Estrogen Withdrawal Research Articles

Sex-Specific Differences in Kidney Function and Blood Pressure Regulation.

Premenopausal women generally exhibit lower blood pressure and a lower prevalence of hypertension than men of the same age, but these differences reverse postmenopause due to estrogen withdrawal. Sexual dimorphism has been described in different components of kidney physiology and pathophysiology, including the renin-angiotensin-aldosterone system, endothelin system, and tubular transporters. This review explores the sex-specific differences in kidney function and blood pressure regulation. Understanding these differences provides insights into potential therapeutic targets for managing hypertension and kidney diseases, considering the patient's sex and hormonal status.

Targeting aberrant fatty acid synthesis and storage in endocrine resistant breast cancer cells.

Lipid metabolic reprogramming is an emerging characteristic of endocrine therapy (ET) resistance in estrogen receptor-positive (ER+) breast cancer. We explored changes in lipid metabolism in ER+ breast cancer cell lines following acquired resistance to common endocrine treatments and tested efficacy of an inhibitor in current clinical trials. We derived ER+ breast cancer cell lines resistant to Tamoxifen (TamR), Fulvestrant (FulvR), and long-term estrogen withdrawal (EWD). Parental and ET resistant cells were subjected to global gene expression and unbiased lipidomic profiling. Lipid storage changes were assessed via neutral lipid staining with Oil Red O (ORO). The impact of the fatty acid synthase (FASN) inhibitor TVB-2640 on the growth and lipid storage of these cell lines was evaluated. Additionally, 13 C 2 -acetate tracing was used to examine FASN activity in parental and ET resistant cells in the absence or presence of TVB-2640. Compared to parental cells, lipid metabolism and processing pathways were notably enriched in ET resistant cells, which exhibited distinct lipidomes characterized by increased triglyceride and polyunsaturated FA (PUFA) species. ET-resistant cells displayed enhanced cytoplasmic lipid droplets. Increased FASN protein levels were observed in ET-resistant cells, and TVB-2640 effectively inhibited FASN activity. FASN inhibition reduced cell growth in some but not all cell lines and ET resistance types and did not correlate to lipid storage reduction. 13 C 2 -acetate tracing confirmed reduced palmitate synthesis and enhanced PUFA synthesis in ET-resistant cells, especially when combined with FulvR. ET resistant breast cancer cells exhibit a shift towards enhanced triglyceride storage and complex lipids enriched with PUFA acyl chains. While targeting FASN alongside ET may not fully overcome ET resistance in our models, focusing on the unique lipid metabolic dependencies, such as PUFA pathways, may present a promising alternative strategy for treating ET resistant breast cancer.

Neoadjuvant aromatase inhibitor therapy for ER+ breast cancer: The NAOMI trial.

e12646 Background: Neoadjuvant Endocrine Therapy (NET) is an effective and well tolerated treatment for locally advanced ER+ breast cancer (BC) yet it is underutilized. It can induce comparable clinical response rates to chemotherapy but not pathologic complete response. There is a knowledge gap regarding its effect on residual cancer cells and fatty acid oxidation which is essential for survival of dormant ER+BC cells after estrogen withdrawal in vitro. The NAOMI trial is a study testing the effects of NET with the AI letrozole in early ER+ BC. It aimed to assess the effect of letrozole on residual cancer cells as well as markers of mitochondrial and fatty acid metabolism. Herein we present the results of the molecular and clinical analyses. Methods: NAOMI is a phase II single-arm open-label study with use of letrozole for 4 to 12 weeks prior to surgical resection. Eligible patients were women over 18 years of age, post-menopausal, with stage I-III ER+, HER2- BC. Exclusion criteria included prior ET use or other neoadjuvant options. The primary objective was to assess whether residual cancer cells exhibit upregulation of the fatty acid transporter CPT1A compared to baseline. Secondary objectives included the proportion of subjects who had clinical response and whether residual cancer cells exhibit markers of altered mitochondrial and fatty acid metabolism compared to baseline tumors. Protocol was amended to include adherence rates to adjuvant AI. Results: A total of 77 women have consented to study participation since December 2022 including 57 evaluable patients. Molecular analyses were performed on diagnostic and post-letrozole tumor specimens from the first 33 patients. Immunohistochemistry was done using antibodies against Ki67, CPT1A, FASN, perilipin, CD36, and MT-CO2. CPT1A histoscore between baseline and post letrozole specimens were similar (paired t-test p=0.2636). Markers of altered mitochondrial and fatty acid metabolism including MT-CO2, CD36, FASN, Perilipin were unchanged when comparing pre and post-AI tissues. Post hoc comparisons of changes in Ki67 (marker of biological response to letrozole) vs. changes in metabolic markers were performed. Ki67+ cells were substantially decreased during neoadjuvant letrozole therapy (paired t-test p<0.0001). Linear regression analysis of residual tumor cell proliferation [post-letrozole log2(Ki67+1)] vs. letrozole-induced change in log2(MT-CO2) showed that tumors with more residual cell proliferation had greater levels of MT-CO2 (p=0.0278). Patients enrolled in NAOMI had a rate of adherence to adjuvant ET of 82%, higher than historical averages. Conclusions: NET is associated with a decrease in Ki67+ breast cancer cells however it has minimal impact on fatty acid and mitochondrial metabolism. It is suggested to increase adherence to adjuvant ET. Additional studies are planned to elucidate mechanisms of resistance in those samples that did not show a decrease in Ki67. Clinical trial information: NCT04568616 .

