LSD1 Research Articles

Silencing DDX3 Attenuates Interleukin-1β-Induced Intervertebral Disc Degeneration Through Inhibiting Pyroptosis.

Intervertebral disc degeneration (IVDD) is a common disorder associated with chronic inflammation and cell death. In this study, an IVDD rat model was created through Interleukin-1β (IL-1β) injection. The degeneration of intervertebral disc tissues was assessed using magnetic resonance imaging (MRI), followed by hematoxylin and eosin (H&E) and terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) staining. RNA sequencing was performed to identify differentially expressed genes (DEGs) between the IVDD model and control rats. The expression levels of DEGs (DEAD-box polypeptide 3 (DDX3), lysine-specific demethylase 5D (KDM5D), interferon-induced gene-1 (IFIT1), ribosomal protein S10 (RPS10), tenomodulin (TNMD), and pentraxin 3 (PTX3)) were measured by real-time quantitative polymerase chain reaction (RT-qPCR). The regulatory effect of DDX3 on pyroptosis in IL-1β-treated nucleus pulpous (NP) cells was assessed after transfection with siRNA of DDX3. A total of 601 DEGs were identified from the IVDD model rat, and were abundant in extracellular matrix (ECM) organization, ECM-receptor interaction, and inflammatory pathways, including the PI3K-Akt, TNF, and AMPK signaling pathways. DDX3, KDM5D, and IFIT1 levels were notably elevated, whereas RPS10, TNMD, and PTX3 levels were decreased in the IL-1β-induced IVDD rat model. Moreover, silencing DDX3 promoted cell proliferation and abolished IL-1β-induced cell apoptosis and pyroptosis. This study revealed the role of DDX3 in IVDD pyroptosis, providing potential target for IVDD management.

SP2509, a specific antagonist of LSD1, exhibits antiviral properties against Porcine epidemic diarrhea virus

BackgroundPorcine epidemic diarrhea virus (PEDV), a type of coronavirus, is one of the main pathogens that can infect pigs of all ages. It causes diarrhea and acute death of newborn piglets, resulting in massive economic losses to the worldwide swine industry. While vaccination remains the primary approach in combating PEDV, it often fails to address all the challenges posed by the infection, particularly in light of the emergence of evolving mutant strains. Therefore, there is a critical need to identify potent antiviral drugs that can effectively safeguard pigs against PEDV infection.ResultsIn this study, the antiviral efficacy of SP2509, a specific antagonist of Lysine-specific demethylase 1(LSD1), was evaluated in vitro. The RT-qPCR, Western blot, TCID50, and IFA showed that at a concentration of 1µmol/L, SP2509 significantly inhibited PEDV infection. Additionally, viral life cycle assays showed that SP2509 operates by impeding PEDV internalization and replication rather than attachment and release. Regarding mechanism, in Huh-7 cells, knockdowns LSD1 can suppress PEDV replication. This indicated that the inhibition effect of SP2509 on PEDV largely depends on the activity of its target protein, LSD1.ConclusionOur results in vitro show that SP2509 can inhibit PEDV infection during the internalization and replication stage and revealed a role of LSD1 as a restriction factor for PEDV. These imply that LSD1 might be a target for interfering with the viral infection, and SP2509 could be developed as an effective anti-PEDV agent.

Inhibition of LSD1 via SP2509 attenuated the progression of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by synovial hyperplasia, pannus formation, and cartilage and bone destruction. Lysine-specific demethylase 1 (LSD1), an enzyme involved in transcriptional regulation, has an unclear role in synovial inflammation, fibroblast-like synoviocytes migration, and invasion during RA pathogenesis. In this study, we observed increased LSD1 expression in RA synovial tissues and in TNF-α-stimulated MH7A cells. SP2509, an LSD1 antagonist, directly reduced LSD1 expression and reversed the elevated levels of proteins associated with inflammation, apoptosis, proliferation, and autophagy induced by TNF-α. Furthermore, SP2509 inhibited the migratory capacity of MH7A cells, which was enhanced by TNF-α. In CIA models, SP2509 treatment ameliorated RA development, reducing the expression of pro-inflammatory cytokines and alleviating joint pathological symptoms. These findings underscore the significance of LSD1 in RA and propose the therapeutic potential of SP2509.

