Lysine-specific Histone Demethylase 1A Research Articles

KDM1A/LSD1 inhibition enhances chemotherapy response in ovarian cancer.

Ovarian cancer (OCa) is the deadliest of all gynecological cancers. The standard treatment for OCa is platinum-based chemotherapy, such as carboplatin or cisplatin in combination with paclitaxel. Most patients are initially responsive to these treatments; however, nearly 90% will develop recurrence and inevitably succumb to chemotherapy-resistant disease. Recent studies have revealed that the epigenetic modifier lysine-specific histone demethylase 1A (KDM1A/LSD1) is highly overexpressed in OCa. However, the role of KDM1A in chemoresistance and whether its inhibition enhances chemotherapy response in OCa remains uncertain. Analysis of TCGA datasets revealed that KDM1A expression is high in patients who poorly respond to chemotherapy. Western blot analysis show that treatment with chemotherapy drugs cisplatin, carboplatin, and paclitaxel increased KDM1A expression in OCa cells. KDM1A knockdown (KD) or treatment with KDM1A inhibitors NCD38 and SP2509 sensitized established and patient-derived OCa cells to chemotherapy drugs in reducing cell viability and clonogenic survival and inducing apoptosis. Moreover, knockdown of KDM1A sensitized carboplatin-resistant A2780-CP70 cells to carboplatin treatment and paclitaxel-resistant SKOV3-TR cells to paclitaxel. RNA-seq analysis revealed that a combination of KDM1A-KD and cisplatin treatment resulted in the downregulation of genes related to epithelial-mesenchymal transition (EMT). Interestingly, cisplatin treatment increased a subset of NF-κB pathway genes, and KDM1A-KD or KDM1A inhibition reversed this effect. Importantly, KDM1A-KD, in combination with cisplatin, significantly reduced tumor growth compared to a single treatment in an orthotopic intrabursal OCa xenograft model. Collectively, these findings suggest that combination of KDM1A inhibitors with chemotherapy could be a promising therapeutic approach for the treatment of OCa.

USP1-mediated deubiquitination of KDM1A promotes the malignant progression of triple-negative breast cancer.

Previous research has indicated the highly expressed lysine-specific histone demethylase 1A (KDM1A) in several human malignancies, including triple-negative breast cancer (TNBC). However, its detailed mechanisms in TNBC development remain poorly understood. The mRNA levels of KDM1A and Yin Yang 1 (YY1) were determined by RT-qPCR analysis. Western blot was performed to measure KDM1A and ubiquitin-specific protease 1 (USP1) protein expression. Cell proliferation, apoptosis, invasion, migration and stemness were evaluated by MTT assay, EdU assay, flow cytometry, transwell invasion assay, wound-healing assay and sphere-formation assay, respectively. ChIP and dual-luciferase reporter assays were conducted to determine the relationship between YY1 and KDM1A. Xenograft tumor experiment and IHC were carried out to investigate the roles of USP1 and KDM1A in TNBC development in vivo. The highly expressed KDM1A was demonstrated in TNBC tissues and cells, and KDM1A knockdown significantly promoted cell apoptosis, and hampered cell proliferation, invasion, migration, and stemness in TNBC cells. USP1 could increase the stability of KDM1A via deubiquitination, and USP1 depletion restrained the progression of TNBC cells through decreasing KDM1A expression. Moreover, YY1 transcriptionally activated KDM1A expression by directly binding to its promoter in TNBC cells. Additionally, USP1 inhibition reduced KDM1A expression to suppress tumor growth in TNBC mice in vivo. In conclusion, YY1 upregulation increased KDM1A expression via transcriptional activation. USP1 stabilized KDM1A through deubiquitination to promote TNBC progression.