In Vitro Cell Culture Model for Osteoclast Activation during Estrogen Withdrawal.

Estrogen (17β-estradiol) deficiency post-menopause alters bone homeostasis whereby bone resorption by osteoclasts exceeds bone formation by osteoblasts, leading to osteoporosis in females. We established an in vitro model to examine the consequences of estrogen withdrawal (E2-WD) on osteoclasts derived from the mouse macrophage RAW 264.7 cell line and utilized it to investigate the mechanism behind the enhanced osteoclast activity post-menopause. We found that a greater population of osteoclasts that underwent E2-WD contained a podosome belt necessary for osteoclasts to adhere and resorb bone and possessed elevated resorptive activity compared to osteoclasts exposed to estrogen (E2) continuously. Our results show that compared to osteoclasts that received E2 continuously, those that underwent E2-WD had a faster rate of microtubule (MT) growth, reduced RhoA activation, and shorter podosome lifespan. Thus, altered podosome and MT dynamics induced by the withdrawal of estrogen supports podosome belt assembly/stability in osteoclasts, which may explain their enhanced bone resorption activity.

Arthralgia and myalgias associated with aromatase inhibitors: Frequency and characterization in real-life patients.

e12504 Background: Adjuvant treatment with aromatase inhibitors (AI) in estrogen receptor (ER) and/or progesterone receptor (PR) positive breast cancer (BC) has been shown to increase overall survival (OS). However, arthralgias and myalgias are common adverse effects in patients treated with AI. Objective: To evaluate the frequency and characteristics of arthralgias and myalgias in patients with early BC, treated adjuvantly with AI at the Mastology Unit of the Oncology Service of the Hospital de Clínicas and the Soriano Departmental Hospital. Methods: A prospective, cross-sectional, descriptive study was conducted. A questionnaire was administered to the patients to assess the presence and characteristics of arthralgias and myalgias associated with AI. Statistical analysis: "Age" was described using measures of central tendency and dispersion. Qualitative variables were presented in absolute and relative frequencies. Logistic models were used to assess the association between patient, tumor, and treatment characteristics and the presence of pain. Results: Results were presented using Odds Ratio and p-value, using R software (version 4.1.2) with a significance threshold of 5%. 83 patients were included, with a median age of 69 years. 75.9% presented treatment-related arthralgias and/or myalgias, with an average intensity of 5 to 7. 80.9% received NSAIDs, achieving satisfactory analgesia. The presence of arthralgias and myalgias was significantly associated with age and time since last menstruation, being more frequent in patients over 50 years old and those with more than 5 years since the last menstruation. Conclusions: Approximately 70% of the patients presented arthralgias or myalgias. These findings suggest a possible role of estrogen withdrawal in their development mechanism. It is crucial to carry out multidisciplinary and translational research to evaluate the etiology and therapeutic options for patients with AI-related arthralgia.

Menstrual Migraine: A Review of Current Research and Clinical Challenges

The term “menstrual migraine” is commonly used to describe migraines that occur in association with menstruation, as distinct from other migraine types. A significant proportion of women of reproductive age experience migraine attacks related to their menstrual cycle. Menstrual migraine is characterized by migraine attacks occurring on day 1±2 (i.e., days −2 to +3) of menstruation in at least two out of three menstrual cycles. Although the reported prevalence of menstrual migraine varies considerably, population-based studies have found that menstrual migraine affects up to 60% of women with migraines. Several hypotheses have been proposed to explain the etiology of menstrual migraine, among which the estrogen withdrawal hypothesis is the most widely accepted. Women who experience menstrual migraines often face considerable disability due to perimenstrual attacks. Studies have reported that perimenstrual attacks are more severe and more difficult to manage. The principles of acute managing perimenstrual attacks are the same as those for managing nonmenstrual attacks. Short-term preventive therapy is needed to prevent menstrual migraines before they occur during the perimenstrual period. This review summarizes the prevalence, distinct clinical features, pathophysiological mechanisms, and management of menstrual migraine.

Arthralgia and myalgia associated with aromatase inhibitors: frequency and characterization in real-life patients.