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.

Lysine-specific demethylase 1 (LSD1) participate in porcine early embryonic development by regulating cell autophagy and apoptosis through the mTOR signaling pathway

Lysine-specific demethylase 1 (LSD1) stands as the pioneering histone demethylase uncovered, proficient in demethylating H3K4me1/2 and H3K9me1/2, thereby governing transcription and participating in cell apoptosis, proliferation, or differentiation. Nevertheless, the complete understanding of LSD1 during porcine early embryonic development and the underlying molecular mechanism remains unclear. Thus, we investigated the mechanism by which LSD1 plays a regulatory role in porcine early embryos. This study revealed that LSD1 inhibition resulted in parthenogenetic activation (PA) and in vitro fertilization (IVF) embryo arrested the development, and decreased blastocyst quality. Meanwhile, H3K4me1/2 and H3K9me1/2 methylase activity was increased at the 4-cell embryo stage. RNA-seq results revealed that autophagy related biological processes were highly enriched through GO and KEGG pathway analyses when LSD1 inhibition. Further studies showed that LSD1 depletion in porcine early embryos resulted in low mTOR and p-mTOR levels and high autophagy and apoptosis levels. The LSD1 deletion-induced increases in autophagy and apoptosis could be reversed by addition of mTOR activators. We further demonstrated that LSD1 inhibition induced mitochondrial dysfunction and mitophagy. In summary, our research results indicate that LSD1 may regulate autophagy and apoptosis through the mTOR pathway and affect early embryonic development of pigs.

Discovery of an efficacious KDM5B PROTAC degrader GT-653 up-regulating IFN response genes in prostate cancer

Epigenetic alterations promote cancer development by regulating the expression of various oncogenes and anti-oncogenes. Histone methylation modification represents a pivotal area in epigenetic research and numerous publications have demonstrated that aberrant histone methylation is highly correlated with tumorigenesis and development. As a key histone demethylase, lysine-specific demethylase 5B (KDM5B) demethylates lysine 4 of histone 3 (H3K4) and serves as a transcriptional repressor of certain tumor suppressor genes. Meanwhile, KDM5B inhibits STING-induced intrinsic immune response of tumor cells or recruits SETDB1 through non-enzymatic function to silence reverse transcription elements to promote immune escape. The conventional small molecule inhibitors can only inhibit the enzymatic function of KDM5B with no effect on the non-enzymatic function. In the article, we present the development of the first series of KDM5B degraders based on CPI-455 to inhibit the non-enzymatic function. Among them, GT-653 showed optimal KDM5B degradation efficiency in a ubiquitin proteasome-dependent manner. GT-653 efficiently reduced KDM5B protein levels without affecting KDM5B transcription. Interestingly, GT-653 increased H3K4me3 levels and activated the type-I interferon signaling pathway in 22RV1 cells without significant phenotypic response on cell proliferation.

Overview and new insights of lysine-specific histone demethylase 1 in colorectal cancer: promoting epithelial-mesenchymal transition and stemness features of cancer stem cells

Abstract Colorectal cancer (CRC) is a common malignant tumor, and research on its pathological mechanism has received increasing attention. Most CRC patients have a poor prognosis, and there is still a lack of effective immunotherapy options. An in-depth exploration of the molecular mechanism of CRC occurrence and development is of great clinical significance for the diagnosis, treatment guidance, and prognosis of CRC. Lysine-specific histone demethylase 1 (LSD1) is highly expressed in CRC, and closely related to the occurrence, invasion, metastasis, and drug resistance of CRC. The histone H3K27 demethylase KDM6A forms an inhibitory complex with LSD1 and other epigenetic regulators, silencing epithelial-mesenchymal transition (EMT) transcription factors and inhibiting EMT-induced cancer stem cells (CSCs) properties. LSD1 is a promising target for CRC therapy, some LSD1 inhibitors are in the experimental stage by blocking its demethylase activity and may benefit CRC patients in the clinical treatment course in the future. This article reviews the latest research progress on the function of LSD1 and its relationship with CRC.