Downregulation of lysine-specific histone demethylase 1A (KDM1A/LSD1) in medial prefrontal cortex facilitates chronic stress–induced pain and emotional dysfunction in female mice

Chronic primary pain, characterized by overlapping symptoms of chronic pain, anxiety, and depression, is strongly associated with stress and is particularly prevalent among females. Recent research has convincingly linked epigenetic modifications in the medial prefrontal cortex (mPFC) to chronic pain and chronic stress. However, our understanding of the role of histone demethylation in the mPFC in chronic stress-induced pain remains limited. In this study, we investigated the function of lysine-specific histone demethylase 1A (KDM1A/LSD1) in the context of chronic overlapping pain comorbid with anxiety and depression in female mice. We employed a chronic variable stress model to induce pain hypersensitivity in the face and hindpaws, as well as anxiety-like and depression-like behaviors, in female mice. Our findings revealed that chronic stress led to a downregulation of KDM1A mRNA and protein expression in the mPFC. Notably, overexpressing KDM1A in the mPFC alleviated the pain hypersensitivity, anxiety-like behaviors, and depression-like behaviors in female mice, without affecting basal pain responses or inducing emotional distress. Conversely, conditional knockout of KDM1A in the mPFC exacerbated pain sensitivity and emotional distress specifically in females. In summary, this study highlights the crucial role of KDM1A in the mPFC in modulating chronic stress-induced overlapping pain, anxiety, and depression in females. Our findings suggest that KDM1A may serve as a potential therapeutic target for treating chronic stress-related overlap pain and associated negative emotional disorders.

Potential role of lipophagy impairment for anticancer effects of glycolysis-suppressed pancreatic ductal adenocarcinoma cells

Although increased aerobic glycolysis is common in various cancers, pancreatic ductal adenocarcinoma (PDAC) cells can survive a state of glycolysis suppression. We aimed to identify potential therapeutic targets in glycolysis-suppressed PDAC cells. By screening anticancer metabolic compounds, we identified SP-2509, an inhibitor of lysine-specific histone demethylase 1A (LSD1), which dramatically decreased the growth of PDAC PANC-1 cells and showed an anti-tumoral effect in tumor-bearing mice. The growth of glycolysis-suppressed PANC-1 cells was also inhibited by another LSD1 inhibitor, OG-L002. Similarly, the other two PDAC cells (PK-1 and KLM-1) with suppressed glycolysis exhibited anticancer effects against SP-2509. However, the anticancer effects on PDAC cells were unrelated to LSD1. To investigate how PDAC cells survive in a glycolysis-suppressed condition, we conducted proteomic analyses. These results combined with our previous findings suggested that glucose-starvation causes PDAC cells to enhance mitochondrial oxidative phosphorylation. In particular, mitochondrial fatty acid metabolism was identified as a key factor contributing to the survival of PDAC cells under glycolysis suppression. We further demonstrated that SP-2509 and OG-L002 disturbed fatty acid metabolism and induced lipid droplet accumulation through the impairment of lipophagy, but not bulk autophagy. These findings indicate a significant potential association of lipophagy and anticancer effects in glycolysis-suppressed PDAC cells, offering ideas for new therapeutic strategies for PDAC by dual inhibition of glycolysis and fatty acids metabolism.

E3 ligase Trim35 inhibits LSD1 demethylase activity through K63-linked ubiquitination and enhances anti-tumor immunity in NSCLC

Targeting lysine-specific histone demethylase 1A (LSD1) can improve tumor immunogenicity of poorly immunogenic tumors, such as non-small cell lung cancer (NSCLC), with elevated Tcell infiltration and sensitize tumors to anti-PD-1 therapy. However, the lack of reliable biomarkers limits utilization of LSD1 inhibitors in cancer therapy. Here, we identify an E3 ligase, Trim35, as an effective biomarker for high activity of LSD1 to predict prognosis of LSD1-targeted therapy as well as immunotherapy. Mechanistically, Trim35 represses LSD1 demethylase activity by mediating K63 ubiquitination at lysine site 422 of LSD1. Suppressed LSD1 activity facilitates ERGIC1 transcription, followed by autophagy inhibition and IFNGR1 stabilization to activate IFN-γ signaling, leading to increased MHC class I expression and immune surveillance of NSCLC cells. Furthermore, combinational use of an LSD1 inhibitor and anti-PD-1 therapy can significantly eradicate poorly immunogenic lung cancer with low Trim35. These findings strongly suggest that Trim35 is a promising biomarker for prediction of immunotherapy outcome in NSCLC.

Salsolinol improves angiotensin II‑induced myocardial fibrosis invitro via inhibition of LSD1 through regulation of the STAT3/Notch‑1 signaling pathway.

