Targeting PELP1 oncogenic signaling in TNBC with the small molecule inhibitor SMIP34.

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.

The LIFR Inhibitor EC359 Effectively Targets Type II Endometrial Cancer by Blocking LIF/LIFR Oncogenic Signaling.

Endometrial cancer (ECa) is the most common female gynecologic cancer. When comparing the two histological subtypes of endometrial cancer, Type II tumors are biologically more aggressive and have a worse prognosis than Type I tumors. Current treatments for Type II tumors are ineffective, and new targeted therapies are urgently needed. LIFR and its ligand, LIF, have been shown to play a critical role in the progression of multiple solid cancers and therapy resistance. The role of LIF/LIFR in the progression of Type II ECa, on the other hand, is unknown. We investigated the role of LIF/LIFR signaling in Type II ECa and tested the efficacy of EC359, a novel small-molecule LIFR inhibitor, against Type II ECa. The analysis of tumor databases has uncovered a correlation between diminished survival rates and increased expression of leukemia inhibitory factor (LIF), suggesting a potential connection between altered LIF expression and unfavorable overall survival in Type II ECa. The results obtained from cell viability and colony formation assays demonstrated a significant decrease in the growth of Type II ECa LIFR knockdown cells in comparison to vector control cells. Furthermore, in both primary and established Type II ECa cells, pharmacological inhibition of the LIF/LIFR axis with EC359 markedly decreased cell viability, long-term cell survival, and invasion, and promoted apoptosis. Additionally, EC359 treatment reduced the activation of pathways driven by LIF/LIFR, such as AKT, mTOR, and STAT3. Tumor progression was markedly inhibited by EC359 treatment in two different patient-derived xenograft models in vivo and patient-derived organoids ex vivo. Collectively, these results suggest LIFR inhibitor EC359 as a possible new small-molecule therapeutics for the management of Type II ECa.

SETDB1 interactions with PELP1 contributes to breast cancer endocrine therapy resistance.

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.

Activation of estrogen receptor beta signaling reduces stemness of glioma stem cells.

Glioblastoma (GBM) is the most common and deadliest tumor of the central nervous system. GBM has poor prognosis and glioma stem cells (GSCs) are implicated in tumor initiation and therapy resistance. Estrogen receptor ß (ERß) is expressed in GBM and exhibit tumor suppressive function. However, the role of ERß in GSCs and the therapeutic potential of ERß agonists on GSCs remain largely unknown. Here, we examined whether ERß modulates GSCs stemness and tested the utility of two ERß selective agonists (LY500307 and Liquiritigenin) to reduce the stemness of GSCs. The efficacy of ERß agonists was examined on GSCs isolated from established and patient derived GBMs. Our results suggested that knockout of ERß increased the proportion of CD133+ and SSEA+ positive GSCs and overexpression of ERß reduced the proportion of GSCs in GBM cells. Overexpression of ERß or treatment with ERß agonists significantly inhibited the GSCs cell viability, neurosphere formation, self-renewal ability, induced the apoptosis and reduced expression of stemness markers in GSCs. RNA sequencing analysis revealed that ERß agonist modulate pathways related to stemness, differentiation and apoptosis. Mechanistic studies showed that ERß overexpression or agonist treatment reduced glutamate receptor signaling pathway and induced apoptotic pathways. In orthotopic models, ERß overexpression or ERß agonists treatment significantly reduced the GSCs mediated tumor growth and improved the mice overall survival. Immunohistochemical studies demonstrated that ERß overexpression decreased SOX2 and GRM3 expression and increased expression of GFAP in tumors. These results suggest that ERß activation could be a promising therapeutic strategy to eradicate GSCs.

FASN inhibition as a potential treatment for endocrine-resistant breast cancer.

