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Purpose Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer. Oncogenic PELP1 is frequently overexpressed in TNBC, and it has been demonstrated that PELP1 signaling is essential for TNBC progression. The therapeutic utility of targeting PELP1 in TNBC, however, remains unknown. In this study, we investigated the effectiveness of SMIP34, a recently developed PELP1 inhibitor for the treatment of TNBC. Methods To ascertain the impact of SMIP34 treatment, we used seven different TNBC models for testing cell viability, colony formation, invasion, apoptosis, and cell cycle analysis. Western blotting and RT-qPCR were used to determine the mechanistic insights of SMIP34 action. Using xenograft and PDX tumors, the ability of SMIP34 in suppressing proliferation was examined both ex vivo and in vivo. Results TNBC cells’ viability, colony formation, and invasiveness were all decreased by SMIP34 in in vitro cell-based assays, while apoptosis was increased. SMIP34 treatment promoted the degradation of PELP1 through the proteasome pathway. RT-qPCR analyses confirmed that SMIP34 treatment downregulated PELP1 target genes. Further, SMIP34 treatment substantially downregulated PELP1 mediated extranuclear signaling including ERK, mTOR, S6 and 4EBP1. Mechanistic studies confirmed downregulation of PELP1 mediated ribosomal biogenesis functions including downregulation of cMyc and Rix complex proteins LAS1L, TEX-10, and SENP3. The proliferation of TNBC tumor tissues was decreased in explant experiments by SMIP34. Additionally, SMIP34 treatment markedly decreased tumor progression in both TNBC xenograft and PDX models. Conclusions Together, these findings from in vitro, ex vivo, and in vivo models show that SMIP34 may be a useful therapeutic agent for inhibiting PELP1 signaling in TNBC.

Leukemia inhibitory factor receptor (LIFR) and its ligand LIF play a critical role in cancer progression, metastasis, stem cell maintenance, and therapy resistance. Here, we describe a rationally designed first-in-class inhibitor of LIFR, EC359, which directly interacts with LIFR to effectively block LIF/LIFR interactions. EC359 treatment exhibits antiproliferative effects, reduces invasiveness and stemness, and promotes apoptosis in triple-negative breast cancer (TNBC) cell lines. The activity of EC359 is dependent on LIF and LIFR expression, and treatment with EC359 attenuated the activation of LIF/LIFR-driven pathways, including STAT3, mTOR, and AKT. Concomitantly, EC359 was also effective in blocking signaling by other LIFR ligands (CTF1, CNTF, and OSM) that interact at LIF/LIFR interface. EC359 significantly reduced tumor progression in TNBC xenografts and patient-derived xenografts (PDX), and reduced proliferation in patient-derived primary TNBC explants. EC359 exhibits distinct pharmacologic advantages, including oral bioavailability, and in vivo stability. Collectively, these data support EC359 as a novel targeted therapeutic that inhibits LIFR oncogenic signaling.See related commentary by Shi et al., p. 1337

Supplementary Figure S1 shows SPR studies and Biotin EC359 pull down assays; Supplementary Figure S2 shows superimposition of hLIFR onto the mLIFR -hLIF complex; Supplementary Figure S3 shows sequence alignment of human (P42702) and mouse (P42703) LIFR ; Supplementary Figure S4 shows five prominent sites identified through sitemap program Schrödinger; Supplementary Figure S5 shows binding poses of EC359 at Site-3; Supplementary Figure S6 shows MM-GBSA scores for different poses obtained from the IFD; Supplementary Figure S7 shows ligand induced conformational changes in the best scored pose; Supplementary Figure S7 shows ligand induced conformational changes in the best scored pose; Supplementary Figure S8 shows the RMSD of protein and ligand; Supplementary Figure S9 shows pharmacological features of EC359; Supplementary Figure S10 shows effect of dox inducible CRISPR/Cas9 mediated KO of LIFR on STAT3 signaling and effect of EC359 on self-renewal of stem cells; Supplementary Table 1 shows primer sequences used for RT-qPCR analysis.

Supplementary methods section contain description of synthesis and characterization of EC330 and EC359, SPR studies, structural and sequence comparison of human and mouse LIFR, energy minimization of hLIFR, Protein and ligand preparation, molecular docking and MM-GBSA calculations, and molecular dynamics simulation.

Supplementary Data from A First-in-Class Inhibitor of ER Coregulator PELP1 Targets ER+ Breast Cancer

Supplementary Figure from A First-in-Class Inhibitor of ER Coregulator PELP1 Targets ER+ Breast Cancer

Supplementary Table from A First-in-Class Inhibitor of ER Coregulator PELP1 Targets ER+ Breast Cancer

Most patients with estrogen receptor alpha–positive (ER+) breast cancers initially respond to treatment but eventually develop therapy resistance with disease progression. Overexpression of oncogenic ER coregulators, including proline, glutamic acid, and leucine-rich protein 1 (PELP1), are implicated in breast cancer progression. The lack of small molecules that inhibits PELP1 represents a major knowledge gap. Here, using a yeast-two-hybrid screen, we identified novel peptide inhibitors of PELP1 (PIP). Biochemical assays demonstrated that one of these peptides, PIP1, directly interacted with PELP1 to block PELP1 oncogenic functions. Computational modeling of PIP1 revealed key residues contributing to its activity and facilitated the development of a small-molecule inhibitor of PELP1, SMIP34, and further analyses confirmed that SMIP34 directly bound to PELP1. In breast cancer cells, SMIP34 reduced cell growth in a dose-dependent manner. SMIP34 inhibited proliferation of not only wild-type (WT) but also mutant (MT) ER+ and therapy-resistant breast cancer cells, in part by inducing PELP1 degradation via the proteasome pathway. RNA sequencing analyses showed that SMIP34 treatment altered the expression of genes associated with estrogen response, cell cycle, and apoptosis pathways. In cell line–derived and patient-derived xenografts of both WT and MT ER+ breast cancer models, SMIP34 reduced proliferation and significantly suppressed tumor progression. Collectively, these results demonstrate SMIP34 as a first-in-class inhibitor of oncogenic PELP1 signaling in advanced breast cancer.Significance:Development of a novel inhibitor of oncogenic PELP1 provides potential therapeutic avenues for treating therapy-resistant, advanced ER+ breast cancer.