The clinical incidence of congestive heart failure (CHF) is very high and it poses a significant threat to the health of patients. The traditional Chinese medicine monomer salsolinol is widely used to treat similar symptoms of CHF. However, there have been no reports on the effect of salsolinol for the management of CHF and its effects on myocardial fibrosis. In the present study, salsolinol was used to treat angiotensin II (AngII)-induced human cardiac fibroblasts (HCFs) and cell proliferation and migration were assessed using a CCK-8, EdU staining assay and wound healing assay. Subsequently, immunofluorescence, western blotting and other techniques were used to detect indicators associated with cell fibrosis and relevant kits were used to detect markers of cellular inflammation and reactive oxygen species (ROS) production. Molecular docking analysis was used to predict the relationship between salsolinol and lysine-specific histone demethylase 1A (LSD1). Subsequently, the expression of LSD1 in the serum of CHF patients was detected by reverse transcription-quantitative PCR. Finally, LSD1 was overexpressed in cells to explore the regulatory mechanism of salsolinol in AngII-induced HFCs. Salsolinol reduced the proliferation and migration. Salsolinol reduced the expression of fibrosis marker proteins α-smooth muscle actin, Collagen I and Collagen III in a concentration-dependent manner, thereby reducing cell fibrosis. In addition, salsolinol reduced the levels of TNF-α and IL-6 in the cell supernatant and ROS production following AngII induction. Salsolinol inhibited LSD1 expression and regulated the STAT3/Notch-1 signaling pathway. Upregulation of LSD1 reversed the effects of salsolinol on AngII-induced HCFs. Salsolinol inhibited LSD1 via regulation of the STAT3/Notch-1 signaling pathway to improve Ang II-induced myocardial fibrosis in vitro.

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 (ERβ) 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 ERβ and regulates its expression/functions in OCa. Analysis of TCGA data sets indicated KDM1A and ERβ expression showed an inverse relationship in OCa. Knockout (KO), knockdown (KD), or inhibition of KDM1A increased ERβ isoform 1 expression in established and patient-derived OCa cells. Further, KDM1A interacts with and functions as a corepressor of ERβ, and its inhibition enhances ERβ target gene expression via alterations of histone methylation marks at their promoters. Importantly, KDM1A-KO or -KD enhanced the efficacy of ERβ agonist LY500307, and the combination of KDM1A inhibitor (KDM1Ai) NCD38 with ERβ 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 ERβ signaling and that genes altered by KDM1A-KO and ERβ 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 ERβ expression/functions, and its inhibition improves ERβ mediated tumor suppression. Overall, our findings suggest that KDM1Ai and ERβ agonist combination therapy is a promising strategy for OCa.

Demethylase-independent roles of LSD1 in regulating enhancers and cell fate transition

The major enhancer regulator lysine-specific histone demethylase 1A (LSD1) is required for mammalian embryogenesis and is implicated in human congenital diseases and multiple types of cancer; however, the underlying mechanisms remain enigmatic. Here, we dissect the role of LSD1 and its demethylase activity in gene regulation and cell fate transition. Surprisingly, the catalytic inactivation of LSD1 has a mild impact on gene expression and cellular differentiation whereas the loss of LSD1 protein de-represses enhancers globally and impairs cell fate transition. LSD1 deletion increases H3K27ac levels and P300 occupancy at LSD1-targeted enhancers. The gain of H3K27ac catalyzed by P300/CBP, not the loss of CoREST complex components from chromatin, contributes to the transcription de-repression of LSD1 targets and differentiation defects caused by LSD1 loss. Together, our study demonstrates a demethylase-independent role of LSD1 in regulating enhancers and cell fate transition, providing insight into treating diseases driven by LSD1 mutations and misregulation.

Lysine-specific histone demethylase 1A mediates the up-regulation of NADPH oxidase expression in the kidney of diabetic mice

Lysine-specific histone demethylase 1A mediates the up-regulation of NADPH oxidase expression in the kidney of diabetic mice

Targeting lysine-specific demethylase 1 (KDM1A/LSD1) impairs colorectal cancer tumorigenesis by affecting cancer cells stemness, motility, and differentiation

