LSD1 Inhibitors Research Articles

A Quinquennial Review of Potent LSD1 Inhibitors Explored for the Treatment of Different Cancers, with Special Focus on SAR Studies.

Cancer bears a significant share of global mortality. The enzyme Lysine Specific Demethylase 1 (LSD1, also known as KDM1A), since its discovery in 2004, has captured the attention of cancer researchers due to its overexpression in several cancers like acute myeloid leukaemia (AML), solid tumours, etc</i>. The Lysine Specific Demethylase (LSD1) downregulation is reported to have an effect on cancer proliferation, migration, and invasion. Therefore, research to discover safer and more potent LSD1 inhibitors can pave the way for the development of better cancer therapeutics. These efforts have resulted in the synthesis of many types of derivatives containing diverse structural nuclei. The present manuscript describes the role of Lysine Specific Demethylase 1 (LSD1) in carcinogenesis, reviews the LSD1 inhibitors explored in the past five years and discusses their comprehensive structural activity characteristics apart from the thorough description of LSD1. Besides, the potential challenges, opportunities, and future perspectives in the development of LSD1 inhibitors are also discussed. The review suggests that tranylcypromine derivatives are the most promising potent LSD1 inhibitors, followed by triazole and pyrimidine derivatives with IC₅₀ values in the nanomolar and sub-micromolar range. A number of potent LSD1 inhibitors derived from natural sources like resveratrol, protoberberine alkaloids, curcumin, etc</i>. are also discussed. The structural-activity relationships discussed in the manuscript can be exploited to design potent and relatively safer LSD1 inhibitors as anticancer agents.

LSD1 inhibition by tranylcypromine hydrochloride reduces alkali burn-induced corneal neovascularization and ferroptosis by suppressing HIF-1α pathway.

Corneal neovascularization (CNV) is a sight-threatening condition that necessitates epigenetic control. The role of lysine-specific demethylase 1 (LSD1) in CNV remains unclear, despite its established significance in tumor angiogenesis regulation. An alkali burn-induced CNV mouse model was used in vivo. The effects of LSD1 inhibitor tranylcypromine hydrochloride (TCP) were examined through slit lamp, histological staining, and immunofluorescence. The expression of malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione (GSH) levels were assessed in corneal tissues. Oxidative stress and ferrous ion expression during CNV were determined using 4-HNE, GPX4, and FerroOrange staining. In vitro, a hypoxia-reoxygenation (H/R) model was established using human umbilical vein endothelial cells (HUVECs) to study LSD1 or hypoxia-inducible factor (HIF-1α) knockdown and lentiviral overexpression of HIF-1α. The effects on HUVECs migration, invasion, and angiogenesis were evaluated through cell scratching assay, transwell migration assay and tube formation assay. The role of ferroptosis was investigated using ROS staining, FerroOrange staining, and key ferroptosis proteins. Further, The JAK2/STAT3 pathway's involvement in CNV regulation was explored through in vivo experiments with subconjunctival injection of AG490. The results showed a substantial correlation between corneal damage and LSD1 levels. In addition, HIF-1α expression was also elevated after alkali burns, and subconjunctival injection of TCP reduced corneal inflammation and neovascularization. Corneal alkali burns increased ROS levels and reduced antioxidative stress indicators, accompanied by elevated ferrous ion levels, which were reversed by TCP injection. In vitro, TCP or siRNAs inhibited H/R-induced ferroptosis and angiogenesis in HUVECs by affecting specific protein expressions and MDA, SOD, and GSH levels. HIF-1α levels, associated with ROS production, ferroptosis, and angiogenesis, increased during H/R, but were reversed by TCP or siRNA administration. HIF-1α overexpression counteracted the effects of LSD1 inhibition. Additionally, AG490 injection effectively reduced HIF-1α and VEGFA expression in the CNV model. These findings suggest that LSD1 inhibition via the HIF-1α-driven pathway prevents angiogenesis, oxidative stress, and ferroptosis in corneal alkali burn-induced CNV, highlighting LSD1 as a potential therapeutic target.

Photocatalytic intermolecular bromonitroalkylation of styrenes: synthesis of cyclopropylamine derivatives and their evaluation as LSD1 inhibitors.

A mild and efficient method for photoredox-catalyzed bromonitroalkylation of alkenes is described herein. In this reaction, bromonitromethane serves as a source of both nitroalkyl and bromine for direct and regioselective formation of C-Br and C-C bonds from alkenes, and additional cyclization provides C-C bonds to the cyclopropylamine core as an LSD1 inhibitor.

LSD1 Regulates Neurogenesis in Human Neural Stem Cells Through the Repression of Human-Enriched Extracellular Matrix and Cell Adhesion Genes.

Neurogenesis begins with neural stem cells undergoing symmetric proliferative divisions to expand and then switching to asymmetric differentiative divisions to generate neurons in the developing brain. Chromatin regulation plays a critical role in this switch. Histone lysine-specific demethylase LSD1 demethylates H3K4me1/2 and H3K9me1/2 but the mechanisms of its global regulatory functions in human neuronal development remain unclear. We performed genome-wide ChIP-seq of LSD1 occupancy, RNA-seq, and Histone ChIP-seq upon LSD1 inhibition to identify its repressive role in human neural stem cells. Novel downstream effectors of LSD1 were identified, including the Notch signaling pathway genes and human-neural progenitor-enriched extracellular matrix (ECM) pathway/cell adhesion genes, which were upregulated upon LSD1 inhibition. LSD1 inhibition led to decreased neurogenesis, and overexpression of downstream effectors mimicked this effect. Histone ChIP-seq analysis revealed that active and enhancer markers H3K4me2, H3K4me1, and H3K9me1 were upregulated upon LSD1 inhibition, while the repressive H3K9me2 mark remained mostly unchanged. Our work identifies the human-neural progenitor-enriched ECM pathway/cell adhesion genes and Notch signaling pathway genes as novel downstream effectors of LSD1, regulating neuronal differentiation in human neural stem cells.