Background: The obesity epidemic is rapidly increasing in the USA and obese women are at a higher likelihood of developing triple negative breast cancer (TNBC). Several studies implicated the importance of the breast microenvironment on the aggressive cancer biology especially obese microenvironment. However, the underlying mechanism(s) by which obesity contributes to the progression of TNBC remains unclear. The objective of this study is to test a novel concept that obesity upregulates leukemia inhibitory factor receptor (LIFR) oncogenic signaling in TNBC and test whether LIFR inhibition blocks TNBC progression. Methods: Established TNBC cell lines were co-cultured with human primary adipocytes or incubated with adipocyte conditioned medium or with high glucose (HG) followed by treatment with LIFR inhibitor EC359. The effect of adiposity on TNBC cells was determined using cell viability, colony formation, and invasion assays. Mechanistic studies were performed using CRISPR/Cas9 KO of LIFR, Western blotting, RT-qPCR, and reporter gene assays. Utility of LIFR inhibitor EC359 was tested using xenografts, and patient derived organoid (PDO) models. Results: Treatment of TNBC cells with adipose conditions or HG increased the proliferation and invasion of TNBC cells. Western blot and RT-qPCR analyses confirmed that increased expression of LIFR correlated with enhanced downstream LIFR signaling such as STAT3 and subsequent activation of STAT3 target genes. CRISPR KO of LIFR or treatment of TNBC cells with EC359 significantly reduced the cell viability, colony formation and invasion under adipose conditions. Western blotting results showed that co-culture with adipocytes significantly enhanced LIFR downstream signaling in TNBC model cells and is effectively blocked by LIFR KO or EC359 treatment. Further, EC359 treatment blocked the adipose environment mediated growth of organoids. Importantly, co-implantation of adipocytes significantly enhanced TNBC xenograft tumor growth, however treatment with EC359 significantly attenuated adipocyte induced TNBC progression. Conclusions: Collectively, these results suggest that adiposity contributes to increased TNBC cell growth via upregulation of the LIF/LIFR pathway. The LIF/LIFR axis represents a potential therapeutic target for adiposity driven TNBC and the LIFR inhibitor EC359 could be used as a new therapeutic agent to treat obesity associated TNBC. Citation Format: Suryavathi Viswanadhapalli, Uday P Pratap, Behnam Ebrahimi, Logan Blankenship, Jaitri Joshi, Zexuan Liu, Kristin A Altwegg, Xiaonan Li, Gangadhara R Sareddy, Bindu Santhamma, Swapna Konda, Manjeet Rao, Edward Kost, Rajeshwar R Tekmal, Hareesh B Nair, Ratna K Vadlamudi. Leukemia inhibitory factor receptor inhibition reduces obesity driven progression of triple negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-10-01.

Most patients with estrogen receptor alpha–positive (ER+) breast cancers initially respond to treatment but eventually develop therapy resistance with disease progression. Overexpression of oncogenic ER coregulators, including proline, glutamic acid, and leucine-rich protein 1 (PELP1), are implicated in breast cancer progression. The lack of small molecules that inhibits PELP1 represents a major knowledge gap. Here, using a yeast-two-hybrid screen, we identified novel peptide inhibitors of PELP1 (PIP). Biochemical assays demonstrated that one of these peptides, PIP1, directly interacted with PELP1 to block PELP1 oncogenic functions. Computational modeling of PIP1 revealed key residues contributing to its activity and facilitated the development of a small-molecule inhibitor of PELP1, SMIP34, and further analyses confirmed that SMIP34 directly bound to PELP1. In breast cancer cells, SMIP34 reduced cell growth in a dose-dependent manner. SMIP34 inhibited proliferation of not only wild-type (WT) but also mutant (MT) ER+ and therapy-resistant breast cancer cells, in part by inducing PELP1 degradation via the proteasome pathway. RNA sequencing analyses showed that SMIP34 treatment altered the expression of genes associated with estrogen response, cell cycle, and apoptosis pathways. In cell line–derived and patient-derived xenografts of both WT and MT ER+ breast cancer models, SMIP34 reduced proliferation and significantly suppressed tumor progression. Collectively, these results demonstrate SMIP34 as a first-in-class inhibitor of oncogenic PELP1 signaling in advanced breast cancer. Significance: Development of a novel inhibitor of oncogenic PELP1 provides potential therapeutic avenues for treating therapy-resistant, advanced ER+ breast cancer.

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 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.

Transcription factor AP‐2 gamma (TFAP2C) is a known regulator of the estrogen receptor, and high expression of TFAP2C is associated with therapy resistance. This study identified PELP1 as a TFAP2C interacting protein. PELP1 is essential for optimal TFAP2C transcriptional functions. TFAP2C interactions with PELP1 confer a growth advantage to breast cancer (BC) cells by activating an oncogenic RET signaling thus contributing to BC progression and therapy resistance., 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 in vivo. 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.

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.