Among all cancers, colorectal cancer (CRC) is the 3rd most common and the 2nd leading cause of death worldwide. New therapeutic strategies are required to target cancer stem cells (CSCs), a subset of tumor cells highly resistant to present-day therapy and responsible for tumor relapse. CSCs display dynamic genetic and epigenetic alterations that allow quick adaptations to perturbations. Lysine-specific histone demethylase 1A (KDM1A also known as LSD1), a FAD-dependent H3K4me1/2 and H3K9me1/2 demethylase, was found to be upregulated in several tumors and associated with a poor prognosis due to its ability to maintain CSCs staminal features. Here, we explored the potential role of KDM1A targeting in CRC by characterizing the effect of KDM1A silencing in differentiated and CRC stem cells (CRC-SCs). In CRC samples, KDM1A overexpression was associated with a worse prognosis, confirming its role as an independent negative prognostic factor of CRC. Consistently, biological assays such as methylcellulose colony formation, invasion, and migration assays demonstrated a significantly decreased self-renewal potential, as well as migration and invasion potential upon KDM1A silencing. Our untargeted multi-omics approach (transcriptomic and proteomic) revealed the association of KDM1A silencing with CRC-SCs cytoskeletal and metabolism remodeling towards a differentiated phenotype, supporting the role of KDM1A in CRC cells stemness maintenance. Also, KDM1A silencing resulted in up-regulation of miR-506-3p, previously reported to play a tumor-suppressive role in CRC. Lastly, loss of KDM1A markedly reduced 53BP1 DNA repair foci, implying the involvement of KDM1A in the DNA damage response. Overall, our results indicate that KDM1A impacts CRC progression in several non-overlapping ways, and therefore it represents a promising epigenetic target to prevent tumor relapse.

Abstract 6263: Lysine-specific histone demethylase 1A (KDM1A/LSD1) inhibition attenuates DNA double-strand break repair and augments efficacy of temozolomide in glioblastoma

Abstract Background: Glioblastoma (GBM) patients face a dismal prognosis, with median survival of ~15 months and 5-year survival rate of 6.8%. Standard of care includes surgical resection, external beam radiation therapy, and adjuvant chemotherapy with temozolomide (TMZ). Unfortunately, all GBM patients succumb to their disease. Glioma stem cells (GSCs), a GBM subpopulation, possess enhanced DNA repair that contributes to chemoradiotherapy resistance. The epigenetic modifier lysine-specific histone demethylase 1A (KDM1A/LSD1) is overexpressed in GBM and emerging studies show KDM1A is implicated in DNA damage response. This study aims to determine the significance of KDM1A to promote DNA repair in GSCs. We hypothesize KDM1A inhibition augments therapeutic efficacy of TMZ via attenuation of DNA repair pathways. Methods: Patient-derived GSCs were obtained via IRB-approved protocol from patient samples at UT Health San Antonio. KDM1A knockdown and knockout cells were generated by transduction of validated KDM1A-specific shRNA or gRNA, respectively. Brain bioavailability of KDM1A inhibitor NCD38 was established using LS-MS/MS. Effect of combination KDM1A knockdown or inhibition with TMZ was studied using cell viability, neurosphere, and self-renewal assays. Mechanistic studies were conducted using CUT&Tag-seq, RNA-seq, immunofluorescence, comet, Western blotting, RT-qPCR, homologous recombination (HR), and non-homologous end-joining (NHEJ) reporter assays. In vivo efficacy of KDM1A knockdown or inhibitor alongside TMZ was determined using orthotopic murine GBM models. Results: GBM patient data sets showed KDM1A is elevated in recurrent GBM versus primary tumors. KDM1A knockdown, knockout, or pharmacological inhibition increased efficacy of TMZ in reducing cell viability and self-renewal of GSCs. Pharmacokinetic studies demonstrated KDM1A inhibitor NCD38 is readily brain penetrable. CUT&Tag-seq studies showed KDM1A is enriched at the promoters of DNA repair genes. RNA-seq studies confirmed that KDM1A inhibition reduced expression of DNA repair-related genes. RT-qPCR and Western blotting validated downregulation of DNA double-strand break repair genes. Knockdown, knockout, or inhibition of KDM1A attenuated HR and NHEJ-mediated DNA repair capacity. Immunofluorescence and comet assay demonstrated increased TMZ-mediated DNA damage in NCD38-treated GSCs. Importantly, KDM1A knockdown or inhibition enhanced efficacy of TMZ and significantly improved survival of mice bearing orthotopic GBM tumors. Conclusions: Our results provide compelling evidence KDM1A is essential for DNA repair in GSCs and KDM1A inhibition sensitizes GBM to TMZ via attenuation of DNA repair pathways. These findings suggest combination therapy of KDM1A inhibitor NCD38 with TMZ is a potential novel therapeutic strategy to improve GBM outcomes. Citation Format: Salvador Alejo, Bridgitte Palacios, Prabhakar Pitta Venkata, He Yi, Wenjing Li, Jessica Johnson, Sridharan Jayamohan, Weixing Zhao, Siyuan Zheng, Takayoshi Suzuki, Rajeshwar R. Tekmal, Andrew Brenner, Ratna K. Vadlamudi, Gangadhara R. Sareddy. Lysine-specific histone demethylase 1A (KDM1A/LSD1) inhibition attenuates DNA double-strand break repair and augments efficacy of temozolomide in glioblastoma [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 6263.