PURPOSE: The majority of breast cancers are estrogen receptor (ERα) positive making endocrine therapy a mainstay for these patients. Unfortunately, resistance to endocrine therapy is a common occurrence. Fatty acid synthase (FASN) is a key enzyme in lipid biosynthesis and its expression is commensurate with tumor grade and resistance to numerous therapies. METHODS: The effect of the FASN inhibitor TVB-3166 on ERα expression and cell growth was characterized in tamoxifen-resistant cell lines, xenografts, and patient explants. Subcellular localization of ERα was assessed using subcellular fractionations. Palmitoylation and ubiquitination of ERα were assessed by immunoprecipitation. ERα and p-eIF2α protein levels were analyzed by Western blotting after treatment with TVB-3166 with or without the addition of palmitate or BAPTA. RESULTS: TVB-3166 treatment leads to a marked inhibition of proliferation in tamoxifen-resistant cells compared to the parental cells. Additionally, TVB-3166 significantly inhibited tamoxifen-resistant breast tumor growth in mice and decreased proliferation of primary tumor explants compared to untreated controls. FASN inhibition significantly reduced ERα levels most prominently in endocrine-resistant cells and altered its subcellular localization. Furthermore, we showed that the reduction of ERα expression upon TVB-3166 treatment is mediated through the induction of endoplasmic reticulum stress. CONCLUSION: Our preclinical data provide evidence that FASN inhibition by TVB-3166 presents a promising therapeutic strategy for the treatment of endocrine-resistant breast cancer. Further clinical development of FASN inhibitors for endocrine-resistant breast cancer should be considered.

KDM1A inhibition is effective in reducing stemness and treating triple negative breast cancer.

PURPOSE: Cancer stem cells (CSCs) are highly tumorigenic, spared by chemotherapy, sustain tumor growth, and are implicated in tumor recurrence after conventional therapies in triple negative breast cancer (TNBC). Lysine-specific histone demethylase 1A (KDM1A) is highly expressed in several human malignancies and CSCs including TNBC. However, the precise mechanistic role of KDM1A in CSC functions and therapeutic utility of KDM1A inhibitor for treating TNBC is poorly understood. METHODS: The effect of KDM1A inhibition on cell viability, apoptosis, and invasion were examined by Cell Titer Glo, Caspase 3/7 Glo, and matrigel invasion assays, respectively. Stemness and self-renewal of CSCs were examined using mammosphere formation and extreme limiting dilution assays. Mechanistic studies were conducted using RNA-sequencing, RT-qPCR, Western blotting and reporter gene assays. Mouse xenograft and patient derived xenograft models were used for preclinical evaluation of KDM1A inhibitor. RESULTS: TCGA data sets indicated that KDM1A is highly expressed in TNBC. CSCs express high levels of KDM1A and inhibition of KDM1A reduced the CSCs enrichment in TNBC cells. KDM1A inhibition reduced cell viability, mammosphere formation, self-renewal and promoted apoptosis of CSCs. Mechanistic studies suggested that IL6-JAK-STAT3 and EMT pathways were downregulated in KDM1A knockdown and KDM1A inhibitor treated cells. Importantly, doxycycline inducible knockout of KDM1A reduced tumor progression in orthotopic xenograft models and KDM1A inhibitor NCD38 treatment significantly reduced tumor growth in patient derived xenograft (PDX) models. CONCLUSIONS: Our results establish that KDM1A inhibition mitigates CSCs functions via inhibition of STAT3 and EMT signaling, and KDM1A inhibitor NCD38 may represent a novel class of drug for treating TNBC.

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

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

Estrogen receptor coregulator binding modulator (ERX-11) enhances the activity of CDK4/6 inhibitors against estrogen receptor-positive breast cancers.