Adjuvant treatment with aromatase inhibitors (AI) in oestrogen receptor-positive and/or progesterone receptor-positive breast cancer (BC) has been shown to increase overall survival. However, arthralgias and myalgias are common adverse effects in patients treated with AI. To evaluate the frequency and characteristics of arthralgias and myalgias in patients with early BC-treated adjuvantly with AI in the Mastology Unit of the Oncology Service of the Hospital de Clínicas and the Departmental Hospital of Soriano. A prospective, cross-sectional and descriptive study was performed. A questionnaire was administered to patients to assess the presence and characteristics of arthralgias and myalgias associated with AI. 'Age' was described with measures of central tendency and dispersion. Qualitative variables were presented in absolute and relative frequencies. Logistic models were used to evaluate the association between patient characteristics, tumour characteristics, treatment characteristics and the presence of pain. Results were presented by odds ratio and p-value, using R software (version 4.1.2) with a significance threshold of 5%. 83 patients were included, with a median age of 69 years. 75.9% presented arthralgias and/or myalgias related to treatment, with an average intensity of 5-7. 80.9% received non-steroidal anti-inflammatory drugs (NSAIDs), achieving satisfactory analgesia. The presence of arthralgias and myalgias was significantly associated with age and time since the last menstrual period (LMP), being more frequent in patients older than 50 years and those with more than 5 years since the LMP. Approximately 70% of the patients presented arthralgias or myalgias. These findings suggest a possible role of oestrogen withdrawal in its mechanism of development. Multidisciplinary and translational research is crucial to evaluate the ethology and therapeutic options for patients with AI-related arthralgia.

Migraines in Women: A Focus on Reproductive Events and Hormonal Milestones

Migraine, a prevalent neurological disorder, is more common in women than in men. This sex difference is more pronounced after menarche and diminishes after menopause. Migraines in women are influenced by the menstrual cycle, pregnancy, and lactation, suggesting a connection to sex hormones, known as the estrogen withdrawal theory. Beyond endogenous hormonal changes accompanying reproductive events, exogenous hormonal factors such as contraceptives or hormone replacement therapy may also affect migraines. The hormonal influence cannot be explained simply by serum estrogen levels; instead, it involves a complex interplay of various factors. Here, we delineate aspects of migraines associated with endogenous and exogenous hormonal changes over the course of a woman’s life, exploring the mechanisms and contributing factors through which sex hormones influence migraines.

Menstrual Psychosis with Premenstrual Onset: A case presentation

IntroductionMenstrual psychosis has an acute onset and is characterised by confusion, stupor and mutism, delusions, hallucinations, or a manic syndrome lasting for a brief duration, with full recovery. These symptoms maintain periodicity in rhythm with the menstrual cycle. The symptoms may appear in the premenstrual phase or may begin with the onset of menstrual flow (catamenial psychoses). Usually, menstrual psychosis has a polymorphism of both psychotic and affective symptoms (Brockington I. Menstrual psychosis. World Psychiatry. 2005;4(1):9-17.). In this article we present a case of menstrual psychosis with premenstrual onset.ObjectivesA 26 years old, nulliparous single female with one mentally ill relative on her mother’s side (her grandmother) presented with an episodic illness characterized by mood swings, irrelevant speech, irritability, suspiciousness and thought disorder related to her menstrual cycles. She had earlier suffered prolonged attacks of mania, developed a recurrent episodic illness which returned every month for five years. Her menses began at 15. She presented with the history of a few episodes of manic illness starting five days before and ending suddenly with the onset of the menses.MethodsOn mental status evaluation during the index episode, the patient was agitated, had labile affect, grandiose and referential delusions and erotomania. A detailed physical examination, routine biochemistry, and gonadal hormonal assay were unremarkable.ResultsShe was started on olanzapine 10 mg/day, lithium 1200 mg/day and low-dose clonazepam. Although the severity of the psychotic and affective symptoms gradually reduced during the future menstrual cycles, they did not completely resolve.ConclusionsThe pathophysiology of menstrual psychosis is not exactly understood, but it has been postulated that fluctuation of the sex hormones occurring during the menstrual cycle is responsible. Previous studies have reported the association of psychosis with estrogen withdrawal (Mahé V, Dumaine A. Oestrogen withdrawal associated psychoses. Acta Psychiatr Scand. 2001;104(5):323-331.). Treatment strategies for menstrual psychosis include the use of oral contraceptive pills for the regulation of hormones during the menstrual cycle, in our case patient did not want to use oral contraceptive pills.Disclosure of InterestNone Declared