Unveiling Arformoterol as a potent LSD1 inhibitor for breast cancer treatment: A comprehensive study integrating 3D-QSAR pharmacophore modeling, molecular docking, molecular dynamics simulations and in vitro assays

Lysine Specific Demethylase 1 (LSD1) has been identified as a chromatin-modifying enzyme implicated in various cancer pathogeneses, highlighting the potential for novel epigenetic cancer treatments through the development of effective inhibitors. We employed 3D-QSAR pharmacophore modeling, molecular docking, and molecular dynamics simulations to identify a promising drug candidate for LSD1 inhibition. RMSD, RMSF, H-bond, and DSSP analysis demonstrated that ZINC02599970 (Arformoterol) and ZINC13453966 exhibited the highest LSD1 inhibitory potential.Experimental validation using MCF-7 and MDA-MB-231 cell lines revealed that Arformoterol displayed potent antiproliferative activity with IC50 values of 12.30 ± 1.48 μM and 19.69 ± 1.15 μM respectively. In contrast, the IC50 values obtained for the control (tranylcypromine) in exposure to MCF-7 and MDA-MB-231 cells were 104.6 ± 1.69 μM and 77 ± 0.67 μM, respectively. Arformoterol demonstrated greater LSD1 inhibitory potency in MCF-7 cells compared to MDA-MB-231 cells. Also, the expression of genes involved in chromatin rearrangement (LSD1), angiogenesis (VEGF1), cell migration (RORα), signal transduction (S100A8), apoptosis, and cell cycle (p53) were investigated. Arformoterol enhanced apoptosis and induced cell cycle arrest at the G2/M phase, both in MCF-7 and MDA-MB-231 cancer cells. Based on our findings, we propose that Arformoterol represents a promising candidate for breast cancer treatment, owing to its potent LSD1 inhibitory activity.

Uncovering New Conformational States of the Substrate Binding Pocket of LSD1 Potential for Inhibitor Design via Funnel Metadynamics.

Lysine-specific demethylase 1 (LSD1) is a promising therapeutic target for cancer therapy. So far, over 80 crystal structures of LSD1 in different complex states have been deposited in the Protein Data Bank, which are valuable resources for performing structure-based drug design. However, among all of the crystal structures of LSD1, the substrate binding pocket, which is the most efficient druggable site for designing LSD1 inhibitors at present, is very similar no matter whether LSD1 is in the apo or any holo forms, which is inconsistent with its versatile demethylase functions. To investigate whether the substrate binding pocket is rigid or exhibits other representative conformations different from the crystal conformations that are feasible for designing new LSD1 inhibitors, we performed funnel metadynamics simulations to study the conformation dynamics of LSD1 in the binding process of two effective LSD1 inhibitors (CC-90011 and 6X0, CC-90011 undergoing clinical trials). Our results showed that the entrance of the substrate binding pocket is very flexible. Two representative entrance conformations of LSD1 counting against binding with the substrate of histone H3 were detected, which may be used for structure-based LSD1 inhibitor design. Besides, alternative optimal binding modes and prebinding modes for both inhibitors were also detected, which depicted that the key interactions changed along with the binding process. Our results should provide great help for LSD1 inhibitor design.

Effect of the LSD1 inhibitor RN-1 on γ-globin and global gene expression during erythroid differentiation in baboons (Papio anubis).

Elevated levels of Fetal Hemoglobin interfere with polymerization of sickle hemoglobin thereby reducing anemia, lessening the severity of symptoms, and increasing life span of patients with sickle cell disease. An affordable, small molecule drug that stimulates HbF expression in vivo would be ideally suited to treat the large numbers of SCD patients that exist worldwide. Our previous work showed that administration of the LSD1 (KDM1A) inhibitor RN-1 to normal baboons increased Fetal Hemoglobin (HbF) and was tolerated over a prolonged treatment period. HbF elevations were associated with changes in epigenetic modifications that included increased levels of H3K4 di-and tri-methyl lysine at the γ-globin promoter. While dramatic effects of the loss of LSD1 on hematopoietic differentiation have been observed in murine LSD1 gene deletion and silencing models, the effect of pharmacological inhibition of LSD1 in vivo on hematopoietic differentiation is unknown. The goal of these experiments was to investigate the in vivo mechanism of action of the LSD1 inhibitor RN-1 by determining its effect on γ-globin expression in highly purified subpopulations of bone marrow erythroid cells enriched for varying stages of erythroid differentiation isolated directly from baboons treated with RN-1 and also by investigating the effect of RN1 on the global transcriptome in a highly purified population of proerythroblasts. Our results show that RN-1 administered to baboons targets an early event during erythroid differentiation responsible for γ-globin repression and increases the expression of a limited number of genes including genes involved in erythroid differentiation such as GATA2, GFi-1B, and LYN.

Inhibition of the lysine demethylase LSD1 modulates the balance between inflammatory and antiviral responses against coronaviruses.

Innate immune responses to coronavirus infections are highly cell specific. Tissue-resident macrophages, which are infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in patients but are inconsistently infected in vitro, exert critical but conflicting effects by secreting both antiviral type I interferons (IFNs) and tissue-damaging inflammatory cytokines. Steroids, the only class of host-targeting drugs approved for the treatment of coronavirus disease 2019 (COVID-19), indiscriminately suppress both responses, possibly impairing viral clearance. Here, we established in vitro cell culture systems that enabled us to separately investigate the cell-intrinsic and cell-extrinsic proinflammatory and antiviral activities of mouse macrophages infected with the prototypical murine coronavirus MHV-A59. We showed that the nuclear factor κB-dependent inflammatory response to viral infection was selectively inhibited by loss of the lysine demethylase LSD1, which was previously implicated in innate immune responses to cancer, with negligible effects on the antiviral IFN response. LSD1 ablation also enhanced an IFN-independent antiviral response, blocking viral egress through the lysosomal pathway. The macrophage-intrinsic antiviral and anti-inflammatory activity of Lsd1 inhibition was confirmed in vitro and in a humanized mouse model of SARS-CoV-2 infection. These results suggest that LSD1 controls innate immune responses against coronaviruses at multiple levels and provide a mechanistic rationale for potentially repurposing LSD1 inhibitors for COVID-19 treatment.