LSD1 inhibition circumvents glucocorticoid-induced muscle wasting of male mice

Synthetic glucocorticoids (GC), such as dexamethasone, are extensively used to treat chronic inflammation and autoimmune disorders. However, long-term treatments are limited by various side effects, including muscle atrophy. GC activities are mediated by the glucocorticoid receptor (GR), that regulates target gene expression in various tissues in association with cell-specific co-regulators. Here we show that GR and the lysine-specific demethylase 1 (LSD1) interact in myofibers of male mice, and that LSD1 connects GR-bound enhancers with NRF1-associated promoters to stimulate target gene expression. In addition, we unravel that LSD1 demethylase activity is required for triggering starvation- and dexamethasone-induced skeletal muscle proteolysis in collaboration with GR. Importantly, inhibition of LSD1 circumvents muscle wasting induced by pharmacological levels of dexamethasone, without affecting their anti-inflammatory activities. Thus, our findings provide mechanistic insights into the muscle-specific GC activities, and highlight the therapeutic potential of targeting GR co-regulators to limit corticotherapy-induced side effects.

LSD1 drives intestinal epithelial maturation and controls small intestinal immune cell composition independent of microbiota in a murine model

Postnatal development of the gastrointestinal tract involves the establishment of the commensal microbiota, the acquisition of immune tolerance via a balanced immune cell composition, and maturation of the intestinal epithelium. While studies have uncovered an interplay between the first two, less is known about the role of the maturing epithelium. Here we show that intestinal-epithelial intrinsic expression of lysine-specific demethylase 1A (LSD1) is necessary for the postnatal maturation of intestinal epithelium and maintenance of this developed state during adulthood. Using microbiota-depleted mice, we find plasma cells, innate lymphoid cells (ILCs), and a specific myeloid population to depend on LSD1-controlled epithelial maturation. We propose that LSD1 controls the expression of epithelial-derived chemokines, such as Cxcl16, and that this is a mode of action for this epithelial-immune cell interplay in local ILC2s but not ILC3s. Together, our findings suggest that the maturing epithelium plays a dominant role in regulating the local immune cell composition, thereby contributing to gut homeostasis.

Characterization of structural, biochemical, pharmacokinetic, and pharmacodynamic properties of the LSD1 inhibitor bomedemstat in preclinical models.

Lysine-specific demethylase 1 (LSD1) is emerging as a critical mediator of tumor progression in metastatic castration-resistant prostate cancer (mCRPC). Neuroendocrine prostate cancer (NEPC) is increasingly recognized as an adaptive mechanism of resistance in mCRPC patients failing androgen receptor axis-targeted therapies. Safe and effective LSD1 inhibitors are necessary to determine antitumor response in prostate cancer models. For this reason, we characterize the LSD1 inhibitor bomedemstat to assess its clinical potential in NEPC as well as other mCRPC pathological subtypes. Bomedemstat was characterized via crystallization, flavine adenine dinucleotide spectrophotometry, and enzyme kinetics. On-target effects were assessed in relevant prostate cancer cell models by measuring proliferation and H3K4 methylation using western blot analysis. In vivo, pharmacokinetic (PK) and pharmacodynamic (PD) profiles of bomedemstat are also described. Structural, biochemical, and PK/PD properties of bomedemstat, an irreversible, orally-bioavailable inhibitor of LSD1 are reported. Our data demonstrate bomedemstat has >2500-fold greater specificity for LSD1 over monoamine oxidase (MAO)-A and -B. Bomedemstat also demonstrates activity against several models of advanced CRPC, including NEPC patient-derived xenografts. Significant intra-tumoral accumulation of orally-administered bomedemstat is measured with micromolar levels achieved in vivo (1.2 ± 0.45 µM at the 7.5 mg/kg dose and 3.76 ± 0.43 µM at the 15 mg/kg dose). Daily oral dosing of bomedemstat at 40 mg/kg/day is well-tolerated, with on-target thrombocytopenia observed that is rapidly reversible following treatment cessation. Bomedemstat provides enhanced specificity against LSD1, as revealed by structural and biochemical data. PK/PD data display an overall safety profile with manageable side effects resulting from LSD1 inhibition using bomedemstat in preclinical models. Altogether, our results support clinical testing of bomedemstat in the setting of mCRPC.

