and BT549) models. Additionally, SMIP34 showed no activity in human mammary epithelial cells. Specificity of SMIP34 was confirmed using PELP1 knockdown BCa cell lines. Mechanistic studies using Western blot analysis confirmed that SMIP34 binding to PELP1 contributes to its degradation. In matrigel invasion chamber assays, SMIP34 significantly reduced the invasiveness of TR-BC and TNBC models. In combination studies, SMIP34 displayed synergy and enhanced the efficacy of current chemotherapeutics Cisplatin and Paclitaxel. In PDEX assays, Ki67 staining revealed SMIP34 significantly decreased tumor proliferation. In xenograft models, SMIP34 (10mg/kg/s.c.) treatment resulted in significant reduction in tumors compared to vehicle treatment. Furthermore, overall mouse body weight in both control and SMIP34 treated groups were similar, suggesting no overt signs of toxicity. Conclusion: We have developed a first-in-class small molecule inhibitor of PELP1 (SMIP) displaying effectivity against TR-BC and TNBC in vitro and in vivo. Supported by CPRIT Predoctoral Fellowship CPRIT RTA; RP170345 (K.A. Altwegg) Citation Format: Kristin A Altwegg, Suryavathi Viswanadhapalli, Monica Mann, Uday P Pratap, Mengxing Li, Junhao Liu, Yiliao Luo, Gangadhara R Sareddy, Hariprasad Vankayalapati, Ratna K. Vadlamudi. Development and characterization of a first-in-class small molecule inhibitor of PELP1 [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-10-01.

Abstract P6-04-14: The role of PELP1-TFAP2C crosstalk in mediating endocrine-therapy-resistance in breast cancer

Abstract Background: A significant proportion of estrogen receptor (ER) positive (ER+) breast cancer (BC) initially respond to endocrine therapies, such as antiestrogens or aromatase inhibitors. However, ER+ BC can build up resistance to treatment progressing into therapy resistant-BC (TR-BC). Development of effective therapeutics for endocrine-therapy-resistant BC represents a significant unmet need in BC treatment options. Proline-, glutamic acid-, and leucine-rich protein 1 (PELP1) is oncogenic nuclear receptor coregulator, commonly overexpressed in BC. PELP1 overexpression is correlated with poorer patient survival and is associated with development of TR-BC. TFAP2C (AP2Gamma) is a known regulator of ER activity. In addition, high expression of TFAP2C is associated with a decreased response to the steroidal antiestrogen, Fulvestrant. However, it remains unknown whether PELP1 and TFAP2C crosstalk and if the PELP1/TFAP2C axis behaves synergistically to contribute to the development of therapy resistance in TR-BC. Methods: To gain insight into PELP1 signaling mechanisms, we used yeast two-hybrid screen to identify proteins that interact with PELP1. The interaction between PELP1 and TFAP2C was confirmed by immunoprecipitation using both endogenous and GFP tagged proteins. GST fusions of various domains of PELP1 were used to identify the TFAP2C interacting domain. Functional significance of the crosstalk was tested using Celltiter Glo, MTT, apoptosis, and invasion assays. Mechanistic studies were conducted using shRNA, overexpression, Western Blot, reporter gene assays, RT-qPCR, ChIP and RNA-Seq analysis. Biological significance of PELP1 and TFAP2C in endocrine-therapy-resistance was examined using overexpression and under-expression models of PELP1 and TFAP2C in multiple ER+ BC and TR-BC model cells (MCF-7, ZR-75, T-47D, MCF-7-TamR, MCF7-LTLT and ZR75-ERMT537S. Results: Screening of a mammary gland cDNA expression library using PELP1 as the bait identified TFAP2C as a novel interacting protein of PELP1. Immunoprecipitation assays utilizing multiple BC cell lysates confirmed interaction of PELP1 with TFAP2C. We also confirmed PELP1 and TFAP2C interactions using GFP and GST epitope tagged proteins. Using GST fusion of various domains of PELP1, we identified the PELP1 N-terminal domain (aa 400-600) as the major interaction site for TFAP2C. Using PELP1 knockdown BC model cell lines with RNA-Seq analysis, we identified a set of genes regulated by PELP1. The GSEA results from the RNA-Seq data predicted TFAP2C as an enriched transcription factor in a subset of PELP1 regulated genes. RT-qPCR analysis confirmed that PELP1 is needed for optimal regulation of the ER and associated ER target genes by TFAP2C. Reporter gene assays confirmed that PELP1 functions as a coregulator of TFAP2C. Overexpression of TFAP2C contributed to endocrine-therapy-resistance in BC model cells, while knockdown of PELP1 abolished TFAP2C mediated therapy resistance. Conclusions: Collectively, our studies identified PELP1 functions as a coregulator of TFAP2C in modulating ER target genes. TFAP2C functions as a transcription factor of PELP1 regulated genes and blocking the PELP1-TFAP2C axis will have therapeutic utility for treating TR-BC Citation Format: Junhao Liu, Suryavathi Viswanadhapalli, Mengxing Li, Uday P Pratap, Yiliao Luo, Kristin A Altwegg, Xiaonan Li, Gangadhara R Sareddy, Rajeshwar R Tekmal, Ratna K Vadlamudi. The role of PELP1-TFAP2C crosstalk in mediating endocrine-therapy-resistance in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-04-14.