Background: Triple-negative breast cancer (TNBC) is a heterogeneous disease. TNBC lacks targeted therapies and represents a disproportional share of the breast cancer (BC) mortality rate. Histone deacetylase inhibitors (HDACIs) are emerging as promising multifunctional agents in TNBC to elicit cytotoxic actions. Recent studies have shown that cancer cells elucidate feedback activation of leukemia inhibitory factor receptor (LIFR) which in turn curtails response to HDACIs. We developed a first-in-class inhibitor of LIFR, EC359 that directly interacts with LIFR and effectively blocks LIFR downstream signaling. Here, we examined whether the novel LIFR inhibitor, EC359, has the ability to counteract negative effects of LIFR signaling to enhance HDACIs therapeutic efficacy in the treatment of TNBC. Methods: We tested multiple HDACIs currently in clinical trials including vorinostat, panobinostat, romidepsin, and givinostat using multiple TNBC models. The effect of combination therapy of HDACIs and EC359 on TNBC cell viability and invasion was examined using MTT assays and matrigel invasion assays respectively. The efficacy of combination therapy on cell survival and apoptosis was determined using clonogenic assays and Caspase 3/7 assays, respectively. Mechanistic studies were performed using Western blotting, qRT-PCR, and reporter gene assays. The efficacy of combination therapy in vivo was examined using Xenograft, patient-derived xenograft (PDX), and patient-derived explant (PDEX) models. Results: We demonstrated that the treatment of TNBC models with HDACIs increased the expression of LIFR. Immunohistochemistry analyses of breast tumors using tissue microarrays revealed significant expression of LIFR in TNBC samples. Knockdown of LIFR or treatment with a small molecule inhibitor of LIFR (EC359) significantly enhanced the efficacy of HDACIs in reducing cell viability, colony formation ability, and invasiveness as well as promoted apoptosis compared to monotherapy of HDACIs or EC359 in TNBC cell lines. Mechanistic studies, reporter gene assays and biochemical studies using multiple TNBC models exhibited activation of the LIFR signaling pathway upon HDACIs treatment but was attenuated by EC359+HDACI combination therapy. Treatment of human breast tumors utilizing PDEX assays showed that EC359 enhanced the ability of HDACIs to decrease the proliferation (Ki-67 positivity) compared to monotherapy. Furthermore, using TNBC xenografts and PDX models, we demonstrated that EC359 treatment enhanced the ability of HDACIs to reduce in vivo tumor growth compared to monotherapy. Conclusions: Our results suggest that the combination therapy of HDACIs and EC359 provides greater therapeutic efficacy than monotherapy. In addition, treatment with EC359 can overcome the feedback activation of LIFR currently observed in the treatment of TNBC with HDACIs. Citation Format: Suryavathi Viswanadhapalli, Mengxing Li, Bindu Santhamma, Uday P Pratap, Yiliao Luo, Junhao Liu, Kristin A Altwegg, Xiaonan Li, Hui Yan, Zhenming Xu, Andrew Brenner, Gangadhara R Sareddy, Rajeshwar R Tekmal, Hareesh B Nair, Klaus J Nickisch, Ratna K Vadlamudi. Targeting LIFR enhances the activity of HDAC inhibitors for the treatment of triple negative 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 P3-11-08.

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.

Background Methyltransferase 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. Methods We 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. Results RNA-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. Conclusions This 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.

Histone deacetylase inhibitors (HDACi) are identified as novel therapeutic agents, however, recent clinical studies suggested that they are marginally effective in treating triple negative breast cancer (TNBC). Here, we show that first-in-class Leukemia Inhibitory Factor Receptor (LIFRα) inhibitor EC359 could enhance the therapeutic efficacy of HDACi against TNBC. We observed that both targeted knockdown of LIFR with CRISPR or treatment with EC359 enhanced the potency of four different HDACi in reducing cell viability, cell survival, and enhanced apoptosis compared to monotherapy in TNBC cells. RNA-seq studies demonstrated oncogenic/survival signaling pathways activated by HDACi were attenuated by the EC359 + HDACi therapy. Importantly, combination therapy potently inhibited the growth of TNBC patient derived explants, cell derived xenografts and patient-derived xenografts in vivo. Collectively, our results suggest that targeted inhibition of LIFR can enhance the therapeutic efficacy of HDACi in TNBC., Li, Viswanadhapalli et al utilized multiple in vitro, ex vivo and in vivo models of TNBC to investigate LIFR inhibition. The authors reported that HDAC inhibition in TNBC cells led to an increase of LIFR expression and over activation of the downstream signaling elements (e.g., STAT3, mTOR, AKT), enhancing the aggressive potential of TNBC cells, and an LIFR inhibitor, EC359, synergistically enhanced the efficacy of HADC inhibitors in suppressing TNBC in vitro and in vivo.

Endometrial cancer (EC) is the fourth most common cancer in women. Advanced-stage EC has limited treatment options with a poor prognosis. There is an unmet need for the identification of actionable drivers for the development of targeted therapies in EC. Leukemia inhibitory factor receptor (LIFR) and its ligand LIF play a major role in cancer progression, metastasis, stemness, and therapy resistance. However, little is known about the functional significance of the LIF/LIFR axis in EC progression. In this study using endometrial tumor tissue arrays, we identified that expression of LIF, LIFR is upregulated in EC. Knockout of LIFR using CRISPR/Cas9 in two different EC cells resulted in a significant reduction of their cell viability and cell survival. In vivo studies demonstrated that LIFR-KO significantly reduced EC xenograft tumor growth. Treatment of established and primary patient-derived EC cells with a novel LIFR inhibitor, EC359 resulted in the reduction of cell viability with an IC50 in the range of 20–100 nM and induction of apoptosis. Further, treatment with EC359 reduced the spheroid formation of EC cancer stem cells and reduced the levels of cancer stem cell markers SOX2, OCT4, NANOG, and Axin2. Mechanistic studies demonstrated that EC359 treatment attenuated the activation of LIF-LIFR driven pathways, including STAT3 and AKT/mTOR signaling in EC cells. Importantly, EC359 treatment resulted in a significant reduction of the growth of EC patient-derived explants ex vivo, EC cell line-derived xenografts, and patient-derived xenografts in vivo. Collectively, our work revealed the oncogenic potential of the LIF/LIFR axis in EC and support the utility of LIFR inhibitor, EC359, as a novel targeted therapy for EC via the inhibition of LIF/LIFR oncogenic signaling.