Abstract 3081: KDM1A/LSD1 inhibition enhances estrogen receptor beta mediated tumor suppression in ovarian cancer

Abstract Introduction: Ovarian cancer (OCa) is the deadliest of all gynecologic cancers. Recent studies suggest that OCa cells express estrogen receptor beta (ERb), which functions as a tumor suppressor. However, ERb expression decreases during tumor progression and under the selection pressure of chemotherapy; this decrease occurs via epigenetic mechanisms and is inversely correlated with OCa progression. Lysine-specific histone demethylase 1A (KDM1A/LSD1), an epigenetic modifier, is highly overexpressed in OCa. However, it is not known if KDM1A regulates ERb expression and its corresponding tumor suppressor functions. In this study, we explored the hypotheses that KDM1A inhibits ERb expression and function and that ERb-mediated tumor suppression in OCa is enhanced by KDM1A inhibition. Methods: Transduction of KDM1A-specific shRNA or gRNA was used to generate KDM1A knockdown (KD) or knockout (KO) cells, respectively. Cell viability, survival, apoptosis, and invasion assays were used to determine the impact of KDM1A knockdown or inhibitor therapy on ERb agonist response in established and patient-derived OCa cells. Mechanistic studies were conducted using RNA-seq, discovery proteomics, chromatin immunoprecipitation (ChIP), co-immunoprecipitation (co-IP), proximity ligation, ERE-Luc reporter, RT-qPCR, and western blot analysis. The efficacy of the combination of KDM1A inhibitor and ERb agonist therapy was investigated using patient-derived explant (PDeX) models. In vivo efficacy of KDM1A inhibitor and ERb agonist was determined using orthotopic OCa and patient-derived xenograft (PDX) murine models. Results: Analysis of TCGA datasets revealed a negative correlation between ERb and KDM1A expression in OCa patients. ChIP experiments showed that KDM1A is enriched at the ERb 0N promoter and that KDM1A KD, KO, or inhibition specifically upregulates the expression of ERb isoform 1. Co-IP and proximity ligation assays demonstrated that KDM1A interacts with ERb. Combining the ERb agonist LY500307 with KDM1A inhibitor NCD38 decreased the cell viability, survival, and invasion of both established and patient-derived primary OCa cells while increasing apoptosis. KDM1A KD- or inhibition potentiated the efficacy of ERb agonist in inducing ERb target gene expression via altered histone methylation marks. RNA-seq and proteomic analysis demonstrated that KDM1A KO and ERb agonist treatment increased the expression of apoptotic genes and downregulated cell cycle, EMT, and DNA repair genes. Importantly, combination therapy significantly reduced OCa cell proliferation in PDeX models and reduced in vivo tumor growth in orthotopic and PDX models. Conclusions: Our findings demonstrate that KDM1A inhibition enhances ERb expression and tumor suppressive functions, suggesting that combination therapy of a KDM1A inhibitor and ERb agonist may be a promising therapeutic option for treating OCa. Citation Format: Prabhakar P. Venkata, Sridharan Jayamohan, Yi He, Salvador Alejo, Jessica D. Johnson, Uday P. Pratap, Suryavathi Viswanadhapalli, Susan Weintraub, Rajeshwar R. Tekmal, Ratna K. Vadlamudi, Edward Kost, Gangadhara R. Sareddy. KDM1A/LSD1 inhibition enhances estrogen receptor beta mediated tumor suppression in ovarian 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 3081.