Epigenetic repression of Cend1 by lysine-specific demethylase 1 is essential for murine heart development

Epigenetic regulation of heart development remains incompletely understood. Here we show that LSD1, a histone demethylase, plays a crucial role in regulating cardiomyocyte proliferation during heart development. Cardiomyocyte-specific deletion of Lsd1 in mice inhibited cardiomyocyte proliferation, causing severe growth defect of embryonic and neonatal heart. Invivo RNA-seq and invitro functional studies identified Cend1 as a target suppressed by LSD1. Lsd1 loss resulted in elevated Cend1 transcription associated with increased active histone mark H3K4me2 at Cend1 promoter. Cend1 knockdown relieved the cell-cycle arrest and proliferation defect caused by LSD1 inhibition in primary rat cardiomyocytes. Moreover, genetic deletion of Cend1 rescued cardiomyocyte proliferation defect and embryonic lethality in Lsd1 null embryos. Consistently, LSD1 promoted the cell cycle of cardiomyocytes derived from human-induced pluripotent stem cells by repressing CEND1. Together, these findings reveal an epigenetic regulatory mechanism involving the LSD1-CEND1 axis that controls cardiomyocyte proliferation essential for murine heart development.

LSD1 inhibitor hinders the demethylation of FOXA1 to inhibit prostate cancer progression

Abnormal activation of androgen receptor (AR) occurs in prostate cancer (PC) progression and metastasis. Lysine-specific demethylase 1 (LSD1), the first demethylating enzyme, regulates AR-mediated transcriptional activity. Considering the effect of Forkhead box protein A1 (FOXA1) on the expression of AR, estrogen receptor (ER) and tumor suppressor genes, this study investigated the demethylation of FOXA1 upon treatment with LSD1 inhibitors and assessed the biological behaviors of PC cells. PC cells were cultured and infected with viruses. After transient transfection, CWR22-RV1-Cas9 cells were selected by puromycin with expression of LSD1 detected by Western blot. Apart from measurement of formaldehyde production, immunoprecipitation and chromatin immunoprecipitation (ChIP) were performed, followed by ATAC-seq detection, and Western blot. The data indicated the association between LSD1-binding sites and high levels of FOXA1. LSD1 inhibitor treatment resulted in a dramatic decline in overall FOXA1 binding, significantly reducing chromosomal accessibility and also increasing lysine-methylated FOXA1 level, but it failed to affect H3K4me2 levels at LSD1-FOXA1 occupied sites. Overexpression of LSD1-WT obtained reverse outcome. Besides, LSD1 inhibition diminished binding of FOXA1 and restored lysine-methylation of FOXA1 in methylation-deficient cells with mutant K270R. Moreover, silencing of LSD1 suppressed CWR22-RV1 tumor growth, resulting in increased H3K4me2 and decreased AR-FL/V7 gene expression. K270me is demethylated by LSD1. LSD1 inhibitor disrupts FOXA1 chromatin association, blocks FOXA1 K270-demethylation and hinders AR binding, thereby suppressing PC cell growth.

Abstract A061: The LSD1 inhibitor iadademstat shows preclinical efficacy in malignant peripheral nerve sheath tumor cells and synergistic effects in combination

Abstract Background: Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive and rare malignancies accounting for 10% of all tissue sarcomas. Around 50% develop in patients with neurofibromatosis type 1 carrying mutations in the NF1 gene and the other 50% occur sporadically. NF1 is a tumor suppressor gene and a negative regulator of RAS signaling pathway. However, mutation of the NF1 gene is not sufficient to drive MPNST and a series of further mutational events must accumulate in genes other than NF1, such as in CDKN2A and PRC2 complex components. Mutations in p53 or PTEN and/or mutations or copy number variations in oncogenes, such as EGFR or c-Met can also be detected in MPNST. Herein we explore the effects of LSD1 inhibition with iadademstat as a single agent in a panel of MPNST cell lines, both NF1-related and sporadic, and in combination with inhibitors of the MEK-ERK and PI3K/AKT signaling pathways. Materials and Methods: The cell viability in 12 MPNST cell lines, both commercial and derived from patients’ primary tumors, was assessed after 6 days of treatment with iadademstat as a single agent. Synergisms between iadademstat and the MEK inhibitor selumetinib or the PI3K inhibitor copanlisib were also evaluated after 6 days of treatment in three selected responsive cell lines and analyzed with Calcusyn and Combenefit software. Additionally, the effects of iadademstat in RAS pathways (pAKT and pERK levels) were also evaluated in these cell lines. Results: Iadademstat displayed sub-nanomolar activity as a single agent in 8 out of 12 cell lines tested, both NF1-related and sporadic, with different degrees of viability reduction. This was further confirmed with two additional LSD1 inhibitors, with iadademstat being the most potent inhibitor tested. Highly-responsive cell lines showed a decrease in pAKT levels while medium- and low-responsive cell lines displayed no significant differences. pERK levels were not affected. Moreover, the combination of iadademstat and selumetinib or copanlisib displayed strong synergy in cell viability assays in both commercial and patient-derived iadademstat-sensitive cell lines. Conclusions: The LSD1 inhibitor iadademstat is efficacious as single agent in nearly 70% of the MPNST cell lines tested, both NF1-related and sporadic and this efficacy increases synergistically in combination with selumetinib or copanlisib. Therefore, LSD1 inhibitors alone or in combination emerge as a potential treatment for MPNST, an indication with high unmet needs with no approved targeted therapies available. Citation Format: Natalia Sacilotto, Cristina Mascaró, Edgar Creus, Juana Fernández, Conxi Lázaro, Ana Limón, Robert Soliva, Jordi Xaus. The LSD1 inhibitor iadademstat shows preclinical efficacy in malignant peripheral nerve sheath tumor cells and synergistic effects in combination [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A061.