Leukemia inhibitory factor receptor (LIFR) and its ligand LIF play a critical role in cancer progression, metastasis, stem cell maintenance, and therapy resistance. Here, we describe a rationally designed first-in-class inhibitor of LIFR, EC359, which directly interacts with LIFR to effectively block LIF/LIFR interactions. EC359 treatment exhibits antiproliferative effects, reduces invasiveness and stemness, and promotes apoptosis in triple-negative breast cancer (TNBC) cell lines. The activity of EC359 is dependent on LIF and LIFR expression, and treatment with EC359 attenuated the activation of LIF/LIFR-driven pathways, including STAT3, mTOR, and AKT. Concomitantly, EC359 was also effective in blocking signaling by other LIFR ligands (CTF1, CNTF, and OSM) that interact at LIF/LIFR interface. EC359 significantly reduced tumor progression in TNBC xenografts and patient-derived xenografts (PDX), and reduced proliferation in patient-derived primary TNBC explants. EC359 exhibits distinct pharmacologic advantages, including oral bioavailability, and in vivo stability. Collectively, these data support EC359 as a novel targeted therapeutic that inhibits LIFR oncogenic signaling. See related commentary by Shi et al., p. 1337

Background: Leukemia inhibitory factor (LIF) and its receptor LIFR are over-expressed in multiple solid tumors and play a key role in tumor growth, progression, and resistance to standard anti-cancer treatments. Triple-negative breast cancer (TNBC) lacks targeted therapies and represents a disproportional share of breast cancer (BCa) mortality. TNBC exhibits autocrine stimulation of the LIF/LIFR axis and overexpression of LIF is associated with poorer relapse-free survival in BCa patients. LIF signaling also promotes maintenance of stem cells. Therefore, targeting the LIF/LIFR axis may have therapeutic utility in TNBC. Methods: We rationally designed a small organic molecule (EC359) that emulates the LIF/LIFR binding site and functions as a LIFR inhibitor from a library of compounds. In silico docking studies were used to identify the putative interaction of the EC359 and LIF/LIFR complex. Direct binding of EC359 to LIFR was confirmed using surface plasmon resonance (SPR) and microscale thermophoresis technique (MST) assays. In vitro activity was tested using Cell-Titer Glo, MTT, invasion, and apoptosis assays. Mechanistic studies were conducted using Western blot, reporter gene assays, and RNA-seq analysis. Xenograft, patient-derived xenograft (PDX), and patient-derived explant (PDEX) models were used for preclinical evaluation and toxicity. Results: Molecular docking studies showed that EC359 interacts at the LIF/LIFR binding interface. SPR and MST studies confirmed direct interaction of EC359 to LIFR. EC359 reduced the growth of TNBC cells with high potency (IC50 50-100nM) and promoted apoptosis. Further, EC359 treatment reduced invasion and stemness of TNBC cells. EC359 activity is dependent on the expression levels of LIFR and showed little or no activity on TNBC cells that have low levels of LIFR or ER+ve BCa cells. Further, EC359 significantly reduced the viability of cisplatin and taxane-resistant TNBC cells and enhanced the efficacy of HDAC inhibitors. Mechanistic and biochemical studies showed that EC359 interacts with LIFR and effectively blocking LIF/LIFR interactions. EC359 also blocked LIFR interactions with other LIFR ligands such as oncostatin M, ciliary neurotrophic factor, and cardiotrophin-1. EC359 treatment attenuated the activation of LIF/LIFR driven pathways including STAT3, mTOR, AKT, and MAPK. RNA-seq analysis identified regulation of apoptosis as one of the important pathway modulated by EC359. In TNBC xenograft and PDX assays, EC359 significantly reduced tumor progression. Further, using human primary BCa PDEX cultures, we demonstrated that EC359 has the potential to substantially reduce the proliferation of human BCa. Pharmacologically, EC359 exhibited high oral bioavailability and long half-life with a wide therapeutic window. Conclusions: EC359 is a novel targeted therapeutic agent that inhibits LIF/LIFR oncogenic signaling in TNBC via a unique mechanism of action. EC359 has the distinct pharmacologic advantages of oral bioavailability, in vivo stability, and is associated with minimal systemic side effects. (DOD BCRP grant #BC170312) Citation Format: Viswanadhapalli S, Luo Y, Sareddy GR, Santhamma B, Zhou M, Li M, Pratap UP, Altwegg KA, Li X, Srinivasan U, Ma S, Chang A, Riveros AC, Zhang KY, Dileep KV, Pan X, Murali R, Bajda M, Raj G, Brenner A, Manthati V, Rao M, Tekmal RR, Nair HB, Nickisch KJ, Vadlamudi RK. Development of a first-in-class small molecule inhibitor (EC359) targeting oncogenic LIF/LIFR signaling for the treatment of triple negative 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 P2-06-02.

