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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.

Diversity of participants in biomedical research with respect to race, ethnicity, and biological sex is crucial, particularly given differences in disease prevalence, recovery, and survival rates between demographic groups. The objective of this systematic review was to report on the demographics of neuroimaging studies using magnetic resonance imaging (MRI). The Web of Science database was used and data collection was performed between June 2021 to November 2021; all articles were reviewed independently by at least two researchers. Articles utilizing MR data acquired in the United States, with n ≥ 10 human subjects, and published between 2010–2020 were included. Non-primary research articles and those published in journals that did not meet a quality control check were excluded. Of the 408 studies meeting inclusion criteria, approximately 77% report sex, 10% report race, and 4% report ethnicity. Demographic reporting also varied as function of disease studied, participant age range, funding, and publisher. We anticipate quantitative data on the extent, or lack, of reporting will be necessary to ensure inclusion of diverse populations in biomedical research.

Triple-negative breast cancer (TNBC) has a poor clinical outcome, due to a lack of actionable therapeutic targets. Herein we define lysosomal acid lipase A (LIPA) as a viable molecular target in TNBC and identify a stereospecific small molecule (ERX-41) that binds LIPA. ERX-41 induces endoplasmic reticulum (ER) stress resulting in cell death, and this effect is on target as evidenced by specific LIPA mutations providing resistance. Importantly, we demonstrate that ERX-41 activity is independent of LIPA lipase function but dependent on its ER localization. Mechanistically, ERX-41 binding of LIPA decreases expression of multiple ER-resident proteins involved in protein folding. This targeted vulnerability has a large therapeutic window, with no adverse effects either on normal mammary epithelial cells or in mice. Our study implicates a targeted strategy for solid tumors, including breast, brain, pancreatic and ovarian, whereby small, orally bioavailable molecules targeting LIPA block protein folding, induce ER stress and result in tumor cell death.

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.

Thermodynamics has strong predictive power for materials synthesis by identifying the stability region of target phases, but does not give explicit information about the relative competitiveness of undesired byproduct phases in synthesis. In this work, we propose a quantitative and computable measure to guide the selection of synthesis conditions. Defining thermodynamic competition as the difference in driving force between a target phase and its competing phases, we hypothesize that phase-pure synthesis becomes more likely when this thermodynamic competition is minimized. We systematically validate this hypothesis with two approaches: (1) we analyze large-scale solution synthesis procedures as text-mined from the literature, and show that experimentally-optimized synthesis conditions are near the predicted thermodynamic optimum point, and (2) direct experimental evaluation of synthesis in LiIn(IO3)4 and LiFePO4, which show that phase-pure synthesis occurs only when thermodynamic competition is minimized. Our work demonstrates that a quantitative assessment of thermodynamic competition is an effective descriptor for synthesis optimization and a promising tool for optimizing aqueous solution-based experimental synthesis conditions.

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.

Introduction: Estrogen receptor gene (ESR1) drives approximately 70-80% of breast cancer cases, which are primarily treated with endocrine therapies. Mutations in the ESR1 exclusively occur in the ligand binding domain (LBD) and are significantly enriched following endocrine therapy. These mutations retain the estrogen receptor (ER) expression thus conferring ligand-independent growth, which is a paramount contributor to endocrine therapy resistant disease. Worse progression-free survival is consistently associated with ESR1 mutation status; therefore, there is an unmet need for therapies against these mutations. To this end, we recently developed TX-245, a bis-benzamide molecule that hampers the function of the two most common ESR1 mutations - Y537S and D538G. Methods: TX-245 was evaluated in vitro using MTT cell proliferation assay and colony formation assay carried out in multiple ER+ breast cancer cells harboring wild type (WT) ESR1 or mutant (MT) ESR1. Approaches such as tumor spheroid cultures were also implemented to study the effect of TX-245 compared to selected SERDs/SERMs via CellTiter-Glo 3D viability assay. Mechanistic insights were obtained performing TR-FRET assay, fluorescence polarization assay, RNA-seq, ChIP-seq, western blot, and qRT-PCR. The therapeutic efficacy in vivo was evaluated using cell xenografts, patient derived xenografts (PDX), patient derived explants (PDE), and metastatic mouse models of MT-ER breast cancer. Results: TX-245 demonstrated greater anti-proliferative dose-dependent activity in models harboring WT and MT ESR1, including those resistant to fulvestrant and tamoxifen, when compared to other SERDs/SERMs. RNAseq data suggests that TX-245 dramatically alters the transcription of ERα-regulated genes, with repression of canonical estradiol-upregulated and induction of estradiol-repressed genes. TX-245 was able to antagonize ER-chromatin interaction and ER-transcriptional signaling in breast cancer cells driven by ESR1 mutation. TX-245 treatment downregulated ER protein expression and decreased expression of ER targeted genes (i.e., GREB and PGR) in multiple ER+ cell lines. TX-245 treatment as a single agent or in combination with palbociclib resulted in tumor regression in xenografts expressing WT, Y537S or D538G ESR1 mutation. PDX and PDE models consistently exhibited substantial tumor growth inhibition as well as decreased proliferation (Ki67 staining). Metastases studies using intracardiac and intratibial injection demonstrated that TX-245 inhibited the progression of established metastatic niches in mice injected with Y537S or D538G ER+ breast cancer cells. Conclusion: Our results indicate that TX-245 inhibits cell proliferation in cell lines that express WT or mutant ESR1, induce ER degradation, suppress ER-mediated signaling and reduce tumor burden in mouse models. These studies highlight the utility of TX-245 in targeting mutations in the ER-LBD. Citation Format: Karla Parra, Suryavathi Viswanadhapalli, Tanner Reese, Xihui Liu, JunHao Liu, Zexuan Liu, Tae-Kyung Lee, Chia Yuan Chen, Kara Kassees, Jung-Mo Ahn, Ratna Vadlamudi, Ganesh Raj, Carlos Roggero. Targeting the mutant estrogen receptor in metastatic breast 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 3440.