LSD1 Inhibition Enhances Antigen Presentation and Co-Stimulation in AML to Promote T Cell-Mediated Anti-Leukemia Immune Responses

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) can provide a curative treatment option for acute myeloid leukemia (AML). Donor T cells play an important role in the curative potential of allo-HSCT by recognizing and eliminating residual leukemic cells, inducing the desirable graft-versus-leukemia (GVL) effect. Evading from this effect of donor T cell immune surveillance through the downregulation of human leukocyte antigen class II (HLA-II) molecules has been shown to contribute to AML relapse after allo-HSCT. Lysine-specific demethylase 1 (LSD1) is an emerging epigenetic therapeutic target in AML due to its role in regulating myeloid differentiation pathways. We hypothesized that LSD1 inhibition has the potential to enhance anti-leukemic immune responses by differentiating AML cells and inducing the acquisition of markers typical for antigen-presenting cells. We have studied these effects by treating human AML cell lines with the LSD1 inhibitor bomedemstat and investigated the expression of antigen-presenting molecules by flow cytometry and quantitative PCR (qPCR). At a concentration of 100 nM, bomedemstat upregulated the expression of CIITA by a log2 fold change of 2.23 ± 0.68 (p &amp;lt; 0.01) in OCI-AML3, 2.17 ± 0.28 (p &amp;lt; 0.01) in HL60 and 1.80 ± 0.67 (p &amp;lt; 0.05) in MOLM-13. To assess if the observed upregulation of CIITA can lead to a re-expression of HLA-DR in these HLA-DR negative cell lines, we then treated these cell lines with 100 nM of bomedemstat with additional IFN-γ stimulation. Expression of HLA-DR and the co-stimulatory molecule CD86 was determined by flow cytometry. Bomedemstat increased the expression of HLA-DR and CD86 across all three cell lines leading to the generation of 26.63 ± 3.07% (p &amp;lt; 0.0001) HLA-DR +/CD86 + cells in OCI-AML3, 4.19 ± 1.68% (p &amp;lt; 0.001) HLA-DR +/CD86 + cells in HL-60 and 14.67 ± 2.03% (p &amp;lt; 0.001) HLA-DR +/CD86 + cells in MOLM-13. We further assessed the effects of bomedemstat treatment on cytokine production by qPCR. Notably, bomedemstat treatment increased the expression of CXCL-10 by a log2 fold change of 2.59 ± 0.66 (p &amp;lt; 0.001) in MOLM-13 and 4.12 ± 0.88 (p &amp;lt; 0.001) in HL-60 at 100 nM concentration. The upregulation of CXCL-10 was also validated using bead-based ELISA assays. To investigate the molecular mechanism of the observed concomitant upregulation of MHC-II molecules and CD86, we then used murine myeloid progenitor cells transformed by overexpression of Hoxa9 and Meis1 (H9M) or MN1. In H9M-transformed cells, bomedemstat concurrently upregulated MHC-II (I-A/I-E) and CD86 even in the absence of IFN-γ stimulation, leading to an increase in MHC-II +/CD86 + cells from 2.52 ± 1.22% at baseline to 29.95 ± 14.11 (p &amp;lt; 0.05). This effect was not observed in MN1-transformed cells. H9M-transformed progenitor cells from Irf8 knockout mice (H9M- Irf8-KO) did not show any upregulation of MHC-II, pointing towards a role for Irf8 in this process. To interrogate whether the concurrent upregulation of MHC-II and CD86 can enhance CD4 + T cell activation, we used ovalbumin-expressing H9M-transformed cells that were co-cultured with OT-II T cells after bomedemstat treatment. We could show that bomedemstat-treatment enhanced the ability of H9M-OVA cells to activate T cells as demonstrated by an enhanced expression of the T cell activation markers CD25 and CD69. Furthermore, bomedemstat also increased the immune killing of OVA-peptide pulsed H9M cells at an effector-to-target (E:T) ratio of 1:1 from 1.12 ± 4.01 to 19.70 ± 6.20% (p &amp;lt; 0.01). It also enhanced the killing of OVA-expressing H9M cells from 5.80 ± 2.95% to 17.70 ± 3.16% (p &amp;lt; 0.05). No significant killing effect was observed for MN1 cells. In conclusion, we demonstrate both phenotypically and functionally that LSD1 inhibition by bomedemstat can enhance the immunogenicity of human and murine AML models. These findings point towards a potential role of LSD1 inhibition as a maintenance therapy after allo-HSCT.

The LSD1 Inhibitor Ory-1001 (ladademstat) in Combination with Menin Inhibitor SNDX-5613 (revumenib) Has Synergistic in Vitro Activity in KMT2A-Rearranged AML Models