PELP1 signaling contributes to medulloblastoma progression by regulating the NF-κB pathway.

Medulloblastoma (MB) is the most common and deadliest brain tumor in children. Proline-, glutamic acid-, and leucine-rich protein 1 (PELP1) is a scaffolding protein and its oncogenic signaling is implicated in the progression of several cancers. However, the role of PELP1 in the progression of MB remains unknown. The objective of this study is to examine the role of PELP1 in the progression of MB. Immunohistochemical analysis of MB tissue microarrays revealed that PELP1 is overexpressed in the MB specimens compared to normal brain. Knockdown of PELP1 reduced cell proliferation, cell survival, and cell invasion of MB cell lines. The RNA-sequencing analysis revealed that PELP1 knockdown significantly downregulated the pathways related to inflammation and extracellular matrix. Gene set enrichment analysis confirmed that the PELP1-regulated genes were negatively correlated with nuclear factor-κB (NF-κB), extracellular matrix, and angiogenesis gene sets. Interestingly, PELP1 knockdown reduced the expression of NF-κB target genes, NF-κB reporter activity, and inhibited the nuclear translocation of p65. Importantly, the knockdown of PELP1 significantly reduced in vivo MB progression in orthotopic models and improved the overall mice survival. Collectively, these results suggest that PELP1 could be a novel target for therapeutic intervention in MB.

Abstract 3485: PELP1 is a novel mediator of medulloblastoma progression

Abstract Background: Medulloblastoma (MB) is the most common and deadliest primary brain tumor in children that accounts for 15-20% of all pediatric brain tumors. Despite recent advances in multimodal treatment, the 5-year overall survival of MB patients is approximately 60-70%. Unfortunately, improved outcome have been associated with significant long-term toxicities. Identifying novel targets that drive MB progression is urgently needed. Proline-, glutamic acid-, and leucine-rich protein 1 (PELP1) is a scaffolding protein that functions as a coregulator of several nuclear receptors. Oncogenic PELP1 signaling is implicated in the progression of several cancers including breast, ovarian, prostate, lung, pancreas and colon. However, its role in the progression of medulloblastoma remains unknown. Here, we examined the role of proto-oncogene PELP1 in the progression of MB. Methods: The expression of PELP1 in tumor micro arrays was analyzed using validated PELP1 antibodies for immunohistochemistry. PELP1 expression was determined in vitro by western blotting. PELP1 knockdown cells were generated using PELP1 shRNA lentiviral particles or PELP1 siRNA. The effect of PELP1 knockdown or overexpression was studied using cell proliferation, colony formation and migration using established in vitro assays. Mechanistic studies were conducted using RNA-seq, RT-qPCR, immunohistochemistry, reporter gene assays and signaling analysis. Interaction of PELP1 with NF-κB was examined by immunoprecipitation. Mouse orthotopic xenografts models were used for preclinical evaluation of PELP1 knock down. Results: Immunohistochemical analysis of MB tissue microarrays revealed that PELP1 is overexpressed in MB specimens compared to normal brain specimens. Knockdown of PELP1 using PELP1 specific siRNA or shRNA significantly reduced cell proliferation, cell survival, and cell migration of MB cell lines. RNA-seq analysis revealed that PELP1 knockdown significantly downregulated the pathways related to inflammation, angiogenesis and extracellular matrix. Further, gene set enrichment analysis (GSEA) confirmed that the PELP1-regualted genes were negatively correlated with NF-κB, extracellular matrix and angiogenesis. Mechanistic studies showed that PELP1 knockdown reduced the expression of NF-κB reporter gene activity and its target genes. Additionally, knockdown of PELP1 significantly reduced in vivo MB tumor progression in orthotopic models, and improved overall mice survival. IHC analysis demonstrated that the proliferation marker Ki67 and NF- κB targets were significantly downregulated in PELP1 knockdown tumors compared to controls. Conclusions: Taken together, these results provide the evidence that PELP1 could be a potential therapeutic target for therapeutic intervention in medulloblastoma. Citation Format: Yiliao Luo, Gangadhara Reddy Sareddy, Uday P. Pratap, Mengxing Li, Junhao Liu, Prabhakar Pitta-Venkata, Suryavathi Viswanadhapalli, Xiaonan Li, Manjeet Rao, Rajeshwar Rao Tekmal, Ratna K. Vadlamudi. PELP1 is a novel mediator of medulloblastoma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3485.