Time-resolved particle image velocimetry is used to study the flow characteristics of rotating U-shaped channels with different types of bend sections: one with both inner and outer walls square, one with an inner circular wall and an outer square wall, and one with both inner and outer walls circular. The rotation number varies from 0 to 0.25, at a Reynolds number of 11 500. The present work aims at providing a detailed insight of the flow field occurring within a rotating U-shaped channel, typically resembling internal cooling channel embedded into first stages of turbine blades in aeroengines. A validated numerical simulation is carried out to determine the flow mechanism. A proper orthogonal decomposition and the Ω-criterion vortex identification method are used to study the vortical distribution and flow characteristics. The results show that a bend with both inner and outer square walls produces corner vortices on the outside of the bend section, and both the separation vortex and reattachment vortex are larger than those of the other two geometrical configurations. In the channels with a circular inner wall of the bend, separation is delayed, and both the separation vortex and reattachment vortex are smaller. When both walls of the bend are square, the peak Reynolds shear stress is twice than when they are both circular. With the increase in the rotation number, the size of vortical structures changes. The Coriolis force also changes the relative size of the secondary flow in the bend section, and the vortex near the leading surface becomes larger.

We examine the effects of quantity‐based approaches to airport congestion management such as slot restrictions on price dispersion and on‐time performance. From the evidence at Newark Liberty International Airport, we find that slot restrictions increase price dispersion and improve on‐time performance. Price dispersion originates from heterogeneous price effects, as ticket prices for high‐value passengers increase more than that for low‐value passengers. Our novel finding is that the share of passengers who have purchased tickets at a top‐end price increase despite the price hike. We use various empirical specifications and an analysis on leads and lags of the policy for robustness.

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.

Chinese local pigs have abundant phenotypes as a result of different cultures and habits of Chinese populations, geographic constraints and the long history of pig domestication. A comprehensive investigation of local Chinese pigs will benefit biodiversity research and future breeding practices. However, their classification and demographic history are not yet clear. We studied 91 Chinese local pigs from 14 breeds and 15 Chinese wild boars to reveal the dispersal of Chinese pigs, genetic groups and the demographic history. Based on spatial feature analyses, we believe that the geographic landscape played an important role in the dispersal of local pigs. According to genetic studies, Chinese pigs are divided into three groups where each group appears to have a distinct background. The nucleotide diversity, observed heterozygosity, runs of homozygosity and inbreeding coefficient varied among the groups and widespread migration also existed between the groups. Furthermore, demographic models have been constructed to explain the evolutionary relationship between the groups using the approximate Bayesian computation approach. These suggested that Chinese local pigs are inherited from an extinct Sus scrofa population from ~22 000 years ago. Then, the three groups diverged from ancestors ~16 000, ~11 000 and ~8700 years ago respectively. This study advances our understanding of the genetic variation and demographic history of Chinese local pigs.

We numerically investigate the superconvergence property of the discontinuous Galerkin method by patch reconstruction. The convergence rate \begin{document}$ 2m+1 $\end{document} can be observed at the grid points and barycenters in one dimensional case with uniform partitions. The convergence rate \begin{document}$ m + 2 $\end{document} is achieved at the center of the element faces in two and three dimensions. The meshes are uniformly partitioned into triangles/tetrahedrons or squares/hexahedrons. We also demonstrate the details of the implementation of the proposed method. The numerical results for all three dimensional cases are presented to illustrate the propositions.