Introduction: Menin is a scaffold protein that binds to gene promoters, enhancers and transcription factors, thereby influencing transcription. Leukemias with rearrangement of KMT2A known as mixed-lineage leukemia (MLL), as well as other abnormalities such as NPM1c, respond to Menin inhibition with promising early clinical results. MLL leukemias affect children and adults and are associated with high rates of resistance to conventional chemotherapy. In addition to these alterations, other leukemia subsets with similar transcriptional dependency could be targeted through Menin inhibition. LSD1 is an essential regulator of murine MLL-AF9 leukemia stem cells (LSCs) and maintains the LSC potential of MLL-AML cells through blocking of differentiation and apoptosis ( Harris et al, 2012). LSD1 acts at genomic loci that are bound by MLL-AF9 to maintain expression of the associated leukemogenic program, thus preventing differentiation and apoptosis. We investigated the pre-clinical efficacy of combined Menin and LSD1 inhibition in AML. PC4 and SF2 interacting protein 1 (PSIP1)/p75, also known as LEDGF, interacts with the N-terminus of MLL and Menin. This complex contributes to the association of MLL and MLL-fusion multiprotein complexes with the chromatin. Several studies have shown that both PSIP1 and Menin are required for efficient MLL-fusion-mediated leukemogenesis and for the expression of MLL-regulated genes. A proposed model of LSD1 interaction with KMT2A-Menin-PSIP1 suprcomplex is depicted in Fig. 1A. Methods: To assess in vitro LSD1 inhibitor (ORY-1001) and Menin inhibitor (SNDX) potency, cell lines were plated and treated with the respective doses of ORY-1001, SNDX, and the combination of these drugs in 96-well plates. We performed synergy assays using increasing concentrations of SNDX and 3µM of ORY-1001. Cell viability and IC50 were measured after 4 days of treatment. The SynergyFinder 2.0 web application was used to calculate synergy scores and generate 2D synergy maps. Western blotting of LSD1, co-immunoprecipitation, and ChIP-qPCR studies were used to assess the depletion of the LSD1 from chromatin. The interaction network of LSD1 interactome proteins was assessed by mass spectrometry. Results: We showed that LSD1 degrades with increasing concentrations of ORY-1001 after 48h of treatment (Fig.1 B). IC 50 curves for ORY-1001, SNDX and synergy effect is shown with increasing concentrations of SNDX and 3µM ORY-1001 in MV4-11 cells (expressing MLL-AF4) (Fig.1 C). ORY-1001 alone shows an IC 50 of ~0.5 µM, and SNDX alone shows an IC 50 of ~10 nM. The combination of LSD1 and SNDX inhibitors synergistically reduced cell viability in MLL-rearranged MOLM-13 and MV4-11 cells (Fig.1 C). We also assayed the LSD1 endogenous protein interactome in MOLM-13 cells (expressing MLL-AF9) by mass spectrometry. Our LSD1 interactome analysis revealed an association of LSD1 with previously identified interactors such as RCOR1, HDAC1, HDAC2, CARM1 and HMG20B. We discovered an unexpected association between LSD1 and PSIP1. Interestingly, PSIP1 interact with MLL1-MEN1 complex and its crucial for assembling MLL-fusion proteins to their gene targets. Conclusion: LSD1 Inhibitor ladademstat in Combination with Menin Inhibitor revumenib has synergistic effect in KMT2A-Rearranged AML models. Surprisingly, we also found that PSIP1, which is essential for inducing MLL-rearranged leukemia, interacts with LSD1. This suggests that PSIP1 may modulate gene expression through its ability to interact with both MLL1 and LSD1. This study opens a new avenue to investigate the role of KMT2A-MEN1 in relation to other epigenetic modifiers, such as LSD1, and guide new potential combination therapies that may further potentiate differentiation and elimination of leukemia LSCs.

Effect of the Oral LSD1 Inhibitor Ory-1001 on F-Retics in Baboons ( Papio anubis)

Elevated levels of Fetal Hemoglobin (HbF) reduce the severity of symptoms and increase life span in patients with sickle cell disease (SCD). Experiments performed in baboons ( P. anubis) have been instrumental in discovering the potent pharmocological agents that increase HbF in vivo. Experimental baboon studies have been directly translated to clinical trials in patients with sickle cell disease. Drugs such as DNMT1 and LSD1 inhibitors that attack the repressive epigenetic regulatory mechanisms that silence expression of the γ-globin gene in adult erythroid cells increase HbF in baboons to levels necessary to provide therapeutic relief in SCD patients. Dose-dependent adverse events associated with the use of these drugs are minimized by modifications of dose and schedule as shown by a clinical trial of oral THU-decitabine in 25 SCD patients (Molokie et al, Plos Med 2017;14(9):e10022382). Our recent results have shown that subcutaneous administration of the DNMT1 inhibitor decitabine and the LSD1 inhibitor RN-1 2d/week produced synergistic increases in F-retics, F cells and γ-globin mRNA in normal baboons and increased HbF to therapeutic levels in phlebotomized anemic baboons after administration of only four doses (Ibanez et al, Blood Adv 2023;7(15):3891-3902). The goal of this current study is to develop an oral combinatorial drug regimen using THU-decitabine and the LSD1 inhibitor ORY-1001 that could be directly translated to clinical trials. Our previous studies demonstrated the effectiveness of oral THU-decitabine to increase HbF levels in baboons (Lavelle et al, Blood 2012;119(5):1240-1247). In this study we have performed 16 dose-response experiments in baboons to test the effect of oral ORY-1001 at doses ranging between 8 and 24µg/kg/d. ORY-1001 was chosen because it exhibited a good safety profile with known pharmacokinetic and pharmacodynamic parameters in a recent study in patients with relapsed/refractory acute myeloid leukemia (Salamero et al, J Clin Oncol 2020; 38(36):4260-4273). Four doses were tested (24µg/kg, n=2; 16µg/kg n=4; 12µg/kg, n-=7; 8µg/kg, n=3). The drug was administered on a single day to facilitate further combinatorial studies with THU-decitabine. Preliminary data from a current clinical trial of THU-decitabine in SCD patients (NCT04055818) suggests that administering the drug on a 1d per week schedule effectively increased HbF levels in patients. ORY-1001 was administered by gavage on a single day (d1) and the effect on F-retics measured by flow cytometry in peripheral blood samples obtained daily between days 6 and 10. The peak F-retic response was observed on d8. A linear dose-response relationship on F-retics was observed (Figure 1, r=0.79). Complete blood counts (CBC) were also performed daily between d6 and d10. Adverse hematological effects were not observed with no incidences of neutropenia or thrombocytopenia (Table 1). No consistent effects on reticulocyte counts were observed (Table 1). These results show that a single oral dose of ORY-1001 is sufficient to increase HBF in baboons without any adverse hematological effects and provide the necessary dose-response data to pursue further combinatorial studies with oral THU-decitabine

Phase 1/2a Trial of Bomedemstat with or without All- Trans Retinoic Acid (ATRA) in Advanced Myeloid Malignancies