BACKGROUND: CDK4/6 inhibitors in combination with endocrine therapy (AE/AI/SERDs) are approved for the treatment of ER+ advanced breast cancer (BCa). However, not all patients benefit from CDK4/6 inhibitors therapy. We previously reported a novel therapeutic agent, ERX-11, that binds to the estrogen receptor (ER) and modulates ER-coregulator interactions. Here, we tested if the combination of ERX-11 with agents approved for ER+ BCa would be more potent. METHODS: We tested the effect of combination therapy using BCa cell line models, including those that have acquired resistance to tamoxifen, letrozole, or CDK4/6 inhibitors or have been engineered to express mutant forms of the ER. In vitro activity was tested using Cell Titer-Glo, MTT, and apoptosis assays. Mechanistic studies were conducted using western blot, reporter gene assays, RT-qPCR, and mass spectrometry approaches. Xenograft, patient-derived explants (PDEs), and xenograft-derived explants (XDE) were used for preclinical evaluation and toxicity. RESULTS: ERX-11 inhibited the proliferation of therapy-resistant BCa cells in a dose-dependent manner, including ribociclib resistance. The combination of ERX-11 and CDK4/6 inhibitor was synergistic in decreasing the proliferation of both endocrine therapy-sensitive and endocrine therapy-resistant BCa cells, in vitro, in xenograft models in vivo, xenograft-derived explants ex vivo, and in primary patient-derived explants ex vivo. Importantly, the combination caused xenograft tumor regression in vivo. Unbiased global mass spectrometry studies demonstrated profound decreases in proliferation markers with combination therapy and indicated global proteomic changes in E2F1, ER, and ER coregulators. Mechanistically, the combination of ERX-11 and CDK4/6 inhibitor decreased the interaction between ER and its coregulators, as evidenced by immunoprecipitation followed by mass spectrometry studies. Biochemical studies confirmed that the combination therapy significantly altered the expression of proteins involved in E2F1 and ER signaling, and this is primarily driven by a transcriptional shift, as noted in gene expression studies. CONCLUSIONS: Our results suggest that ERX-11 inhibited the proliferation of BCa cells resistant to both endocrine therapy and CDK4/6 inhibitors in a dose-dependent manner and that the combination of ERX-11 with a CDK4/6 inhibitor may represent a viable therapeutic approach.

NADPH oxidase 4 induces cardiac fibrosis and hypertrophy through activating Akt/mTOR and NFκB signaling pathways.

BACKGROUND: NADPH oxidase 4 (Nox4) has been implicated in cardiac remodeling, but its precise role in cardiac injury remains controversial. Furthermore, little is known about the downstream effector signaling pathways activated by Nox4-derived reactive oxygen species in the myocardium. We investigated the role of Nox4 and Nox4-associated signaling pathways in the development of cardiac remodeling. METHODS AND RESULTS: Cardiac-specific human Nox4 transgenic mice (c-hNox4Tg) were generated. Four groups of mice were studied: (1) control mice, littermates that are negative for hNox4 transgene but Cre positive; (2) c-hNox4 Tg mice; (3) angiotensin II (AngII)-infused control mice; and (4) c-hNox4Tg mice infused with AngII. The c-hNox4Tg mice exhibited an ≈10-fold increase in Nox4 protein expression and an 8-fold increase in the production of reactive oxygen species, and manifested cardiac interstitial fibrosis. AngII infusion to control mice increased cardiac Nox4 expression and induced fibrosis and hypertrophy. The Tg mice receiving AngII exhibited more advanced cardiac remodeling and robust elevation in Nox4 expression, indicating that AngII worsens cardiac injury, at least in part by enhancing Nox4 expression. Moreover, hNox4 transgene and AngII infusion induced the expression of cardiac fetal genes and activated the Akt-mTOR and NFκB signaling pathways. Treatment of AngII-infused c-hNox4Tg mice with GKT137831, a Nox4/Nox1 inhibitor, abolished the increase in oxidative stress, suppressed the Akt-mTOR and NFκB signaling pathways, and attenuated cardiac remodeling. CONCLUSIONS: Upregulation of Nox4 in the myocardium causes cardiac remodeling through activating Akt-mTOR and NFκB signaling pathways. Inhibition of Nox4 has therapeutic potential to treat cardiac remodeling.

Activation of AMP-activated protein kinase prevents TGF-ß1-induced epithelial-mesenchymal transition and myofibroblast activation.