A significant proportion of estrogen receptor positive breast cancers (ER+BC) will initially respond to treatment, but many eventually develop therapy resistance (TR-BC), and progress to incurable metastases. Oncogenic ER coregulators overexpressed in BC can contribute to constitutive, ligand-independent and ligand-dependent signaling which drives growth, resistance to therapy and metastasis. Proline-, glutamic acid, and leucine-rich protein 1 (PELP1), is a known coregulator that plays a critical role in ER oncogenic functions. Its expression is deregulated in BC and is a prognostic indicator of poor BC survival. The lack of a small molecule inhibitor that directly targets PELP1 represents a major knowledge gap. Therefore, we conducted a large scale peptide library screening and identified novel Peptide Inhibitor of PELP1 (PIP1). We demonstrated that PIP1 directly interacts with PELP1, promotes its degradation and has the potential to block PELP1 oncogenic functions in vitro. Using innovative peptidomimetic technology, we modeled PIP1 and synthesized several derivatives as Small Molecule Inhibitors of PELP1 (SMIPs). Using MTT assay and multiple BC cell lines, we identified a lead compound, SMIP34 with an IC50 of 5-10µM and with minimal effect on human mammary epithelial cells. SMIP34 in vitro activity was assessed by colony formation and Matrigel invasion assays. Knockdown of PELP1 using shRNA in BC cells significantly reduced SMIP34 activity, indicating target specificity. Further, MST and biotin-SMIP34 pulldown confirmed direct binding of SMIP34 to PELP1. Using ER+WT, mtER (mutant ER), and TR-BC cell lines we demonstrated that SMIP34 exhibits antiproliferative effects and reduces invasiveness. Mechanistic studies using Western blot analysis confirmed that SMIP34 binding to PELP1 contributes to its degradation via the proteasome pathway. Thus MG132 treatment attenuated SMIP34 mediated degradation. RTqPCR analyses confirmed SMIP34 treatment reduced expression of PELP1 target genes. RNAseq analyses showed SMIP34 treatment altered the expression of genes associated with Estrogen response, Cell cycle and Apoptosis pathways. Cell cycle analyses revealed SMIP34 treatment promoted S phase arrest of BC cell lines. Using ER+WT, mtER, and PDX tumor tissues ex vivo, we demonstrated that SMIP34 significantly decreased tumor proliferation as measured by Ki67 staining. Further, SMIP34 (20mg/kg/IP) treatment in vivo significantly reduced tumor progression in mouse models of ER+WT, mtER, and mtER patient-derived xenograft BC. Our results using in vitro, ex vivo, and in vivo models showcase SMIP34 as a first-in-class inhibitor of oncogenic PELP1 signaling and may serve as a potential therapeutic molecule for treating ER+, mtER, and TR-BC. Supported by NIH 1F31CA257298 (KA) and VA I01BX004545 (RV). Citation Format: Kristin Ann Altwegg, Monica Mann, Dimple Chakravarty, Zexuan Liu, Junhao Liu, Uday P. Pratap, Behnam Ebrahimi, John R. Sanchez, Ben H. Park, Hariprasad Vankayalapati, Gangadhara R. Sareddy, Suryavathi Viswanadhapalli, Ratna K. Vadlamudi. A novel small molecule targeting oncogenic PELP1 demonstrates anti-tumor activity in wild-type and mutant ER-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 648.

Background Glioblastomas (GBMs) are the most lethal primary brain tumors. Estrogen receptor β (ESR2/ERβ) function as a tumor suppressor in GBM, however, ERβ expression is commonly suppressed during glioma progression. In this study, we examined whether drugs that reverse epigenetic modifications will enhance ERβ expression and augment ERβ agonist-mediated tumor suppression. Methods We tested the utility of epigenetic drugs which act as an inhibitor of histone deacetylases (HDACs), histone methylases, and BET enzymes. Mechanistic studies utilized RT-qPCR, chromatin immunoprecipitation (ChIP), and western blotting. Cell viability, apoptosis, colony formation, and invasion were measured using in vitro assays. An orthotopic GBM model was used to test the efficacy of in vivo. Results Of all inhibitors tested, HDACi (panobinostat and romidepsin) showed the potential to increase the expression of ERβ in GBM cells. Treatment with HDACi uniquely upregulated ERβ isoform 1 expression that functions as a tumor suppressor but not ERβ isoform 5 that drives oncogenic functions. Further, combination therapy of HDACi with the ERβ agonist, LY500307, potently reduced cell viability, invasion, colony formation, and enhanced apoptosis. Mechanistic studies showed that HDACi induced ERβ is functional, as it enhanced ERβ reporter activities and ERβ target genes expression. ChIP analysis confirmed alterations in the histone acetylation at the ERβ and its target gene promoters. In orthotopic GBM model, combination therapy of panobinostat and LY500307 enhanced survival of tumor-bearing mice. Conclusions Our results suggest that the combination therapy of HDACi and LY500307 provides therapeutic utility in overcoming the suppression of ERβ expression that commonly occurs in GBM progression.

Objectives: The incidence of endometrial cancer (EC) is estimated to increase by 1-2% annually. More than half of all EC diagnoses are currently attributable to obesity, which is an independent risk factor for this cancer. Obesity is suspected to function as a potent inducer of leukemia inhibitory factor (LIF), the most pleiotropic member of the interleukin-6 family of cytokines. Our recent studies utilizing public databases revealed high levels of LIF and its receptor LIFR are associated with poor survival in EC patients (KM plotter). The objective of this study is to determine whether alterations in the LIF/LIFR signal cascade occur in EC cells under conditions of adiposity; and whether this pathway could be a target for therapy. Methods: All patient samples, EC tissue, serum, and adipose tissue, were collected through the UT Health San Antonio tumor registry under IRB approval. Expression of LIF in EC patient samples was analyzed using immunohistochemistry (IHC) using a UT Health generated tumor tissue array (TMA) and scored by UT Health Pathology. Primary EC tissue derived cells and established EC cell lines were treated with primary adipocyte conditioned medium and non-obese patient serum or obese patient serum. The effect of adiposity on EC cells was determined using MTT cell viability assay and colony formation assay, in addition to utilization of organoids derived from primary EC tissue. Mechanistic studies were conducted using Western blotting, RT-qPCR, and reporter assays thus confirming the activation of LIF/LIFR downstream pathways including STAT3, mTOR, Akt, and MAPK. The recently developed LIFR inhibitor, EC359, was used to elucidate the therapeutic utility of targeting the enhanced LIF/LIFR pathway generated by high adiposity. Results: Treatment of established and primary EC cells with adipose environment increased the expression of LIFR protein, enhanced LIFR reporter activity, and increased cellular proliferation. Western blot and RT-qPCR analyses confirmed that increased expression of LIFR correlated with enhanced downstream LIFR signaling and subsequent activation of LIFR target genes. Treatment of EC cells with the LIFR inhibitor EC359 significantly reduced cell viability under adipose conditions. EC359 treatment also decreased adipose environment mediated organoid growth. IHC results using TMA that contained normal tissue (n=33) and EC tumors (n=45) with BMI >30 showed significantly increased expression of LIF in EC compared to normal endometrium. Further, LIF expression was upregulated in EC from obese patients compared to non-obese EC control patients. Conclusions: Collectively, our results suggest that increased adiposity contributes to accelerated EC cell growth via upregulation of the LIF/LIFR pathway. The LIF/LIFR axis represents a potential therapeutic target for adiposity driven EC and the LIFR inhibitor EC359 may be useful in modulating LIF/LIFR signaling in obese EC patients.