Background: Acute myeloid leukemia (AML) is a devastating disease difficult to treat. Although &amp;gt; 60% of patients (pts) with AML treated with standard therapy achieve complete remission, at least 70% relapse. Overexpression of lysine-specific demethylase 1 (LSD1) mRNA and excess LSD1 protein have been observed in several cancer types, including progenitor populations of leukemic cells in AML. In preclinical studies, LSD1 inhibition impaired tumor growth and induced apoptosis of leukemic cells (Harris et al., 2012). In combination with ATRA, LSD1 inhibition enhanced survival in mouse models of AML (Shenck et al., 2014). In this first-in-human trial, we examine the safety, pharmacokinetics (PK), and pharmacodynamics (PD) of bomedemstat with or without ATRA in pts with high-risk AML and myelodysplastic syndrome (MDS). Methods: In this multicenter, open-label phase 1/2a trial, eligible pts were aged ≥18 years old, with high-risk AML or MDS, ECOG PS ≤2 (AML), and International Prognostic Score (IPSS) of at least intermediate-2 or Revised International Prognostic Score (IPSS-R) of at least intermediate (MDS). Pts could not have received immunotherapy within &amp;lt;8 weeks, chemotherapy within &amp;lt;3 weeks (except hydroxyurea), and radiation to &amp;gt;30% of marrow within &amp;lt;2 weeks of treatment. In the dose-finding phase, pts received bomedemstat at a starting dose of 0.75, 1.5, 3.0, or 6.0 mg/kg once-daily in 14-day cycles (7 days on, 7 days off), either alone or in combination with 45 mg/m 2ATRA for up to 4 cycles. Treatment was allowed to continue upon evidence of clinical benefit. In the duration-finding phase, pts received 6.0 mg/kg bomedemstat with ATRA in 21-day (14 days on, 7 days off) or 28-day (21 days on, 7 days off) cycles. Treatment continued until progression, unacceptable toxicity, or withdrawal. Primary endpoint was safety and tolerability. Secondary endpoints included PK parameters and PD effect on hematopoiesis. Clinical responses were documented per revised/modified Cheson criteria. Data cut-off date was December 13, 2018. Results: A total of 45 pts enrolled with a median age 73 years (range, 42 to 88); 19 (42%) were female. A total of 39 pts had AML with 27 (69%) having an ECOG PS score ≤1. Six pts had MDS with 2 (33%) pts having an IPSS of Very High Risk and IPSS-R of High Risk. A total of 33 pts were enrolled in the dose-finding phase (0.75 mg/kg/d bomedemstat [n=3], 1.5 mg/kg/d [n=6], 3.0 mg/kg/d [n=4], 6.0 mg/kg [n=6], 3.0 mg/kg + ATRA [n=5], 6.0 mg/kg + ATRA [n=9]). Twelve pts enrolled in the duration finding phase, 4 received bomedemstat plus ATRA for 14/28 days, and 8 received bomedemstat plus ATRA for 21/28 days. A total of 21 (47%) pts discontinued, 8 (18%) due to death, 5 (11%) due to withdrawal and 4 (9%) due to physician decision. Any grade adverse events (AE) related to bomedemstat occurred in 42 (93%) pts. The most common were diarrhea (42%), nausea (42%), and thrombocytopenia (38%). Grade ≥3 bomedemstat-related AEs occurred in 30 (66.7%) pts. One pt receiving 6.0 m/kg plus ATRA had a grade 5 AE related to bomedemstat due to respiratory failure. No patient experienced dose-limiting toxicity and no maximum tolerated dose was determined. The table summarizes PK parameters in whole plasma for the dose-finding phase. Based on plasma exposures, the Data Safety Monitoring Committee designated 6.0 mg/kg bomedemstat as the PD dose. Mean percent change (standard deviation) in blast counts was 368% (400) for pts receiving 0.75 mg/kg bomedemstat, 2478% (3279) for 1.5 mg/kg, 10% (25) for 3 mg/kg, 2841% (6047) for 6 mg/kg/d. For pts with AML, overall median EFS was 0.97 months (mo) (95% CI, 0.93-1.46), and median OS was 18.8 mo (95% CI, 3.86 to NR). The 6-mo rate for EFS and OS were 9% (95% CI, 0-46%) and 64% (95% CI, 25%-86%), respectively. The overall response rate was 28.2% (95% CI, 15.0-44.9, [1 PR, 10 SD]). For pts with MDS, median EFS was 0.93 mo (95% CI, 0.57 to NR) and median OS was NR (95% CI, 0.57 to NR). One pt with MDS had SD as best response (95% CI, 0.4-64.1). Conclusions: Bomedemstat up to 6.0 mg/kg alone or in combination with ATRA was well-tolerated in pts with high-risk AML or MDS. Change in blast count seen in treated pts could be due to differentiation to monocytes induced by bomedemstat rather than blast count reduction per se. Objective responses were seen in a subset of pts with AML (28%). OS was numerically higher to historical data with standard of care. Bomedemstat is currently under investigation in myeloproliferative neoplasms.

Differentiation of Acute Myeloid Leukemia Cells upon Pharmacological Inhibition of LSD1 Requires Its N-Terminal Intrinsically Disordered Region