Effect of Low-Level Tragus Stimulation on Cardiac Metabolism in Heart Failure with Preserved Ejection Fraction: A Transcriptomics-Based Analysis.

Abnormal cardiac metabolism precedes and contributes to structural changes in heart failure. Low-level tragus stimulation (LLTS) can attenuate structural remodeling in heart failure with preserved ejection fraction (HFpEF). The role of LLTS on cardiac metabolism is not known. Dahl salt-sensitive rats of 7 weeks of age were randomized into three groups: low salt (0.3% NaCl) diet (control group; n = 6), high salt diet (8% NaCl) with either LLTS (active group; n = 8), or sham stimulation (sham group; n = 5). Both active and sham groups received the high salt diet for 10 weeks with active LLTS or sham stimulation (20 Hz, 2 mA, 0.2 ms) for 30 min daily for the last 4 weeks. At the endpoint, left ventricular tissue was used for RNA sequencing and transcriptomic analysis. The Ingenuity Pathway Analysis tool (IPA) was used to identify canonical metabolic pathways and upstream regulators. Principal component analysis demonstrated overlapping expression of important metabolic genes between the LLTS, and control groups compared to the sham group. Canonical metabolic pathway analysis showed downregulation of the oxidative phosphorylation (Z-score: -4.707, control vs. sham) in HFpEF and LLTS improved the oxidative phosphorylation (Z-score = -2.309, active vs. sham). HFpEF was associated with the abnormalities of metabolic upstream regulators, including PPARGC1α, insulin receptor signaling, PPARα, PPARδ, PPARGC1β, the fatty acid transporter SLC27A2, and lysine-specific demethylase 5A (KDM5A). LLTS attenuated abnormal insulin receptor and KDM5A signaling. HFpEF is associated with abnormal cardiac metabolism. LLTS, by modulating the functioning of crucial upstream regulators, improves cardiac metabolism and mitochondrial oxidative phosphorylation.

Baicalein promotes KDM4E to induce BICD1 and inhibit triple-negative breast cancer progression by blocking PAR1 signaling.

Baicalein has been implicated in the chemotherapy overcoming triple-negative breast cancer (TNBC). However, many unanswered questions remain regarding its role in treating TNBC. Here, we sought to demonstrate the molecular pathway mediated by baicalein in TNBC. Lysine-specific demethylase 4E (KDM4E), reduced in TNBC cells, was identified as a target protein of baicalein, and baicalein enhanced the protein expression and stability of KDM4E in TNBC cells. Knockdown of KDM4E attenuated the inhibitory effect of baicalein on TNBC cell activity, as demonstrated by intensified mobility, viability, and apoptosis resistance in TNBC cells. KDM4E activated protein bicaudal D homolog 1 (BICD1) expression by reducing the deposition of histone H3 lysine 9 trimethylation (H3K9me3) in its promoter, whereas BICD1 promoted protease-activated receptor-1 (PAR1) endocytosis and blocked PAR1 signaling through physical interaction with PAR1. Knockdown of KDM4E strengthened the PAR1-dependent activity of TNBC cells in response to thrombin activation, whereas TNBC progression activated by PAR1 signaling was blocked by combined overexpression of BICD1. Taken together, our data indicate that baicalein-promoted KDM4E enhanced the expression of BICD1 and activated the inhibitory effect of BICD1 on PAR1 signaling, thereby inhibiting TNBC progression.