301 Inhibition of lysine-specific histone demethylase 1A (KDM1A/LSD1) attenuates DNA double strand break repair and enhances efficacy of temozolomide in glioblastoma

OBJECTIVES/GOALS: Glioblastoma (GBM) patients face a poor prognosis. Glioma stem cells (GSCs), a chemo resistant GBM subpopulation, possess enhanced DNA repair and elevated levels of epigenetic modifier KDM1A. This study aims to establish the significance of KDM1A in DNA repair and determine the potential of novel KDM1A inhibitor NCD38 to enhance TMZ efficacy in GSCs. METHODS/STUDY POPULATION: Patient derived GSCs were obtained via IRB-approved protocol from patient samples at UT Health San Antonio. KDM1A knockdown and knockout cells were generated by transduction of validated KDM1A-specific shRNA or gRNA, respectively. Brain bioavailability of KDM1A inhibitor NCD38 was established using LS-MS/MS. Effect of combination of KDM1A knockdown, knockout, or inhibition with TMZ was studied using cell viability, neurosphere, and self-renewal assays. Mechanistic studies were conducted using CUT&Tag-seq, RNA-seq, immunofluorescence, comet, Western blotting, RT-qPCR, homologous recombination (HR) or non-homologous end-joining (NHEJ) DNA repair reporter assays. In vivo efficacy of KDM1A knockdown or inhibitor alongside TMZ treatment was determined using orthotopic murine GBM models. RESULTS/ANTICIPATED RESULTS: KDM1A knockdown, knockout, or inhibition increased efficacy of TMZ in reducing cell viability and self-renewal of GSCs. Pharmacokinetic studies demonstrated KDM1A inhibitor NCD38 is readily brain penetrable. CUT&Tag-seq studies revealed KDM1A is enriched at DNA repair gene promoters. RNA-seq studies suggest KDM1A inhibition reduces DNA double strand break repair gene expression, with these findings validated using RT-qPCR and Western blotting. Knockdown, knockout, or inhibition of KDM1A attenuated HR and NHEJ-mediated DNA repair capacity. Immunofluorescence and comet assay support findings of increased DNA damage in NCD38/TMZ combination treated GSCs. Importantly, KDM1A knockdown or inhibition enhanced efficacy of TMZ and significantly improved survival of orthotopic GBM tumor-bearing mice. DISCUSSION/SIGNIFICANCE: Our results show compelling evidence that KDM1A is essential for DNA repair in GSCs and that KDM1A inhibition sensitizes GBM to TMZ via attenuation of DNA repair pathways. These findings suggest combination of KDM1A inhibitor NCD38 with TMZ could serve as a promising novel therapeutic strategy that can be translated to improve GBM patient outcomes.

Abstract P6-12-08: Identifying FOXA1 Binding Partners using Proximity Labeling

Abstract Approximately 75% of breast cancers are driven by the estrogen receptor alpha (ER), and despite the advent of endocrine therapy to block ER signaling pathways, a significant portion of women develop resistance to these drugs. The pioneer factor FOXA1 has been shown to facilitate nearly all DNA-binding events of ER in response to estrogen in ER+ breast cancer (ER+BC). Notably, up-regulation of FOXA1 is a hallmark of endocrine-resistant phenotypes and has been shown to reprogram enhancer elements, leading to an altered transcriptome. However, FOXA1 is a critical pioneer factor for multiple nuclear hormone receptors, aside from ER, and is implicated in regulation of important factors such as HER2 and the androgen receptor (AR). With the diverse array of breast cancer molecular subtypes displaying complex interplay between ER, HER2, AR, PR, and other hormone receptors, describing the complete ensemble of FOXA1 binding partners in various contexts, such as endocrine-resistant tumors, is of increasing importance. To define a comprehensive catalog of FOXA1 binding partners under basal conditions, we generated MCF-7 cell lines stably expressing constructs of FOXA1 fused at its N- or C-terminus to the biotin ligase miniTurbo. Using proximity labeling coupled with mass-spectrometry, we have comprehensively cataloged binding partners of FOXA1, including many expected proteins such as ER, AR, MLL3, YAP1, and GATA-3. Moreover, we have discovered more than 150 previously unidentified binding partners of FOXA1, which may exert profound effects on FOXA1 function. Importantly, high hazard ratios and significant dependencies are associated with several of these new binding partners, such as subunits of a previously described histone deacetylase (HDAC) complex containing genetic suppressor element 1 (GSE1) and lysine-specific histone demethylase 1A (KDM1A). Genomic approaches are currently underway to characterize where in the genome FOXA1 is interacting with these novel proteins and to guide future exploration into the physiological significance of these interactions. Integrating biochemical, molecular, and genomic approaches, we have potentially highlighted new mechanisms of FOXA1, which could have significant clinical impact in the future. Citation Format: Rosemary N. Plagens, Shen Li, Christine A. Mills, Laura Herring, Hector L. Franco. Identifying FOXA1 Binding Partners using Proximity Labeling [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 P6-12-08.