Lysine-specific histone demethylase 1 (LSD1/KDM1A) governs hematopoiesis by regulating hematopoietic stem cell renewal and proper differentiation. Additionally, LSD1 is required for the maintenance of acute myeloid leukemia (AML) cells and has emerged as a key therapeutic target. Prior work from our group and others have revealed that the LSD1 demethylase activity is not required for AML proliferation. Instead, the interaction between LSD1 and GFI1/GFI1B is necessary to sustain AML cell survival. Thus, beyond its canonical demethylase activity, LSD1 has nonenzymatic, transcription factor (TF)-specific scaffolding functions critical for its function. Most LSD1 inhibitors function by blocking protein-protein interactions between LSD1 and TFs, even though they were originally designed as enzyme inhibitors. While studies indicate that LSD1 has important scaffolding functions, how LSD1 can coordinate with TFs to control gene expression programs is not well understood. Here, we used CRISPR-suppressor scanning to identify regions of LSD1 outside the active site that when mutated can lead to resistance to the covalent LSD1 inhibitor, GSK-LSD1. The understudied N-terminal intrinsically disordered region (IDR) of LSD1 was among the most enriched regions. To explore this further, we generated clonal AML cell lines harboring in-frame deletions within the IDR. These IDR-mutant cell lines were drug resistant and failed to differentiate upon GSK-LSD1 treatment. Moreover, knockdown of endogenous LSD1 followed by overexpression of LSD1 IDR-mutants rescued growth in the presence of GSK-LSD1, confirming that a fully functional IDR is necessary for differentiation. We performed LSD1 co-immunoprecipitation followed by mass spectrometry in wild-type and IDR-mutant cells to measure changes in LSD1 interactors upon GSK-LSD1 treatment. GSK-LSD1 treatment disrupted the LSD1-GFI1B interaction in both wild-type and mutant cells at comparable levels. Since GSK-LSD1 still induces dissociation of the mutant complex but has minimal effect on proliferation, our results suggest that the IDR-mutant cells no longer require GFI1B. In support, knockout of GFI1B inhibited growth of wild-type but not IDR-mutant cells. Strikingly, GSK-LSD1 treatment promoted LSD1's interaction with several master regulator TFs in hematopoiesis, including C/EBPα, PU.1, and RUNX1. Notably, C/EBPα was the most up-regulated interactor in both cell lines, with modest enrichment in IDR-mutant over wild-type. Using live-cell imaging assays, we found that deletions within the LSD1 IDR enhance association with these TFs. These results suggest that GSK-LSD1 reprograms LSD1-TF interactions, disrupting LSD1-GFI1B to induce LSD1-C/EBPα association, and that the IDR mutations alters this LSD1 redistribution to other TF partners. LSD1 is known to silence gene enhancers during differentiation in a process termed “enhancer decommissioning”. The mechanisms by which LSD1 occupies and controls enhancers remain elusive. To test if the LSD1 IDR regulates enhancer elements, we profiled LSD1 and H3K27ac using ChIP-seq in wild-type and IDR-mutant cells upon GSK-LSD1 treatment. Upon GSK-LSD1 treatment, disruption of LSD1-GFI1B binding was accompanied by increased LSD1 levels at other TF and H3K27ac sites, supporting the notion that loss of LSD1-GFI1B interaction induces LSD1 redistribution to other TF partner sites. A concomitant increase in H3K27ac levels was observed across LSD1 sites co-bound by C/EBPα, PU.1, or RUNX1 in wild-type cells. By contrast, in IDR-mutant cells, increases in H3K27ac levels were blocked at these sites upon GSK-LSD1 treatment, suggesting that full enhancer activation is impaired by mutations in the IDR, potentially due to altered association with master regulator TFs. This points to a function of LSD1 IDR in controlling enhancer activation for differentiation. Collectively, our study reveals a role for IDR in regulating LSD1-TF interactions, controlling enhancer activation that is necessary for AML cell differentiation. These data provide deep functional and mechanistic insights into the role of LSD1 IDR and offer important considerations for pharmacological targeting of protein-protein interactions.

Epigenetic Modulation Enhances the Therapeutic Potential of All-Trans Retinoic Acid in Acute Myeloid Leukemia

Epigenetic dysregulation has been strongly associated with the development and progression of acute myeloid leukemia (AML). While all-trans retinoic acid (ATRA), a derivative of vitamin A, has demonstrated significant anti-cancer effects in acute promyelocytic leukemia (APL-AML), its clinical efficacy in non-APL AMLs has been limited. This limited response can be attributed, in part, to the epigenetic silencing of genes targeted by or involved in the ATRA pathway, rendering them unresponsive to the drug. Consequently, our research aims to overcome the epigenetic barriers in AML by combining ATRA with small molecules that possess epigenetic modulating properties. To investigate the activation of the retinoic acid pathway in response to ATRA and a library of over 650 small molecules with epigenetic activity, we transduced HEK-293 cells with the p-GreenFire-RARE-Tk-Luc reporter construct. Through the screening process, we have identified 11 compounds that show promise as potential candidates for combination therapy with ATRA in non-APL AML. Notably, when combined with ATRA, two specific compounds, a dual PI3K HDAC inhibitor, and a pan-PKC inhibitor, demonstrated a significant 4-5 fold increase in RARE activity, indicating a synergistic effect. To determine the impact of selected compounds on the functional level, we evaluated the viability, differentiation, and phosphorylation status of ERK1/2, AKT1, MAPK9, p38, and MAPK9 in various cell lines, including HL-60, KG1a, BMNC, and primary MSC and AML cells. The dual PI3K HDAC inhibitor and a pan-PKC inhibitor were noncytotoxic to HL60, BMNC, and primary MSC. Interestingly, dual PI3K HDAC inhibitor with and without ATRA reduced the viability of KG1α by more than 95%. Moreover, dual PI3K HDAC inhibitor and a pan-PKC inhibitor, when combined with ATRA, lead to a 2-3 fold increase in the differentiation of HL60 cells and a 2-4 fold increase in the differentiation of AML cells ex vivo compared to ATRA in combination with the LSD1 inhibitor TCP (treatment control protocol) and untreated cells from AML subtypes M1, M2, and M4. PI3K HDAC inhibitor combined with ATRA significantly reduced p-MAPK and p-p38 levels while increasing the phosphorylation of p53 in HL-60 cells. Furthermore, RNA sequencing and chromatin profiling by Cleavage Under Targets and Tagmentation (CUT&amp;Tag) were performed to gain additional insights into the molecular mechanisms involved. Based on obtained results, the combination of ATRA with specific dual PI3K HDAC and pan-PKC inhibitors holds promise as a potential therapeutic approach for AML. Nevertheless, further investigations, including studies utilizing patient-derived xenograft models, are warranted to validate the efficacy and feasibility of this proposed treatment strategy.