Protein degradation of Lsd1 is mediated by Bre1 yet opposed by Lsd1-interacting lncRNAs during fly follicle development

Tissue development, homeostasis, and repair all require efficient progenitor expansion. Lysine-specific demethylase 1 (Lsd1) maintains plastic epigenetic states to promote progenitor proliferation while overexpressed Lsd1 protein causes oncogenic gene expression in cancer cells. However, the precise regulation of Lsd1 protein expression at the molecular level to drive progenitor differentiation remains unclear. Here, using Drosophila melanogaster oogenesis as our experimental system, we discovered molecular machineries that modify Lsd1 protein stability invivo. Through genetic and biochemical analyses, an E3 ubiquitin ligase, Bre1, was identified as required for follicle progenitor differentiation, likely by mediating Lsd1 protein degradation. Interestingly, specific Lsd1-interacting long non-coding RNAs (LINRs) were found to antagonize Bre1-mediated Lsd1 protein degradation. The intricate interplay discovered among the Lsd1 complex, LINRs and Bre1 provides insight into how Lsd1 protein stability is fine-tuned to underlie progenitor differentiation invivo.

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.

SP2509 functions as a novel ferroptosis inhibitor by reducing intracellular iron level in vascular smooth muscle cells

Previous studies have shown that ferroptosis of vascular smooth muscle cells (VSMCs) is involved in the development of aortic dissection (AD) and that histone methylation regulates this process. SP2509 acts as a specific inhibitor of lysine-specific demethylase 1 (LSD1), which governs a variety of biological processes. However, the effect of SP2509 on VSMC ferroptosis and AD remains to be elucidated. This aim of this study was to investigate the role and underlying mechanism of SP2509-mediated histone methylation on VSMC ferroptosis. Here, a mouse model of AD was established, and significantly reduced levels of H3K4me1 and H3K4me2 (target of SP2509) were found in the aortas of AD mice. In VSMCs, SP2509 treatment led to a dose-dependent increase in H3K4me2 levels. Furthermore, we found that SP2509 provided equivalent protection to ferrostatin-1 against VSMC ferroptosis, as evidenced by increased cell viability, decreased cell death and lipid peroxidation. RNA-sequencing analysis and subsequent experiments revealed that SP2509 counteracted cystine deficiency-induced response to inflammation and oxidative stress. More importantly, we demonstrated that SP2509 inhibited the expression of TFR and ferritin to reduce intracellular iron levels, thereby effectively blocking the process of ferroptosis. Therefore, our findings indicate that SP2509 protects VSMCs from multiple stimulus-induced ferroptosis by reducing intracellular iron levels, thereby preventing lipid peroxidation and cell death. These findings suggest that SP2509 may be a promising drug to alleviate AD by reducing iron deposition and VSMC ferroptosis.

GFI1B and LSD1 repress myeloid traits during megakaryocyte differentiation

The transcription factor Growth Factor Independence 1B (GFI1B) recruits Lysine Specific Demethylase 1 A (LSD1/KDM1A) to stimulate gene programs relevant for megakaryocyte and platelet biology. Inherited pathogenic GFI1B variants result in thrombocytopenia and bleeding propensities with varying intensity. Whether these affect similar gene programs is unknow. Here we studied transcriptomic effects of four patient-derived GFI1B variants (GFI1BT174N,H181Y,R184P,Q287*) in MEG01 megakaryoblasts. Compared to normal GFI1B, each variant affected different gene programs with GFI1BQ287* uniquely failing to repress myeloid traits. In line with this, single cell RNA-sequencing of induced pluripotent stem cell (iPSC)-derived megakaryocytes revealed a 4.5-fold decrease in the megakaryocyte/myeloid cell ratio in GFI1BQ287* versus normal conditions. Inhibiting the GFI1B-LSD1 interaction with small molecule GSK-LSD1 resulted in activation of myeloid genes in normal iPSC-derived megakaryocytes similar to what was observed for GFI1BQ287* iPSC-derived megakaryocytes. Thus, GFI1B and LSD1 facilitate gene programs relevant for megakaryopoiesis while simultaneously repressing programs that induce myeloid differentiation.