Transforming growth factor (TGF)-ß contributes to tubulointerstitial fibrosis. We investigated the mechanism by which TGF-ß exerts its profibrotic effects and specifically the role of AMP-activated protein kinase (AMPK) in kidney tubular epithelial cells and interstitial fibroblasts. In proximal tubular epithelial cells, TGF-ß1 treatment causes a decrease in AMPK phosphorylation and activation together with increased fibronectin and α-smooth muscle actin expression and decreased in E-cadherin. TGF-ß1 causes similar changes in interstitial fibroblasts. Activation of AMPK with 5-aminoimidazole-4-carboxamide 1-ß-d-ribofuranoside, metformin, or overexpression of constitutively active AMPK markedly attenuated TGF-ß1 functions. Conversely, inhibition of AMPK with adenine 9-ß-d-arabinofuranoside or siRNA-mediated knockdown of AMPK (official name PRKAA1) mimicked the effect of TGF-ß1 and enhanced basal and TGF-ß1-induced phenotypic changes. Importantly, we found that tuberin contributed to the protective effects of AMPK and that TGF-ß1 promoted cell injury by blocking AMPK-mediated tuberin phosphorylation and activation. In the kidney cortex of TGF-ß transgenic mice, the significant decrease in AMPK phosphorylation and tuberin phosphorylation on its AMPK-dependent activating site was associated with an increase in mesenchymal markers and a decrease in E-cadherin. Collectively, the data indicate that TGF-ß exerts its profibrotic action in vitro and in vivo via inactivation of AMPK. AMPK and tuberin activation prevent tubulointerstitial injury induced by TGF-ß. Activators of AMPK provide potential therapeutic strategy to prevent kidney fibrosis and progressive kidney disease.

Pharmacological inhibition of the LIF/LIFR autocrine loop reveals vulnerability of ovarian cancer cells to ferroptosis.

Of all gynecologic cancers, epithelial-ovarian cancer (OCa) stands out with the highest mortality rates. Despite all efforts, 90% of individuals who receive standard surgical and cytotoxic therapy experience disease recurrence. The precise mechanism by which leukemia inhibitory factor (LIF) and its receptor (LIFR) contribute to the progression of OCa remains unknown. Analysis of cancer databases revealed that elevated expression of LIF or LIFR was associated with poor progression-free survival of OCa patients and a predictor of poor response to chemotherapy. Using multiple primary and established OCa cell lines or tissues that represent five subtypes of epithelial-OCa, we demonstrated that LIF/LIFR autocrine signaling is active in OCa. Moreover, treatment with LIFR inhibitor, EC359 significantly reduced OCa cell viability and cell survival with an IC50 ranging from 5-50 nM. Furthermore, EC359 diminished the stemness of OCa cells. Mechanistic studies using RNA-seq and rescue experiments unveiled that EC359 primarily induced ferroptosis by suppressing the glutathione antioxidant defense system. Using multiple in vitro, ex vivo and in vivo models including cell-based xenografts, patient-derived explants, organoids, and xenograft tumors, we demonstrated that EC359 dramatically reduced the growth and progression of OCa. Additionally, EC359 therapy considerably improved tumor immunogenicity by robust CD45+ leukocyte tumor infiltration and polarizing tumor-associated macrophages (TAMs) toward M1 phenotype while showing no impact on normal T-, B-, and other immune cells. Collectively, our findings indicate that the LIF/LIFR autocrine loop plays an essential role in OCa progression and that EC359 could be a promising therapeutic agent for OCa.

Novel LIPA-Targeted Therapy for Treating Ovarian Cancer.

Ovarian cancer (OCa) is the most lethal form of gynecologic cancer, and the tumor heterogeneities at the molecular, cellular, and tissue levels fuel tumor resistance to standard therapies and pose a substantial clinical challenge. Here, we tested the hypothesis that the heightened basal endoplasmic reticulum stress (ERS) observed in OCa represents an exploitable vulnerability and may overcome tumor heterogeneity. Our recent studies identified LIPA as a novel target to induce ERS in cancer cells using the small molecule ERX-41. However, the role of LIPA and theutility of ERX-41 to treat OCa remain unknown. Expression analysis using the TNMplot web tool, TCGA data sets, and immunohistochemistry analysis using a tumor tissue array showed that LIPA is highly expressed in OCa tissues, compared to normal tissues. ERX-41 treatment significantly reduced the cell viability and colony formation ability and promoted the apoptosis of OCa cells. Mechanistic studies revealed a robust and consistent induction of ERS markers, including CHOP, elF2α, PERK, and ATF4, upon ERX-41 treatment. In xenograft and PDX studies, ERX-41 treatment resulted in a significant reduction in tumor growth. Collectively, our results suggest that ERX-41 is a novel therapeutic agent that targets the LIPA with a unique mechanism of ERS induction, which could be exploited to treat heterogeneity in OCa.