Hormones and Psychosis: The Role of Estrogen in Schizophrenia

IntroductionSchizophrenia is a complex psychiatric disorder in which biological sex differences, have been extensively documented and researched. What is less well described, is what motivates these differences. Of the various proposed and explored reasons, estrogen appears to be one that has maintained some interest and promise. An increase in symptoms of schizophrenia has been observed to correspond with decreasing levels of estrogen in menopausal women, this, allied to the later symptom onset, culminated in the interest in this hormone and its role in psychotic illness.ObjectivesThe authors aim to briefly explore the current evidence on the association between estrogen and schizophrenia. Its relevance in symptom onset, protective status and eventual therapeutic applications will also be discussed.MethodsThe authors conducted a brief non-structured narrative literature review using articles published in the Medline/Pubmed, ScienceDirect and Google Scholar databases. The keywords used during the research, alone or in combination, included: sex hormones, estrogen, schizophrenia and psychiatry.The studies consulted in this work included: cross-sectional studies, cohort studies, literature reviews and clinical case reports.ResultsThe literature exploring the relationship between the sex hormone, estrogen, and schizophrenia is extensive. Various studies confirm that during periods of estrogen withdrawal, women appear more susceptible to psychotic episodes. Results also demonstred that those with low estrogen, respond poorly to anti-psychotic drugs, whereas estrogen increased the efficiency of antipsychotics. In regards to symptoms, estrogen has been demonstrated to reduce the positive and cognitive symptoms of schizophrenia in the short term, thus being proposed as an eventual complementary treatment in those suffering from the disorder. It is known that estrogen regulates important pathophysiological pathways in schizophrenia, including dopamine activity, mitochondrial function, and the stress system.One of the explanations for this beneficial effect has been proposed to be action on cerebral blood flow and glucoce metabilism, as well as sensitizing postsynaptic dopamine receptors, thus serving as a protective agent against schizophrenia.ConclusionsThe research appears to be pointing in the direction that estrogen appears to have an effect on psychosis in women, serving as a protective factor in these conditions as well as playing a significant part of the pathophysiology in schizophrenia. This influence on the pathophysiology, promises clinical pertinence, not only in a possible application so to attenuate positive and cognitive symptoms but also as a method to influence antipsychotic efficacy. Continued study in regards to the effects of sex hormones on the psychotic disorders is merited so as to further expand the tools in the mental health professional’s repertoire in the treatment of these serious mental illnesses.Disclosure of InterestNone Declared

Effects of alendronate on cartilage lesions and micro-architecture deterioration of subchondral bone in patellofemoral osteoarthritic ovariectomized rats with patella-baja

BackgroundPatellofemoral osteoarthritis (PFJOA) is a subtype of knee OA, which is one of the main causes of anterior knee pain. The current study found an increased prevalence of OA in postmenopausal women, called postmenopausal OA. Therefore, we designed the ovariectomized rat model of patella baja-induced PFJOA. Alendronate (ALN) inhibits osteoclast-mediated bone loss, and has been reported the favorable result of a potential intervention option of OA treatment. However, the potential effects of ALN treatment on PFJOA in the ovariectomized rat model are unknown and need further investigation prior to exploration in the clinical research setting. In this study, the effects of ALN on articular cartilage degradation and subchondral bone microstructure were assessed in the ovariectomized PFJOA rat model for 10 weeks.MethodsPatella baja and estrogen withdrawal were induced by patellar ligament shortening (PLS) and bilateral ovariectmomy surgeries in 3-month-old female Sprague–Dawley rats, respectively. Rats were randomly divided into five groups (n = 8): Sham + V; OVX + V, Sham + PLS + V, OVX + PLS + V, OVX + PLS + ALN (ALN: 70 μg/kg/week). Radiography was performed to evaluate patellar height ratios, and the progression of PFJOA was assessed by macroscopic and microscopic analyses, immunohistochemistry and micro-computed tomography (micro-CT).ResultsOur results found that the patella baja model prepared by PLS can successfully cause degeneration of articular cartilage and subchondral bone, resulting in changes of PFJOA. OVX caused a decrease in estrogen levels in rats, which aggravated the joint degeneration caused by PFJOA. Early application of ALN can delay the degenerative changes of articular cartilage and subchondral bone microstructure in castrated PFJOA rat to a certain extent, improve and maintain the micrometabolism and structural changes of cartilage and subchondral bone.ConclusionThe early application of ALN can delay the destruction of articular cartilage and subchondral bone microstructure in castrated PFJOA rat to a certain extent.

Estrogen receptor GPR30 in the anterior cingulate cortex mediates exacerbated neuropathic pain in ovariectomized mice

Menopausal women experience neuropathic pain 63% more frequently than men do, which may attribute to the estrogen withdrawal. However, the underlying mechanisms remain unclear. Here, the role of estrogen receptors (ERs) in ovariectomized (OVX) female mice following chronic constriction injury (CCI) was investigated. With 17β-estradiol (E2) supplemented, aggravated mechanical allodynia in OVX mice could be significantly alleviated, particularly after intra-anterior cingulate cortex (ACC) E2 delivery. Pharmacological interventions further demonstrated that the agonist of G-protein-coupled estrogen receptor 30 (GPR30), rather than ERα or ERβ in the ACC, exhibited the similar analgesic effect as E2, whereas antagonist of GPR30 exacerbated allodynia. Furthermore, OVX surgery reduced GPR30 expression in the ACC, which could be restored with estrogen supplementation. Selective downregulation of GPR30 in the ACC of naïve female mice induces mechanical allodynia, whereas GPR30 overexpression in the ACC remarkedly alleviated OVX-exacerbated allodynia. Collectively, estrogen withdrawal could downregulate the ACC GPR30 expression, resulting in exacerbated neuropathic pain. Our findings highlight the importance of GPR30 in the ACC in aggravated neuropathic pain during menopause, and offer a potential therapeutic candidate for neuropathic pain management in menopausal women.