Transcriptomic dysregulation and autistic-like behaviors in Kmt2c haploinsufficient mice rescued by an LSD1 inhibitor.

Recent studies have consistently demonstrated that the regulation of chromatin and gene transcription plays a pivotal role in the pathogenesis of neurodevelopmental disorders. Among many genes involved in these pathways, KMT2C, encoding one of the six known histone H3 lysine 4 (H3K4) methyltransferases in humans and rodents, was identified as a gene whose heterozygous loss-of-function variants are causally associated with autism spectrum disorder (ASD) and the Kleefstra syndrome phenotypic spectrum. However, little is known about how KMT2C haploinsufficiency causes neurodevelopmental deficits and how these conditions can be treated. To address this, we developed and analyzed genetically engineered mice with a heterozygous frameshift mutation of Kmt2c (Kmt2c+/fs mice) as a disease model with high etiological validity. In a series of behavioral analyses, the mutant mice exhibit autistic-like behaviors such as impairments in sociality, flexibility, and working memory, demonstrating their face validity as an ASD model. To investigate the molecular basis of the observed abnormalities, we performed a transcriptomic analysis of their bulk adult brains and found that ASD risk genes were specifically enriched in the upregulated differentially expressed genes (DEGs), whereas KMT2C peaks detected by ChIP-seq were significantly co-localized with the downregulated genes, suggesting an important role of putative indirect effects of Kmt2c haploinsufficiency. We further performed single-cell RNA sequencing of newborn mouse brains to obtain cell type-resolved insights at an earlier stage. By integrating findings from ASD exome sequencing, genome-wide association, and postmortem brain studies to characterize DEGs in each cell cluster, we found strong ASD-associated transcriptomic changes in radial glia and immature neurons with no obvious bias toward upregulated or downregulated DEGs. On the other hand, there was no significant gross change in the cellular composition. Lastly, we explored potential therapeutic agents and demonstrate that vafidemstat, a lysine-specific histone demethylase 1 (LSD1) inhibitor that was effective in other models of neuropsychiatric/neurodevelopmental disorders, ameliorates impairments in sociality but not working memory in adult Kmt2c+/fs mice. Intriguingly, the administration of vafidemstat was shown to alter the vast majority of DEGs in the direction to normalize the transcriptomic abnormalities in the mutant mice (94.3 and 82.5% of the significant upregulated and downregulated DEGs, respectively, P < 2.2 × 10-16, binomial test), which could be the molecular mechanism underlying the behavioral rescuing. In summary, our study expands the repertoire of ASD models with high etiological and face validity, elucidates the cell-type resolved molecular alterations due to Kmt2c haploinsufficiency, and demonstrates the efficacy of an LSD1 inhibitor that might be generalizable to multiple categories of psychiatric disorders along with a better understanding of its presumed mechanisms of action.