PELP1 promotes glioblastoma progression by enhancing Wnt/β-catenin signaling.

BackgroundGlioblastoma (GBM) is a deadly neoplasm of the central nervous system. The molecular mechanisms and players that contribute to GBM development is incompletely understood.MethodsThe expression of PELP1 in different grades of glioma and normal brain tissues was analyzed using immunohistochemistry on a tumor tissue array. PELP1 expression in established and primary GBM cell lines was analyzed by Western blotting. The effect of PELP1 knockdown was studied using cell proliferation, colony formation, migration, and invasion assays. Mechanistic studies were conducted using RNA-seq, RT-qPCR, immunoprecipitation, reporter gene assays, and signaling analysis. Mouse orthotopic models were used for preclinical evaluation of PELP1 knock down.ResultsNuclear receptor coregulator PELP1 is highly expressed in gliomas compared to normal brain tissues, with the highest expression in GBM. PELP1 expression was elevated in established and patient-derived GBM cell lines compared to normal astrocytes. Knockdown of PELP1 resulted in a significant decrease in cell viability, survival, migration, and invasion. Global RNA-sequencing studies demonstrated that PELP1 knockdown significantly reduced the expression of genes involved in the Wnt/β-catenin pathway. Mechanistic studies demonstrated that PELP1 interacts with and functions as a coactivator of β-catenin. Knockdown of PELP1 resulted in a significant increase in survival of mice implanted with U87 and GBM PDX models.ConclusionsPELP1 expression is upregulated in GBM and PELP1 signaling via β-catenin axis contributes to GBM progression. Thus, PELP1 could be a potential target for the development of therapeutic intervention in GBM.

Abstract 532: PELP1 and AIB1 (SRC-3) complexes promote cancer stem cell-associated phenotypes in ER+ breast cancer models

Abstract Proline, glutamic acid, and leucine rich protein 1 (PELP1) is overexpressed in approximately 80% of invasive breast tumors. PELP1 dynamically shuttles between the nucleus and cytoplasm, but is primarily nuclear in normal breast tissue. However, altered localization of PELP1 to the cytoplasm is an oncogenic event that promotes breast cancer initiation and progression. Herein, we sought to identify interacting partners unique to cytoplasmic PELP1 and determine the mechanisms by which these interactions promote oncogenic PELP1 signaling. We identified AIB1 (amplified in breast cancer 1; also known as SRC-3 or NCOA3) as a novel binding partner of cytoplasmic PELP1 in both estrogen receptor-positive (ER+) and ER-negative cell lines. Cytoplasmic PELP1 expression elevated basal phosphorylation levels (i.e., activation) of AIB1, enhanced ALDH+ tumorsphere formation, and upregulated specific target genes independently of hormone stimulation. Direct manipulation of AIB1 levels using shRNA abrogated cytoplasmic PELP1-induced tumorsphere formation and downregulated cytoplasmic PELP1-specific target genes. SI-2, an AIB1 inhibitor, inhibited the PELP1/AIB1 interaction and decreased cytoplasmic PELP1-induced tumorsphere formation. Similar results were observed in a murine-derived MMTV-AIB1 tumor cell line (J110). Furthermore, in vivo syngeneic tumor studies showed that PELP1 knockdown in J110 cells resulted in increased survival of tumor-bearing mice as compared to mice injected with control cells. These studies suggest that manipulating PELP1 location or levels has the potential to mitigate tumor progression, particularly in the context of AIB1-mediated tumorigenesis. Taken together, our data demonstrate that cytoplasmic PELP1/AIB1-containing complexes function to promote advanced cancer phenotypes, including outgrowth of stem-like cells, associated with estrogen-independent breast cancer progression. Citation Format: Thu H. Truong, Hsiangyu Hu, Nuri A. Temiz, Kyla M. Hagen, Brian J. Girard, Nicholas J. Brady, Kathryn L. Schwertfeger, Carol A. Lange, Julie H. Ostrander. PELP1 and AIB1 (SRC-3) complexes promote cancer stem cell-associated phenotypes in ER+ breast cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 532.