Hereditary angioedema with normal C1-inhibitor: Clinical and genetic characterization of 15 Portuguese unrelated families

BackgroundHereditary angioedema with normal C1-inhibitor (HAE-nC1-INH) is a rare genetic disease with similar phenotype to HAE-C1-INH but different genetic background. Currently, 6 subtypes are recognized, based on the underlying mutations. Several aspects need further clarification. ObjectiveTo assess clinical features of patients with genetically characterized HAE-nC1-INH from the North of Portugal. MethodsRetrospective assessment of clinical data from all patients with HAE-nC1-INH followed at a HAE Reference Center. ResultsA total of 41 patients were identified, 4 with no family history. The FXII mutation Thr328Lys (38 carriers) was the most prevalent. There were 3 new potentially disease-causing variants linked to HAE-nC1-INH identified (c.529+4A>G:FXII; Cys248*:Kininogen-1; and Arg261His:Plasminogen). The HAE-FXII cohort included 82% females and 71.8% symptomatic patients. Penetrance rate was significantly higher in females (81.3% vs 28.6%; P = .012). A hormonal influence was observed in 96.2% of the symptomatic females, although 62.5% remained symptomatic after oral estrogen withdrawal. Trauma and dental procedures were frequent triggers (82.6% and 45.5%, respectively). Main locations were facial (described by 96%), lips (82.1%), and eyelids (64.3%). One patient reported erythema marginatum as prodrome. Plasma-derived C1-INH was effective as short-term prophylaxis in all treated patients, but only in 80% as on-demand treatment. Icatibant was effectively used on demand in 9 patients, but with relapses in 5 (57%). ConclusionWe described a large Portuguese series of patients with HAE-nC1-INH genetically characterized. Differences with others may contribute to improve current unmet needs and raise awareness of this rare disease. We highlighted the identification of 3 new variants (additional molecular studies are ongoing) and the report of erythema marginatum in HAE-nC1-INH.

Ferroptosis in Osteocytes as a Target for Protection Against Postmenopausal Osteoporosis.

Ferroptosis is a necrotic form of iron-dependent regulatory cell death. Estrogen withdrawal can interfere with iron metabolism, which is responsible for the pathogenesis of postmenopausal osteoporosis (PMOP). Here, it is demonstrated that estrogen withdrawal induces iron accumulation in the skeleton and the ferroptosis of osteocytes, leading to reduced bone mineral density. Furthermore, the facilitatory effect of ferroptosis of osteocytes is verified in the occurrence and development of postmenopausal osteoporosis is associated with over activated osteoclastogenesis using a direct osteocyte/osteoclast coculture system and glutathione peroxidase 4 (GPX4) knockout ovariectomized mice. In addition, the nuclear factor erythroid derived 2-related factor-2 (Nrf2) signaling pathway is confirmed to be a crucial factor in the ferroptosis of osteocytic cells. Nrf2 regulates the expression of nuclear factor kappa-B ligand (RANKL) by regulating the DNA methylation level of the RANKL promoter mediated by DNA methyltransferase 3a (Dnmt3a), which is as an important mechanism in osteocytic ferroptosis-mediated osteoclastogenesis. Taken together, this data suggests that osteocytic ferroptosis is involved in PMOP and can be targeted to tune bone homeostasis.

Cone-Beam Computed Tomography to evaluate changes in trabecular lower jawbone microstructure caused by bone loss and antiresorptive treatment.