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

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). 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. 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. Acombination of KDM1A knockdown or inhibition and TMZ treatment significantly enhanced the survival of tumor-bearing mice. Our results provide evidence that KDM1A inhibition sensitizes GBM to TMZ via attenuation of DNA DSB repair pathways.

YTHDF2 Regulates Cell Growth and Cycle by Facilitating KDM1A mRNA Stability

Breast cancer is the leading cause of cancer death in women. The physiological functions of N6-methyladenosine methylation in cancer have been the focus of studies in recent years. Herein, four data sets (GSE70947, GSE45827, GSE42586, and The Cancer Genome Atlas Breast Cancer) were analyzed to confirm the differentially expressed N6-methyladenosine genes. YTH N6-methyladenosine RNA-binding protein 2 (YTHDF2) was found to be highly expressed in breast cancer tissues and cells. Invitro, YTHDF2 affects cell proliferation, cell cycle, and invasive ability. Tumorigenesis in xenograft nude mice confirmed that YTHDF2 interference reduced the tumor formation ability of cancer cells. Pearson correlation analysis demonstrated a positive correlation between YTHDF2 and lysine-specific histone demethylase 1A (KDM1A) expression. An online tool, Sequence-based RNA Adenosine Methylation Site Predictor (SRAMP), predicted eight methylation sites in the KDM1A mRNA sequence. The expression of KDM1A was dramatically increased in breast cancer tissues and cells. Down-regulation of YTHDF2 reduced KDM1A expression and the methylation level of KDM1A mRNA. YTHDF2 interference promoted the degradation of KDM1A mRNA, which suggested an interaction between YTHDF2 and KDM1A. KDM1A interference altered cell proliferation, cell cycle, and invasive ability, whereas YTHDF2 overexpression rescued KDM1A interference-induced cell phenotypic changes. In conclusion, YTHDF2 promotes breast cancer cell growth and cell cycle progression by facilitating KDM1A mRNA stability. This study provides new therapeutic targets for breast cancer treatment in the future.

Epigenetic modification mechanism of histone demethylase KDM1A in regulating cardiomyocyte apoptosis after myocardial ischemia-reperfusion injury.

Hypoxia and reoxygenation (H/R) play a prevalent role in heart-related diseases. Histone demethylases are involved in myocardial injury. In this study, the mechanism of the lysine-specific histone demethylase 1A (KDM1A/LSD1) on cardiomyocyte apoptosis after myocardial ischemia-reperfusion injury (MIRI) was investigated. Firstly, HL-1 cells were treated with H/R to establish the MIRI models. The expressions of KDM1A and Sex Determining Region Y-Box Transcription Factor 9 (SOX9) in H/R-treated HL-1 cells were examined. The cell viability, markers of myocardial injury (LDH, AST, and CK-MB) and apoptosis (Bax and Bcl-2), and Caspase-3 and Caspase-9 protein activities were detected, respectively. We found that H/R treatment promoted cardiomyocyte apoptosis and downregulated KDM1A, and overexpressing KDM1A reduced apoptosis in H/R-treated cardiomyocytes. Subsequently, tri-methylation of lysine 4 on histone H3 (H3K4me3) level on the SOX9 promoter region was detected. We found that KDM1A repressed SOX9 transcription by reducing H3K4me3. Then, HL-1 cells were treated with CPI-455 and plasmid pcDNA3.1-SOX9 and had joint experiments with pcDNA3.1-KDM1A. We disclosed that upregulating H3K4me3 or overexpressing SOX9 reversed the inhibitory effect of overexpressing KDM1A on apoptosis of H/R-treated cardiomyocytes. In conclusion, KDM1A inhibited SOX9 transcription by reducing the H3K4me3 on the SOX9 promoter region and thus inhibited H/R-induced apoptosis of cardiomyocytes.