Cancer Stem Cell Phenotypes in ER+ Breast Cancer Models Are Promoted by PELP1/AIB1 Complexes.

Proline, glutamic acid, leucine-rich protein 1 (PELP1) is overexpressed in approximately 80% of invasive breast tumors. PELP1 dynamically shuttles between the nucleus and cytoplasm, but is primarily nuclear in normal breast tissue. However, altered localization of PELP1 to the cytoplasm is an oncogenic event that promotes breast cancer initiation and progression. Herein, interacting partners unique to cytoplasmic PELP1 and the mechanisms by which these interactions promote oncogenic PELP1 signaling were sought. AIB1 (amplified in breast cancer 1; also known as SRC-3 or NCOA3) was identified as a novel binding partner of cytoplasmic PELP1 in both estrogen receptor-positive (ER+) and ER-negative cell lines. Cytoplasmic PELP1 expression elevated basal phosphorylation levels (i.e., activation) of AIB1 at Thr24, enhanced ALDH+ tumorsphere formation, and upregulated specific target genes independently of hormone stimulation. Direct manipulation of AIB1 levels using shRNA abrogated cytoplasmic PELP1-induced tumorsphere formation and downregulated cytoplasmic PELP1-specific target genes. SI-2, an AIB1 inhibitor, limited the PELP1/AIB1 interaction and decreased cytoplasmic PELP1-induced tumorsphere formation. Similar results were observed in a murine-derived MMTV-AIB1 tumor cell line. Furthermore, in vivo syngeneic tumor studies revealed that PELP1 knockdown resulted in increased survival of tumor-bearing mice as compared with mice injected with control cells.Implications: These data demonstrate that cytoplasmic PELP1/AIB1-containing complexes function to promote advanced cancer phenotypes, including outgrowth of stem-like cells, associated with estrogen-independent breast cancer progression. Mol Cancer Res; 16(4); 707-19. ©2018 AACR.

Minireview: Deciphering the Cellular Functions of PELP1

Proline, glutamic acid, and leucine-rich protein 1 (PELP1) has been ascribed a large number of cellular functions, including roles in regulation of steroid receptor (SR) signaling (1), SR cross talk, cell cycle regulation (2), ribosome biogenesis (3), apoptosis, and differentiation (4). PELP1 is critical during development, as evidenced by embryonic lethality of PELP1 knockdown. PELP1 may act as a protooncogene, with its dysregulation linked to therapy resistance in hormonal cancers (1, 5–9). How does PELP1 perform these various cellular roles? PELP1 neither binds specific sequences on the DNA nor is involved in signal transduction at the plasma membrane not has any known enzymatic activity. The explanation for the ascribed roles of PELP1 may lie in its extensive social network with other protein partners. PELP1 has multiple structural motifs that enable its direct interaction with a number of key proteins (Figure 1) (10); by functioning as a scaffold, PELP1 “enables” cross talk between its interacting proteins (11–14). The interactome of PELP1 is extensive and includes SRs, chromatin-modifying enzymes, cellular kinases, splicing factors, and transcriptional regulators (Figure 2). PELP1 likely indirectly but critically influences cell signaling and cellular processes. This review critically evaluates the molecular basis of the role of PELP1 in hormonal signaling and cancer.

Novel role of PELP1 in regulating chemotherapy response in mutant p53-expressing triple negative breast cancer cells.