PELP1 inhibition by SMIP34 reduces endometrial cancer progression via attenuation of ribosomal biogenesis.

Endometrial carcinoma (ECa) is the fourth most common cancer among women. The oncogene PELP1 is frequently overexpressed in a variety of cancers, including ECa. We recently generated SMIP34, a small-molecule inhibitor of PELP1 that suppresses PELP1 oncogenic signaling. In this study, we assessed the effectiveness of SMIP34 in treating ECa. Treatment of established and primary patient-derived ECa cells with SMIP34 resulted in a significant reduction of cell viability, colony formation ability, and induction of apoptosis. RNA-seq analyses showed that SMIP34-regulated genes were negatively correlated with ribosome biogenesis and eukaryotic translation pathways. Mechanistic studies showed that the Rix complex, which is essential for ribosomal biogenesis, is disrupted upon SMIP34 binding to PELP1. Biochemical assays confirmed that SMIP34 reduced ribosomal biogenesis and new protein synthesis. Further, SMIP34 enhanced the efficacy of mTOR inhibitors in reducing viability of ECa cells. SMIP34 is also effective in reducing cell viability in ECa organoids in vitro and explants ex vivo. Importantly, SMIP34 treatment resulted in a significant reduction of the growth of ECa xenografts. Collectively, these findings underscore the potential of SMIP34 in treating ECa.

Preclinical Development of Brain Permeable ERß Agonist for the Treatment of Glioblastoma.

Glioblastoma (GBM) is the most prevalent and aggressive type of adult brain tumors with low 5-year overall survival rates. Epidemiologic data suggest that estrogen may decrease brain tumor growth, and estrogen receptor beta (ERß) has been demonstrated to exert antitumor functions in GBM. The lack of potent, selective, and brain permeable ERß agonist to promote its antitumor action is limiting the therapeutic promise of ERß. In this study, we discovered that Indanone and tetralone-keto or hydroxyl oximes are a new class of ERß agonists. Because of its high activity in ERß reporter assays, specific binding to ERß in polar screen assays, and potent growth inhibitory activity in GBM cells, CIDD-0149897 was discovered as a possible hit by screening a library of compounds. CIDD-0149897 is more selective for ERß than ERα (40-fold). Treatment with CIDD-0149897 markedly reduced GBM cell viability with an IC50 of ∼7 to 15 µmol/L, while having little to no effect on ERß-KO cells and normal human astrocytes. Further, CIDD-0149897 treatment enhanced expression of known ERß target genes and promoted apoptosis in established and patient-derived GSC models. Pharmacokinetic studies confirmed that CIDD-0149897 has systemic exposure, and good bioavailability in the brain. Mice tolerated daily intraperitoneal treatment of CIDD-0149897 (50 mg/kg) with a 7-day repeat dosage with no toxicity. In addition, CIDD-0149897 treatment significantly decreased tumor growth in U251 xenograft model and extended the survival of orthotopic GBM tumor-bearing mice. Collectively, these findings pointed to CIDD-0149897 as a new class of ERß agonist, offering patients with GBM a potential means of improving survival.

Pharmacological inhibition of KDM1A/LSD1 enhances estrogen receptor beta-mediated tumor suppression in ovarian cancer.

Ovarian cancer (OCa) is the most lethal gynecologic cancer. Emerging data indicates that estrogen receptor beta (ERß) functions as a tumor suppressor in OCa. Lysine-specific histone demethylase 1A (KDM1A) is an epigenetic modifier that acts as a coregulator for steroid hormone receptors. However, it remain unknown if KDM1A interacts with ERß and regulates its expression/functions in OCa. Analysis of TCGA data sets indicated KDM1A and ERß expression showed an inverse relationship in OCa. Knockout (KO), knockdown (KD), or inhibition of KDM1A increased ERß isoform 1 expression in established and patient-derived OCa cells. Further, KDM1A interacts with and functions as a corepressor of ERß, and its inhibition enhances ERß target gene expression via alterations of histone methylation marks at their promoters. Importantly, KDM1A-KO or -KD enhanced the efficacy of ERß agonist LY500307, and the combination of KDM1A inhibitor (KDM1Ai) NCD38 with ERß agonist synergistically reduced the cell viability, colony formation, and invasion of OCa cells. RNA-seq and DIA mass spectrometry analyses showed that KDM1A-KO resulted in enhanced ERß signaling and that genes altered by KDM1A-KO and ERß agonist were related to apoptosis, cell cycle, and EMT. Moreover, combination treatment significantly reduced the tumor growth in OCa orthotopic, syngeneic, and patient-derived xenograft models and proliferation in patient-derived explant models. Our results demonstrate that KDM1A regulates ERß expression/functions, and its inhibition improves ERß mediated tumor suppression. Overall, our findings suggest that KDM1Ai and ERß agonist combination therapy is a promising strategy for OCa.