Abstract 5630: Targeting the LSD1/ADNP axis as an effective pro-differentiating strategy against acute myeloid leukemia AML

Abstract Acute Myeloid Leukemia (AML) represents a diverse category of blood cancers marked by the clonal proliferation of atypical myeloid precursor cells within the bone marrow. Lysine Specific Demethylase 1 (LSD1), an epigenetic regulator, exhibits elevated expression in AML. Consequently, inhibiting LSD1 has emerged as a promising therapeutic approach for addressing blood and bone marrow cancers. While initial emphasis centered on LSD1's enzymatic inhibition, compelling evidence highlights its pivotal role as a scaffolding protein, orchestrating transcriptional and chromatin remodeling complexes that control cellular differentiation and proliferation. However, our understanding of the precise mechanisms governing these complexes remains limited, necessitating further investigation. The K562 erythroleukemia cell line has served as a prominent model for investigating the molecular factors influencing cell fate determination. In response to specific cues, these cells exhibit a bi-potential capacity, mirroring the behavior of normal hematopoietic megakaryocyte/erythrocyte progenitors (MEP). Notably, when LSD1 is either depleted or inhibited, the erythroid differentiation of K562 cells is hindered, even when exposed to various pro-differentiating triggers, including the BCR-ABL inhibitor Imatinib. In order to gain deeper insights and identify potential new protein partners involved in the K562 differentiation process that interact with LSD1, we employed the erythroleukemic K562 cell model. Using unbiased, proteome-wide proximity labeling we discovered that LSD1 serves as a scaffold for recruitment of the ChAHP complex, comprised of ADNP, CBX3, and CHD4. The ChAHP complex serves fundamental roles in chromatin remodeling, RNA splicing, and transcriptional control. We confirmed these proximity relationships by immunoblotting biotinylated proteins generated in situ by the promiscuous biotin ligase, (BirA*) when anchored to LSD1. We show that depleting ADNP induces spontaneous erythroid differentiation in K562 erythroleukemia cells, which is augmented by BCR-ABL inhibitor Imatinib treatment. ADNP-deficient K562 cells exhibited accelerated erythroid differentiation, as evidenced by a four-fold increase in the surface expression of erythroid markers (CD71, CD235a) and a significant upregulation of beta-globin gene cluster markers. These findings collectively suggest an antagonistic functional relationship between ADNP and LSD1, potentially counterbalancing LSD1's role in lineage allocation. Furthermore, we demonstrate that the loss of the key ChAHP component, ADNP, substantially hampers proliferation and increases apoptosis in K562 cells. These results underscore the potential of ADNP/ChAHP as an appealing target to modulate the choice between erythroid and megakaryocytic fates and offer promise as a target for pro-differentiation therapies in AML Citation Format: Mehraju Din Lone, Mohd Sayeed, Alaa Habieb, Praveen Kumar, Michael E. Engel. Targeting the LSD1/ADNP axis as an effective pro-differentiating strategy against acute myeloid leukemia AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5630.

Abstract 1690: LSD1 and CoREST2 demethylate STAT3 to promote enteroendocrine cell differentiation in mucinous colorectal cancer

Abstract Across different cancer types, tumor heterogeneity has been shown to drive tumor progression, metastasis, and therapeutic resistance. Adenocarcinoma to neuroendocrine lineage transition is an emergent mechanism of targeted therapy resistance in several cancer types, including lung and prostate cancer. Therefore, understanding the dynamics and mechanisms driving neuroendocrine cell fates in cancer is critical. Mucinous colorectal cancer accounts for upwards of 20% of colorectal cancer cases and is characterized by tumors with mucous accounting for at least 50% of the tumor volume. The normal colon epithelium consists of several specialized cell types, including hormone secreting enteroendocrine cells (EECs), which are the neuroendocrine cell of the intestine. We have previously shown that EEC progenitors are enriched in mucinous colorectal cancer and promote cancer cell survival via secreted factors. Additionally, we have shown that lysine specific demethylase 1 (LSD1) promotes EEC differentiation in these tumors; however, the mechanism by which LSD1 promotes EEC differentiation has remained unknown. Typically to carry out its enzymatic function, LSD1 must be a part of a transcriptional regulatory complex. One such complex is the CoREST complex, which contains LSD1, HDAC1, and one of three CoREST protein family members. Here we report that LSD1 and CoREST2 promote EEC differentiation by demethylating the transcription factor signal transducer and activator of transcription 3 (STAT3) to promote STAT3 chromatin binding. Additionally, we demonstrate that knocking down CoREST2 decreases tumor growth and lung metastases of mucinous colorectal cancer cells injected orthotopically into the colons of immunocompromised mice. Furthermore, we utilized single cell multi-omics that combines single cell RNA sequencing with single cell ATAC sequencing to show that during EEC differentiation there is an increase in chromatin accessibility at regulatory regions of known EEC-promoting transcription factors. Finally, through our single cell multi-omics analysis we identify a new rare cell type with neuron-like but not secretory characteristics. Mucinous colorectal cancer is an aggressive and chemotherapeutically intractable form of CRC that is enriched for EEC progenitors. Our data demonstrates that LSD1 and STAT3 promote EEC differentiation, suggesting that inhibiting LSD1 and or STAT3 may have benefits for patients with mucinous colorectal cancer. Citation Format: Christopher A. Ladaika, Ahmed H. Ghobashi, William C. Boulton, Heather M. O'Hagan. LSD1 and CoREST2 demethylate STAT3 to promote enteroendocrine cell differentiation in mucinous colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1690.