Triple-negative breast cancer (TNBC), the most aggressive breast cancer subtype, occurs in younger women and is associated with poor prognosis. Gain-of-function mutations in TP53 are a frequent occurrence in TNBC and have been demonstrated to repress apoptosis and up-regulate cell cycle progression. Even though TNBC responds to initial chemotherapy, resistance to chemotherapy develops and is a major clinical problem. Tumor recurrence eventually occurs and most patients die from their disease. An urgent need exists to identify molecular-targeted therapies that can enhance chemotherapy response. In the present study, we report that targeting PELP1, an oncogenic co-regulator molecule, could enhance the chemotherapeutic response of TNBC through the inhibition of cell cycle progression and activation of apoptosis. We demonstrate that PELP1 interacts with MTp53, regulates its recruitment, and alters epigenetic marks at the target gene promoters. PELP1 knockdown reduced MTp53 target gene expression, resulting in decreased cell survival and increased apoptosis upon genotoxic stress. Mechanistic studies revealed that PELP1 depletion contributes to increased stability of E2F1, a transcription factor that regulates both cell cycle and apoptosis in a context-dependent manner. Further, PELP1 regulates E2F1 stability in a KDM1A-dependent manner, and PELP1 phosphorylation at the S1033 residue plays an important role in mediating its oncogenic functions in TNBC cells. Accordingly, depletion of PELP1 increased the expression of E2F1 target genes and reduced TNBC cell survival in response to genotoxic agents. PELP1 phosphorylation was significantly greater in the TNBC tumors than in the other subtypes of breast cancer and in the normal tissues. These findings suggest that PELP1 is an important molecular target in TNBC, and that PELP1-targeted therapies may enhance response to chemotherapies.

Abstract 2731: PELP1 promotes DNA double-strand break repair via alternative-NHEJ: Implications in therapy resistance

Abstract Estrogen Receptor positive breast cancer is commonly treated with hormonal therapy; however, many patients acquire therapy resistance, a major clinical problem. Recent evidence suggests that the accumulation of chromosomal abnormalities via increased error-prone Alternative-Non-Homologous End Joining (A-NHEJ) DNA repair pathway could contribute to breast cancer therapy resistance. Proline, Glutamic acid, Leucine rich Protein-1 (PELP1), an oncogenic co-regulator for transcription factors, is commonly overexpressed in breast cancer, and its deregulation has been implicated in therapy resistance. Our recent studies identified PELP1 as a novel substrate of the DNA damage response (DDR) kinases. In this study, we discovered that PELP1 regulates the A-NHEJ pathway, and we developed a novel peptide drug that targets PELP1-A-NHEJ axis for the treatment of therapy resistance. Our results suggest that PELP1 is phosphorylated at Ser1033 by DDR kinases and phosphorylated PELP1 co-localizes with the gamma-H2AX foci. Using Homologous Recombination (HR) and NHEJ pathway specific reporter assays, we found that PELP1 knockdown decreases NHEJ, with no apparent effect on the HR pathway. Further, using A-NHEJ specific reporter cell line, we found that PELP1 knockdown decreased the frequency of A-NHEJ, while PELP1 overexpression increased the repair frequency. PELP1's ability to regulate A-NHEJ pathway was further confirmed by sequencing repaired plasmids and by quantitating the degree of end resection. More importantly, metaphase chromosome spreads revealed gross chromosomal abnormalities in PELP1 deregulated breast cancer cells. Mechanistic studies revealed that PELP1 interacts with Mre11, and modulates the degree of end resection at the DNA double strand breaks. Mapping studies identified the C-terminus of PELP1 as the binding site for Mre11. Using yeast based genetic screen composed of 10 million peptides, we identified a unique peptide, PIP3 that binds the C-terminus of PELP1. We developed a stapled peptide of PIP3 (sPIP3), which functioned as a potent cytotoxic agent for breast cancer cells with little activity on normal cells. Further, sPIP3 efficiently inhibited the A-NHEJ pathway and significantly, reduced survival and promoted apoptosis of therapy resistant model cells. Collectively, these findings suggest that PELP1 plays a critical role in the A-NHEJ pathway and sPIP3 represents a novel drug for the treatment of therapy resistant breast cancer. Citation Format: Samaya R. Krishnan, Binoj C. Nair, Gangadhara R. Sareddy, Monica Mann, Ratna K. Vadlamudi. PELP1 promotes DNA double-strand break repair via alternative-NHEJ: Implications in therapy resistance. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2731. doi:10.1158/1538-7445.AM2014-2731