Background: Endometrial cancer (EC) is the fourth most common cancer in women. Approximately 80% of EC belong to the endometroid-EC subtype and are driven by estrogen signaling. Advanced-stage EC has limited treatment options with poor prognosis. There is an urgent need for the identification of actionable drivers as new targets for treating advance stage EC. Leukemia inhibitory factor receptor (LIFR) and its ligand LIF plays a major role in cancer progression, metastasis, stemness, and therapy resistance. Published and our preliminary data suggest a critical role of the LIF-LIFR signaling axis in EC progression. The objective of this study is to test the utility of targeting the LIF/LIFR axis using a novel LIFR inhibitor, EC359. Methods: We used multiple established and primary EC cells to test the utility LIFR inhibitor, EC359 in treating EC. CRISPR/Cas9 system was used to generate LIFR KO EC cells. In vitro activity was tested using Cell-Titer Glo, MTT, invasion, and apoptosis assays. Mechanistic studies were conducted using Western blot, reporter gene assays, and RNA-seq analysis. EC cell-derived xenograft (CDX) and patient-derived explant (PDEX) models were used for preclinical evaluation and toxicity. Results: EC359 treatment of seven EC cells showed anti-proliferative effects in MTT cell viability assays with an IC50 of 25-100 nM. Further, EC359 treatment reduced invasiveness, stemness, and promoted apoptosis of EC cells. The activity of EC359 is dependent on LIF/LIFR expression in EC cells. CRISPR mediated knockout of LIFR significantly abolished EC359 activity. In vivo xenograft studies using Ishikawa-vector or LIFR-KO cells demonstrated that LIFR-KO significantly reduced EC tumor growth, and tumor weights. Further, EC359 treatment attenuated the activation of LIF/LIFR driven pathways, including STAT3, AKT-mTOR signaling. Mechanistic studies using RNA-seq revealed that EC359 significantly upregulated 213 genes and down regulated 126 genes. Pathway analyses of differential genes revealed enrichment in the apoptotic pathways upon EC359 treatment. EC359 (5mg/kg body weight) treatment significantly reduced CDX tumor progression and reduced proliferation in PDEX models. Conclusions: Collectively, these data support EC359 as a novel targeted therapy for EC by inhibiting LIF/LIFR oncogenic signaling pathway. Citation Format: Weiwei Tang, Kumaraguruparan Ramasamy, Sureshkumar M. Pillai, Bindu Santhamma, Swapna Konda, Prabhakar P. Vekata, Logan Blankenship, Junhao Liu, Zexuan Liu, Kristin A. Altwegg, Behnam Ebrahimi, Uday P. Pratap, Xiaonan Li, Edward Kost, Gangadhara R. Sareddy, Ratna K. Vadlamudi, Hareesh B. Nair, Rajeshwar R. Tekmal, Suryavathi Viswanadhapalli. Therapeutic targeting of endometrial cancer with novel LIFR inhibitor EC359 [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 1253.

Background: Breast cancer (BC) is composed of distinct molecular subtypes, such as ER+ BC and triple negative BC (TNBC). Development of novel effective therapies for patients with TNBC remains the highest unmet need in patient treatment and survivorship. PELP1 plays an essential role in several pathways including hormonal signaling, cell cycle progression, ribosomal biogenesis, and DNA damage response. PELP1 expression is an independent prognostic predictor of shorter BC-specific survival and an independent prognostic factor for predicting poor survivorship in TNBC patients. Recently, we generated a small molecule inhibitor of PELP1 (SMIP34) that binds and inhibits PELP1 oncogenic signaling. The objective of this study is to test the utility of SMIP34 as a novel therapeutic for treating TNBC. Methods: We have selected seven TNBC model cell lines (BT549, MDA-MB-453, MDA-MB-231, MDA-MB-468, SUM-159, HCC1806, and HCC1937) and one human mammary epithelial (HMEC) cell line in this study. In vitro activity was assessed using Cell Titer Glo, MTT, colony formation, and matrigel invasion assays. Mechanistic studies were conducted using Western blot, reporter gene assays, and RNA-seq. Xenograft tumor-derived explant (XDEX) assays and patient-derived tumor explant (PDEX) assays were used for preclinical evaluation. Results: Using a panel of BC model cell lines, we found that SMIP34 treatment reduced cell viability with an IC50 of 3-8µM with no activity in HMEC cells. Knockdown of PELP1 using shRNA in BC cells significantly reduced SMIP34 activity, confirming its target specificity. We confirmed the physical interaction of SMIP34 to PELP1 using biotin-SMIP34 and MST assays. SMIP34 treatment significantly reduced the invasiveness and colony formation of TNBC cell lines. Mechanistic studies using Western blot analysis confirmed that SMIP34 binding to PELP1 contributes to its degradation. Further, RTqPCR analyses confirmed SMIP34 treatment reduced expression of PELP1 target genes. Western analyses confirmed SMIP34 treatment significantly reduced known PELP1 downstream signaling. Mechanistic studies using global RNA-seq identified that SMIP34 treatment alters known PELP1 modulated pathways (ribosomal biogenesis). Using MDA-MB-231 xenograft and PDX tumor tissues in explant assays, we demonstrated that SMIP34 significantly decreased tumor proliferation as measured by Ki67 staining. Accordingly, in xenograft models, SMIP34 (20mg/kg/IP) treatment resulted in a significant reduction in tumor volume compared to vehicle control. Conclusion: Our results using in vitro, ex vivo, and in vivo studies demonstrated that the PELP1 inhibitor SMIP34 has therapeutic efficacy against TNBC. 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, Hariprasad Vankayalapati, Ratna K. Vadlamudi. Evaluation of a novel PELP1 inhibitor for treatment of triple negative breast cancer [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 1333.

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 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.

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.