Mutational Analysis of the Genome Stability Factor BOD1L1 in Myeloproliferative Neoplasm

The myeloproliferative neoplasms (MPN) are a group of clonal hematopoietic stem cell disorders that include essential thrombocythemia (ET), polycythemia vera (PV) and myelofibrosis (MF) which arise from somatic mutations in JAK2, MPL or CALR, but which can evolve to acute myeloid leukemia (AML) by the acquisition of additional somatic mutations. To investigate the contribution that mutations might make to the pathogenesis or clinical course of MPNs, 261 genes recurrently mutated in myeloid malignancies were sequenced in DNA from germline and blood samples from patients with AML, ET and MF enrolled in studies of the LSD1 inhibitor bomedemstat. Earlier work identified somatic mutations inthegene BOD1L1 in isolated megakaryocytes in 6 of 12 patients with MPN with mutations suggestive of loss-of-function (Guo et al., 2017). BOD1L1 plays a key role in multiple pathways of cellular DNA repair in conjunction with the histone methyltransferase SETD1A, which regulates transcription and DNA repair (Higgs et al. 2018; Hoshii et al. 2018. We previously demonstrated that BOD1L1 prevents uncontrolled resection of damaged DNA forks under conditions of replication stress, and that its loss leads to severe genome instability (Higgs et al., 2015, 2018). In addition, BOD1L1 promotes repair of DNA double-strand breaks after ionizing radiation through its interaction with the DNA repair protein RIF1 (Bayley et al., 2022). This sequencing-based analysis identified specific germline mutations in BOD1L1 that were 3-6 times more prevalent in MPN patients compared to a control population (gnomAD). This enrichment of BOD1L1 mutations was confirmed in whole-exome sequencing from an independent data set (EGA) of 183 patients with MPNs. We predicted these highly deleterious mutations, or at least a subset, may compromise the DNA repair functions of BOD1L1 and potentially influence the development and/or progression of MPN. To investigate this prediction, we disrupted BOD1L1 using a doxycycline-inducible CRISPR-Cas9 system in HeLa cells and complemented them with gRNA-resistant wild-type BOD1L1,or one of six mutants distributed throughout the protein. None of the mutants affected BOD1L1 protein expression; however, two of the six variants were defective for RIF1 binding by co-immunoprecipitation/proximity ligation, failed to recruit this key DNA repair factor to radiation-induced double-strand breaks, and compromised repair of these breaks. Moreover, single molecule DNA fiber analyses revealed that these same two mutants were unable to protect stalled replication forks against resection, leading to elevated levels of genomic damage. To explore the functional consequences of BOD1L1 loss-of-function on megakaryocytes, the gene was silenced using shRNA in human megakaryoblastic JAK2 V617F SET-2 cells, differentiated with thrombopoietin, and analyzed by flow cytometry. These cells were CD41-positive, consistent with megakaryocytic differentiation, and showed increased DNA damage (γH2AX) compared to controls. Depletion of BOD1L1 also lead to a greater number of cells with 2n nuclear content, and fewer with ≥8n, compared with controls, suggesting that the loss of BOD1L1 led to impaired polyploidization likely due to unrepaired DNA damage and subsequent cell death. In summary, we have shown that MPN patients have a high prevalence of mutations in the key genome instability factor BOD1L1 and that a subset of these mutations compromised its DNA repair functions and increased genome instability. The presence of BOD1L1 somatic mutations in megakaryocytes in MF patients may deregulate polyploidization, altering megakaryocyte function that may contribute to the development of bone marrow fibrosis. That no patients with BOD1L1 germline mutations progressed to AML in these clinical studies, coupled with the fact that these variants were not enriched in patients with AML, invites the possibility that BOD1L1 mutations may be synthetically lethal with other somatic mutations that result in genome instability and, hence, protect against progression to AML.

Repression of LSD1 potentiates homologous recombination-proficient ovarian cancer to PARP inhibitors through down-regulation of BRCA1/2 and RAD51

Poly (ADP-ribose) polymerase inhibitors (PARPi) are selectively active in ovarian cancer (OC) with homologous recombination (HR) deficiency (HRD) caused by mutations in BRCA1/2 and other DNA repair pathway members. We sought molecular targeted therapy that induce HRD in HR-proficient cells to induce synthetic lethality with PARPi and extend the utility of PARPi. Here, we demonstrate that lysine-specific demethylase 1 (LSD1) is an important regulator for OC. Importantly, genetic depletion or pharmacological inhibition of LSD1 induces HRD and sensitizes HR-proficient OC cells to PARPi in vitro and in multiple in vivo models. Mechanistically, LSD1 inhibition directly impairs transcription of BRCA1/2 and RAD51, three genes essential for HR, dependently of its canonical demethylase function. Collectively, our work indicates combination with LSD1 inhibitor could greatly expand the utility of PARPi to patients with HR-proficient tumor, warranting assessment in human clinical trials.

Inhibition of LSD1 induces ferroptosis through the ATF4-xCT pathway and shows enhanced anti-tumor effects with ferroptosis inducers in NSCLC

Lysine-specific demethylase 1 (LSD1) has been identified as an important epigenetic target, and recent advances in lung cancer therapy have highlighted the importance of targeting ferroptosis. However, the precise mechanisms by which LSD1 regulates ferroptosis remain elusive. In this study, we report that the inhibition of LSD1 induces ferroptosis by enhancing lipid peroxidation and reactive oxygen species (ROS) accumulation. Mechanistically, LSD1 inhibition downregulates the expression of activating transcription factor 4 (ATF4) through epigenetic modification of histone H3 lysine 9 dimethyl (H3K9me2), which sequentially inhibits the expression of the cystine–glutamate antiporter (xCT) and decreases glutathione (GSH) production. Furthermore, LSD1 inhibition transcriptionally upregulates the expression of transferrin receptor (TFRC) and acyl-CoA synthetase long chain family member 4 (ACSL4) by enhancing the binding of histone H3 lysine 4 dimethyl (H3K4me2) to their promoter sequences. Importantly, the combination of an LSD1 inhibitor and a ferroptosis inducer demonstrates an enhanced anti-tumor effect in a xenograft model of non-small cell lung cancer (NSCLC), surpassing the efficacy of either agent alone. These findings reveal new insights into the mechanisms by which LSD1 inhibition induces ferroptosis, offering potential guidance for the development of new strategies in the treatment of NSCLC.