The aim of this preliminary report is to evaluate the usefulness of CBCT to assess trabecular mandible microstructural properties in normal ewes and to compare the quantitative changes associated with ovariectomy and antiresorptive treatment. Twelve adult Corriedale ewes (n=4/group) aged 3-4 years were divided into 3 groups and studied for 28 months. Eight ewes were ovariectomized (OVX) and divided into OVX and OVX+ZOL groups (n=4/group) which were treated as follows, by jugular injection: OVX received saline solution and OVX+ZOL received zoledronate (Zol) (Gador SA, CABA, Argentina) (4 mg/month). Another four ewes were subjected to sham surgery (SHAM group) and received saline solution. Densitometry showed that jaw mineral content (BMC) and density (BMD) were significantly lower in OVX than in SHAM and OVX+ZOL ewes; no difference was observed between OVX+ ZOL and SHAM groups. CBCT analysis showed that bone volume (BV/TV%); trabecular thickness (TbTh); connectivity density (CD) and anisotropy degree (AD) were significantly lower, and trabecular spacing (TbSp), significantly higher in OVX than in SHAM ewes. AD was significantly higher and TbSp significantly lower in OVX+ZOL than in OVX groups. BV/TV%, TbTh and CD showed a clear tendency to being higher in OVX+ZOL than in OVX groups. No statistical difference was observed between OVX+ZOL and SHAM ewes. CBCT in a nondestructive, fast, very precise procedure for measuring bone morphometric indices without biopsies, which are not indicated for morphometric evaluation in osteoporosis. The current study demonstrated the potential of the high-resolution CBCT imaging to assess in vivo quantitative bone morphometry and bone quality of lower jaw cancellous bone under normal conditions and to differentiate changes associated with excessive bone loss induced by estrogen withdrawal and antiresorptive intervention.

KPT-8602 Induced Hematopoietic Differentiation in U2AF1 S34F Mutant Cells and Myelodysplastic Syndromes Bone Marrow Stem and Progenitor Cells

Myelodysplastic syndromes (MDS) are a group of clonal bone marrow (BM) neoplasms characterized by ineffective hematopoiesis, cytopenia's, and a high risk of progression to acute myeloid leukemia. Current standard therapies include erythropoiesis stimulating agents for lower risk disease and DNMTi for higher risk MDS; however, less than half of patients respond and even the best responses are transient and non-curative. The majority of MDS patients harbor at least one of ~40 commonly found somatic gene mutations, most of which occur in splicing factor genes such as U2AF1, which also have non-canonical roles in nuclear export. KPT-8602 (Eltanexor) is a novel selective inhibitor of nuclear export (SINE) that acts by disrupting nuclear export of cargo relevant in MDS. We hypothesized that U2AF1 S34 F mutant cells would be selectively sensitive to treatment with KPT-8602. We created immortalized murine hematopoietic progenitor cells by retroviral transduction of stimulated bone marrow (BM) cells isolated from U2AF1 WT or U2AF1 S34F mice with a HOXB8_ER vector. Estrogen drives the hematopoietic transcription factor immortalizing the cells in a progenitor state, however, upon estrogen withdrawal the cells will differentiate and eventually die. This differentiation can be driven by treatment with specific growth factors such as erythropoietin (EPO) for erythroid differentiation or granulocyte macrophage colony stimulating factor (GM-CSF) for myeloid differentiation. KPT-8602 exposure induced differentiation and increased hematopoiesis in murine cell models and primary MDS samples. U2AF1 WT and U2AF1 S34F cells were plated in medium lacking estrogen and treated with GM-CSF (10nM) or EPO (1nM) with or without KPT-8602 (10nM) for 72h, then stained with Ly6C, CD11b, Ter119, and CD71 for flow cytometry analysis. In addition, we performed colony forming capacity assays in both the murine cells and primary MDS BM stem and progenitor cells and age-comparable healthy donors, with 14 day KPT-8602 exposure. Treatment with GM-CSF significantly increased the expression of Ly6C in both U2AF1 WT and U2AF1 S34F cells (WT fold change untreated vs GMCSF =1.9, p=0.03; S34F fold change untreated vs GMCSF =2.3, p<0.01). However, addition of KPT-8602 caused significantly more Ly6C expression than GM-CSF alone in the mutant cells (WT fold change GMCSF vs combination =3.1, p=0.06; S34F fold change GMCSF vs combination =4.0, p<0.01). There were no significant changes in CD11b expression with KPT-8602 treatment. We did not find a statistically significant increase in erythroid markers assessed by Ter-119 and CD71 after EPO treatment, however, KPT-8602 treatment alone showed a trend of increased expression of Ter-119 and CD71 in the mutant cells that fell just below statistical significance (Ter119 p=0.08 for WT, p=0.09 for S34F; CD71 p=0.21 for WT, p=0.06 for S34F). Additionally, we looked at selective toxicity of U2AF1 S34Fby WST-1 reagent and found no differences in toxicity between the WT or mutant cells with KPT-8602. However, U2AF1 WT and U2AF1 S34F progenitor cells showed an increase in colony forming capacity (n=1 with technical duplicates) when plated in the presence of KPT-8602 showing an average fold change in colony growth of 1.4 for U2AF1 WT and 1.3 for U2AF1 S34F underscoring the ability of KPT-8602 to induce differentiation. To confirm these results were not cell line artifacts, we next treated primary BM stem and progenitor cells from MDS patients (n=2) and healthy donors (n=4). MDS patient 1 (age 73y) was diagnosed with MDS-MLD-EB1 and harbored mutations in TP53, ASXL1, BCOR, TET2 and PPM1D. MDS patient 2 (age 79y) was diagnosed with MDS/MPN and harbored an SF3B1 mutation. Healthy donors (n=4) were all male and had a median age of 65y. Treatment with KPT-8602 (10nM) significantly increased burst and colony forming unit-erythroid (BFU-E/CFU-E) in MDS primary cells (p=0.02) with no significant increase in the healthy donors (p=0.57) (BFU-E/CFU-E fold increase was 1.1 for healthy donors and 3.5 for MDS). Collectively, these findings suggest that KPT-8602 promotes myeloid and erythroid differentiation and further validates the use of KPT-8602 as a therapeutic strategy for MDS. Future studies will fully elucidate the specific mechanisms by which KPT-8602 acts in the context of splicing factor mutations, and other commonly found MDS mutation types.