Background: Breast cancer (BC) is the most commonly diagnosed cancer and the second leading cause of cancer death in American women. BC is composed of distinct molecular subtypes, such as ER positive BC (ER+) and triple negative BC (TNBC). Development of novel effective therapies for patients with therapy resistant BC (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 BC, is a prognostic indicator of poorer BC survival, and its deregulation contributes to TR-BC. The objective of this study is characterization of a small molecule inhibitor of PELP1 (SMIP34) as novel therapeutic for treating advanced BC. Methods: Yeast two-hybrid screening was used to identify PELP1 Inhibitory Peptide 1 (PIP1). Direct binding of PIP1 to PELP1 was confirmed using biotin pull-down assays and inhibition of BC proliferation confirmed using MTT assays. Hit-Ligand interaction site with PIP1 hot spot residues based on 3D alignment and morphology was used to generate a library of small chemical molecules that function as peptidomimetics of PIP1. In vitro activity was assessed using CellTiter Glo, MTT, colony formation and matrigel invasion assays in multiple BC models. Mechanistic studies were conducted using Western blot, and reporter gene assays. Xenograft and patient derived explant (PDEX) assays were used for preclinical evaluation. Results: Screening utilizing MTT assays lead to the selection of SMIP34 as an inhibitor of PELP1. SMIP34 treatment reduced cell viability at an IC50 of 3-10µM in a panel of BC cells. Additionally, SMIP34 showed no activity in human mammary epithelial cells. Knockdown of PELP1 in BC cells significantly reduced the SMIP34 activity, confirming target specificity. Furthermore, SMIP34 treatment significantly reduced the invasiveness and colony formation of TR-BC and TNBC cells. Mechanistic studies using Western blot analysis confirmed that SMIP34 binding to PELP1 contributes to its degradation. In combination studies, SMIP34 displayed synergy and enhanced the efficacy of current chemotherapeutics Cisplatin and Paclitaxel. In PDEX assays, SMIP34 significantly decreased tumor proliferation as measured by Ki67 staining. In xenograft models, SMIP34 (10mg/kg/s.c.) treatment resulted in significant reduction in tumor volume compared to vehicle control. 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 (SMIP34) displaying therapeutic efficacy against TR-BC and TNBC in vitro, ex vivo, and in vivo. Supported by CPRIT Predoctoral Fellowship CPRIT RTA; RP170345 (K.A. Altwegg) and VA grant I01BX004545 (R.K.V) Citation Format: Kristin Ann Altwegg, Suryavathi Viswanadhapalli, Monica Mann, Uday P. Pratap, Mengxing Li, Junhao Liu, Yiliao Luo, Gangadhara R. Sareddy, Hariprasad Vankayalapati, Ratna K. Vadlamudi. Characterization of small molecule inhibitors of PELP1 for treating advanced breast cancer [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 6403.

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.

Background: Ovarian cancer (OCa) is the deadliest gynecologic cancer. Recent studies suggest that OCa cells express estrogen receptor beta (ESR2), which functions as a tumor suppressor. However, ESR2 expression decreases during tumor progression and under the selection pressure of chemotherapy; this decrease occurs via epigenetic mechanisms. The lysine-specific histone demethylase 1A (KDM1A), an epigenetic regulator, is overexpressed in OCa. We reason that agents that restore the expression/functions of ESR2 may provide a novel therapeutic opportunity for suppression of OCa. In this study we tested the hypothesis that KDM1A suppresses ESR2 expression/functions and that inhibition of KDM1A potentiates ESR2 mediated tumor suppression. Methods: We examined the utility of combination therapy of KDM1A inhibitor and ESR2 agonist on established and patient derived OCa cells using cell viability, survival, apoptosis and invasion assays. KDM1A knockout (KDM1A-KO) cells were generated using the CRISPR/Cas9 system. Mechanistic studies were conducted using RNA-seq, ERE-Luc reporter assays, RT-qPCR, co-IP, ChIP and Western blot analysis. The efficacy of combination therapy on primary ovarian tumors was examined using patient derived explant (PDEx) models. In vivo efficacy of KDM1A inhibitor and ESR2 agonist was studied using orthotopic OCa and patient derived xenograft models. Results: Analysis of the gene expression profiles of OCa patients showed that KDM1A negatively correlates with ESR2. Combination of KDM1A inhibitor NCD-38 and ESR2 agonist LY500307 synergistically reduced cell viability, survival and invasion and increased apoptosis of established and patient derived primary OCa cells. Inhibition or knockout of KDM1A sensitized OCa cells to ESR2 agonist treatment and significantly increased the expression of ESR2 and its target genes. Further, we found that KDM1A is recruited to the ESR2 0N promoter and interacts with ESR2. In addition, inhibition of KDM1A increased the active histone methylation mark H3K4-me2 at the ESR2 0N promoter. RNA-seq analysis demonstrated that ESR2 agonist treatment resulted in modulation of pathways related to cell cycle, apoptosis and DNA damage. Importantly, combination treatment significantly reduced the tumor growth in orthotopic and patient derived xenograft models and reduced the proliferation of OCa cells in PDEx models. Conclusions: Our results demonstrate that KDM1A inhibition enhances ESR2 expression/functions and combination therapy of KDM1A inhibitor and ESR2 agonist is an attractive therapy for treating OCa. Citation Format: Prabhakar Pitta Venkata, Bridgitte E. Palacios, Yihong Chen, Salvador Alejo, Yi He, Ilanna Loeffel, Uday Pratap, Kristin Altwegg, Suryavathi Viswanadhapalli, Takayoshi Suzuki, Rajeshwar Tekmal, Edward Kost, Gangadhara Sareddy. KDM1A inhibition activates estrogen receptor beta pathway to suppress ovarian cancer progression [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 1764.

