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 patientderived 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.

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.

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.