Early-life stress and ovarian hormones alter transcriptional regulation in the nucleus accumbens resulting in sex-specific responses to cocaine

Early-life stress and ovarian hormones contribute to increased female vulnerability to cocaine addiction. Here, we reveal molecular substrates in the reward area, the nucleus accumbens, through which these female-specific factors affect immediate and conditioning responses to cocaine. We find shared involvement of X chromosome inactivation-related and estrogen signaling-related gene regulation in enhanced conditioning responses following early-life stress and during the low-estrogenic state in females. Low-estrogenic females respond to acute cocaine by opening neuronal chromatin enriched for the sites of ΔFosB, a transcription factor implicated in chronic cocaine response and addiction. Conversely, high-estrogenic females respond to cocaine by preferential chromatin closing, providing a mechanism for limiting cocaine-driven chromatin and synaptic plasticity. We find that physiological estrogen withdrawal, early-life stress, and absence of one X chromosome all nullify the protective effect of a high-estrogenic state on cocaine conditioning in females. Our findings offer a molecular framework to enable understanding of sex-specific neuronal mechanisms underlying cocaine use disorder.

Estrogen administration and withdrawal in a model of hormone-simulated pregnancy lead to alterations in behavior and gene expression but do not induce depression-like phenotypes in mice

Pregnancy and the post-partum period are associated with substantial fluctuations in hormone levels and are frequently associated with significant stress. Many individuals also experience affective disturbances during the peri‑partum period, including anxiety, the ‘baby blues,’ and post-partum depression. However, the extent to which these affective changes result from rapidly altering hormone levels, increased stress, or the combination of both remains largely unknown. The current study sought to evaluate the consequences of pregnancy-like hormonal changes on behavior and gene expression in c57BL/6 mice in the absence of stress using a hormone-simulated pregnancy model. Our results reveal that animals receiving hormone injections to simulate the high levels of estrogen observed in late pregnancy and animals withdrawn from estrogen to mimic the rapid decline in this hormone following parturition both exhibit increased anxiety-like behavior compared to ovariectomized controls in the novel open field test. However, no other significant anxiety- or depression-like alterations were observed in either hormone-treated group compared to ovariectomized controls. Both hormone administration and estrogen withdrawal were shown to induce several significant alterations in gene expression in the bed nucleus of the stria terminalis and the paraventricular nucleus of the hypothalamus. In contrast to the estrogen withdrawal hypothesis of post-partum depression, our results suggest that this method estrogen withdrawal following hormone-simulated pregnancy in the absence of stress does not induce phenotypes consistent with post-partum depression in c57BL/6 mice. However, given that estrogen withdrawal does lead to significant gene expression changes in two stress-sensitive brain regions, it remains possible that estrogen withdrawal could still contribute to affective dysregulation in the peri-partum period by influencing susceptibility to stress. Future research is required to evaluate this possibility.

Estrogen modulation of cortical spreading depression

Background and aimsCortical spreading depression (CSD), a transient neuronal and glial depolarization that propagates slowly across the cerebral cortex, is the putative electrophysiological event underlying migraine aura and a headache trigger. Migraine is three times more prevalent in women than men, linked to circulating female hormones. High estrogen levels or estrogen withdrawal may be a migraine trigger for many women. We, therefore, aimed to examine whether sex, gonadectomy, and female hormone supplementation and withdrawal affect the susceptibility to CSD.MethodsTo determine CSD susceptibility, we recorded the frequency of CSDs triggered during 2-h topical KCl application in intact or gonadectomized female and male rats, without or with estradiol or progesterone supplementation via daily intraperitoneal injections. Estrogen or progesterone treatment followed by withdrawal was studied in a separate cohort. To take the first step towards identifying potential mechanisms, we studied glutamate and GABAA receptor binding using autoradiography.ResultsThe CSD frequency in intact female rats was higher than intact male and ovariectomized rats. We did not detect a change in CSD frequency during different stages of the estrous cycle in intact females. Daily estrogen injections for three weeks did not change CSD frequency. However, one-week estrogen withdrawal after two weeks of treatment significantly increased CSD frequency compared with the vehicle group in gonadectomized females. The same protocol of estrogen treatment and withdrawal was ineffective in gonadectomized males. In contrast to estrogen, daily progesterone injections for three weeks elevated CSD susceptibility, and one-week withdrawal after two weeks of treatment partially normalized this effect. Autoradiography did not reveal significant changes in glutamate or GABAA receptor binding density after estrogen treatment and withdrawal.ConclusionsThese data suggest that females are more susceptible to CSD, and sexual dimorphism is abrogated by gonadectomy. Moreover, estrogen withdrawal after prolonged daily treatment enhances CSD susceptibility. These findings may have implications for estrogen-withdrawal migraine, although the latter tends to be without aura.