Background: Triple-negative breast cancer (TNBC) lacks targeted therapies and represents a disproportional share of the breast cancer (BC) mortality rate. TNBC exhibits autocrine stimulation of the LIF/LIFR axis and overexpression of LIF is associated with poorer relapse-free survival in BC patients. Histone deacetylase inhibitors (HDACIs) are emerging as promising multifunctional agents in TNBC to elicit cytotoxic actions. Recent studies have shown that cancer cells elicit feedback activation of leukemia inhibitory factor receptor (LIFR) which in turn curtails response to HDACIs. We developed a first-in-class inhibitor of LIFR, EC359 that directly interacts with LIFR and effectively blocks LIFR downstream signaling. The objective of this study is to examine the therapeutic efficacy of combination therapy using preclinical and patient-derived xenograft (PDX) models. Methods: We tested utility of combination therapy using multiple HDACIs that are currently in clinical trails along with EC359. The effect of combination therapy was evaluated using MTT, invasion, colony formation, and Caspase3/7 assays. Mechanistic studies were performed using Western blotting, qRT-PCR, and STAT3 reporter assays. The efficacy of combination therapy in vivo was examined using xenograft, PDX, and patient-derived explant (PDEx) models. Results: Immunohistochemical analyses of breast tumors using tissue microarrays revealed significant expression of LIFR in TNBC tissues. Treatment of TNBC model cells with four different HDACIs increased the expression of LIFR. LIFR inhibitor EC359 at nM concentration is additive to HDACIs in reducing cell viability. Knockdown of LIFR or treatment with EC359 significantly enhanced the efficacy of HDACIs in reducing the cell viability, colony formation ability, and invasiveness as well as promoted apoptosis compared to monotherapy in TNBC model cells. On the contrary, treatment with STAT3 inhibitor requires µM concentrations to reduce the cell viability of TNBC cells and is not additive to HDACIs. Mechanistic studies utilizing STAT3 reporter gene assays and biochemical studies using multiple TNBC model cells exhibited activation of the LIFR signaling pathway upon HDACIs treatment but was attenuated by EC359 therapy. Treatment of human TNBC utilizing PDEx assays showed that EC359 enhanced the ability of HDACIs to decrease proliferation (Ki-67 positivity) compared to monotherapy. Using TNBC xenografts and PDX models, we demonstrated that EC359 treatment enhanced the ability of HDACIs to reduce in vivo tumor growth compared to monotherapy. Conclusions: Our results suggest that the combination therapy of HDACIs and EC359 provides therapeutic utility in overcoming the limitation of feedback activation of LIFR observed in the treatment of HDACIs in treating TNBC. Supported by DOD BCRP grant W81XWH-18-1-0016 (R.K. Vadlamudi; K.J. Nickisch) Citation Format: Suryavathi Viswanadhapalli, Mengxing Li, Bindu Santhamma, Uday P. Pratap, Yiliao Luo, Junhao Liu, Kristin A. Altwegg, Xiaonan Li, Ahmed Gulzar, Hui Yan, Zhenming Xu, Andrew Brenner, Gangadhara R. Sareddy, Manjeet K. Rao, Rajeshwar R. Tekmal, Hareesh B. Nair, Klaus J. Nickisch, Ratna K. Vadlamudi. Novel combination therapy for treating TNBC using LIFR and HDAC Inhibitors [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 562.

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.

Background: Leukemia inhibitory factor receptor (LIFR) and its ligand LIF play a major critical role in cancer progression, metastasis, stem cell maintenance, and therapy resistance. Recent studies in breast cancer have shown that feedback activation of LIFR limits response to histone deacetylase (HDAC) inhibitors and induce resistance. We rationally designed a small molecule (EC359) that emulates the LIF-LIFR binding site and functions as a LIFR inhibitor from a library of compounds. Here, we tested the utility of EC359 as a monotherapy and to effectively block LIF-LIFR interactions in overcoming resistance to HDAC inhibitors. Methods: We have used multiple triple negative breast cancer (TNBC) models that represent all six types of TNBC. In vitro activity was tested using Cell-Titer Glo, MTT, invasion, and apoptosis assays. Mechanistic studies were conducted using western blot, reporter gene assays, and RNA-seq analysis. Xenograft, patient-derived xenograft (PDX), and patient-derived explant (PDeX) models were used for preclinical evaluation and toxicity. Results: EC359 treatment exhibited anti-proliferative effects, reduced invasiveness and stemness, and promoted apoptosis in all six TNBC cell lines. The activity of EC359 is dependent on LIF and LIFR expression and CRISPR mediated knockdown of LIFR significantly abolished EC359 activity. Treatment with EC359 attenuated the activation of LIF-LIFR driven pathways including STAT3, mTOR, and AKT. EC359 significantly reduced tumor progression in TNBC xenografts, PDX models and reduced proliferation in patient derived primary TNBC explants. In MTT based cell viability assays, addition of EC359 enhanced efficacy of SAHA compared to monotherapy of SAHA. In clonogenic survival assays, EC359 significantly enhanced ability of SAHA to reduce the colony formation compared to monotherapy. Mechanistic studies using three different TNBC models using western blot analysis and reporter gene assays confirmed activation of LIFR signaling pathway upon SAHA treatment and its blockage by EC359. Treatment of TNBC PDX explants with EC359 enhanced ability of SAHA to substantially decrease the proliferation (Ki-67 positivity) compared to monotherapy treated tumors. Conclusions: Collectively, these data support EC359 as a novel targeted therapeutic that inhibits LIFR oncogenic signaling as a monotherapy or in combination with HDAC inhibitors. Citation Format: Suryavathi Viswanadhapalli, Mengxing Li, Yiliao Luo, Gangadhara R Sareddy, Bindu Santhamma, Mei Zhou, Shihong Ma, Rajni Sonavane, Uday P. Pratap, Kristin A. Altwegg, Annabel Chang, Alejandra Chávez-Riveros, Kalarickal V. Dileep, Kam Y. Zhang, Marek Bajda, Ganesh V. Raj, Andrew Brenner, Vijaya Manthati, Manjeet Rao, Rajeshwar R. Tekmal, Hareesh B. Nair, Klaus J. Nickisch, Ratna K. Vadlamudi. Therapeutic utility of EC359 for targeting oncogenic LIFR signaling in triple negative breast cancer [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 4716.