Lysine-specific histone demethylase 1A (KDM1A/LSD1) inhibition attenuates DNA double-strand break repair and augments the efficacy of temozolomide in glioblastoma.

BACKGROUND: Efficient DNA repair in response to standard chemo and radiation therapies often contributes to glioblastoma (GBM) therapy resistance. Understanding the mechanisms of therapy resistance and identifying the drugs that enhance the therapeutic efficacy of standard therapies may extend the survival of GBM patients. In this study, we investigated the role of KDM1A/LSD1 in DNA double-strand break (DSB) repair and a combination of KDM1A inhibitor and temozolomide (TMZ) in vitro and in vivo using patient-derived glioma stem cells (GSCs). METHODS: Brain bioavailability of the KDM1A inhibitor (NCD38) was established using LS-MS/MS. The effect of a combination of KDM1A knockdown or inhibition with TMZ was studied using cell viability and self-renewal assays. Mechanistic studies were conducted using CUT&Tag-seq, RNA-seq, RT-qPCR, western blot, homologous recombination (HR) and non-homologous end joining (NHEJ) reporter, immunofluorescence, and comet assays. Orthotopic murine models were used to study efficacy in vivo. RESULTS: TCGA analysis showed KDM1A is highly expressed in TMZ-treated GBM patients. Knockdown or knockout or inhibition of KDM1A enhanced TMZ efficacy in reducing the viability and self-renewal of GSCs. Pharmacokinetic studies established that NCD38 readily crosses the blood-brain barrier. CUT&Tag-seq studies showed that KDM1A is enriched at the promoters of DNA repair genes and RNA-seq studies confirmed that KDM1A inhibition reduced their expression. Knockdown or inhibition of KDM1A attenuated HR and NHEJ-mediated DNA repair capacity and enhanced TMZ-mediated DNA damage. A combination of KDM1A knockdown or inhibition and TMZ treatment significantly enhanced the survival of tumor-bearing mice. CONCLUSIONS: Our results provide evidence that KDM1A inhibition sensitizes GBM to TMZ via attenuation of DNA DSB repair pathways.

Targeting LIF/LIFR signaling in cancer.

Leukemia inhibitory factor (LIF), and its receptor (LIFR), are commonly over-expressed in many solid cancers and recent studies have implicated LIF/LIFR axis as a promising clinical target for cancer therapy. LIF/LIFR activate oncogenic signaling pathways including JAK/STAT3 as immediate effectors and MAPK, AKT, mTOR further downstream. LIF/LIFR signaling plays a key role in tumor growth, progression, metastasis, stemness and therapy resistance. Many solid cancers show overexpression of LIF and autocrine stimulation of the LIF/LIFR axis; these are associated with a poorer relapse-free survival. LIF/LIFR signaling also plays a role in modulating multiple immune cell types present in tumor micro environment (TME). Recently, two targeted agents that target LIF (humanized anti-LIF antibody, MSC-1) and LIFR inhibitor (EC359) were under development. Both agents showed effectivity in preclinical models and clinical trials using MSC-1 antibody are in progress. This article reviews the significance of LIF/LIFR pathways and inhibitors that disrupt this process for the treatment of cancer.

Global Genomic and Proteomic Analysis Identified Critical Pathways Modulated by Proto-Oncogene PELP1 in TNBC.