Abstract P3-11-01: Increasing DAXX Expression in ER+ Breast Cancer Cells to Overcome Endocrine Therapy Resistance

Abstract Background: New treatment paradigms are needed to overcome resistance to endocrine therapy (ET; tamoxifen or aromatase inhibitors, AI) in ER+ breast cancer (BC). ET resistance is due to survival of breast cancer stem cells (BCSCs) that contribute to relapse of ER+ BC. Notch signaling drives BCSCs. In order to identify Notch specific biomarkers for the purpose of patient selection for anti-Notch therapy, we conducted a pre-surgical biomarker window study combining ET plus MK-0752, a -secretase inhibitor (GSI). Death Associated Protein 6 (DAXX) was discovered to be a novel Notch1 target gene and necessary for GSI-mediated inhibition of BCSCs. Subsequently, we found that DAXX alone was sufficient to inhibit BCSCs. In this current study, we investigated the mechanism by which high DAXX expression inhibited growth of ET resistant ER+ BC cells in vitro and in vivo. Methods: Isogenic ER+ BC cell lines (parental MCF-7, ET resistant MCF-7/5C, parental T47D, and ET resistant T47D-ED) were used. Cells were cultured in estrogen-deprived medium for more than 1 year to mimic AI use. DAXX was depleted using siRNA or overexpressed using a pCMV-expression vector. Bulk cell proliferation was analyzed in response to estrogen depletion or increasing concentrations of 17-estradiol. BCSC survival was measured using the mammosphere-forming assay. Tumor onset and burden were measured by injecting DAXX-expressing or depleted mammospheres into mammary fat pads of female, athymic nude mice. Recurrence of an ER+ PDX tumor (BCM 5097) was measured after withdrawal of estrogen. RNA sequencing identified enriched genes and pathways that required DAXX. Based on these results, cell death was assessed using Annexin V/7-AAD flow cytometry, PARP-1 and Caspase 8 cleavage, phosphorylation of JNK, and expression of apoptotic protein regulators, BIM, BAX, Bcl-2, and Bcl-xL. JNK signaling was inhibited using SB600125. Results: Estradiol stimulated proliferation of parental, ER+ MCF-7 and T47D cells. In contrast, estradiol inhibited proliferation of isogenic ET resistant MCF-7/5C and T47D-ED cells in a concentration and ER-dependent manner. Estradiol induced the DAXX protein in both ET sensitive and resistant cells. DAXX was required for BCSC survival in ET sensitive cells. However, once ER+ cells acquired resistance to ET, DAXX was necessary and sufficient to inhibit both bulk cell proliferation and BCSC survival, suggesting that increasing DAXX might be a novel approach to overcome ET resistance. In mice, high DAXX expression significantly inhibited tumor onset and burden of ET resistant tumors compared to DAXX-depleted tumors. Low DAXX expression was significantly associated with recurrence of an ER+ PDX tumor (BCM 5097) after withdrawal of estrogen. RNA sequencing revealed that DAXX activated an anti-neoplastic gene signature, including transcription factors that regulate cell death genes including the Bcl2-family. DAXX was required for high BIM expression and low levels of Bcl-xL. DAXX was necessary and sufficient to induce apoptosis, PARP-1 cleavage, and phosphorylation of JNK in ET resistant cells. A selective JNK inhibitor, SB600125 rescued DAXX-mediated inhibition of ET resistant bulk cell proliferation and BCSC survival, suggesting that high DAXX expression activates JNK signaling to regulate apoptotic proteins to induce cell death of BCSCs-derived from ET resistant BC. Conclusions: Expressing high DAXX levels is a potent method to inhibit ET-resistant BC cell proliferation and BCSC survival. The mechanism by which DAXX inhibits ET-resistant BC is through activation of JNK signaling, regulation of pro-apoptotic genes, and induction of apoptosis. The translational impact of this research is to identify novel agents that can increase DAXX expression and test them pre-clinically and in clinical trials for patients with ET-resistant breast cancer. Citation Format: Clodia Osipo, Kathy S. Albain, Daniel Peiffer, Debra Wyatt. Increasing DAXX Expression in ER+ Breast Cancer Cells to Overcome Endocrine Therapy Resistance [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-11-01.