Abstract 307: Generation and characterization of a glioblastoma stem cell model with acquired resistance to LSD1 inhibition

Abstract Epigenetic modifications play an important role in the initiation of various cancer types, including glioblastoma (GBM), and in the development of therapeutic resistance. Patients with GBM, a common malignant brain tumor, have an average survival of 12-15 months after first-line therapy. Lysine-specific demethylase 1 (LSD1) is a histone demethylase that is overexpressed in glioblastoma stem cells (GSCs) and functions to promote proliferation, inhibit differentiation, and enhance motility. Previous work from our group has investigated LSD1 as a therapeutic target in glioblastoma and identified genes that may predict resistance to pharmacological LSD1 inhibitors using RNA-seq. Five upregulated genes, HKDC1, RAB3IL1, RAB39B, FTH1, and FAM213A, were associated with LSD1 inhibitor (LSD1i) resistance and were validated in brain tissue of a GBM model treated with GSK-LSD1 after succumbing to tumor burden. In order to determine whether selection pressure from LSD1 inhibitor treatment caused upregulation of these genes, we created a model of acquired resistance in GSC lines in vitro. Isogenic resistant lines were generated by treating MDA-GSC17 cells with increasing concentrations of LSD1i or diluent (DMSO), as tolerated starting at 50 uM (which was five fold below the IC50 for these lines). In parallel, MDA-GSC17 controls were exposed to DMSO. The resistant line was created over 4 weeks and monitored by cell number and viability to reach 90%. To assess the change in gene expression, we conducted quantitative PCR for the five genes in the parental and resistant MDA-GSC17 line and saw upregulation in 3 of the 5 genes. Two genes, HKDC1 and RAB3IL1, were unable to be assessed due to low basal expression. However, 3 genes were upregulated which are RAB39B, FTH1, and FAM213A. RAB39B is a Ras-analog in brain (RAB) small GTPase and a member of the RAS oncogene family, involved in vesicular trafficking. High expression of ferritin heavy chain 1 (FTH1) is correlated with high-grade gliomas and poor prognosis in glioblastoma. Lastly, the redox regulatory protein, FAM213A, can activate antioxidant proteins and elevated expression is associated with worse outcomes for AML patients. This suggests that modulating the RAS signaling pathways and specific redox pathways may overcome LSD1 resistance. Understanding the changes in gene expression as the cells acquire resistance to LSD1 inhibitors will help us understand the mechanism of resistance to LSD1 inhibitors in glioblastoma and to design new treatment strategies. Citation Format: Kareena H. Patel, Lea Stitzlein, Deokhwa Nam, Frederick Lang, Joy Gumin, Joya Chandra. Generation and characterization of a glioblastoma stem cell model with acquired resistance to LSD1 inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 307.