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.

Background: ESR1 mutations are acquired following ERα targeted therapies and are a major determinant of therapy-resistance. These ESR1 mutations maintain ESR1 signaling, albeit in a ligand-independent manner. Effective drugs targeting these mutant (MT) ERα proteins represent a significant unmet clinical need. We had previously shown that ERX-11, an ESR1-coregulator binding inhibitor, could block the function of these MT ERα proteins. In this study, we sought to leverage recently published structures of MT ERα to develop more potent analogues of ERX-11. Methods: Virtual screening of >250,000 derivatives of ERX-11 was performed with simulated docking on the MT ERα to identify and design analogues of ERX-11. Several hundred analogues were synthesized and tested in vitro using multiple BC model cells that express wild type (WT) ESR1 or mutant (MT) ESR1 (Y537S or D538G). Mechanistic studies were performed using RNA-Seq, Western blotting, qRT-PCR and reporter gene assays. The in vivo efficacy of the most potent ERX-11 analogue ERX-245 was examined using xenograft, PDX and metastatic models of MT-ER driven BC. Results: From our virtual and functional screen, we identified an ERX-11 analogue, ERX-245 as the most potent hit to target MT-ERα. Docking studies modeled a better fit of ERX-245 into the ligand binding domain of both the Y537S and D538G MT-ERα. ERX-245 potently reduced (IC50 ~250 nM) the cell viability of both WT-ERα and MT-ERα driven BC cells but not ERα negative BC cells. ERX-245 significantly reduced the growth (colony formation, clonogenic and mammosphere assays) of MT-ERα BC cells. ERX-245 exhibited synergistic activity in combination with CDK4/6 inhibitors. In distinction to classic SERDs like fulvestrant (which degrade ERα with in 4h), ERX-245 treatment decreased MT-ERα protein levels over 24 hours. PK studies indicated that ERX-245 is more polar and has better solubility and pharmacokinetic properties than ERX-11. ERX-245 reduced tumor growth of subcutaneous xenograft and PDX models driven by MT-ERα as well as the proliferation of xenograft derived MT-ERα explant models. ERX-245 significantly reduced the invasive capability of MT-ERα BC cells in vitro and inhibited both the metastatic capability and growth of metastatic tumors derived from MT-ERα BC cells injected by intracardiac or intratibial routes. Conclusions: Taken together, these results indicate that ERX-245 is a potent and pharmacologically translatable analog of ERX-11, with activity against both primary and metastatic tumors driven by MT-ERα. Citation Format: Ganesh V Raj, Suryavathi Viswanadhapalli, Karla Parra, Shihong Ma, Tae-Kyung Lee, Xihui Liu, Kara Kassees, Weiwei Tang, Junhao Liu, Zexuan Liu, Uday P Pratap, Behnam Ebrahimi, Rajeshwar Rao Tekmal, Jung-Mo Ann, Ratna K Vadlamudi. Development of a potent mutant-ESR1 targeted agent, ERX-245, for treating metastatic therapy-resistant 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 PS17-09.

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.

Transcription factors (TFs) are the mainstay of cancer and have a widely reported influence on the initiation, progression, invasion, metastasis, and therapy resistance of cancer. However, the prognostic values of TFs in breast cancer (BC) remained unknown. In this study, comprehensive bioinformatics analysis was conducted with data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) database. We constructed the co-expression network of all TFs and linked it to clinicopathological data. Differentially expressed TFs were obtained from BC RNA-seq data in TCGA database. The prognostic TFs used to construct the risk model for progression free interval (PFI) were identified by Cox regression analyses, and the PFI was analyzed by the Kaplan-Meier method. A receiver operating characteristic (ROC) curve and clinical variables stratification analysis were used to detect the accuracy of the prognostic model. Additionally, we performed functional enrichment analysis by analyzing the differential expressed gene between high-risk and low-risk group. A total of nine co-expression modules were identified. The prognostic index based on 4 TFs (NR3C2, ZNF652, EGR3, and ARNT2) indicated that the PFI was significantly shorter in the high-risk group than their low-risk counterpart (p < 0.001). The ROC curve for PFI exhibited acceptable predictive accuracy, with an area under the curve value of 0.705 and 0.730. In the stratification analyses, the risk score index is an independent prognostic variable for PFI. Functional enrichment analyses showed that high-risk group was positively correlated with mTORC1 signaling pathway. In conclusion, the TF-related signature for PFI constructed in this study can independently predict the prognosis of BC patients and provide a deeper understanding of the potential biological mechanism of TFs in BC.