The PELP1 oncogene is commonly overexpressed in many cancers, including triple negative breast cancer (TNBC). However, the mechanisms by which PELP1 contributes to TNBC progression are not well understood. To elucidate these mechanisms, we generated CRISPR-Cas9 mediated PELP1 knockout TNBC cell lines, and alterations in the proteome were examined using global data-independent acquisition mass spectrometry (DIA-MS). Further mechanistic studies utilized shRNA knockdown, Western blotting, and RNA-seq approaches. TCGA data sets were utilized for determining the status of PELP1 in TNBC patient tumors and for examining its correlation with ribosomal proteins. Global DIA-MS studies revealed that 127 proteins are upregulated while 220 proteins are downregulated upon PELP1-KO. Bioinformatic analyses suggested that the oncogenic activities of PELP1 involve regulation of expression of ribosomal proteins and ribosomal complexes. RNA-seq studies further suggested PELP1 modulates the functions of transcription factor c-Myc in TNBC. TCGA data confirmed PELP1 has high expression in TNBC patient tumors, and this high expression pattern correlates with c-Myc, a regulator of ribosomal proteins. Collectively, our global approach studies suggest that PELP1 contributes to TNBC progression by modulation of cell cycle, apoptosis, and ribosome biogenesis pathways.

The antidepressant imipramine inhibits breast cancer growth by targeting estrogen receptor signaling and DNA repair events.

Aberrant activities of various cell cycle and DNA repair proteins promote cancer growth and progression and render them resistant to therapies. Here, we demonstrate that the anti-depressant imipramine blocks growth of triple-negative (TNBC) and estrogen receptor-positive (ER+) breast cancers by inducing cell cycle arrest and by blocking heightened homologous recombination (HR) and non-homologous end joining-mediated (NHEJ) DNA repair activities. Our results reveal that imipramine inhibits the expression of several cell cycle- and DNA repair-associated proteins including E2F1, CDK1, Cyclin D1, and RAD51. In addition, we show that imipramine inhibits the growth of ER + breast cancers by inhibiting the estrogen receptor- α (ER-α) signaling. Our studies in preclinical mouse models and ex vivo explants from breast cancer patients show that imipramine sensitizes TNBC to the PARP inhibitor olaparib and endocrine resistant ER + breast cancer to anti-estrogens. Our studies suggest that repurposing imipramine could enhance routine care for breast cancer patients. Based on these results, we designed an ongoing clinical trial, where we are testing the efficacy of imipramine for treating patients with triple-negative and estrogen receptor-positive breast cancer. Since aberrant DNA repair activity is used by many cancers to survive and become resistant to therapy, imipramine could be used alone and/or with currently used drugs for treating many aggressive cancers.

KDM1A inhibition augments the efficacy of rapamycin for the treatment of endometrial cancer.

Endometrial cancer (EC) often exhibit aberrant activation of PI3K/Akt/mTOR signaling and targeted therapies using mTOR inhibitors showed limited success. The epigenetic modifier, lysine-specific histone demethylase-1A (KDM1A/LSD1) is overexpressed in EC, however, the mechanistic and therapeutic implications of KDM1A in EC are poorly understood. Here, using 119 FDA-approved drugs screen, we identified that KDM1A inhibition is highly synergistic with mTOR inhibitors. Combination therapy of KDM1A and mTOR inhibitors potently reduced the cell viability, survival, and migration of EC cells. Mechanistic studies demonstrated that KDM1A inhibition attenuated the activation of mTOR signaling cascade and abolished rapamycin induced feedback activation of Akt. RNA-seq analysis identified that KDM1A inhibition downregulated the expression of genes involved in rapamycin induced activation of Akt, including the mTORC2 complex. Chromatin immunoprecipitation experiments confirmed KDM1A recruitment to the promoter regions of mTORC2 complex genes and that KDM1A inhibition promoted enrichment of repressive H3K9me2 marks at their promoters. Combination therapy of KDM1A inhibitor and rapamycin reduced the tumor growth in EC xenograft and patient derived xenograft models in vivo and patient derived tumor explants ex vivo. Importantly, in silico analysis of TCGA EC patients data sets revealed that KDM1A expression positively correlated with the levels of PI3K/Akt/mTOR genes. Collectively, our results provide compelling evidence that KDM1A inhibition potentiates the activity of mTOR inhibitors by attenuating the feedback activation of Akt survival signaling. Furthermore, the use of concurrent KDM1A and mTOR inhibitors may be an attractive targeted therapy for EC patients.