Lysine-specific histone demethylase 1A mediates oxidative stress, inflammation, and atherogenesis in apolipoprotein E-deficient mice; prospective implications for human atherosclerosis

Background and Aims : Epigenetic mechanisms play a major role in the pathophysiology of cardiovascular diseases. The implication of histone methylation-based regulatory mechanisms in atherosclerosis remains poorly understood. We aimed at investigating the potential role of lysine-specific histone demethylase 1A (KDM1A) in the regulation of key down-stream molecular effectors and pathways linked to atherosclerotic plaque formation.

QSAR Evaluations to Unravel the Structural Features in Lysine-Specific Histone Demethylase 1A Inhibitors for Novel Anticancer Lead Development Supported by Molecular Docking, MD Simulation and MMGBSA.

Using 84 structurally diverse and experimentally validated LSD1/KDM1A inhibitors, quantitative structure–activity relationship (QSAR) models were built by OECD requirements. In the QSAR analysis, certainly significant and understated pharmacophoric features were identified as critical for LSD1 inhibition, such as a ring Carbon atom with exactly six bonds from a Nitrogen atom, partial charges of lipophilic atoms within eight bonds from a ring Sulphur atom, a non-ring Oxygen atom exactly nine bonds from the amide Nitrogen, etc. The genetic algorithm–multi-linear regression (GA-MLR) and double cross-validation criteria were used to create robust QSAR models with high predictability. In this study, two QSAR models were developed, with fitting parameters like R2 = 0.83–0.81, F = 61.22–67.96, internal validation parameters such as Q2LOO = 0.79–0.77, Q2LMO = 0.78–0.76, CCCcv = 0.89–0.88, and external validation parameters such as, R2ext = 0.82 and CCCex = 0.90. In terms of mechanistic interpretation and statistical analysis, both QSAR models are well-balanced. Furthermore, utilizing the pharmacophoric features revealed by QSAR modelling, molecular docking experiments corroborated with the most active compound’s binding to the LSD1 receptor. The docking results are then refined using Molecular dynamic simulation and MMGBSA analysis. As a consequence, the findings of the study can be used to produce LSD1/KDM1A inhibitors as anticancer leads.

Uncovering the Therapeutic Potential of Purified Protein Extract From Allium sativum and Ibervillea sonorae Greene as Treatment of Non-infectious and Infectious Diseases.

ObjectivesThe main objective was to examine the therapeutic effects of purified proteins from Allium sativum (Garlic) and Ibervillea sonorae Greene (Wereke) inhibit human Dipeptidyl peptidase-4 (DPP-4) Lysine-specific histone demethylase 1A (LSD1) known to play a role in treating chronic diseases. Also, cytotoxic effect on cell model studies to reveal the possibility of repurposing proteins from plants as a source to improve the therapeutic opportunities of chronic diseases. MethodsMass spectrometric validated purified proteins were obtained using the pulverization method. Fluorometric assays were carried out to determine the inhibitory activity of DPP4 and LSD1. In cell-based models, we had evaluated cell proliferation and cytotoxic effect on skeletal muscle cells, kidney cells, and lung cancer cells to reveal the potential of purified proteins. ResultsThe purified proteins (0.005 mg/ml) effect on therapeutic drug targets revealed that Wereke protein extract doesn’t inhibit DPP-4, whereas Garlic does inhibit significantly by 70%. Further, prepared proteins are non-toxic to insulin-stimulated skeletal muscles (Figure 1). Indeed, muscle cells adopted a proliferation state, revealing their ability to induce glucose homeostasis of differentiated skeletal muscle cells and anti-diabetic potential. To further demonstrate its anticancer potential on cancer cells, we had evaluated its effect on the lung cancer model. It is observed that both plant extract possesses an anticancer effect (Figure 2), revealing its possibility to repurpose for cancer treatments. ConclusionsOur results demonstrate that the therapeutic potential of total protein extracts from Garlic and Wereke are not toxic to skeletal muscle cells, induce muscle cell differentiation, induce glucose homeostasis, and as well possess the potential to be anticancer activity. Therefore, the proposed protein extracts in this study must be considered for further developing plants as future opportunistic therapy to control non-infectious and infectious diseases. Indeed, plant proteins can be given as supplements to improve existing treatment outcomes of aging and chronic diseases. Funding SourcesAll authors wish to express their gratitude to Escuela Nacional de Ciencias Biológicas-Instituto Politécnico Nacional (ENCB-IPN) and CONACYT for providing financial support for the present study.